U.S. patent application number 11/770456 was filed with the patent office on 2007-11-08 for detection of mutational frequency and related methods.
This patent application is currently assigned to The Government of the USA as Represented by the Secretary of the Dept of Health and Human Services. Invention is credited to Sachiko Kajigaya, Neal S. Young.
Application Number | 20070259370 11/770456 |
Document ID | / |
Family ID | 38337000 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070259370 |
Kind Code |
A1 |
Young; Neal S. ; et
al. |
November 8, 2007 |
DETECTION OF MUTATIONAL FREQUENCY AND RELATED METHODS
Abstract
Methods are provided herein for measuring the mutational
frequency of a DNA molecule in cells, for example stem cells or
hematopoietic cells such as CD34.sup.+ cells or granulocytes. The
method includes sequencing corresponding regions of mtDNA from a
set of hematopoietic cells, or a set of clonal populations of
hematopoietic cells, and comparing the sequence of the
corresponding regions of mtDNA from the cells, or clonal
populations of cells. The method also includes the comparison of
mtDNA sequences with genomic DNA sequences. Also provided are
methods for screening for an agent that has a mutagenic effect on a
cell. The method includes contacting, or treating, clonal
populations of cells with an agent and comparing the sequence of
the mtDNA obtained from the treated clonal populations of cells,
with the sequence of the corresponding region of mtDNA obtained
from a control clonal populations of cells.
Inventors: |
Young; Neal S.; (Washington,
DC) ; Kajigaya; Sachiko; (Rockville, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET
SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the USA as
Represented by the Secretary of the Dept of Health and Human
Services
|
Family ID: |
38337000 |
Appl. No.: |
11/770456 |
Filed: |
June 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10704283 |
Nov 6, 2003 |
7255993 |
|
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11770456 |
Jun 28, 2007 |
|
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60424515 |
Nov 6, 2002 |
|
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60463077 |
Apr 14, 2003 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of screening for an agent that increases the mutational
frequency of a hematopoietic cell, comprising: contacting isolated
hematopoietic cells with the agent to produce treated hematopoietic
cells, wherein the isolated hematopoietic cells each contain at
least one mitochondrion comprising mitochondrial DNA; determining
the mutational frequency of the treated hematopoietic cells using a
method of measuring a mutational frequency of a mitochondrial DNA
sequence, wherein measuring comprises: separately sequencing the
same regions of the mitochondrial DNA from individual, non-clonally
expanded treated hematopoietic cells or from individual, clonal
populations of the treated hematopoietic cells; and determining a
proportion of the treated hematopoietic cells exhibiting
mitochondrial DNA heterogeneity within the sequenced regions of the
mitochondrial DNA, wherein the proportion corresponds to the
mutational frequency of the mitochondrial DNA sequence, thereby
determining the mutational frequency of the treated hematopoietic
cells; and comparing the mutational frequency of the treated
hematopoietic cells to a mutational frequency of hematopoietic
cells that were not contacted with the agent, wherein an increase
in the mutational frequency of the treated hematopoietic cells,
compared to the mutational frequency of hematopoietic cells that
were not contacted with the agent, indicates that the agent
increases the mutational frequency of the hematopoietic cell,
thereby screening for the agent that increases the mutational
frequency of the hematopoietic cell.
2. The method of claim 1, further comprising: expanding individual
treated hematopoietic cells into individual clonal populations of
treated hematopoietic cells; extracting mitochondrial DNA from each
of the clonal populations of treated hematopoietic cells; and
determining a proportion of the clonal populations of treated
hematopoietic cells exhibiting mitochondrial DNA heterogeneity
within the sequenced regions of the mitochondrial DNA, wherein the
proportion corresponds to the mutational frequency of the
mitochondrial DNA sequence.
3. The method of claim 1, wherein the agent comprises a small
molecule, chemical compound, a radioisotope, a protein, a peptide,
or a peptidomimetic.
4. The method of claim 3, wherein the chemical compound comprises a
chemotherapeutic drug.
5. The method of claim 1, wherein isolated hematopoietic cells
comprise CD34.sup.+ cells, granulocytes, monocytes or
macrophages.
6. The method of claim 1, wherein the hematopoietic cells are
isolated from bone marrow, peripheral blood, or umbilical cord
blood.
7. The method of claim 1, wherein the mutation comprises a point
mutation, a polymorphism, a frame-shift mutation, a missense
mutation, a nonsense mutation, a silent mutation, or a deletion
mutation.
8. The method of claim 1, wherein the mutation is in a
homopolymeric C tract of a mitochondrial DNA control region or in a
gene in a mitochondrial DNA coding region.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No.
10/704,283, filed Nov. 6, 2003, which claims the benefit of U.S.
Provisional Application No. 60/424,515, filed Nov. 6, 2002, and
U.S. Provisional Application No. 60/463,077, filed Apr. 14, 2003,
which are all incorporated herein by reference.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to the field of detection of genetic
mutations, more specifically to the mutational frequency of genomic
and mitochondrial DNA (mt DNA) in hematopoietic cells, such as
hematopoietic stem cells.
BACKGROUND OF THE DISCLOSURE
[0003] The human mitochondrial genome is an approximately 16
kilobase circular, double stranded DNA that encodes 13 polypeptides
of the mitochondrial respiratory chain, 22 transfer RNAs, and two
ribosomal RNAs required for protein synthesis. Multiple copies of
mtDNA exist in a single cell.
[0004] The mitochondrial genome is particularly susceptible to
mutations. It is believed that the elevated mtDNA mutation rate is
a result of the high level of reactive oxygen species generated in
the organelle, coupled with a low level of DNA repair. Mutations in
the mtDNA sequence that affect all copies of mtDNA in an individual
are known as homoplasmic. Mutations that affect only some copies of
mtDNA are known as heteroplasmic, thus cells can contain a mixture
of both mutant and wildtype mtDNA species. Since multiple copies of
mtDNA exist in a single cell, a mtDNA mutation may only become
physiologically relevant once the particular mtDNA mutation
accumulates in the cell and exceeds a certain threshold.
[0005] Somatic cell mtDNA mutations have been intensely studied
because of their proposed involvement in the development of
diseases. For example, large heteroplasmic deletions of mtDNA have
been identified in muscle biopsies from patients suffering from a
relatively common mitochondrial myopathy. Missense and silent
mutations in the genes encoding cytochrome c oxidase subunits have
been shown to segregate with Alzheimer's disease and it has been
demonstrated that a high proportion of human tumors contain one or
more mtDNA mutations. It is known that the Pearson marrow-pancreas
syndrome involves hematopoietic, as well as pancreatic,
abnormalities arising from a defect of oxidative phosphorylation
which is associated with deletions in the mtDNA. Finally, it is
believed that the number of somatic mtDNA mutations in humans
increases with the age of the individual and thus mtDNA mutations
may be involved in the aging process.
[0006] Currently, there is no adequate method that allows the
measurement of the mutational frequency in the mitochondrial or
genomic (nuclear) DNA of intact animals, including humans. Thus,
there remains a need in the art to develop methods for measuring
and monitoring the mutational frequency of DNA in cells.
SUMMARY OF THE DISCLOSURE
[0007] It is now surprisingly revealed that mtDNA mutations can be
used to measure the mutational frequency of a mitochondrial DNA
sequence. For instance, the frequency can be determined by
measuring the number (or percent or proportion) of cells, or clonal
populations of cells, with at least one mutation in a corresponding
region of mtDNA within a set of test cells, or within a set of
clonal populations of test cells. The mtDNA mutations can also be
used to estimate (by correlation) the mutational frequency of a
genomic (nuclear) DNA sequence. It is also surprisingly revealed
that the measurement of a mtDNA mutational frequency can be used to
screen for agents that increase the mutational frequency of a cell,
such as a hematopoietic cell. In addition, the measurement of mtDNA
mutational frequency can be used to track mtDNA and genomic DNA
mutagenesis, for instance mutagenesis caused by disease,
therapeutic treatments, environmental exposure, and other
influences, using hematopoietic cells, such as CD34.sup.+ cells,
granulocytes, monocytes, or macrophages.
[0008] A first embodiment is a method of measuring the mutational
frequency of a mtDNA sequence, which method involves isolating a
set (for example two or more) of test cells (such as hematopoietic
cells), for example test cells from a subject; sequencing a region
of the mtDNA; and determining the proportion of test cells
exhibiting mtDNA heterogeneity within the sequenced region of the
mtDNA, thereby measuring the mutational frequency of the mtDNA
sequence in the subject.
[0009] In specific examples of this method, the cells are
hematopoietic cells, for exampleisolated CD34.sup.+ cells,
granulocytes, monocytes, or macrophages. In particular examples,
the cells are hematopoietic stem cells, such as CD34.sup.+ cells.
The hematopoietic cell is in some embodiments isolated from a bone
marrow aspirate, from umbilical cord blood, or from peripheral
blood.
[0010] In other examples, the cells are non-tumor cells, such as
skin cells or intestinal epithelial cells, particularly relatively
undifferentiated skin cells or intestinal epithelial cells.
[0011] In more specific embodiments, the cells are each expanded
into a separate clonal population of cells. In such an embodiment,
mtDNA is extracted from each of the clonal populations, a
corresponding region of the mtDNA of each population is sequenced,
and the proportion of clonal populations (or colonies) exhibiting
mtDNA heterogeneity within the sequenced region is determined.
Other specific, non-limiting examples of the method further involve
amplification of the mtDNA prior to determining the proportion of
cells possessing at least one mtDNA mutation that distinguishes it
from cells with a mtDNA sequence containing only polymorphisms.
[0012] In any of the provided methods, the subject from whom the
cells (for example the hematopoietic cells) are isolated can be a
subject who has a disease, or who is suspected of having a disease
in which mtDNA mutations are present or more prevalent. In other
embodiments, the subject has been subjected to a mutagenic
treatment, for instance a treatment that involves chemotherapy or
radiation, such that mutagenic damage from the treatment may be
assessed. Alternatively, the subject in some examples is exposed to
a man-made or a natural mutagenic agent, for instance an agent
found in the environment. Alternatively, a relative increase in
mtDNA mutations is used as an indicator that a subject has been
exposed to a suspected or known mutagenic agent, or than an
environment is contaminated with mutagens. In still other
embodiments, the subject has (or is suspected of having) a genetic
defect, for instance a defect in a component of a DNA repair
mechanism or DNA replication mechanism. Optionally, the subject has
(or is suspected of having) a genetic disease, such as Fanconi
anemia, Bloom's syndrome, or certain types of tumors of the colon.
Hence the method can be used as a diagnostic test to screen for or
supplement other diagnostic tests in the evaluation of a disease in
which mtDNA mutations have an increased prevalence.
[0013] Another embodiment is a method of estimating the mutational
frequency of a genomic DNA sequence, which method involves
isolating the cells (such as hematopoietic cells) from a subject;
sequencing a corresponding region of the mtDNA in multiple of the
isolated cells; determining the proportion of the cells exhibiting
mtDNA heterogeneity within the sequenced region; and correlating
the mutational frequency of the mtDNA to an estimated mutational
frequency of genomic DNA from the same subject, thereby estimating
the mutational frequency of the genomic DNA sequence. Optionally,
the cells are expanded into multiple clonal populations of cells.
In such an embodiment, mtDNA is extracted from each of the clonal
populations, a corresponding region of the mtDNA of each population
is sequenced, and the proportion of clonal populations (or
colonies) possessing at least one mtDNA mutation within the
sequenced region that distinguishes it from clonal populations with
a mtDNA sequence containing only polymorphisms is determined. Other
specific, non-limiting examples of the method further involve
amplification of the mtDNA prior to determining the proportion of
cells possessing at least one mtDNA mutation.
[0014] Yet another specific embodiment is a method of screening for
an agent that has a mutagenic effect on a cell. Examples of such
methods include contacting, or treating, theisolated cells with an
agent. A region of the mtDNA, for example the mtDNA control region
or another region in which mitochondrial mutations are known to
frequently occur, is sequenced. In a further embodiment, the mtDNA
is amplified prior to sequencing. The sequence of the mtDNA from
the treated cells (such as hematopoietic cells) is compared to the
sequence of a corresponding region of a control mtDNA from cells
that were not contacted with the agent, thereby determining a
proportion of treated cells exhibiting mtDNA heterogeneity in the
sequenced region of the mitochondrial DNA and comparing it to a
proportion of cells that were not contacted with the agent
exhibiting mtDNA heterogeneity in the sequenced corresponding
region in the control mitochondrial DNA. An increase in the
proportion of treated cells exhibiting mtDNA heterogeneity,
compared to the proportion of cells that were not contacted with
the agent, indicates that the agent increases the mutational
frequency of the cell. In one embodiment, isolated hematopoietic
cells are expanded into multiple clonal populations of
hematopoietic cells. In such an embodiment, the mtDNA is extracted
from the clonal populations of hematopoietic cells.
[0015] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic drawing of a linearized map, including
the location of certain functional domains, of a human mtDNA
control region.
[0017] FIG. 2 is a graph showing the proportion of mtDNA showing
heteroplasmic patterns in various donors. The proportion of
heterogenous mtDNA in CD34.sup.+ clones was 8.6% (n=8), 8.3% (n=3),
6.8% (n=7), 5.0% (n=5), 17.3% (n=13) and 5.6% (n=6) in donors 1
through 6, respectively (8.6.+-.4.5, mean .+-.SD).
[0018] FIG. 3 is a series of drawings and digital images. FIG. 3A
is a flow chart showing the steps involved in the isolation and
analysis of CD34.sup.+ cells and mononuclear cells from peripheral
blood. FIG. 3B is a schematic drawing of a mtDNA, including the
control region, the CO1 and Cytb genes, as well as a linearized map
and function location of the mtDNA control region between
nucleotide 16,024 and nucleotide 16569 and between nucleotide 1 and
nucleotide 576 (D-loop). FIG. 3C is a series of digital images
showing the number and morphology of circulating CD34.sup.+ clones
after 5 day-suspension culture. Each image shows a different grade:
image 1, less than 5 cells per well (grade 1); image 2, between 6
and 10 cells per well (grade 2); image 3, 11 to 20 cells per well
(grade 3); image 4, more than 21 cells per well (grade 4).
SEQUENCE LISTING
[0019] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. .sctn.1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0020] SEQ ID NO: 1 is an upstream PCR primer of an outer nested
primer pair for a control region of human mtDNA.
[0021] SEQ ID NO: 2 is a downstream PCR primer of an outer nested
primer pair for a control region of human mtDNA.
[0022] SEQ ID NO: 3 is an upstream PCR primer of an inner nested
primer pair for a control region of human mtDNA.
[0023] SEQ ID NO: 4 is a downstream PCR primer of an inner nested
primer pair for a control region of human mtDNA.
[0024] SEQ ID NO: 5 is an upstream sequencing primer for the
control region of human mtDNA.
[0025] SEQ ID NO: 6 is a downstream sequencing primer for the
control region of human mtDNA.
[0026] SEQ ID NO: 7 is an upstream sequencing primer for the
control region of human mtDNA.
[0027] SEQ ID NO: 8 is a downstream sequencing primer for the
control region of human mtDNA.
[0028] SEQ ID NO: 9 is an upstream sequencing primer for the
control region of human mtDNA.
[0029] SEQ ID NO: 10 is a downstream sequencing primer for the
control region of human mtDNA.
[0030] SEQ ID NO: 11 is an upstream PCR primer of an outer nested
primer pair for the Cytb gene of human mtDNA.
[0031] SEQ ID NO: 12 is a downstream PCR primer of an outer nested
primer pair for the Cytb gene of human mtDNA.
[0032] SEQ ID NO: 13 is an upstream PCR primer of an inner nested
primer pair for the Cytb gene of human mtDNA.
[0033] SEQ ID NO: 14 is a downstream PCR primer of an inner nested
primer pair for the Cytb gene of human mtDNA.
[0034] SEQ ID NO: 15 is an upstream PCR primer of an outer nested
primer pair for the CO 1 gene of human mtDNA.
[0035] SEQ ID NO: 16 is a downstream PCR primer of an outer nested
primer pair for the CO1 gene of human mtDNA.
[0036] SEQ ID NO: 17 is an upstream PCR primer of an inner nested
primer pair for the CO1 gene of human mtDNA.
[0037] SEQ ID NO: 18 is a downstream PCR primer of an inner nested
primer pair for the CO1 gene of human mtDNA.
[0038] SEQ ID NO: 19 is a downstream sequencing primer for the
control region of human mtDNA.
[0039] SEQ ID NO: 20 is an upstream sequencing primer for the Cytb
gene of human mtDNA.
[0040] SEQ ID NO: 21 is a downstream sequencing primer for the Cytb
gene of human mtDNA.
[0041] SEQ ID NO: 22 is an upstream sequencing primer for the CO1
gene of human mtDNA.
[0042] SEQ ID NO: 23 is a downstream sequencing primer for the CO1
gene of human mtDNA.
DETAILED DESCRIPTION
I. Abbreviations
[0043] AA amino acid
[0044] Ala Alanine
[0045] BM bone marrow
[0046] CB cord blood
[0047] CH constant heavy
[0048] CL constant light
[0049] CO1 cytochrome c oxidase 1
[0050] CSB conserved sequence block
[0051] C tract cytosine tract
[0052] Cytb cytochrome b
[0053] F forward primer
[0054] FACS fluorescence activated cell sorting
[0055] G grade
[0056] G-CSF granulocyte-colony stimulating factor
[0057] HVS hypervariable segment
[0058] I inner primer
[0059] Ile isoleucine
[0060] Leu leucine
[0061] M mutation
[0062] mtDNA mitochondrial DNA
[0063] NC nucleotide change
[0064] np nucleotide position
[0065] O outer primer
[0066] OH H-strand origin
[0067] P polymorphism
[0068] PB peripheral blood
[0069] PBS phosphate buffered saline
[0070] PCR polymerase chain reaction
[0071] PE phycoerythrin
[0072] PEf plating efficiency
[0073] PH1 major H-strand promoter
[0074] PL L-strand promoter
[0075] R reverse primer
[0076] SCF stem cell factor
[0077] TAS termination-associated sequence
[0078] TFB transcription factor binding site
[0079] Thr threonine
[0080] TPO thrombopoeitin
[0081] TS transition
[0082] TV transversion
[0083] Val valine
[0084] VH variable heavy
[0085] VL variable light
II. Terms
[0086] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0087] In order to facilitate review of the various embodiments of
the invention, the following explanations of specific terms are
provided:
[0088] Agent: Any substance, including, but not limited to, a
chemical compound, small molecule, peptide mimetic, peptide or
protein. An agent can be a mutagen and thus will have a mutagenic
effect on a cell by causing mutations in the mtDNA or the genomic
DNA of the cell.
[0089] Amplification: An increase in the amount of (number of
copies of) nucleic acid sequence, wherein the increased sequence is
the same as or complementary to the existing nucleic acid template.
An example of amplification is the polymerase chain reaction (PCR),
in which a biological sample collected from a subject is contacted
with a pair of oligonucleotide primers, under conditions that allow
for the hybridization (annealing) of the primers to nucleic acid
template in the sample. The primers are extended under suitable
conditions (though nucleic acid polymerization). If additional
copies of the nucleic acid are desired, the first copy is
dissociated from the template, and additional copies of the primers
(usually contained in the same reaction mixture) are annealed to
the template, extended, and dissociated repeatedly to amplify the
desired number of copies of the nucleic acid.
[0090] The products of amplification may be characterized by
electrophoresis, restriction endonuclease cleavage patterns,
hybridization, ligation, and/or nucleic acid sequencing, using
standard techniques.
[0091] Other examples of in vitro amplification techniques include
reverse-transcription PCR(RT-PCR), strand displacement
amplification (see U.S. Pat. No. 5,744,311); transcription-free
isothermal amplification (see U.S. Pat. No. 6,033,881); repair
chain reaction amplification (see WO 90/01069); ligase chain
reaction amplification (see EP-A-320 308); gap filling ligase chain
reaction amplification (see U.S. Pat. No. 5,427,930); coupled
ligase detection and PCR (see U.S. Pat. No. 6,027,889); and
NASBA.TM. RNA transcription-free amplification (see U.S. Pat. No.
6,025,134).
[0092] Animal: Living multicellular organisms, a category which
includes, for example, mammals and birds.
[0093] Antibody: Immunoglobulin (Ig) molecules and immunologically
active portions of Ig molecules, for instance, molecules that
contain an antigen binding site which specifically binds
(immunoreacts with) an antigen. In one embodiment the antigen is
CD34. Monoclonal and polyclonal immunoglobulins are encompassed by
the disclosure.
[0094] A naturally occurring antibody (for example, IgG) includes
four polypeptide chains, two heavy chains and two light chains
inter-connected by disulfide bonds. However, it has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a naturally occurring antibody. Thus, these
antigen-binding fragments are also intended to be designated by the
term "antibody". Examples of binding fragments encompassed within
the term antibody include (i) an Fab fragment consisting of the
variable light (VL), variable heavy (VH), constant light (CL) and
constant heavy (CH)1 domains; (ii) an Fd fragment consisting of the
VH and CH1 domains; (iii) an Fv fragment consisting of the VL and
VH domains of a single arm of an antibody, (iv) a dAb fragment
(Ward et al., (1989) Nature 341:544-546) which consists of a VH
domain; and (v) an F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region. Furthermore, although the two domains of the Fv
fragment are coded for by separate genes, a synthetic linker can be
made that enables them to be made as a single protein chain (known
as single chain Fv (scFv); Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883) by
recombinant methods. Such single chain antibodies, as well as dsFv,
a disulfide stabilized Fv (Bera et al. (1998) J. Mol. Biol.
281:475-483), and dimeric Fvs (diabodies), that are generated by
pairing different polypeptide chains (Holliger et al. (1993) Proc.
Natl. Acad. Sci. 90:6444-6448), are also included.
[0095] In one embodiment, antibody fragments for use in this
disclosure are those which are capable of cross-linking their
target antigen, for example, bivalent fragments such as
F(ab').sub.2 fragments. Alternatively, an antibody fragment which
does not itself cross-link its target antigen (for example, a Fab
fragment) can be used in conjunction with a secondary antibody
which serves to cross-link the antibody fragment, thereby
cross-linking the target antigen. Antibodies can be fragmented
using conventional techniques and the fragments screened for
utility in the same manner as described for whole antibodies. An
antibody is further intended to include humanized monoclonal
molecules that specifically bind the target antigen.
[0096] "Specifically binds" refers to the ability of individual
antibodies to specifically immunoreact with an antigen. This
binding is a non-random binding reaction between an antibody
molecule and the antigen. In one embodiment, the antigen is CD34.
Binding specificity is typically determined from the reference
point of the ability of the antibody to differentially bind the
antigen of interest and an unrelated antigen, and therefore
distinguish between two different antigens, particularly where the
two antigens have unique epitopes. An antibody that specifically
binds to a particular epitope is referred to as a "specific
antibody".
[0097] A variety of methods for attaching detectable labels to
antibodies are well known in the art. Detectable labels useful for
such purposes are also well known in the art, and include
radioactive isotopes such as .sup.32P, fluorophores,
chemiluminescent agents, and enzymes.
[0098] Antigen: Any molecule that can bind specifically with an
antibody. An antigen is also a substance that antagonizes or
stimulates the immune system to produce antibodies. Antigens are
often foreign substances such as allergens, bacteria or viruses
that invade the body.
[0099] Binding or stable binding: An oligonucleotide binds or
stably binds to a target nucleic acid if a sufficient amount of the
oligonucleotide forms base pairs or is hybridized to its target
nucleic acid, to permit detection of that binding. Binding can be
detected by either physical or functional properties of the
target:oligonucleotide complex. Binding between a target and an
oligonucleotide can be detected by any procedure known to one
skilled in the art, including both functional and physical binding
assays. Binding may be detected functionally by determining whether
binding has an observable effect upon a biosynthetic process such
as expression of a coding sequence, DNA replication, transcription,
amplification and the like.
[0100] Physical methods of detecting the binding of complementary
strands of DNA or RNA are well known in the art, and include such
methods as DNase I or chemical footprinting, gel shift and affinity
cleavage assays, Northern blotting, dot blotting and light
absorption detection procedures. For example, one method that is
widely used, because it is so simple and reliable, involves
observing a change in light absorption of a solution containing an
oligonucleotide (or an analog) and a target nucleic acid at 220 to
300 nm as the temperature is slowly increased. If the
oligonucleotide or analog has bound to its target, there is a
sudden increase in absorption at a characteristic temperature as
the oligonucleotide (or analog) and target disassociate from each
other, or melt.
[0101] The binding between an oligomer and its target nucleic acid
is frequently characterized by the temperature (T.sub.m) (under
defined ionic strength and pH) at which 50% of the target sequence
remains hybridized to a perfectly matched probe or complementary
strand. A higher (T.sub.m) means a stronger or more stable complex
relative to a complex with a lower (T.sub.m).
[0102] Bloom's syndrome: An autosomal recessive genetic disease
caused by mutation of the BLM gene (GenBank Accession No.
NM.sub.--000057). Patients with this disease show a range of
symptoms which include a small body size, sun-sensitive facial
reddening, sub- or infertility, immunodeficiency and a
predisposition to the full range of human cancers. Cells from
patients with Bloom's syndrome are genomically unstable and show
elevated levels of both homologous recombination and sister
chromatid exchange. Cells that are defective for BLM also show DNA
replication defects. The BLM gene encodes a protein that displays
3' to 5' DNA helicase activity and promotes branch migration of
Holliday junctions.
[0103] CD34: A cell surface antigen formerly known as hematopoietic
progenitor cell antigen 1, and MY10, is a known marker of human
hematopoietic stem cells. The human CD34 gene, which maps to
chromosome 1q32, spans 26 kb and has 8 exons. CD34 is a 67 kDa
transmembrane glycoprotein. CD34 is expressed selectively on human
hematopoietic progenitor cells. The biological function of CD34 is
still unknown.
[0104] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory
sequences. cDNA may also contain untranslated regions (UTRs) that
are responsible for translational control in the corresponding RNA
molecule. cDNA is usually synthesized in the laboratory by reverse
transcription from messenger RNA extracted from cells or other
samples.
[0105] Clonal population of cells: A group of genetically identical
cells all descended from a single cell. A clone of cells. For
example, such a clonal population may include five or fewer cells,
at least 2 cells, at least 5 cells, at least 10 cells, at least 20
cells, at least 100 cells, at least 200 cells, at least 500 cells,
at least 1000 cells or at least 10,000 cells. In particular
examples, the clonal population includes at least a sufficient
number of cells to be informative about the presence of the mtDNA
mutation.
[0106] Corresponding regions of mtDNA: A region located between the
same two nucleotide positions in more than one mtDNA sequence. For
example, the corresponding region of HVS 2 located between
nucleotide position 57 and nucleotide position 372 in a first mtDNA
sequence is the HVS 2 located between nucleotide position 57 and
nucleotide position 372 in a second mtDNA sequence.
[0107] Fanconi anemia (FA): An inherited anemia that leads to bone
marrow failure (aplastic anemia). It is a recessive disorder, thus
if both parents carry a mutation in the same FA gene, each of their
children has a 25% chance of inheriting both defective genes and
acquiring the disease.
[0108] There are at least seven FA genes: A (GenBank Accession No.
NM.sub.--000135), C (GenBank Accession No. NM.sub.--000136), D2
(GenBank Accession No. NM.sub.--033084), E (GenBank Accession No.
NM.sub.--021922), F (GenBank Accession No. NM.sub.--022725), G
(GenBank Accession No. NM.sub.--004629) and BRAC2 (GenBank
Accession No.). Six of these genes have been cloned. These six
account for more than 85% of the cases of FA. Specifically,
mutations in FA-A and FA-C account for FA in 76% of patients
worldwide.
[0109] FA occurs equally in males and females and is found in all
ethnic groups. Although it is considered primarily a blood disease,
it may affect all systems of the body. Many patients eventually
develop acute myelogenous leukemia. Older patients are extremely
likely to develop head and neck, esophogeal, gastrointestinal,
vulvar, or rectal tumors.
[0110] Fluorphores: Chemical compounds, which when excited by
exposure to a particular wavelength of light, emit light (for
instance, fluoresce), for example at a different wavelength than
that to which they were exposed.
[0111] Also encompassed by the term "fluorophore" are luminescent
molecules, which are chemical compounds which do not require
exposure to a particular wavelength of light to fluoresce;
luminescent compounds naturally fluoresce. Therefore, the use of
luminescent signals eliminates the need for an external source of
electromagnetic radiation, such as a laser. An example of a
luminescent molecule includes, but is not limited to, aequorin
(Tsien, Ann. Rev. Biochem. 67:509, 1998).
[0112] Hematopoiesis: The formation and development of blood cells.
Hematopoiesis involves the proliferation and differentiation of
hematopoietic cells from stem cells. In adult mammals,
hematopoiesis is known to occur in bone marrow. Mammalian
hematopoietic cells are an extraordinarily diverse collection of
cell types, including B cells, T cells, granulocytes, macrophages,
megakaryocytes and erythroid cells, and each cell type has a unique
morphology and function. Despite the diversity of the nature,
morphology, characteristics and function of hematopoietic cells, it
is believed that there exists a single progenitor, known as a
pluripotent hematopoietic stem cell, which is capable of
self-renewal as well as the generation of committed progenitors
that give rise to mature hematopoietic cells.
[0113] A human hematopoietic stem cell has been identified that is
a CD34.sup.+ cell, in that it expresses CD34 on its surface. The
CD34.sup.+ cell population constitutes only a small percentage of
the total number of hematopoietic cells in adult human bone marrow,
however CD34.sup.+ cells are more abundant in umbilical cord
blood.
[0114] Hematopoietic cell: A blood cell, for example a cell
involved in or produced by hematopoiesis. Hematopoietic cells can
be, for example, red blood cells (erythrocytes) or white blood
cells (leukocytes). Cells useful in the disclosed methods are blood
cells that carry mtDNA, for example hematopoietic stem cells and
leukocytes (such as granulocytes). There are five main types of
white blood cell, subdivided between two main groups:
polymorphonuclear leukocytes, or granulocytes, (neutrophils,
eosinophils, basophils) and mononuclear leukocytes (monocytes and
lymphocytes). There are two main classes of lymphocytes: B cells
and T cells. A third class of lymphocytes includes Natural Killer
(NK) cells. Cytotoxic T lymphocytes (CTL), helper T cells, and NKT
cells are types of T cells. Also included among hematopoietic cells
are megakaryocytes, which produce platelets (a particle found in
the bloodstream that binds to fibrinogen at the site of a wound to
begin the blood clotting process), and macrophages, which arise
from monocytes and have phagocytic activity.
[0115] Mammalian blood cells provide for an extraordinarily diverse
range of activities. The blood cells are divided into several
lineages, including lymphoid, myeloid and erythroid. The lymphoid
lineage, comprising B cells and T cells, provides for the
production of antibodies, regulation of the cellular immune system,
detection of foreign agents in the blood, detection of cells
foreign to the host, and the like. The myeloid lineage, which
includes monocytes, granulocytes, megakaryocytes as well as other
cells, monitors for the presence of foreign bodies in the blood
stream, provides protection against neoplastic cells, scavenges
foreign materials in the blood stream, produces platelets, and the
like. The erythroid lineage provides the red blood cells, which act
as oxygen carriers. Despite the diversity of the nature,
morphology, characteristics and function of the blood cells, there
appears to be a single progenitor, which is capable of self
regeneration and by exposure to growth factors becomes dedicated to
a specific lineage.
[0116] Recently, the mouse stem cell has been obtained in at least
highly concentrated, if not a purified form, where fewer than about
30 cells obtained from bone marrow were able to reconstitute all of
the lineages of the hematopoietic system of a lethally irradiated
mouse. Indeed, one injected cell should be able to reconstitute all
of the hematopoietic lineages. A human stem cell has also been
identified that is a CD34.sup.+Thy1.sup.+lin.sup.- cell. This stem
cell population constitutes only a small percentage of the total
number of leukocytes in bone marrow. In particular, B cells
(CD19.sup.+) and myeloid cells (CD33.sup.+) make up 80-90% of the
CD34.sup.+ population. Moreover, a combination of CD3, 8, 10, 15,
19, 20, and 33 will mark >90% of all CD34.sup.+ cells.
[0117] Hereditary colon cancer: Genetic disease that often features
colonic polyps or growths that eventually become cancerous. There
are several kinds of colon cancer, including familial adenomatous
polyposis (FAP) and a variant called Gardner's syndrome. Another
type of colon cancer, hereditary non-polyposis colon cancer
(HNPCC), features few, if any, polyps.
[0118] Genes found to be involved in colorectal cancer include MSH2
(GenBank Accession No. NM.sub.--000251), MSH6 (GenBank Accession
No. NM.sub.--000179) (both on chromosome 2) and MLH1 (GenBank
Accession No. NM.sub.--000249) (on chromosome 3). Normally, the
protein products of these genes help to repair errors in DNA
replication, however mutations in these genes can prevent DNA
repair, eventually leading to colon cancer.
[0119] Heteroplasmy: Mutations that affect only some copies of
mtDNA. Thus, cells that are heteroplasmic can contain a mixture of
both mutant and wildtype mtDNA species. A tissue that is
heteroplasmic can contain a mixture of both mutant and wildtype
mtDNA species in one or more cells of the tissue.
[0120] Homoplasmy: Mutations in the mtDNA sequence that affect all
copies of mtDNA in a cell (a cell is homoplasmic) or in a tissue (a
tissue is homoplasmic).
[0121] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or portion of a tissue) that has
been substantially separated or purified away from other biological
components in the cell of the organism in which the component
naturally occurs. An "isolated" cell is a cell that has been
purified from the other cellular components of a tissue. An
"isolated" population of cells is a population of cells that has
been purified from other populations of cells in a tissue. For
example, a population of hematopoietic cells is a population of
cells that has been substantially purified from other cell types
and/or cellular components in a tissue. Cells can be isolated by,
for instance mechanical and/or enzymatic methods.
[0122] In one embodiment, an isolated population of cells includes
greater than about 95%, or greater than about 99%, of the cells of
interest. In another embodiment, an isolated population of cells is
one in which no other cells of a different phenotype or genotype
can be detected. In a further embodiment, an isolated population of
cells is a population of cells that includes less than about 5%, or
less than about 1% of a cells of a different phenotype than the
cells of interest.
[0123] Nucleic acids and proteins that have been "isolated" include
nucleic acids and proteins purified by standard purification
methods. The term also embraces nucleic acids and proteins prepared
by recombinant expression in a host cell as well as chemically
synthesized nucleic acids.
[0124] Labeled: A biomolecule, such as a specific binding agent,
attached covalently or noncovalently to a detectable label or
reporter molecule. Typical labels include radioactive isotopes,
enzyme substrates, co-factors, ligands, chemiluminescent or
fluorescent agents, haptens, and enzymes. Methods for labeling and
guidance in the choice of labels appropriate for various purposes
are discussed, for example, in Sambrook et al., Molecular Cloning:
A Laboratory Manual, CSHL, New York, 1989 and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publ. Assoc. and
Wiley-Intersciences, 1998.
[0125] Lineage specific marker: A marker that is expressed by a
specific population of cells. In one embodiment, the cells are
hematopoietic cells expressing the cell surface marker CD34. In
other embodiments, the cells are hematopoietic cells expressing,
but not limited to, the CD13, CD14, CD15, CD33, or CD66 cell
surface markers. Cocktails of antibodies that bind lineage specific
markers have been produced.
[0126] Leukocyte: Cells in the blood, also termed "white cells,"
that are involved in defending the body against infective organisms
and foreign substances. Leukocytes are produced in the bone marrow.
There are five main types of white blood cell, subdivided between
two main groups: polymorphonuclear leukocytes, or granulocytes,
(neutrophils, eosinophils, basophils) and mononuclear leukocytes
(monocytes and lymphocytes). When an infection is present, the
production of leukocytes increases.
[0127] Lymphocytes: A type of white blood cell that is involved in
the immune defenses of the body. There are two main types of
lymphocytes: B cell and T cells. A third class of lymphocytes is
Natural Killer (NK) cells. Cytotoxic T lymphocytes (CTL), helper T
cells, and NKT cells are types of T cells.
[0128] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
[0129] Mitochondrial DNA heterogeneity: Any change of the mtDNA
sequence of a cell, or clonal population of cells, that is due to a
mutation (as distinct from any polymorphism) within a cell, when
compared to known mtDNA sequence. A cell, or a clonal population of
cells, with mitochondrial DNA heterogeneity has at least one
mutation in the mtDNA, that distinguishes it from a cell, or a
clonal population of cells, with a mtDNA sequence containing only
polymorphisms. In one specific embodiment, mitochondrial
heterogeneity is the divergence of the mtDNA sequence of an
individual cell from the mtDNA sequence in the general population.
Methods of detecting such a divergence, for example by comparison
to a public database, are disclosed in U.S. Pat. No. 6,344,322. In
another specific embodiment, mitochondrial heterogeneity is the
sequence variation in an individual cell, compared to the sequence
of a bulk, aggregate, or total, sample.
[0130] Mitochondrion: An organelle bound by a double membrane in
which the reactions of the Krebs cycle and electron transport chain
take place, resulting in the formation of ATP, CO.sub.2, and water
from acetyl CoA and ADP. Mitochondria are the organelles in which
most of the ATP of the eukaryotic cell is produced. Mitochondria
have their own DNA, mitochondrial DNA (mtDNA), and are thought to
have evolved when an early eukaryote engulfed some primitive
bacteria, but instead of digesting them, harnessed them to produce
energy. Offspring inherit their mothers' mitochondria, and thus
mtDNA has been useful in tracing human lineages.
[0131] The human mitochondrial genome is an approximately 16
kilobase circular, double stranded DNA that encodes 13 polypeptides
of the mitochondrial respiratory chain, 22 transfer RNAs, and two
ribosomal RNAs required for protein synthesis. The human
mitochondrial genome contains a 1.1 kilobase noncoding control
region, or D-loop. The D-loop is a stable intermediate formed
during the replication of double-stranded mtDNA. Most of the sites
within the control region do not vary among humans, however, the
human mitochondrial genome has two hypervariable regions that are
particularly susceptible to mutation and are hotspots for both
germline and somatic mutations (Stoneking, Am. J. Hum. Genet.
67:1029-1032, 2000). Hypervariable segment (HSV) 1 is located
between nucleotides 16024 and 16383, whereas HSV2 is located
between nucleotides 57 and 372, of the mtDNA control region
(between nucleotides 16,024 and 576), according to the reference
sequence presented in Anderson et al. (Nature, 290:457-465, 1981)
(see FIG. 1 and Table 1). TABLE-US-00001 TABLE 1 Map position of
the genes in the mtDNA control region Map Position Description
(function) Shorthand (nucleotide position) Hypervariable segment 1
HV1 16,024-16,383 Hypervariable segment 2 HV2 57-372 7S DNA 7S
16,106-191 H-strand origin OH 110-441 Termination-associated
sequence TAS 16,157-16,172 Control element mt5 16,194-16,208
L-strand control element mt3L 16,499-16,506 mt4 H-strand control
element mt4H 371-379 mt3 H-strand control element mt3H 384-391
Conserved sequence block 1 CSB1 213-235 Conserved sequence block 2
CSB2 299-315 Conserved sequence block 3 CSB3 346-363 mtTF1 binding
site 1 TFB1 233-260 mtTF1 binding site 2 TFB2 276-303 mtTF1 binding
site 3 TFB3 418-445 mtTF1 binding site 4 TFB4 523-550 Replication
primer PR 317-321 L-strand promoter PL 392-445 Major H-strand
promoter PH1 545-567 Abbreviations: mtTF1, mitochondrial
transcription factor
[0132] Other regions within the mtDNA control region include (but
are not limited to) a homopolymeric cytosine (C) tract located
between nucleotides 303 and 315 in HVS2, the H-stand origin (OH,
between nucleotides 110 and 441), the conserved sequence block
(CSB, between nucleotides 213 and 235, 299 and 315, 346 and 363),
the mt5 control element (between nucleotides 16,194 and 16,208),
the L-strand control element (mt3L, between nucleotides 16,499 and
16,506), the termination-associated sequence (TAS, between
nucleotides 16,157 and 16,172), the L-strand promoter (PL, between
nucleotides 392 and 445), the major H-strand promoter (PH1, between
nucleotides 545 and 567), the mitochondrial transcription factor
binding site (TFB, between nucleotides 233 and 260, 276 and 303,
418 and 445, 523 and 550), the H-strand control elements (mt4H,
between nucleotides 371 and 379, and mt3H, between nucleotides 384
and 391) and 7S DNA (between nucleotides 16,106 and 191).
[0133] Mutagen: An agent that gives rise to mutations in DNA. A
mutagenic agent can be man-made or natural. Common mutagens include
chemotherapeutic drugs, ethyl bromide, or 5-bromouracil. Mutagens
can also be, for instance, a radioactive compound, such as radon
found in the soil, ultraviolet radiation from the sun, X-rays, or
radioisotopes used in nuclear medicine.
[0134] Mutation: A change of the DNA sequence within a gene or
chromosome. In some instances, a mutation will alter a
characteristic or trait (phenotype), but this is not always the
case. Types of mutations include base substitution point mutations
(for example, transitions or transversions), deletions, and
insertions. Missense mutations are those that introduce a different
amino acid into the sequence of the encoded protein; nonsense
mutations are those that introduce a new stop codon. In the case of
insertions or deletions, mutations can be in-frame (not changing
the frame of the overall sequence) or frame shift mutations, which
may result in the misreading of a large number of codons (and often
leads to abnormal termination of the encoded product due to the
presence of a stop codon in the alternative frame).
[0135] This term specifically encompasses variations that arise
through somatic mutation, for instance those that are found only in
disease cells, but not constitutionally, in a given individual.
Examples of such somatically-acquired variations include the point
mutations that frequently result in altered function of various
genes that are involved in development of cancers. This term also
encompasses DNA alterations that are present constitutionally, that
alter the function of the encoded protein in a readily demonstrable
manner, and that can be inherited by the children of an affected
individual. In this respect, the term generally refers to the
subset of constitutional alterations that have arisen within the
past few generations in a kindred and that are not widely
disseminated in a population group.
[0136] Mutational frequency: A measure of the number of cells
possessing at least one mtDNA mutation that distinguishes it from a
cell with a mtDNA sequence containing only polymorphisms. The cells
can be in vivo or in vitro. For example, mutational frequency is
the number of hematopoietic cells, such as granulocytes or
monocytes, which exhibit mtDNA heterogeneity in corresponding
regions of mtDNA within a set of hematopoietic cells. A mutational
frequency can also be a measure of the number of clonal populations
(clones) of cells possessing at least one mtDNA mutation within
corresponding regions of mtDNA that distinguishes it from a clonal
population of cells with a mtDNA sequence containing only
polymorphisms. For example, mutational frequency is a proportion of
clonal populations of hematopoietic cells, such as clonal
populations of CD34.sup.+ cells, which exhibit mtDNA heterogeneity
in corresponding regions of mtDNA within a set of clonal
populations of hematopoietic cells. Mutational frequency can be
represented as a proportion (for example, cells with at least one
mutation in corresponding regions of mtDNA versus the total number
of cells in a set) or as a percentage (for example, cells with at
least one mutation in corresponding regions of mtDNA divided by the
total number of cells in a set.times.100). For example, mutational
frequency is the percentage of clonal populations of CD34.sup.+
cells in a set of clonal populations of CD34.sup.+ cells with one
or more mutations within corresponding regions of mtDNA that
distinguishes it from a clonal population of CD34.sup.+ cells
within the set containing a mtDNA sequence with only polymorphisms.
In other examples, the mutational frequency is the proportion of
cells or clonal populations of cells with a mutation in a
particular mtDNA gene or at a particular mtDNA nucleotide position
that distinguishes it from a cell or clonal population of cells
containing a mtDNA sequence with only polymorphisms.
[0137] Since mtDNA is ten to 100 times more sensitive than genomic
(nuclear) DNA to mutagenesis, the mutational frequency of a segment
of mtDNA can be used to measure or estimate the frequency of
mutations that occur in genomic DNA, or in other mtDNA sequences.
In particular embodiments a measurement of the actual frequency
that occurs in mtDNA is used to estimate (by correlation) the
frequency in genomic DNA mutations. In other embodiments,
mutational frequency is the measure of the number of mutations in
the DNA (mitochondrial or genomic) of a cell over a period of time,
compared to a control cell.
[0138] Myeloblast: An immature cell found in the bone marrow; it is
the most primitive precursor in the granulocytic series, which
matures to develop into the promyleocyte and eventually the
granular leukocyte. Myeloblasts have fine, evenly distributed
chromatin, several nucleoli, and a non-granular basophilic
cytopalsm.
[0139] Nucleotide: "Nucleotide" includes, but is not limited to, a
monomer that includes a base linked to a sugar, such as a
pyrimidine, purine or synthetic analogs thereof, or a base linked
to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide
is one monomer in an oligonucleotide/polynucleotide. A nucleotide
sequence refers to the sequence of bases in an
oligonucleotide/polynucleotide.
[0140] The major nucleotides of DNA are deoxyadenosine
5'-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP
or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine
5'-triphosphate (dTTP or T). The major nucleotides of RNA are
adenosine 5'-triphosphate (ATP or A), guanosine 5'-triphosphate
(GTP or G), cytidine 5'-triphosphate (CTP or C) and uridine
5'-triphosphate (UTP or U). Inosine is also a base that can be
integrated into DNA or RNA in a nucleotide (dITP or ITP,
respectively).
[0141] Polymorphism: The occurrence together in the same population
of more than one allele or genetic marker at the same locus with
the least frequent allele or marker occurring more frequently than
can be accounted for by mutation alone. A variant in a sequence of
a gene, usually carried from one generation to another in a
population. Polymorphisms include those variations (nucleotide
sequence differences) that, while having a different nucleotide
sequence, produce functionally equivalent gene products, such as
those variations generally found between individuals, different
ethnic groups, geographic locations. The term polymorphism also
encompasses variations that produce gene products with altered
function, for instance, variants in the gene sequence that lead to
gene products that are not functionally equivalent. This term also
encompasses variations that produce no gene product, an inactive
gene product, or increased or decreased activity gene product.
[0142] Polymorphisms can be referred to, for instance, by the
nucleotide position at which the variation exists, by the change in
amino acid sequence caused by the nucleotide variation, or by a
change in some other characteristic of the nucleic acid molecule or
protein that is linked to the variation (for example, an alteration
of a secondary structure such as a stem-loop, or an alteration of
the binding affinity of the nucleic acid for associated molecules,
such as polymerases, RNases, and so forth).
[0143] Progenitor cell: A "progenitor cell" is a cell that gives
rise to progeny in a defined cell lineage. A "hematopoietic
progenitor cell" is a cell that gives rise to cells of the
hematopoietic lineage. One specific non-limiting example of a
hematopoietic progenitor cell is a pluripotent stem cell expressing
the CD34 cell surface marker.
[0144] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified cell preparation is one in which the cell
referred to is more pure than the cell in its natural environment
within a tissue. In one embodiment, a "substantially purified"
population of a specific cell type is a composition of cells that
includes less than about 20%, less than about 15%, or less than
about 10% of cell of a different genotype or phenotype. Thus, a
substantially purified population of cells includes greater than
80%, greater than 85%, or greater than 90% of the cells of
interest. In another embodiment, a process that produces purified
population of cells is a process that produces a population of
cells so that at least 50% (or not less than 50%) of the resulting
population is the cell type of interest.
[0145] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids, for
example, by genetic engineering techniques.
[0146] Sample: Includes biological samples such as those derived
from a human or other animal source (for example, blood, bone
marrow, stool, sera, urine, saliva, tears, biopsy samples,
histology tissue samples, cellular smears, moles, warts, etc.);
bacterial or viral preparations; cell cultures; forensic samples;
agricultural products; waste or drinking water; milk or other
processed foodstuff; air; and so forth. Samples containing a small
number of cells can be acquired by any one of a number of methods,
such as needle aspiration, biopsy, or tissue scrapes.
[0147] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a hematopoietic cell specific
binding agent is an agent that binds substantially to a
hematopoietic cell. In one embodiment, the specific binding agent
binds a cell surface marker on a hematopoietic cell, such as CD13,
CD14, CD15, CD33, CD34, or CD64. In other embodiments, the specific
binding agent is a monoclonal antibody or a polyclonal antibody
that specifically binds a cell surface marker on a hematopoietic
cell.
[0148] A variety of methods for making labeled specific binding
agents are well known in the art. Detectable labels useful for such
purposes are also well known in the art, and include radioactive
isotopes such as .sup.32P, fluorophores, chemiluminescent agents,
and enzymes.
[0149] Stem cell: A "stem cell" is a pluripotent cell that gives
rise to progeny in all defined lineages, for example hematopoietic
lineages. In addition, limiting numbers of hematopoietic stem cells
are capable of fully reconstituting a seriously immunocompromised
subject in all blood cell types and their progenitors, including
the pluripotent hematopoietic stem cell, by cell renewal.
[0150] Hematopoietic stem cells are self-regenerating, and also
pluripotent in that they differentiate into several lineages,
including lymphoid, myeloid and erythroid lineages. The lymphoid
lineage, comprising B-cells and T-cells, provides for the
production of antibodies, regulation of the cellular immune system,
detection of foreign agents in the blood, detection of cells
foreign to the host, and the like. The myeloid lineage, which
includes monocytes, granulocytes, megakaryocytes as well as other
cells, monitors for the presence of foreign bodies in the blood
stream, provides protection against neoplastic cells, scavenges
foreign materials in the blood stream, produces platelets, and the
like. The erythroid lineage provides the red blood cells, which act
as oxygen carriers. Exposure to growth factors is believed to
induce a stem cell to be dedicated to differentiate into a specific
lineage. The stem cell population is known to constitute only a
small percentage of the total number of leukocytes in bone marrow.
Recently, the mouse stem cell has been obtained in at least highly
concentrated, if not a purified form, where fewer than about 30
cells obtained from bone marrow were able to reconstitute all of
the lineages of the hematopoietic system of a lethally irradiated
mouse. Indeed, one injected cell should be able to reconstitute all
of the hematopoietic lineages.
[0151] Subject: Any vertebrate that has a vascular system and has
hematopoietic cells in the wild-type organism. The subject includes
non-human mammals such as a monkey, mouse, rat, rabbit, pig, goat,
sheep or cow. It also includes humans. It is understood that a cell
or cell line in culture can be referred to as obtained from a
subject even though the cell has been in culture for a length of
time, even years.
[0152] Treatment: Refers to both prophylactic inhibition of a
disease, and therapeutic interventions to alter the natural course
of an untreated disease process, such as a tumor growth.
[0153] Tumor: A neoplasm that may be either malignant or
non-malignant. Tumors originating in a particular organ (such as
breast, prostate, bladder or lung) are primary tumors. Tumors of
the same tissue type may be divided into tumor of different
sub-types (a classic example being bronchogenic carcinomas (lung
tumors) which can be an adenocarcinoma, small cell, squamous cell,
or large cell tumor).
[0154] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Description of Several Specific Embodiments
[0155] Provided herein are methods of using mtDNA mutations to
measure the mutational frequency of a mitochondrial DNA sequence.
Also provided herein are methods for measuring the mutational
frequency in hematopoietic cells.
[0156] One embodiment is a method of measuring a mutational
frequency of a mitochondrial DNA sequence in a subject, which
method involves isolating the test cells (such as stem cells or
hematopoietic cells) from the subject, wherein the cells each
contain at least one mitochondrion. In one specific example of the
method, the cells are individual, non-clonally expanded
hematopoietic cells. Although these specific examples are discussed
with respect to hematopoietic test cells, it is understood that the
method is not limited only to the use of hematopoietic cells. The
method also includes sequencing corresponding regions of the
mitochondrial DNA of the hematopoietic cells and determining a
proportion of the hematopoietic cells exhibiting mtDNA
heterogeneity within the sequenced corresponding regions of the
mitochondrial DNA, wherein the proportion corresponds to the
mutational frequency of the mitochondrial DNA sequence in the
subject. In one specific example of the method, the mitochondrial
DNA is amplified prior to sequencing.
[0157] Specific examples of the method involve multiple clonal
populations of hematopoietic cells, wherein determining the
proportion of the hematopoietic cells exhibiting mtDNA
heterogeneity within corresponding regions involves determining the
proportion of clonal populations possessing at least one
mitochondrial DNA mutation that distinguishes it from clonal
populations of cells with a mtDNA sequence containing only
polymorphisms. In one example of the method, the mitochondrial DNA
from the multiple clonal populations is extracted without
amplification prior to sequencing.
[0158] The hematopoietic cells of the method include, for example,
CD34.sup.+ cells, granulocytes, monocytes, or macrophages. The
hematopoietic cells are isolated from bone marrow, peripheral
blood, or umbilical cord blood. In particular examples, the cells
are stem cells, such as hematopoietic stem cells, such as
CD34.sup.+ cells.
[0159] Another specific example of the method involves isolating
the cells, which method involves obtaining a biological sample from
the subject, contacting hematopoietic cells in the biological
sample with a specific binding agent attached to a detectable
label, and purifying the hematopoietic cells contacted with the
specific binding agent. The biological sample includes bone marrow,
peripheral blood, or umbilical cord blood. The detectable label
includes a fluorescent agent, a chemiluminescent agent, or a
radioisotope.
[0160] In specific examples of the method, the subject is a human.
The subject can have a disease (or be suspected of having a
disease) that is or may be associated with a mtDNA or a
non-mitochondrial DNA mutation. In another example of the method,
the subject has been subjected to a mutagenic treatment, and the
method assesses the mutagenic effect of the treatment. Mutagenic
treatment includes chemotherapy or radiation.
[0161] In one example of the method the mitochondrial DNA sequence
from the cells (such as hematopoietic cells) has at least one
mutation that distinguishes it from cells with a mtDNA sequence
containing only polymorphisms and that is not present in the
mitochondrial DNA sequence from a control cell (such as a
hematopoietic cell). The mutation includes a point mutation, a
polymorphism, a frame-shift mutation, a missense mutation, a
nonsense mutation, a silent mutation, or a deletion mutation. The
mutation can be, for example, in the mtDNA control region or in the
mtDNA coding region. In one specific example of the method, the
mutation is in a homopolymeric C tract of a mitochondrial DNA
control region. In other specific examples, the mutation is in the
CO1 gene or in the Cytb gene of the mtDNA coding region. In certain
examples of the method, a mutation is selected that is known to be
associated with a particular disease, and the frequency of that
mutation in the mtDNA is determined by this method as a measure of
the prevalence of that mutation in the cell population from which
the mtDNA was obtained.
[0162] This disclosure further provides a method of measuring a
mutational frequency of a mitochondrial DNA sequence in a subject,
which method involves isolating the test cells (such as the
hematopoietic cells) from the subject, wherein the test cells each
contain at least one mitochondrion, expanding the test cells into
multiple clonal populations of hematopoietic cells, extracting
mitochondrial DNA from the multiple clonal populations of
hematopoietic cells, sequencing corresponding regions of the
mitochondrial DNA of the multiple clonal populations, and
determining a proportion of the multiple clonal populations of
hematopoietic cells exhibiting mtDNA heterogeneity within the
corresponding regions of the mitochondrial DNA, wherein the
proportion corresponds to the mutational frequency of the
mitochondrial DNA sequence in the subject. In one specific example
of the method, the mitochondrial DNA is amplified prior to
sequencing.
[0163] The hematopoietic cells of the method include CD34.sup.+
cells, granulocytes, monocytes, or macrophages. The hematopoietic
cells are isolated from bone marrow, peripheral blood, or umbilical
cord blood.
[0164] Another specific example of the method involves isolating
the hematopoietic cells, which method involves obtaining a
biological sample from the subject, contacting hematopoietic cells
in the biological sample with a specific binding agent attached to
a detectable label, and purifying the hematopoietic cells contacted
with the specific binding agent. The biological sample includes
bone marrow, peripheral blood, or umbilical cord blood. The
detectable label includes a fluorescent agent, a chemiluminescent
agent, or a radioisotope.
[0165] In specific examples of the method, the subject is a human.
The subject can have a disease that is or may be associated with a
mtDNA or a non-mitochondrial DNA mutation. In another example of
the method, the subject has been subjected to a mutagenic
treatment. Mutagenic treatment includes chemotherapy or
radiation.
[0166] In one example of the method, the mitochondrial DNA sequence
from the clonal populations of hematopoietic cells has at least one
mutation that distinguishes it from clonal populations of
hematopoietic cells with a mtDNA sequence containing only
polymorphisms and that is not present in the mitochondrial DNA
sequence from a control clonal population of hematopoietic cells.
The mutation includes a point mutation, a polymorphism, a
frame-shift mutation, a missense mutation, a nonsense mutation, a
silent mutation, or a deletion mutation. The mutation can be, for
example, in the mtDNA control region or in the mtDNA coding region.
In one specific example of the method, the mutation is in a
homopolymeric C tract of a mitochondrial DNA control region. In
other specific examples, the mutation is in the CO1 gene or in the
Cytb gene of the mtDNA coding region.
[0167] Another embodiment is a method of estimating a mutational
frequency of a genomic DNA sequence in a subject, which method
involves isolating hematopoietic cells in the subject, wherein the
hematopoietic cells each contain at least one mitochondrion,
expanding the hematopoietic cells into multiple clonal populations
of hematopoietic cells, extracting mitochondrial DNA from the
multiple clonal populations of hematopoietic cells, sequencing a
region of the mitochondrial DNA, determining a proportion of the
multiple clonal populations of hematopoietic cells exhibiting mtDNA
heterogeneity within the sequenced region, and correlating the
mutational frequency of the mitochondrial DNA to an estimated
mutational frequency of genomic DNA from the same subject, thereby
estimating the mutational frequency of the genomic DNA sequence in
the subject. In one specific example of the method, the
mitochondrial DNA is amplified prior to sequencing.
[0168] The hematopoietic cells of the method include CD34.sup.+
cells, granulocytes, monocytes, or macrophages. The hematopoietic
cells are isolated from bone marrow, peripheral blood, or umbilical
cord blood.
[0169] Another specific example of the method involves isolating
the hematopoietic cells, which method involves obtaining a
biological sample from the subject, contacting hematopoietic cells
in the biological sample with a specific binding agent attached to
a detectable label, and purifying the hematopoietic cells contacted
with the specific binding agent. The biological sample includes
bone marrow, peripheral blood, or umbilical cord blood. The
detectable label includes a fluorescent agent, a chemiluminescent
agent, or a radioisotope.
[0170] In specific examples of the method, the subject is a human.
The subject can have a disease that is or may be associated with a
mtDNA or a non-mitochondrial DNA mutation. In another example of
the method, the subject has been subjected to a mutagenic
treatment. Mutagenic treatment includes chemotherapy or
radiation.
[0171] In one example of the method, the mitochondrial DNA sequence
from the clonal populations of hematopoietic cells has at least one
mutation that distinguishes it from clonal populations with a mtDNA
sequence containing only polymorphisms and that is not present in
the mitochondrial DNA sequence from a control clonal population of
hematopoietic cells. The mutation includes a point mutation, a
polymorphism, a frame-shift mutation, a missense mutation, a
nonsense mutation, a silent mutation, or a deletion mutation. The
mutation can be, for example, in the mtDNA control region or in the
mtDNA coding region. In one specific example of the method, the
mutation is in a homopolymeric C tract of a mitochondrial DNA
control region. In other specific examples, the mutation is in the
CO1 gene or in the Cytb gene of the mtDNA coding region.
[0172] Also provided is a method of screening for an agent that
increases the mutational frequency of a hematopoietic cell, which
method involves contacting isolated hematopoietic cells with the
agent to produce treated hematopoietic cells, wherein the
hematopoietic cells each contain at least one mitochondrion. The
method also involves sequencing corresponding regions of the
mitochondrial DNA of the hematopoietic cells and determining a
proportion of the hematopoietic cells that distinguishes it from
cells with a mtDNA sequence containing only polymorphisms within
the sequenced corresponding regions of the mitochondrial DNA,
wherein the proportion corresponds to the mutational frequency of
the mitochondrial DNA sequence in the treated hematopoietic cells.
In one specific example of the method, the mitochondrial DNA is
amplified prior to sequencing.
[0173] The method further involves comparing the mutational
frequency of the treated hematopoietic cells to a mutational
frequency of hematopoietic cells that were not contacted with the
agent, wherein an increase in the mutational frequency of the
treated hematopoietic cells, compared to the mutational frequency
of hematopoietic cells that were not contacted with the agent,
indicates that the agent increases the mutational frequency of the
hematopoietic cell, thereby screening for the agent that increases
the mutational frequency of the hematopoietic cell.
[0174] Another embodiment is a method of screening for an agent
that increases the mutational frequency of a hematopoietic cell,
which method involves contacting isolated hematopoietic cells with
the agent to produce treated hematopoietic cells, wherein the
hematopoietic cells each contain at least one mitochondrion, and
expanding the hematopoietic cells into multiple clonal populations
of hematopoietic cells. The method also involves extracting
mitochondrial DNA from the multiple clonal populations of
hematopoietic cells, sequencing corresponding regions of the
mitochondrial DNA of the multiple clonal populations, and
determining a proportion of the multiple clonal populations of
hematopoietic cells that distinguishes it from clonal populations
with a mtDNA sequence containing only polymorphisms within the
corresponding regions of the mitochondrial DNA, wherein the
proportion corresponds to the mutational frequency of the
mitochondrial DNA sequence in the subject. In one specific example
of the method, the mitochondrial DNA is amplified prior to
sequencing.
[0175] The method further involves comparing the mutational
frequency of the treated hematopoietic cells to a mutational
frequency of hematopoietic cells that were not contacted with the
agent, wherein an increase in the mutational frequency of the
treated clonal populations of hematopoietic cells, compared to the
mutational frequency of clonal populations of hematopoietic cells
that were not contacted with the agent, indicates that the agent
increases the mutational frequency of the hematopoietic cell,
thereby screening for the agent that increases the mutational
frequency of the hematopoietic cell.
[0176] In specific examples of the method, the agent includes a
small molecule, a chemical compound, a radioisotope, a protein, a
peptide, or a peptidomimetic. The chemical compound includes a
chemotherapeutic drug.
[0177] The hematopoietic cells of the method include CD34.sup.+
cells, granulocytes, monocytes, or macrophages. The hematopoietic
cells are isolated from bone marrow, peripheral blood, or umbilical
cord blood.
[0178] In specific examples, the mutation includes a point
mutation, a polymorphism, a frame-shift mutation, a missense
mutation, a nonsense mutation, a silent mutation, or a deletion
mutation. The mutation can be, for example, in the mtDNA control
region or in the mtDNA coding region. In one specific example of
the method, the mutation is in a homopolymeric C tract of a
mitochondrial DNA control region. In other specific examples, the
mutation is in the CO1 gene or in the Cytb gene of the mtDNA coding
region.
IV. Method of Detecting Mutational Frequency in Cells
[0179] Prior to this disclosure, there had not been an adequate
method available to determine the frequency of genetic mutations in
intact animals, including humans. Disclosed herein are methods of
detecting and/or measuring the mutational frequency in cells, such
as hematopoietic cells. The cells can be in vivo or in vitro. In
one embodiment, the mutational frequency is the number of cells
within a set of cells exhibiting mtDNA heterogeneity within
corresponding regions of mtDNA. In another embodiment, the
mutational frequency is the number of cells within a set of cells
exhibiting at least one DNA mutation that distinguishes it from
other cells in the set containing a mtDNA sequence with only
polymorphisms within the corresponding region of mtDNA. For
example, the mutational frequency may be the number of granulocytes
within a set of granulocytes exhibiting at least one DNA mutation
that distinguishes it from other granulocytes in the set containing
a mtDNA sequence with only polymorphisms within the corresponding
region of mtDNA. In another embodiment, the mutational frequency is
the number of clonal populations of cells exhibiting mtDNA
heterogeneity within corresponding regions of mtDNA. In another
embodiment, the mutational frequency is the number of clonal
populations of cells within a set of clonal populations of cells
exhibiting at least one DNA mutation that distinguishes it from
other clonal populations in the set containing a mtDNA sequence
with only polymorphisms within corresponding regions of mtDNA.
Mutational frequency can be represented as a proportion (for
instance cells in a set with at least one mutation that
distinguishes it from other cells in the set containing a mtDNA
sequence with only polymorphisms in corresponding regions of mtDNA
versus the total number of cells in a set) or as a percentage (for
instance cells in the set with at least one mutation that
distinguishes it from other cells in the set containing a mtDNA
sequence with only polymorphisms in corresponding regions of mtDNA,
divided by the total number of cells in a set.times.100). In a
specific, non-limiting example, the mutational frequency is the
number of CD34.sup.+ clones (clonal populations of cells) which
exhibit mtDNA heterogeneity in corresponding regions of mtDNA
within the set of CD34.sup.+ clones. In another specific,
non-limiting example, the mutational frequency is the percentage of
clonal populations of CD34.sup.+ cells which exhibit mtDNA
heterogeneity in corresponding regions of mtDNA within a set of
clonal populations of CD34.sup.+ cells. In other specific,
non-limiting examples, the mutational frequency is the number or
the percentage of granulocytes which exhibit mtDNA heterogeneity in
corresponding regions of mtDNA within a set of granuloctyes.
[0180] Disclosed methods involve isolating a cell from a subject,
wherein the isolated cell contains at least one mitochondrion. In
one embodiment, at least two hematopoietic cells are isolated. In
other embodiments, at least 2, at least 5, at least 10, at least
20, at least 50, at least 75, at least 96, at least 100, at least
500, at least 1000, or more hematopoietic cells are isolated. Each
cell isolated can contain hundreds of mitochondria and each
mitochondrion can contain multiple copies of mtDNA. In one
embodiment, the cells are isolated from a healthy (for example,
normal for a specific condition) subject. In another embodiment,
the cells are isolated from a subject having a disease. The disease
can be any disease, including for instance a neoplasia, an
autoimmune disease, or a congenital disease. In another embodiment,
the subject is suffering from senescence (aging). In certain
embodiments, the isolated cells are human cells. The cell in
preferred embodiments is viable, such that it can be replicated for
at least one generation outside of the subject (in vitro).
[0181] Cells can be isolated directly from a subject or from a
biological sample that is obtained from the subject. Biological
samples can be obtained from any part of the body of the subject,
by any means known to one of ordinary skill in the art. The
biological sample can include blood, urine, stool, sera, saliva,
tears, biopsy samples, histological samples, and the like.
Hematopoietic cells (blood cells), for example, can be obtained
from bone marrow, peripheral blood, or umbilical cord blood. In one
embodiment, hematopoietic cells are obtained by bone marrow
aspiration, for example from the pelvic bone of the subject. In
another embodiment, hematopoietic cells are obtained by
phlebotomy.
[0182] Once the hematopoietic cells are obtained, a specific
hematopoietic cell, or class of cells of interest can be isolated.
Methods of isolating cells, and particularly individual types or
classes of cells, are well known in the art. In one embodiment,
cells expressing a particular cell surface marker, or a collection
of cell surface markers, are selected. Examples of cell surface
markers include, but are not limited to, Sca-1, CD11b, CD13, CD14,
CD15, CD33, CD34, and CD64. In one embodiment, a method of
selecting a cell that expresses specific cell surface markers
involves the use of specific binding agents, such as antibodies,
that recognize the cell surface marker. Antibodies useful in
sorting methods can be monoclonal or polyclonal antibodies.
[0183] In order to identify the cells to be isolated, detectable
labels can be attached to a specific binding agent, such as an
antibody that specifically binds a cell surface marker. Attachment
of the detectable label to an antibody can be accomplished using
any number of means known to those of ordinary skill in the art. In
some embodiments, the antibody is attached to a detectable label by
covalent or non-covalent means. Examples of detectable labels
include radioactive isotopes, chemiluminescent agents, or
fluorophores. In some embodiments, cells are selected using FACS
(fluorescence activated cell sorting).
[0184] In one embodiment, purified populations of CD34.sup.+ cells
are isolated with immobilized antibodies, for example anti-CD34
antibodies. In other embodiments, granulocytes, monocytes, or
macrophages are isolated with immobilized antibodies, for example
granulocytes are isolated with anti-CD13 or anti-CD33 antibodies
and mature granulocytes are isolated with anti-CD15 antibodies.
Myeloblasts are isolated with anti-CD34 antibodies and anti-CD13 or
anti-CD33 antibodies. Monocytes are isolated with anti-CD14 or
anti-CD64 antibodies. Antibodies can be immobilized on particles,
such as sepharose beads or magnetic beads. The particles can be in
the form of a slurry or contained within an apparatus, such as a
column. A particular embodiment uses magnetic cell sorting such as,
for example, the BD IMag Cell Separation System (Becton Dickinson,
San Diego, Calif.) or the MACS Separation System (Miltenyi Biotec,
Auburn, Calif.). Magnetic cell sorting involves the use of a
monoclonal antibody covalently bound to the surface of a magnetic
bead, where the antibody can specifically bind an epitope on a cell
surface marker present on the cells being selected. For example, to
isolate a cell expressing CD34 (a CD34.sup.+ cell) from a mixture
of cells, anti-CD34 monoclonal antibodies bound to magnetic beads
are used. Only CD34.sup.+ cells are specifically bound by the
antibodies and retained by the beads, thereby separating, or
isolating, CD34.sup.+ cells from the other cells in the mixture.
The remaining, unbound cells do not express CD34 and are CD34.sup.+
cells. Immobilized CD34.sup.+ cells can be released from the
magnetic beads under the appropriate conditions.
[0185] In another embodiment, purified populations of CD34.sup.+
cells are isolated using flow cytometry. By way of example, in flow
cytometry, CD34 antibodies attached to a detectable label
specifically bind surface CD34 on CD34.sup.+ expressing cells. In a
further embodiment, granulocytes that express CD13 or CD33 are
isolated by flow cytometry using anti-CD13 or anti-CD33 antibodies
attached to a detectable label. An example of a detectable label
used in flow cytometry is a fluorophore, such as fluorescein or
phycoerythrin. Labeled CD34-expressing cells can then be identified
by a flow cytometer and "sorted." Sorting involves the mechanical
separation of fluorescently-labeled cells from cells that do not
fluoresce, such as, for example, those cells that do not express
the CD34 cell surface marker. Flow cytometry can sort cells that
are labeled with at least one, at least two, at least three, or
more antibodies attached to distinguishable detectable labels, thus
cells can be isolated based on the expression of one or more or any
combination of two or more cell surface markers.
[0186] In some embodiments, CD34.sup.+ cells or CD33.sup.+ cells
are sorted into a culture plate, and optionally individual cells
are sorted into individual growing chambers. The culture plate can
be any shape or size. In one embodiment, the culture plate is a
multi-chamber (multi-well) plate. A multi-well plate can have any
number of individual wells, or chambers. A specific, non-limiting
example of a multi-well plate is a microtiter plate containing 96
wells, though other examples are known to those of ordinary skill
in the art. A flow cytometer can be programmed to sort any number
of cells into a single well; in particular embodiments, cells are
sorted so that only one is placed in each well or chamber.
[0187] When a cell is deposited in a particular growth chamber, a
colony of cells can be formed under the appropriate growth
conditions. A group, or colony, of cells that is derived, or
expanded, from a single cell is referred to as a clonal population
of cells (for instance a clone). Thus, the deposition of single
cells in individual growth chambers ensures that the cells
comprising the resultant clonal population are genetically alike
and the DNA of the clonal population is a reflection of the DNA of
the cell initially deposited in the well. Such a clonal population
may include, for example, five or fewer cells, at least 2 cells, at
least 5 cells, at least 10 cells, at least 20 cells, at least 100
cells, at least 200 cells, at least 500 cells, at least 1000 cells
or at least 10,000 cells.
[0188] By way of example, CD34.sup.+ cells can be sorted by single
cell deposition into a 96 well microtiter plate, or in
microcentrifuge tubes. In one specific, non-limiting example,
CD34.sup.+ cells are labeled with a phycoerythrin (PE) anti-CD34
monoclonal antibody and sorted with a MoFlo cytometer and CyClone
automated cloner in a 0.5 single drop mode, such that only a single
cell is deposited in each well of the microtiter plate. In another
specific, non-limiting example, granulocytes are labeled with a PE
anti-CD33 monoclonal antibody and sorted with a MoFlo cytometer and
CyClone automated cloner in a 0.5 single drop mode, such that only
a single cell is deposited in individual microcentrifuge tubes.
[0189] The wells of the microtiter plate or other culture plate in
many embodiments contain a growth medium. The growth medium can be
any growth medium known to one of ordinary skill in the art that
will support the growth of cells in culture. The growth medium can
contain, for example, agar or methylcellulose (or another polymer)
if the growth of cell colonies is desired. Alternatively, the
growth medium can contain liquid growth medium if the growth of
cells in suspension is desired. In addition, the growth medium can
contain growth factors, or cytokines, such as, but not limited to,
stem cell factor, Flt-3, thrombopoietin, or G-CSF. The growth
medium can also contain other components, for example fetal calf
serum, which are beneficial to support the optimized growth of
cells. The particular combination of components included in the
growth medium will vary depending on the cell type deposited in the
well and/or the type of cell desired at the end of the culture
period.
[0190] The cells can be maintained in culture for any length of
time, depending in part on the degree of expansion desired and the
generational time of the cells. In one embodiment, the cells are
maintained in culture at least one day. In other embodiments, the
cells are maintained in culture at least two days, at least three
days, at least four days, at least five days, at least six days, at
least ten days, at least 15 days, or at least 20 days.
Alternatively, the time in culture can be calculated based on the
number of cell divisions or generations.
[0191] In order for mtDNA heterogeneity and/or mutational frequency
to be determined, cell colonies that grow in the culture wells can
be removed and DNA extracted therefrom. Optionally, it is believed
to be beneficial to isolate the mitochondria prior to extracting
the mtDNA and/or the genomic (nuclear) DNA. Methods of mitochondria
isolation are well known to those of ordinary skill in the art
(Barja, J. Bioenerg. Biomembr. 34:227-233, 2002; Rajapakse et al.,
Brain Res. Brain Res. Protoc., 8:176-183, 2001). In one embodiment,
individual, isolated hematopoietic cells are lysed, such as by
sonication, prior to sequencing the mtDNA.
[0192] In order to determine the heterogeneity of mtDNA sequences
from individual cells, such as granulocytes, or cells deposited and
expanded to form a colony in the culture wells, such as CD34.sup.+
cells, mtDNA is sequenced and the presence of mutations in a
specific region (for instance, the hypervariable region, the
homopolymeric C tract, or a gene in the coding region, such as CO1
or Cytb) or at a specific nucleotide position of the mtDNA can be
assessed. Primers that stably bind, or hybridize, to mtDNA can be
used. Any region of the mtDNA or any number of base pairs can be
sequenced. In one embodiment, the mtDNA control region is
sequenced, for instance 1121 base pairs are sequenced. In another
embodiment, the mtDNA coding region, for instance the Cytb gene
(910 base pairs) or the CO1 gene (1390 base pairs), are sequenced.
Alternatively, at least 50 base pairs, at least 100 base pairs, at
least 200 base pairs, at least 300 base pairs, at least 500 base
pairs, at least 750 base pairs, at least 1000 base pairs, at least
1100 base pairs, at least 1200 base pairs, at least 1500 base
pairs, at least 2000 base pairs, or at least 3000 base pairs are
sequenced. In certain specific embodiments, the entire mtDNA is
sequenced.
[0193] Optionally, at least some of the DNA isolated from the
cultured cells is amplified, for example by polymerase chain
reaction (PCR), prior to sequencing. In one embodiment, the DNA is
subjected to nested gene amplification (Erickson and Castora,
Biochim. Biohphys. Acta 1181:77, 1993; Khrapko et. al., Nucleic
Acids Res. 27:2434, 1999). In nested gene amplification, at least
two pairs of primers are used to amplify the DNA, where one pair of
primers hybridizes to a region that is within the region amplified
by a different pair of primers. When increased specificity of the
amplified product is desired, nested gene amplification can also be
performed with at least three pairs, at least four pairs, or more
pairs of primers.
[0194] Once a mtDNA sequence from an individual cell or from a
clonal population of cells is determined, it can be compared to a
corresponding region of a known mtDNA sequence such as, for
example, the revised Human mtDNA Cambridge Reference sequence
(Andrews et al., Nat. Genet. 23:147, 1999; Kogelnik et al., Nucleic
Acids Res. 26:112, 1998) in order to identify changes in the mtDNA
sequence that are due to mutations (mtDNA heterogeneity), as
opposed to polymorphisms. The mutational frequency of mtDNA can
then be determined by calculating the number of cells within a set
of cells, or the number of clonal populations of cells within the
set of clonal populations of cells, having at least one mutation
(the number of cells or clonal populations of cells exhibiting
mtDNA heterogeneity) in corresponding regions of a mtDNA sequence,
when compared to the mtDNA polymorphisms described in the revised
Human mtDNA Cambridge Reference sequence. In some embodiments, the
mutational frequency is the number of cells or clonal populations
of cells with a mutation in a particular mtDNA gene or at a
particular mtDNA nucleotide position that distinguishes it from a
cell or clonal population of cells containing a mtDNA sequence with
only polymorphisms.
[0195] A mutation in the mtDNA and/or the genomic DNA can be a
point mutation, a polymorphism, a deletion mutation, a silent
mutation, a frame-shift mutation, a nonsense mutation or a missense
mutation (not all of which are mutually exclusive categories). The
mutation can arise in any way, for instance as a result of a cell
being exposed to a mutagenic agent, such as during the course of
treatment for a disease. The mutagenic agent can be man-made or
natural. In one embodiment, the mutagenic agent is a chemical
agent, such as a chemotherapeutic drug. In other embodiments, the
mutagenic agent is a radioactive agent or ultraviolet light. A
mutagenic agent, such as a toxic drug or chemical, can result in
the generation of at least one reactive oxygen species. Mutations
can also be due to a genetic disease or defect, such as a defect in
a component of a DNA repair or maintenance mechanism of the
cell.
[0196] Provided embodiments of the method to detect mutations are
performed using hematopoietic cells isolated from the subject.
Hematopoietic cells are readily obtained from the bone marrow, the
umbilical cord, or the peripheral blood and thus are a convenient
source of DNA. However, the method is not limited to the use of
hematopoietic cells.
[0197] Any method can be used to detect differences between
sequences. In one embodiment, differences between sequences are
determined by the direct sequencing of two or more sequences,
followed by the comparison of the sequences. In a particular
embodiment, amplification of the DNA molecules is performed prior
to sequencing. In a specific, non-limiting example, sequencing is
verified by subcloning the PCR products into pCR2.1-TOPO.RTM.
vector and transforming competent E. coli (TOP 10 cells) using the
TOPO TA.TM. cloning kit (Invitrogen, Carlsbad, Calif.). In another
embodiment, comparative sequencing based upon heteroduplex
detection is used to identify sequence differences that exist
between different DNA sequences, such as mtDNA sequences from two
or more clones, for instance between one or more samples and a
control. An example of a technique that assays for heteroduplex
formation after DNA denaturation is WAVE.TM. denaturing
high-performance liquid chromatography (Transgenomics, Inc., Omaha,
Nebr.).
[0198] Optionally, analysis of the amplified mtDNA sequence from a
first cell or clonal population, and its comparison to the
amplified mtDNA of a corresponding region from a second, or more,
cell or clonal population, reveals that the mtDNA isolated from a
first cell or clonal population has at least one mutation that is
not present in the corresponding region of the mtDNA isolated from
the second, or more, cell or clonal population. The second, or
more, clonal population can be derived from the same biological
sample as the first clonal population, or from a second biological
sample. The second biological sample can be obtained from the same
subject, or from a different subject, as the first biological
sample. In one embodiment, the first biological sample is obtained
from a subject that has undergone treatment of a disease (for
example by the administration of chemotherapy for a tumor), whereas
the second biological sample is obtained from a control subject who
has not undergone the treatment. In another embodiment, the first
biological sample is obtained from a subject before undergoing
treatment and the second biological sample is obtained from a
subject following treatment. In a particular embodiment, the first
and second biological samples are obtained from the same
subject.
[0199] Since mtDNA is ten to 100 times more sensitive than genomic
(nuclear) DNA to mutagenesis, the mutational frequency of mtDNA can
be used to measure the frequency of mutations that occur in genomic
DNA, or in other mtDNA sequences. In particular embodiments a
measurement of the actual mutational frequency that occurs in mtDNA
is used to estimate (by correlation) the frequency in genomic
DNA.
[0200] In one embodiment, hematopoietic stem cells (CD34.sup.+
cells), which are known to circulate in the peripheral and
umbilical cord blood, are isolated from the peripheral or umbilical
cord blood in order to detect the mutational frequency of the
mtDNA, the genomic DNA and, in some embodiments, the mutational
frequency of the cell. The isolated cells can then be sorted (for
instance, using a method similar to those described above) into
individual growth chambers, for instance individual wells of a
96-well microtiter plate. The cells are then cultured to produce
clonal populations of cells, from which DNA (for instance, mtDNA)
can then be amplified and analyzed, for instance by sequencing a
region of the DNA, to determine the mutational frequency of mtDNA,
genomic DNA, or both. Due to the relatively low density of
CD34.sup.+ cells in peripheral blood compared to bone marrow,
special precautions are usually taken during the isolation of
CD34.sup.+ cells from peripheral blood to ensure a maximum yield of
these cells. Such steps include the collection of large volumes of
peripheral blood and lengthy sorting times.
[0201] The provided methods can be used to monitor, or track,
individual stem cells as they contribute to hematopoiesis. For
example, a unique sequence of mtDNA (a mtDNA profile) in a
CD34.sup.+ cell is also present in a proportion of circulating
hematopoietic cells, such as granulocytes, lymphocytes, or
erythrocytes, that are the progeny of the CD34.sup.+ cell
expressing that particular mtDNA profile. In one embodiment,
different CD34.sup.+ cells isolated from a subject have different
mtDNA profiles. Clonal expansion of the CD34.sup.+ cells with
different mtDNA profiles, under conditions that allow for the
differentiation of the CD34.sup.+ cells into all hematopoietic cell
types, can be performed for different lengths of time. Thus, the
number of hematopoietic cells expressing a particular mtDNA profile
at different time points in the differentiation process is an
indication of the proportion and/or types of hematopoietic cells
that are descendants of a particular CD34.sup.+ stem cell. The
proportion of circulating hematopoietic cells expressing a
particular mtDNA profile, for example the proportion of
granulocytes within the population of hematopoietic cells in a
peripheral blood sample obtained from a subject, can then be used
to determine the proportion of CD34.sup.+ stem cells contributing
to blood cell production in that subject. The proportion of
circulating hematopoietic cells expressing the particular mtDNA
profile can be measured at any time in the subject, such that the
proportion of circulating hematopoietic cells expressing the
particular mtDNA profile can be monitored, or tracked, over time in
a subject. The proportion of stem cells (or the proportion of
active stem cells), based on the degree of mtDNA heterogeneity of
circulating granulocytes, can be statistically measured (see, for
example, Abkowitz et al., Proc. Natl. Acad. Sci. U.S.A.,
92:2031-2035, 1995)
[0202] The provided methods can also be used to isolate, or
identify, a cell in a sample obtained from a subject, where the
cell has a set of mtDNA mutations (mtDNA profile) linked to a
particular disease. A mtDNA profile can be causative or
associative, with respect to a disease. In one embodiment, a cell
isolated from a subject and expanded into a clonal population of
cells, using the methods described herein, has a mtDNA profile that
indicates the subject is predisposed to a disease. In other
embodiments, a cell isolated from a subject and expanded into a
clonal population of cells has a mtDNA profile that indicates the
subject has a disease or is having a recurrence of a disease. In a
specific, non-limiting example, a subject with a disease, such as
leukemia, has a proportion of abnormal hematopoietic cells, within
a set of hematopoietic cells obtained from the subject, which
abnormal cells have a particular mtDNA profile specific to the
abnormal cells (the mtDNA mutational frequency of the subject).
Following treatment for the disease, the mtDNA profile is monitored
in cells obtained from the subject, where detection of the same
proportion or more of cells expressing the particular mtDNA profile
following treatment (for instance the mtDNA mutational frequency is
unchanged in the subject) is an indication that the treatment was
not effective in eradicating the abnormal cells. Detection of a
lower proportion of cells expressing the particular mtDNA profile
following treatment (for example, the mtDNA frequency has decreased
in the subject) is an indication that the treatment has an effect
on eradicating the abnormal cells. The cells can be obtained from
the subject after any period of time subsequent to a treatment, or
a series of treatments, for instance about one week, about two
weeks, about one month, about two months, about four months, about
six months, about one year, about five years or more following
treatment of the subject for the disease.
V. Methods of Determining Mutational Effect of Mutagens on mtDNA in
Cells
[0203] Also disclosed herein are methods of determining the effect
of mutagens on cells. These methods involve the exposure of a cell,
or a clonal population of cells, to a mutagen, or a potential or
suspected mutagen, and the subsequent measurement of the mutational
frequency of the DNA of the cell, or clonal population of cells, by
a method described herein. The mutational frequency determined in
the cell, or in clonal population of cells, that is exposed to a
mutagen can be compared to the mutational frequency of a control
cell, or clonal population of cells, that has not been exposed to
the mutagen, or that has been exposed to a different condition,
such as the mutagen and a blocking agent, or a different amount of
the mutagen, or another mutagen, etc.
[0204] These methods can be used to measure the mutational effect
of any agent on the DNA (genomic (nuclear), or mtDNA, or both) of a
cell. The agent can be a drug that is mutagenic, for example by
directly damaging DNA, such as by DNA cross-linkage, or by
increasing the generation of at least one reactive oxygen species
in the cell. For instance, the agent can be a chemical compound
used to treat a disease, such as a chemotherapeutic drug. Examples
of drugs that can have a mutational effect on the DNA include
alkylating agents, such as busulfan, methylmethane sulfonate, or
dimethylnitrosamine; cross-linking agents, such as mitomycin C; or
agents that inhibit DNA replication by intercalative (for example,
actinomycin D) or non-intercalative (for example, hydroxyurea)
mechanisms. Another specific, non-limiting example of a DNA
damaging agent is etoposide. DNA damaging agents also include
radiation from an X-ray, or a radioisotope. Optionally, the method
of determining the effect of mutagens on cells can be used to
predict the prevalence of mutations in the cells of a subject
following treatment of a disease, for instance, a treatment that
involves application of any of these agents.
[0205] The method also can be used to screen for the potential
mutagenic character of chemicals and drugs. In such embodiments,
the clonal expansion and mtDNA analysis method described herein is
used to determine the mutational frequency in a cell (for example,
a cell grown in vitro) before and after the cell (or a clonally
identical or similar cell) is exposed to a known or putative
mutagenic chemical or drug. The cell, or a clonal population of
cells, to be exposed to the putative mutagenic chemical or drug is
in some instances obtained from a relatively young subject, such as
a child, or a middle-aged subject, or an elderly subject, in order
to determine the effect of the mutagenic agent on cells of
different generational age. The cell, or clonal population of
cells, can be obtained from a healthy (for example, normal for a
specific condition) subject or from a subject that has a known or
suspected background mutation(s) in their mtDNA or their genomic
(nuclear) DNA. Typically, the cell, or clonal population of cells,
has not previously been exposed to a mutagenic agent. In some
instances the cell, or clonal population of cells, is obtained from
a subject that has had prior exposure to a mutagenic agent. The
prior exposure can be to the same mutagenic agent or to a different
mutagenic agent. The relative mutagenic potential of the chemical
or drug is then calculated based on the increase in mutational
frequency in the mtDNA that results from the exposure, compared to
a control mtDNA or genomic DNA.
[0206] Optionally, the known or putative mutagenic chemical or drug
can be administered to a whole animal, such as a rabbit or a mouse
or a primate. DNA from cells obtained from the animal subsequent to
administration of the mutagenic chemical or drug can be compared to
DNA from cells obtained from the animal before administration of
the mutagenic chemical or drug, or to cells from a control
animal.
[0207] In one embodiment of the method of screening for a mutagenic
agent, at least two isolated cells are contacted, or treated, with
an agent. In other embodiments, at least 5, at least 10, at least
20, at least 50, at least 75, at least 96, at least 100, at least
500, at least 1000, or more isolated hematopoietic cells are
contacted, or treated, with an agent. Optionally, the treated cells
are subsequently expanded into clonal populations of cells to
produce treated clonal populations of cells. Untreated cells or
clonal populations of cells can serve as control, or reference,
cells or clonal populations of cells. In order to identify or
quantify mutations in the nucleic acid sequence of the mtDNA of the
treated cells, DNA is extracted from treated and untreated clonal
populations of cells, then the mtDNA is sequenced. In another
embodiment, the mtDNA is amplified prior to sequencing. In yet
another embodiment, the treated and untreated cells are lysed and
the DNA is amplified prior to sequencing. Comparison of the
sequence of the mtDNA from the treated cells or clonal populations
of cells with the sequence of a corresponding region of mtDNA from
a control cell or control clonal populations of cells that was not
contacted with the agent can determine the number (or proportion or
percentage) of clonal populations of cells exhibiting mtDNA
heterogeneity (for example, the number of clonal populations with
at least one mutation that distinguishes it from clonal populations
containing a mtDNA sequence with only polymorphisms) in the
sequenced region of the mtDNA from the treated cells and from the
untreated cells. An increase in the percent or proportion of the
treated clonal populations of cells with at least one mutation in
the corresponding regions of mtDNA (an increase in mtDNA mutational
frequency), compared to the untreated clonal populations of cells,
indicates that the agent has a mutagenic effect on the cell and
increases the mutational frequency of the cell.
[0208] In another embodiment of the method of screening for a
mutagenic agent, a single cell is obtained by limiting dilution, as
described herein, and expanded to obtain a uniform clonal
population of cells with a homogenous mtDNA sequence. In this
embodiment, the clonal population is divided into experimental
(treated) and control groups. The cells in the experimental group
are treated with an agent, for instance a mutagen, or a potential
or suspected mutagen, whereas cells in the control group are not
treated. Cells in the experimental group can be treated for various
time intervals, or repeatedly. mtDNA heterogeneity of the control
and treated cells is determined at any time following treatment.
mtDNA heterogeneity is determined using any of the methods
described herein, for example by sequencing, and the mutational
frequency of the mtDNA and/or genomic DNA in the control and
treated groups is determined.
[0209] Since mtDNA is thought to be 10-100 times more sensitive
than genomic DNA to mutagenesis (Bianchi et al., Mutat. Res.,
488:9-23, 2001; Gattermann, Leuk. Res, 24:141-151, 2000; Golden and
Melov, Mech. Ageing Dev., 122:1577-1589, 2001; Gerhard et al.,
Mech. Ageing Dev., 123:155-166, 2002; Battye et al., J. Immunol.
Methods, 243:25-32, 2000), measuring the mutational frequency of
mtDNA in the presence or absence of various agents is a sensitive
method by which to identify agents that are potentially mutagenic
to genomic DNA.
[0210] Specific aspects of the invention are illustrated by the
following non-limiting Examples.
EXAMPLE 1
Bulk Genotype of the mtDNA Control Region
[0211] This example provides a description of a commonly employed
method of identifying the genotype of mtDNA in a subject (bulk
genotype) by sequencing total bone marrow cells obtained from the
subject.
[0212] Bone marrow and peripheral blood specimens were collected
from normal donors. Mononuclear cells from bone marrow and
peripheral blood were separated by density gradient centrifugation
and washed twice in phosphate buffered saline. DNA was extracted
using QIAamp DNA blood mini kit (Qiagen, Valencia, Calif.).
Extracted DNA was resuspended in TE buffer (pH 7.5) containing 10
mM Tris and 1 mM EDTA.
[0213] In order to directly sequence the control region of mtDNA,
where the incidence of somatic mutations per nucleotide is
approximately 10-fold higher than anywhere else in the mtDNA
genome, the DNA extracted from total bone marrow cells was
subjected to nested gene PCR amplification using the following
nested primers: outer primer pair 5'-CGCCTACACAATTCTCCGATC-3' (SEQ
ID NO: 1) and 5'-ACTTGGGTTAATCGTGTGACC-3' (SEQ ID NO: 2), which
amplify the region between nucleotide 15,974 and nucleotide 921 of
the human mtDNA genome, and inner primer pair
5'-TTAACTCCACCATTAGCACC-3' (SEQ ID NO: 3) and
5'-GAAAGGCTAGGACCAAACCTA-3' (SEQ ID NO: 4), which amplify the
region between nucleotide 15,971 and nucleotide 670 of the human
mtDNA genome. Sequencing was performed on an ABI Prism 3100 Genetic
Analyzer in both orientations.
[0214] There was a marked variation in the number of nucleotide
changes among individual normal donors, with a range of 5 (donor 1)
to 24 (donor 4) (11.7.+-.6.6, mean .+-.SD) nucleotide changes per
donor (Table 2). TABLE-US-00002 TABLE 2 Nucleotide sequence changes
of mtDNA control region from total bone marrow cells. Donor
Polymorphism (Age/Sex) (Mutation) Affected mtDNA gene 1 C150T HV2 +
7S + OH (47/F) A263G HV2 + OH 8C/6C* HV2 + OH + CSB2 C16,192T HV1 +
7S C16,270T HV1 + 7S 2 A73G HV2 + 7S (38/F) G185A HV2 + 7S + OH
A263G HV2 + OH 7C/6C* HV2 + OH + CSB2 (A478G) T16,093C HV1 A16,158G
HV1 + TAS T16,172C HV1 + TAS A16,183C HV1 + 7S T16,189C HV1 + 7S
A16,219G HV1 + 7S C16,278T HV1 + 7S 3 A73G HV2 + 7S (43/M) T146C
HV2 + 7S + OH T152C HV2 + 7S + OH T195C HV2 + OH A263G HV2 + OH
9C/6C HV2 + OH + CSB2 del 514 C del 515 A C16,223T HV1 + 7S
C16,278T HV1 + 7S C16,294T HV1 + 7S G16,390A 7S 4 A93G HV2 + 7S
(34/M) A95C HV2 + 7S G185A HV2 + 7S + OH A189G HV2 + 7S + OH T236C
HV2 + OH 8C/6C HV2 + OH + CSB2 G247A HV2 + TFB1 A263G HV2 + OH del
C 514 del A 515 T16,093C HV1 G16,129A HV1 + 7S C16,148T HV1 + 7S
C16,168T HV1 + 7S + TAS T16,172C HV1 + 7S + TAS C16,187T HV1 + 7S
C16,188G HV1 + 7S T16,189C HV1 + 7S C16,223T HV1 + 7S A16,230G HV1
+ 7S C16,278T HV1 + 7S A16,293G HV1 + 7S T16,311C HV1 + 7S C16,320T
HV1 + 7S 5 A73G HV2 + 7S (54/M) A263G HV2 + OH 7C/6C HV2 + OH +
CSB2 del 514C del 515A T16,126C HV1 + 7S C16,294T HV1 + 7S C16,296T
HV1 + 7S T16,519C 7S 6 A73G HV2 + 7S (34/F) C150T HV2 + 7S + OH
A263G HV2 + OH 8C/6C HV2 + OH + CSB2 (A517G) C16,270T HV1 + 7S
C16,292T HV1 + 7S T16,362C HV1 + 7S
For example, when a sample containing total bone marrow cells from
donor 1 was analyzed, six mtDNA polymorphisms were found (C150T,
A263G, 8C/6C at nucleotide position 303 and 311, C16,192T and
C16,270T). When a sample containing total bone marrow cells from
donor 2 was analyzed, 11 such polymorphisms were identified (A73G,
G185A, A263G, 6C at nucleotide position 311 of the mtDNA genome,
T16,093C, A16,158G, T16,172C, A16,183C, T16,189C, A16,219G and
C16,278T), in addition to an apparently new mutation (A478G).
[0215] A total of 70 mtDNA-sequence variants were found in bulk
bone marrow cells from six normal donors. Among these variants, 68
variants were previously listed in the Emory University online
mtDNA polymorphism database and are thus considered polymorphisms.
These results were compatible with data on bulk analysis of normal
marrow specimens (Shin et al. Blood, prepublished online Nov. 21,
2002; DOI 10.1182/blood-2002-06-1825). Two of the nucleotide
variants were new and were thus classified as mutations (A478G and
A517G in donors 1 and 6, respectively).
EXAMPLE 2
[0216] Unexpected MtDNA Heterogeneity in Single CD34.sup.+ Cell
Clones from Normal Bone Marrow
[0217] This example provides a description of one embodiment in
which the mutational frequency in mtDNA is measured in clonal
populations of CD34.sup.+ cells obtained from bone marrow.
[0218] Abnormalities of mtDNA have been hypothesized to play
important roles in senescence, malignancy and autoimmune disease.
mtDNA mutations have also been implicated in myelodysplasia.
However, in the previous studies, numerous point mutations were
found in bulk samples of bone marrow cells from normal controls, as
well as in patients.
[0219] To determine the heterogeneity of mtDNA sequences in normal
bone marrow, bone marrow specimens were collected from six normal
(do not have any apparent hematologic disease or symptom) donors.
Alternatively, peripheral blood samples are collected from the
donors either instead of, or in addition to, the bone marrow
samples. Mononuclear cells were separated from other hematopoietic
cells by standard Ficoll separation and washed twice in
phosphate-buffered saline (PBS). Cells suspended in PBS were
adjusted to 2.times.10.sup.7 cells/ml. To each 12.times.75 mm tube
containing 100 .mu.l of the cell suspension, 10 .mu.l of
phycoerythrin (PE)-conjugated anti-CD34, or 10 .mu.l of
PE-conjugated IgG1 (BD Bioscience, Franklin Lakes, N.J.) were
added. Following a 30 minute incubation at 4.degree. C., cells were
washed using cold PBS and resuspended in 0.5 ml PBS.
[0220] Human CD34.sup.+ cells were sorted using a MoFlo cytometer
(Dako-Cytomation, Fort Collins, Colo.) and an 1-90 argon laser
(emitting at 488 nm, Coherent Inc., Palo Alto, Calif.) for
excitation. Forward scatter was used as the triggering parameter.
PE fluorescence was detected using a 580/30 bandpass filter. Single
cell deposition was performed using a CyClone automated cloner
(Dako-Cytomation) in the 0.5 single drop mode. Gating of the cells
was based on forward scatter and PE fluorescence. Individual
CD34.sup.+ cells were plated into each well of a 96-well culture
plate with 100 .mu.l of serum-free medium containing 100 ng/ml of
stem cell factor (SCF), 100 ng/ml of Flt-3, 100 ng/ml of
thrombopoietin (TPO), in the presence or absence of 50 ng/ml of
granulocyte-colony stimulating factor (G-CSF).
[0221] After five days of culture, each well of the microtiter
plate was carefully examined with an inverted microscope (Olympus
IX50; Melville, N.Y.) in order to examine growth and plating
efficiency of single CD34.sup.+ cells. Cells in each well were
graded based on the number of cells present in the well following
the five-day culture. Grade 1 represented five or fewer cells per
well; Grade 2, between six and ten cells per well; Grade 3, between
11 and 20 cells per well; and Grade 4, more than 21 cells per well.
Cloning (plating) efficiency was defined as the number of positive
wells (any cells present)/total wells.times.100. Although there was
some variation of plating efficiency of CD34.sup.+ cells among six
normal donors, overall average efficiency was 30% (30.+-.11.7, mean
.+-.SD) (Table 3). Plating efficiency was not affected by G-CSF in
the growing medium, however colony size was increased in the
presence of G-CSF. TABLE-US-00003 TABLE 3 The number and
distribution of CD34.sup.+ clones from bone marrow after 5 day
culture. Donor 1 2 3 4 5 6 Subtotal No. G-CSF Total Grade + - + - +
- + - + - + - + - No. 1 14 19 27 22 103 176 45 71 98 208 82 131 369
627 996 2 9 12 1 2 65 84 33 74 67 110 43 103 218 385 603 3 16 14 1
3 49 42 50 53 60 59 79 70 255 241 496 4 36 2 6 0 122 5 112 21 135 5
150 16 561 49 610 Total clone No. 75 47 35 27 339 307 240 219 360
382 354 320 1403 1302 2705 Microplate No.* 2 2 5 5 10 10 10 10 10
10 10 10 47 47 94 Plating 39.1 24.5 7.3 5.6 35.3 32.0 25.0 22.8
37.5 39.8 36.9 33.3 31.1 28.9 30.0 Efficiency (%) Culture medium
with (+) or without (-) G-CSF. *the number of 96-well plates used
for culture.
[0222] To assess heterogeneity of the mtDNA sequences among
CD34.sup.+ cells from normal donors, the 1,121 base pair mtDNA
control region (a region known to contain multiple mutational
hotspots, nucleotide position 16024 through nucleotide position 576
as shown in FIG. 1 and FIG. 3) in a set of 611 CD34.sup.+ clones
(clonal populations) from the normal donors was subjected to
sequencing analysis (Table 4). TABLE-US-00004 TABLE 4 The number of
CD34.sup.+ clones from bone marrow subjected to mtDNA sequencing
analysis. Donor 1 2 3 4 5 6 G-CSF Total Grade + - + - + - + - + - +
- No. 1 14 14 13 10 15 15 13 13 12 14 14 11 158 2 8 12 1 2 15 15 14
14 15 13 15 15 139 3 16 14 1 3 15 15 15 13 15 15 15 15 152 4 35 2 6
0 25 5 15 14 25 5 15 15 162 Subtotal 73 42 21 15 70 50 57 54 67 47
59 56 611 No. Total 115 36 120 111 114 115 No.
[0223] To prepare for sequencing analysis, each CD34.sup.+ clone
was harvested from the culture well into a 1.5 ml microcentrifuge
tube by vigorous pipetting and dispensing, followed by a rinse of
the well with 200 .mu.l PBS. Cells were collected after
centrifugation at 300.times.g for 5 minutes, and then washed with
PBS. Cell pellets were stored at -80.degree. C. In order to extract
DNA from individual CD34.sup.+ clones, each cell pellet was covered
with 30 .mu.l of 1.times.TE buffer and lysed by incubating the
cells at 95.degree. C. for 10 minutes, with occasional shaking. The
lysate was briefly microfuged and stored at -20.degree. C.
[0224] In order to directly sequence the control region of mtDNA,
where the incidence of somatic mutations per nucleotide is
approximately 10-fold higher than anywhere else in the mtDNA genome
(Coller et al., Ann. N.Y. Acad. Sci. 959:434-447, 2002), DNA in the
clonal populations was subjected to nested gene PCR amplification
using the following nested primers: outer primer pair
5'-CGCCTACACAATTCTCCGATC-3' (SEQ ID NO: 1) and
5'-ACTTGGGTTAATCGTGTGACC-3' (SEQ ID NO: 2), which amplify the
region between nucleotide 15,974 and nucleotide 921 of the human
mtDNA genome as represented by the revised Human mtDNA Cambridge
Reference sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998), and inner primer
pair 5'-TTAACTCCACCATTAGCACC-3' (SEQ ID NO: 3) and
5'-GAAAGGCTAGGACCAAACCTA-3' (SEQ ID NO: 4), which amplify the
region between nucleotide 15,971 and nucleotide 670 of the human
mtDNA genome as represented by the revised Human mtDNA Cambridge
Reference sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998). Amplification of
mtDNA was performed with the TaKaRa LA PCR kit (PanVera, Madison,
Wis.).
[0225] The primary PCR mixture contained 400 .mu.M of each dNTP, 2
units of TaKaRa LA Taq.TM. (PanVera, Madison, Wis.), 0.8 .mu.M
outer primers and 2 .mu.l of cell lysate in a total volume of 30
.mu.l. PCR amplification was carried out in a thin-wall 0.5 ml PCR
tube using the DNA thermal cycler 480 (Perkin-Elmer, Foster City,
Calif.): one cycle at 96.degree. C. for 1 minute; 35 cycles at
94.degree. C. for 30 seconds, 52.degree. C. for 50 seconds and
72.degree. C. for 1 minute with a 5-second increase per cycle; one
cycle of 72.degree. C. for 5 minutes. The secondary PCR was
performed in 50 .mu.l of reaction mixture containing 400 .mu.M of
each dNTP, 2 units of TaKaRa LA Taq.TM. (PanVera, Madison, Wis.),
0.8 .mu.M inner nested primers and 1 .mu.l of primary PCR product
under the same amplification procedure as described above.
Secondary PCR samples were electrophoresed on 1% agarose gels and
stained with ethidium bromide to assess the purity and size of DNA
fragments, and subsequently purified using the QIAquick.TM. PCR
purification kit (Qiagen, Valencia, Calif.). As negative controls,
reaction mixtures without DNA templates were subjected to PCR
amplification. These samples were consistently negative for PCR
product. To prevent DNA cross-contamination, special precautions
were taken for each procedure of cell harvest, DNA extraction, PCR
amplification and DNA sequencing.
[0226] The purified-PCR products were subjected to cycle sequencing
with the appropriate primers using the BigDye Terminator v3.0 Ready
Reaction kit (Applied Biosystems, Foster City, Calif.) according to
the manufacturer's protocol, and then applied to the ABI Prism 3100
Genetic Analyzer (Applied Biosystems). The following
oligonucleotide primers were used for sequencing:
5'CAGTGTATTGCTTTGAGGAGG3' (SEQ ID NO: 5),
5'CATCTGGTTCCTACTTCAGGGTC3' (SEQ ID NO: 6),
5'TTAACTCCACCATTAGCACC3' (SEQ ID NO: 7),
5'GCATGGAGAGCTCCCGTGAGTGG3' (SEQ ID NO: 8),
5'CACCCTATTAACCACTCACG3' (SEQ ID NO: 9) and
5'TACATTACTGCCAGCCACCATG3' (SEQ ID NO: 10). The mtDNA sequences
experimentally obtained were compared to the 2001 Revised Cambridge
Reference Sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998) using the
National Center for Biotechnology Information (NCBI) Blast2 program
and the database search tool, MitoAnalyzer (National Institute of
Standards and Technology, Gaithersburg, Md.), in order to identify
polymorphisms and mutations. All automated results were manually
confirmed. To exclude potential artifacts, PCR amplifications from
original cell lysates were additionally replicated one or two more
times: when nucleotide changes were reproduced in all independent
PCR amplifications, they were considered to be confirmed.
[0227] To confirm the mtDNA control region sequence, PCR products
were directly inserted into pCR 2,1-TOPO.RTM. vector and
transformed into competent E. coli (TOP10 cells) using the TOPO
TA.TM. cloning kit (Invitrogen, Carlsbad, Calif.). Recombinant
plasmids isolated from 8 to 12 white colonies were subjected to
sequencing.
[0228] Analysis of the set of 611 CD34.sup.+ clones (clonal
populations) from six normal donors revealed that a total of 138
clones (22.6%.+-.13.6%) of the total assayed clones (611 clones)
displayed mtDNA heterogeneity distinct from the donor's
corresponding bulk mtDNA sequences and 47.8% (66/138 clones) of the
heterogeneity was localized to a mutational hot spot in the poly C
tract between nucleotides 303-315 (Table 5). TABLE-US-00005 TABLE 5
Summary of mtDNA heterogeneity among single CD34.sup.+ clones from
bone marrow Donor/Heteroplasmy No. pattern mtDNA gene Clone Total %
1 BM poly alone 85 85 73.9 + 8C/6C* + 9C/6C HV2, OH, CSB2 22 30
26.1 + 9C/6C* HV2, OH, CSB2 2 + 7C/6C* HV2, OH, CSB2 1 + A189G/A
HV2, OH, 7S 1 + T204C HV2, OH 1 + C277T HV2, OH, TFB1 1 + C514CAC 1
+ C16,114T HV1, 7S 1 Subtotal 115 115 100.0 2 BM poly alone 33 33
91.7 + T16,131C/T HV1, 7S 1 3 8.3 + G16,145A HV1, 7S 1 A73G, A191AA
HV2, OH, 7S 1 A263G C194T HV2, OH T199C HV2, OH G207A HV2, OH
8C/6C* HV2, OH, CSB2 T489C C16,147T HV1, 7S C16,173T HV1, 7S
C16,245T HV1, 7S T16,362C HV1, 7S Subtotal 36 36 100.0 3 BM poly
alone 96 96 80.0 + 9C/6C* + 10C/6C* HV2, OH, CSB2 11 24 20.0 +
8C/6C* + 9C/6C* HV2, OH, CSB2 6 + 8C/6C* HV2, OH, CSB2 2 + C182T/C
HV2, OH, 7S 2 8C/6C* + 9C/6C* HV2, OH, CSB2 + del 71G HV2, 7S 1
9C/6C* + 10C/6C* HV2, OH, CSB2 + T279C/T HV2, OH, TFB1 1 + G16,153A
HV1, 7S 1 Subtotal 120 120 100.0 4 BM poly alone 95 95 85.6 +
8C/6C* + 9C/6C* HV2, OH, CSB2 11 16 14.4 + 7C/6C* + 8C/6C* HV2, OH,
CSB2 2 + 9C/6C* + 10C/6C* HV2, OH, CSB2 1 + T89C HV2, 7S 1 + 8C/6C*
+ 9C/6C* HV2, OH, CSB2 1 16,093T HV1 Subtotal 111 111 100.0 5 BM
poly alone 62 62 54.4 + 514C, 515A 19 52 45.6 + C264T HV2, OH 18 +
C264T/C HV2, OH 3 + 7C/6C* + 8C/6C* HV2, OH, CSB2 2 + T146C HV2,
OH, 7S 2 514C, 515A + T146C HV2, OH, 7S 1 C264T/C HV2, OH + T146C/T
HV2, OH, 7S 1 514C, 515A + T146C HV2, OH, 7S 1 + A189G HV2, OH, 7S
1 + C264T/C HV2, OH 1 514C, 515A + T161C/T HV2, OH, 7S 1 C264T HV2,
OH 514C, 515A + T16,189C HV1, 7S 1 + C16,296C/T HV1 1 Subtotal 114
114 100.0 6 BM poly alone 102 102 88.7 + 8C/6C* + 9C/6C* HV2, OH,
CSB2 5 13 11.3 + A200G/A HV2, OH 3 + A200G HV2, OH 2 + A200G/A HV2,
OH 1 7C/6C* + 8C/6C* HV2, OH, CSB2 + A200G/A HV2, OH 1 8C/6C* +
9C/6C* HV2, OH, CSB2 + 7C/6C* + 8C/6C* HV2, OH, CSB2 1 Subtotal 115
115 100.0 No and % of CD34.sup.+ clones having `the same as total
BM 473 77.4 polymorphism alone` (77.4 .+-. 13.6%, mean .+-. SD) No
and % of CD34.sup.+ clones showing `mtDNA 138 22.6 heterogeneity`
(22.6 .+-. 13.6%, mean .+-. SD) Total No of assayed CD34.sup.+
clones 611 100.0
Abbreviations and symbols used in Table 5 include: BM poly, mtDNA
polymorphism from total bone marrow cells; +, mtDNA nucleotide
changes in addition to the polymorphisms detected in the respective
bulk mtDNA; *, poly C tract localized between nucleotide 303 and
315.
[0229] Common patterns of mtDNA heterogeneity in the CD34.sup.+
clonal populations among the set of CD34.sup.+ clonal populations
from the six donors included one or two nucleotide changes (for
instance substitution, insertion or deletion), in addition to the
polymorphisms detected in the respective bulk mtDNA. Among the
nucleotide changes identified in the corresponding regions of
mtDNA, most were due to single nucleotide substitutions at various
positions and length alterations in the homopolymeric C tract
located between nucleotide position 303 and 315 of the mtDNA
sequence. The mtDNA heterogeneity of CD34.sup.+ clones in six
donors was classified into several patterns according to nucleotide
changes: 8, 3, 7, 5, 13 and 6 patterns in donor 1 to 6,
respectively. The mean proportion of heterogenous pattern of mtDNA
heterogeneity among single CD34.sup.+ clones was 8.6% (8.6.+-.4.5,
mean .+-.SD) (FIG. 2). These results were unexpected, as rapidly
dividing tissues such as bone marrow have not been thought to
permit the homoplasmic resolution of mtDNA mutations over time.
Specifically, donors 1 and 3 showed clones with homopolymeric C
tract length heteroplasmy (nucleotide 303-nucleotide 315). In donor
5, nucleotide changes at nucleotide positions 264, 514, and 515
were common. Donor 5 also showed heteroplasmy of substituted
nucleotides at four nucleotide positions (T146C/T, T161C/T,
C264T/C, and C16296T/C).
[0230] The mtDNA sequence of a single CD34.sup.+ clonal population
from donor 2 exhibited a pattern (A73G, A191AA, C194T, T199C,
G207A, 8C/6C at nucleotide 303 and 311, T489C, C16,147T, C16,173T,
C16,245T, T16,362C) that was distinct from bulk bone marrow mtDNA,
as well as from other CD34.sup.+ clonal populations from the same
donor. Overall, the pattern of mtDNA heterogeneity was remarkably
different among the six donors. Neither the presence of G-CSF in
the growing medium, nor the colony size was statistically
correlated with the proportion of CD34.sup.+ clones with variant
mtDNA (Table 6). TABLE-US-00006 TABLE 6 Distribution of mtDNA
heterogeneity according to each grade and culture media Grade 1 2 3
4 Subtotal No. G-CSF Donor + - + - + - + - + - Total No. 1 3 3 1 1
5 7 10 0 19 11 30 2 2 0 1 0 0 0 0 0 3 0 3 3 2 3 3 3 3 4 5 1 13 11
24 4 1 1 1 3 4 2 0 4 6 10 16 5 6 6 6 6 10 7 10 1 32 20 52 6 0 1 3 5
2 0 1 1 6 7 13 Subtotal No. 14 14 15 18 24 20 26 7 79 59 138
Assayed colony No. 81 77 68 71 77 75 121 41 347 264 611 Proportion
(%)* 17.3 18.2 22.1 25.4 31.2 26.7 21.5 17.1 22.8 22.3 22.6 *No
statistical significant difference between culture media with
G-CSF, those without G-CSF and each grade
[0231] A high incidence of nucleotide variations was observed in
both HV2 (44/62, 71%) and HV1 (11/62, 18%) segments. Thus, most
heteroplasmic mutations were found in HV2, which includes mutations
within the homopolymeric C tract. This narrow region of the
homopolymeric C tract was a "hot spot" for heteroplasmic events in
isolated CD34.sup.+ cells, where 69% (18/44) and 29% (18/62) of the
heteroplasmic mutations were present in the hypervariable regions
and in the mtDNA control region, respectively. The results indicate
that in vivo clonal expansion of mtDNA is a general and common
process in hematopoietic tissue.
[0232] The general mechanism for the clonal expansion of mtDNA
mutations in hematopoietic stem cells is believed to include random
segregation or random genetic drift via unbiased mtDNA replication
and sorting during cell divisions. Based on these theories, the
pattern of length heteroplasmy (the relative proportion of length
variants) in poly (C) tracts could show a different pattern in
sister cells. However, studies have indicated that the pattern of
length heteroplasmy in poly C tracts is actively maintained in
daughter cells.
[0233] The data presented above indicate that several homopolymeric
tracts in the mtDNA exhibit length polymorphisms (the presence in
cells of multiple mtDNA species with various lengths of a
homonucleotide run). For example, a mtDNA variant (T16,189C)
introduces a cytosine, thereby generating an unstable homopolymeric
tract of ten cytosines between nucleotide positions 16,184 and
16,193. Some studies have indicated that the pattern of the length
heteroplasmy associated with the T16,189C mutation is maternally
inherited in that the pattern of length heteroplasmy co-segregates
among maternally related members. In addition, recent evidence
demonstrates the de novo regeneration of the pattern of length
heteroplasmy associated with the T 16,189C variant (Malik et al.,
J. Hum. Genet., 47:122-130, 2002).
EXAMPLE 3
MtDNA Heterogeneity in Single CD34.sup.+ Cell Clones from Normal
Umbilical Cord Blood
[0234] This example provides a description of one embodiment in
which the mutational frequency in mtDNA is measured in clonal
populations of CD34.sup.+ cells.
[0235] To determine the heterogeneity of mtDNA sequences in
CD34.sup.+ cells from umbilical cord blood, normal umbilical cord
blood was collected from five normal (do not have any apparent
hematologic disease or symptom) donors. Alternatively, peripheral
blood, or bone marrow samples are collected from the donors either
instead of, or in addition to, the umbilical cord blood
samples.
[0236] DNA extracted from total umbilical cord blood cells was
subjected to nested gene PCR amplification as described in Example
1, above. Sequencing was performed on an ABI Prism 3100 Genetic
Analyzer in both orientations. Nucleotide sequence changes of the
mtDNA control region in bulk samples of umbilical cord blood are
listed in Table 7.
[0237] The mtDNA sequence changes from bulk umbilical cord blood
showed individual variations but, interestingly, three (cord blood
donors 2, 3, and 5) of the five cord blood donors had length
variations of the poly (C) tract at nucleotide positions 303-315
and 16,183-16, 193 (Table 7). These findings were not observed in
bulk genotype from adult total bone marrow cells. TABLE-US-00007
TABLE 7 Nucleotide sequence changes of mtDNA control region from
total umbilical cord blood. Donors Polymorphism (Age/Sex)
(Mutation) Affected mtDNA gene 1 A73G HV2, 7S T146C HV2, 7S, OH
T195A HV2, OH A263G HV2, OH 7C/6C HV2, OH, CSB2 T489C del CA 514-5
A16,166T (del C 16169) HV1, 7S, TAS T16,172C HV1, 7S, TAS C16,223T
HV1, 7S C16,354T HV1, CSB3 T16,519C 7S 2* T72C HV2, 7S C253T HV2,
OH, TFB1 A263G HV2, OH 9C/6C HV2, OH, CSB2 10C/6C 8C/6C C16,256T
HV1, 7S 3.sup.# T16,298C HV1, 7S T16,519C 7S A73G HV2, 7S T146C
HV2, 7S, OH A189G HV2, 7S, OH C194T HV2, OH T195C HV2, OH T204C
HV2, OH G207A HV2, OH A263G HV2, OH 3* T279C HV2, OH 8C/6C HV2, OH,
CSB2 9C/6C C16,223T HV1, 7S C16,292T HV1, 7S T16,519C 7S 4 A73G
HV2, 7S del 249A HV2, OH, TFB1 del 290,291AA HV2, OH, TFB2 7C/6C
HV2, OH, CSB2 T489C A493G del 514,515CA C16,223T HV1, 7S T16,298C
HV1, 7S T16,325C HV1, 7S C16,327T HV1, 7S T16,519C 7S 5.sup.# A73G
HV2, 7S C150T HV2, 7S, OH T195C HV2, OH A263G HV2, OH 7C/6C HV2,
OH, CSB2 A16,171G HV1, 7S, TAS T16,172C HV1, 7S, TAS T16,189C HV1,
7S C16,193C/CC HV1, 7S C16,223T HV1, 7S C16,320T HV1, 7S T16,519C
7S *Mixed nucleotide signal (mixed peak on sequencing chromatogram)
at nucleotide position between 303 and 315; .sup.#np
16,183-16193
[0238] In order to determine the heterogeneity of single CD34.sup.+
clones from umbilical cord blood, mononuclear cells were separated
from other hematopoietic cells by standard Ficoll separation and
washed twice in phosphate-buffered saline (PBS). Cells suspended in
PBS were adjusted to 2.times.10.sup.7 cells/ml. To each 12.times.75
mm tube containing 100 .mu.l of the cell suspension, 10 .mu.l of
phycoerythrin (PE)-conjugated anti-CD34, or 10 .mu.l of
PE-conjugated IgG1 (BD Bioscience, Franklin Lakes, N.J.) were
added. Following a 30 minute incubation at 4.degree. C., cells were
washed using cold PBS and resuspended in 0.5 ml PBS.
[0239] Human CD34.sup.+ cells were sorted using a MoFlo cytometer
(Dako-Cytomation, Fort Collins, Colo.) and an 1-90 argon laser
(emitting at 488 nm, Coherent Inc., Palo Alto, Calif.) for
excitation. Forward scatter was used as the triggering parameter.
PE fluorescence was detected using a 580/30 bandpass filter. Single
cell deposition was performed using a CyClone automated cloner
(Dako-Cytomation) in the 0.5 single drop mode. Gating of the cells
was based on forward scatter and PE fluorescence. Individual
CD34.sup.+ cells were plated into each well of a 96-well culture
plate with 100 .mu.l of serum-free medium containing 100 ng/ml of
stem cell factor (SCF), 100 ng/ml of Flt-3, 100 ng/ml of
thrombopoietin (TPO), in the presence or absence of 50 ng/ml of
granulocyte-colony stimulating factor (G-CSF).
[0240] After five days of culture, each well of the microtiter
plate was carefully examined with an inverted microscope (Olympus
IX50; Melville, N.Y.) in order to examine growth and plating
efficiency of single CD34.sup.+ cells. As described above in
Example 2, cells in each well were graded based on the number of
cells present in the well following the five-day culture. Cloning
(plating) efficiency was defined as the number of positive wells
(any cells present)/total wells.times.100. Although there was some
variation of plating efficiency of CD34.sup.+ cells among five
normal donors, overall average efficiency was 79% (78.6.+-.11.7,
mean .+-.SD) (Table 8). Plating efficiency was not affected by
G-CSF in the growing medium, however colony size was increased in
the presence of G-CSF. TABLE-US-00008 TABLE 8 The number and
distribution of CD34.sup.+ clones from umbilical cord blood after 5
day culture. Donor 1 2 3 4 Subtotal No Total No Microplate* PE (%)
G-CSF Grade + - + - + - + - + - + - + - 1 46 85 79 155 167 309 398
124 690 673 1363 10 10 71.9 70.1 2 8 8 6 21 23 78 369 309 406 416
822 5 5 84.6 86.7 3 28 55 33 45 57 44 280 245 398 389 787 5 5 82.9
81.0 4 23 30 21 26 30 59 346 299 420 414 834 5 5 87.5 86.3 5 22 33
23 36 29 52 286 243 360 364 724 5 5 75.0 75.8 Subtotal 127 211 162
283 306 542 1679 1220 2274 2256 4530 30 30 79.0 78.3 Total 338 445
848 2899 4530 60 78.6 Culture medium with (+) or without (-) G-CSF.
*the number of 96-well plates used for culture.
[0241] To assess heterogeneity of the mtDNA sequences among
CD34.sup.+ cells from each of the normal umbilical cord blood
donors, the 1,121 base pair mtDNA control region in a set of 580
CD34.sup.+ clones (clonal populations) was subjected to sequencing
analysis (Table 9). TABLE-US-00009 TABLE 9 The number of CD34.sup.+
clones from umbilical cord blood subjected to mtDNA sequencing
analysis. Donor 1 2 3 4 Subtotal No Total G-CSF assayed Grade + - +
- + - + - + - No 1 15 15 14 15 15 15 15 15 59 60 119 2 8 8 6 15 15
16 26 26 55 65 120 3 14 13 14 15 15 15 5 13 48 56 104 4 15 14 15 15
15 15 15 15 60 59 119 5 14 15 15 14 15 15 15 15 59 59 118 Subtotal
66 65 64 74 75 76 76 84 281 299 580 Total 131 138 151 160 580
[0242] To prepare for sequencing analysis, each CD34.sup.+ clone
was harvested from the well into a 1.5 ml microcentrifuge tube by
vigorous pipetting and dispensing followed by rinse of the well
with 200 .mu.l of PBS. Cells were collected after centrifugation at
300.times.g for 5 minutes, and then washed with PBS. Cell pellets
were stored at -80.degree. C. In order to extract DNA from
individual CD34.sup.+ clones, each cell pellet was covered with 30
.mu.l of 1.times.TE buffer and lysed by incubating the cells at
95.degree. C. for 10 minutes, with occasional shaking. The lysate
was briefly microfuged and stored at -20.degree. C.
[0243] DNA in the CD34.sup.+ clonal populations of cells was
amplified to directly sequence the mtDNA control region. Cell
lysates of individual CD34.sup.+ clones were subjected to
amplification of mtDNA using the LA PCR kit (TaKaRa, Panvera,
Madison, Wis.).
[0244] Nested (two-step) PCR amplification was performed with outer
and inner pairs of primers in order to generate sufficient template
from CD34.sup.+ clones for sequencing of the mtDNA control region.
The outer pair of primers (5'-CGCCTACACAATTCTCCGATC-3' (SEQ ID NO:
1) and 5'-ACTTGGGTTAATCG TGTGACC-3' (SEQ ID NO: 2)) was used for
amplification of the fragment between nucleotide 15974 and 921 of
the revised Human mtDNA Cambridge Reference Sequence (Andrews et
al., Nat. Genet. 23:147, 1999; Kogelnik et al., Nucleic Acids Res.
26:112, 1998). The inner nested pair of primers
(5'-TTAACTCCACCATTAGCACC-3' (SEQ ID NO: 3) and
5'-GAAAGGCTA-GGACCAAA CCTA-3' (SEQ ID NO: 4)) amplified the
fragment between nucleotide 15,971 and 670 of the revised Human
mtDNA Cambridge Reference Sequence (Andrews et al., Nat. Genet.
23:147, 1999; Kogelnik et al., Nucleic Acids Res. 26:112,
1998).
[0245] The primary PCR mixture contained 400 .mu.M of each dNTP, 2
units of LA Taq.TM., 0.8 .mu.M outer primers and 3 .mu.l of cell
lysate in a total volume of 30 .mu.l. PCR amplification was carried
out in a thin-wall 0.5 ml PCR tube using the DNA thermal cycler 480
(Perkin-Elmer, Foster City, Calif.): one cycle of 96.degree. C. for
1 minute; 35 cycles of 94.degree. C. for 30 seconds, 52.degree. C.
for 50 seconds and 72.degree. C. for 1 minute with a 5-second
increase per cycle; one cycle of 72.degree. C. for 5 minutes. The
secondary PCR was performed in 50 .mu.l of reaction mixture
containing 400 .mu.M of each dNTP, 2 units of LA Taq.TM., 0.8 .mu.M
inner nested primers and 1 .mu.l of primary PCR product under the
same amplification procedure as described above. Secondary PCR
samples were electrophoresed on 1% agarose gels and stained with
ethidium bromide to assess the purity and size of DNA fragments,
and subsequently purified using the QIA quick PCR purification kit
(Qiagen, Valencia, Calif.). As negative controls, reaction mixtures
without DNA templates were subjected to PCR amplification. These
samples were consistently negative for PCR product. To prevent DNA
cross-contamination, special precautions were taken for each
procedure of cell harvest, DNA extraction, PCR amplification and
DNA sequencing.
[0246] The purified-PCR products were subjected to cycle sequencing
with the appropriate primers using the BigDye Terminator v3.0 Ready
Reaction kit (Applied Biosystems, Foster City, Calif.) according to
the manufacturer's protocol, and then applied to the ABI Prism 3100
Genetic Analyzer (Applied Biosystems). The following
oligonucleotide primers were used for sequencing:
5'CAGTGTATTGCTTTGAGGAGG3' (SEQ ID NO: 5),
5'CATCTGGTTCCTACTTCAGGGTC3' (SEQ ID NO: 6),
5'TTAACTCCACCATTAGCACC3' (SEQ ID NO: 7),
5'GCATGGAGAGCTCCCGTGAGTGG3' (SEQ ID NO: 8),
5'CACCCTATTAACCACTCACG3' (SEQ ID NO: 9) and
5'TACATTACTGCCAGCCACCATG3' (SEQ ID NO: 10). The mtDNA sequences
experimentally obtained were compared to the 2001 Revised Cambridge
Reference Sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998) using the
National Center for Biotechnology Information (NCBI) Blast2 program
and the database search tool, MitoAnalyzer (National Institute of
Standards and Technology, Gaithersburg, Md.), in order to identify
polymorphisms and mutations. All automated results were manually
confirmed. To exclude potential artifacts, PCR amplifications from
original cell lysates were additionally replicated one or two more
times: when nucleotide changes were reproduced in all independent
PCR amplifications, they were considered to be confirmed.
[0247] To confirm the mtDNA control region sequence, PCR products
were directly inserted into pCR 2,1-TOPO.RTM. vector and
transformed into competent E. coli (TOP10 cells) using the TOPO
TA.TM. cloning kit (Invitrogen, Carlsbad, Calif.). Recombinant
plasmids isolated from 8 to 12 white colonies were subjected to
sequencing.
[0248] Analysis of the set of 580 CD34.sup.+ clones (clonal
populations) from the five normal donors revealed that few
CD34.sup.+ clones obtained from umbilical cord blood had mtDNA
heterogeneity. Specifically, only nine clones among the set of 580
CD34.sup.+ clones analyzed (1.6%, 1.6.+-.1.5) showed mtDNA
heterogeneity (Tables 10 and 11) and the proportion of clones with
a unique mtDNA heterogeneity pattern within the set of 580
CD34.sup.+ clones was 1.2% (1.2.+-.1.0). Except for clones with
mtDNA heterogeneity, all CD34.sup.+ clones from cord blood donors
2, 3 and 5 had the same pattern of length heteroplasmy in the poly
C tracts between nucleotides 303 and 315 and nucleotides 16,183 and
16193. The mtDNA sequence of a single CD34.sup.+ clone from cord
blood donor 1 represented an extremely distinct pattern from the
bulk BM mtDNA as well as from other CD34.sup.+ clones (Table 10).
TABLE-US-00010 TABLE 10 Summary of mtDNA heterogeneity among single
CD34.sup.+ clones derived from cord blood CB donor Heteroplasmy
pattern No. of clones Total % 1 BM poly (+) alone 118 118 99.2 A73G
A153G 1 1 0.8 A263G A183G 7C/6C 8C/6C del 514-515CA C325T C16,223T
C463T C16,354T T485C T16,519C T489C T16,198C C16,268T T16,381A
Subtotal 119 119 100 2 BM poly (+) alone 120 120 100.0 Subtotal 120
120 100.0 3 BM poly (+) alone 100 100 96.2 + G316G/C 3 4 3.8 +
CCC305-307CCC/AAA 1 Subtotal 104 104 100 4 BM poly (+) alone 118
118 99.2 + 6C/6C 1 1 0.8 + T16,022C Subtotal 119 119 100.00 5 BM
poly (+) alone 114 114 97.4 + C16,193CCC (12C) 1 3 2.6 + del
16,189T 1 + C16,193CCCC (13C) 1 Subtotal 117 117 100.0 Total BM
poly (+) alone 571 571 98.1 (summary) Total No. of heterogeneity 9
9 1.6 Unique heterogeneity 7 1.2 580 580 580 Abbreviations: BM
poly, mtDNA polymorphism from total bone marrow cells; +, mtDNA
nucleotide changes in addition to the polymorphisms detected in the
respective bulk mtDNA; *poly C tract localized between np 303 and
np 315.
[0249] A comparison of the characteristics between CD34.sup.+
clones derived from adult bone marrow and umbilical cord blood is
presented in Table 11. Chi square test was used to test statistical
differences in comparison of the frequency of heteroplasmy from
adult bone marrow and cord blood. The one-way ANOVA (analysis of
variance) test was performed to examine statistical differences in
the effect of each grade and culture media on mtDNA heterogeneity;
P<0.05 was considered significant. Remarkable diversity of the
mtDNA sequence was observed in individual CD34.sup.+ clones from
adult bone marrow, in contrast to the findings from cord blood.
TABLE-US-00011 TABLE 11 Comparison of characteristics between
CD34.sup.+ clones derived from adult bone marrow and umbilical cord
blood. Adult BM CD34.sup.+ Cord Blood CD34.sup.+ Plating Efficiency
30% 78.6%* Bulk cell analysis Uniform pattern Frequently
heteroplasmic Heteroplasmy Total rate 22.6% 1.6% Unique pattern
8.6% 1.2% Substitution (S) 10.6% (65/611) 0.0% (0/580) Poly C
tract.sup.# 10.8% (66/611) 1.1% (7/580) S + C 1.1% (7/611) 0.3%
(2/580) *statistically significant difference (P < 0.05);
.sup.#mtDNA nucleotide sequence position at 303-315 and
16,183-16,193.
EXAMPLE 4
In Vivo Mutational Spectra of Mitochondrial Genomes and Evidence of
the Clonal Expansion in Single Hematopoietic Progenitor Cell
Clones
[0250] This example provides a description of one embodiment in
which the mutational frequency of the mtDNA control region, as well
as genes (CO1 and Cytb) in the mtDNA coding region is measured in
clonal populations of CD34.sup.+ cells and in single
granulocytes.
[0251] Examples 2 and 3 demonstrated that an average of 25% of
individual CD34.sup.+ clones from adult normal bone marrow differ
in the control region of a mtDNA sequence from the bulk cell
sequence and almost 8% of the mutations are uniquely different, in
contrast to virtually homogenous mtDNA sequences in normal
umbilical CB CD34.sup.+ clones. Thus, it is significant that the
mutations detected in the bone marrow CD34.sup.+ clones were also
found in circulating CD34.sup.+ cells and peripheral blood cells
(single granulocytes). This demonstrates that mutational rates and
monitoring of minimal residual disease can be evaluated by blood
sampling rather than bone marrow aspiration. This example describes
how the mutational frequency of mtDNA in circulating CD34.sup.+
cells and single granulocytes was examined.
[0252] To determine the heterogeneity of mtDNA sequences in single
granulocytes and CD34.sup.+ cells, bone marrow (BM) and peripheral
blood (PB) specimens from six normal (do not have any apparent
hematologic disease or symptom) adult donors were collected and
mononuclear cells from BM and PB were separated by density gradient
centrifugation. The cells were then washed twice in PBS and the
number of cells suspended in PBS was adjusted to 2.times.10.sup.7
cells/ml. 10 .mu.l of anti-CD34 phycoerythrin (PE)-conjugated
monoclonal antibody and 10 .mu.l anti-CD33 FITC-conjugated antibody
(BD Bioscience, San Jose, Calif.) were added to each 12.times.75 mm
tube containing 100 .mu.l of cell suspension. After incubation for
30 minutes at 4.degree. C., cells were washed using cold PBS and
resuspended in 0.5 ml of PBS.
[0253] Cell sorting was performed on a MoFlo Cytometer
(Dako-Cytomation, Ft Collins, Colo.), using one hundred milliwatts
of the 488 nm line of an argon laser (1-90, Coherent Inc, Palo
Alto, Calif.) for excitation. Forward scatter was the triggering
parameter. Fluorescence of FITC was detected using a 530/20
bandpass filter and the fluorescence of PE was detected using a
580/30 bandpass filter. Single cell deposition was accomplished
using the CyClone automated cloner (Dako-Cytomation); in the 0.5
single drop mode with gating based on forward scatter and
fluorescence. Individual CD34.sup.+ cells were placed into each
well of a 96-well microplate (Nalge Nunc International, Rochester,
N.Y.) containing 100 .mu.l of culture media and single granulocytes
were deposited in each well of a MicroAmp.RTM. optical 96-well
reaction plate (Applied Biosystems, Foster City, Calif.) containing
30 .mu.l of 1.times. Tris EDTA (TE) buffer (FIG. 3A).
[0254] Individual CD34.sup.+ cells placed into separate wells of
96-well plates were cultured in serum-free medium containing 100
ng/ml of stem cell factor, 100 ng/ml of Flt-3, 100 ng/ml of
thrombopoietin, with 50 ng/ml of G-CSF (all from Stem Cell
Technologies, Vancouver, Canada). After culture for 5 days, each
well of the microtiter plate was carefully observed using an
inverted microscope (Olympus IX50, Melville, N.Y.) in order to
determine growth and plating efficiency of single CD34.sup.+ cells.
Grade growth was quantified with the following scoring system based
on cell number in each CD34.sup.+ clone: grade 1, .ltoreq.5
cells/well; grade 2, 6-10 cells/well; grade 3, 11-20 cells/well;
grade 4, .gtoreq.21 cells/well (FIG. 3C). Plating efficiency (PEf)
was defined as the number of positive (cells were present)
wells/total wells.times.100. Each CD34.sup.+ clone was harvested
from the well by vigorous pipetting and dispensed into a 1.5 ml
microcentrifuge tube and rinsed with 200 .mu.l of PBS. Cells were
collected after centrifugation at 300-.times.g for 5 minutes, and
then washed with PBS. CD34.sup.+ cell pellets were then stored at
-80.degree. C. In order to extract DNA from individual CD34.sup.+
clones, 30 .mu.l of 1.times.TE buffer was placed in each 1.5 ml
tube containing one cell pellet. The cells were lysed by incubation
at 95.degree. C. for 10 minutes with occasional shaking in order to
liberate the total DNA. The resulting lysate was briefly
centrifuged and stored at -20.degree. C.
[0255] In order to extract DNA from single granulocytes deposited
into each well of a 96-well microplate containing 30 .mu.l of
1.times.TE buffer, the 96-well reaction plate (Applied Biosystems)
was placed in a GeneAmp PCR system 9700 (Applied Biosystems) for
incubation at 95.degree. C. for 5 minutes. The resulting lysate was
briefly centrifuged and stored at -20.degree. C.
[0256] Cell lysates of individual CD34.sup.+ clones and single
granulocytes were then subjected to amplification of the mtDNA
control region and coding region (CO1 and Cytb genes) using the LA
PCR kit (TaKaRa LA Taq.TM., Madison, Wis.). Two-step PCR
amplification was performed with outer and inner pairs of primers
in order to generate sufficient template from CD34.sup.+ clones and
single granulocytes for sequencing of the mtDNA control region and
coding region (CO1 and Cytb genes). Primer pairs for targeted mtDNA
gene amplification and fragments spanning nucleotides position
(amplicons size) are represented in Table 12. TABLE-US-00012 TABLE
12 Primer sets for two-step PCR and direct sequencing of mtDNA
control region, CO1 and Cytb genes Amplicon MtDNA gene Sequence (5'
to 3') for two-step PCR Sequencing primers (5' to 3') 1 Control
F15574 (O) CGCCTACACAATTCTCCGATC (O) F15971 TTAACTCCACCATTAGCACC
(1.12 kb) region (SEQ ID NO: 1) (SEQ ID NO: 7) (16024-16569; R921
(O) ACTTGGGTTAATCGTGTGACC (O) R1 CAGTGTATTGCTTTGAGGAGG 1-576) (SEQ
ID NO: 2) (SEQ ID NO: 19) F15971 (I) TTAACTCCACCATTAGCACC (I) SR
GCATGGAGAGCTCCCGTGAGTGG (SEQ ID NO: 3) (SEQ ID NO: 8) R1 (I)
CAGTGTATTGCTTTGAGGAGG (I) SF CATCTGGTTCCTACTTCAGGGTC (SEQ ID NO: 5)
(SEQ ID NO: 6) 2 CytB F14622 (O) CCACAAACCCCATTACTAAACCCAC (O)
F14688 CTACAACCACGACCAATGATATG (1.39 kb) (14688-15996) (SEQ ID NO:
11) (SEQ ID NO: 20) R16084 (O) CGGTTGTTGATGGGTGAGTC (O) R15996
GCTTTGGGTGCTAATGGTGGAG (SEQ ID NO: 12) (SEQ ID NO: 21) F14663 (I)
GCATACATCATTATTCTCGCACGG (I) (SEQ ID NO: 13) R16057 (I)
GGGTGGTACCCAAATCTGCTTCC (I) (SEQ ID NO: 14) 3 CO1 F6541 (O)
GCAACCTCAACACCACCTTCTTCG (O) F6645 CTACCAGGCTTCGGAATAATCTCCC (0.91
kb) (6611-7470) (SEQ ID NO: 15) (SEQ ID NO: 22) R7613 (O)
GTAGACCTACTTGCGCTGCATGTGC (O) R7425 GTTCTTCGAATGTGTGGTAGGGTG (SEQ
ID NO: 16) (SEQ ID NO: 23) F6586 (I) CCATTCTATACCAACACCTATTCTG (I)
(SEQ ID NO: 17) R7498 (I) CCATGGGGTTGGCTTGAAACCAGC (I) (SEQ ID NO:
18) Abbreviations: Cytb, cytochrome b; CO1, cytochrome c oxidase 1;
F, forward primer; R, reverse primer; O, outer primer; I, inner
primer.
[0257] The primary PCR mixture contained 400 .mu.M of each dNTP, 2
units of LA Taq.TM. (TaKaRa LA Taq.TM.), 0.8 .mu.M outer primers, 3
.mu.l and 10 .mu.l of cell lysates from individual CD34.sup.+
clones and single granulocytes, respectively. PCR amplification was
carried out in a MicroAmp.RTM. optical 96-well reaction plate
(Applied Biosystems) using the GeneAmp PCR system 9700 (Applied
Biosystems): one cycle of 95.degree. C. for 1 minute; then 35
cycles of 95.degree. C. for 30 seconds, 52.degree. C. for 50
seconds and 72.degree. C. for 1 minute with a 10 second increase
per cycle; ending with one cycle of 72.degree. C. for 5 minutes.
The secondary PCR was performed in 50 .mu.l of reaction mixture
containing 400 .mu.M of each dNTP, 2 units of LA Taq.TM., 0.8 .mu.M
inner nested primers and 2 .mu.l of primary PCR product under the
same amplification conditions as described above. Secondary PCR
samples were electrophoresed on 1% agarose gels and stained with
ethidium bromide to assess the purity and size of the DNA
fragments, and subsequently purified using the QIA quick PCR
purification kit (Qiagen, Valencia, Calif.). The negative controls,
reaction mixtures without DNA templates, were subjected to the same
PCR amplification conditions and in all cases, confirmed to be
negative. To prevent DNA cross-contamination, special precautions
were taken for each procedure of cell harvest, DNA extraction, PCR
amplification and DNA sequencing. PCR amplification of mtDNA CO1
and Cytb genes was performed in the same manner described above.
Corresponding primers for each gene amplification were listed in
Table 12.
[0258] To assess heterogeneity of the mtDNA sequences in CD34.sup.+
clones and single granulocytes, the amplified control and coding
regions were subjected to sequencing analysis. mtDNA genes were
directly sequenced using the BigDye Terminator v3.1 ready reaction
kit (Applied Biosystems) and the ABI Prism 3100 Genetic Analyzer
(Applied Biosystems). Sequencing primers used in each mtDNA gene
are shown in Table 12. MtDNA sequences experimentally obtained were
compared to the revised Human mtDNA Cambridge Reference sequence
(Andrews et al., Nat. Genet. 23:147, 1999; Kogelnik et al., Nucleic
Acids Res. 26:112, 1998) using the NCBI Blast2 program and the
database search tool, MitoAnalyzer (National Institute of Standards
and Technology, Gaithersburg, Md.) in order to determine
polymorphisms and mutations. All automated results were manually
confirmed. To exclude potential artifacts, PCR amplifications from
original cell lysates were additionally replicated one or two more
times and when nucleotide changes were reproduced in all
independent PCR amplifications, they were considered to be
confirmed.
[0259] In preliminary experiments, 285 base pair amplicons were
generated by gene amplification of wild type mtDNA and mtDNA
altered in a single base and then mixed in varying proportions. On
sequencing of the mixtures, the lower limit of detection of a minor
species of mtDNA was approximately 20%. Mixed nucleotide signals on
sequencing chromatograms, when observed in the current study, were
assumed to represent at least 20% heteroplasmy but could also
result from gene amplification artifacts. To confirm heteroplasmy
and mixed nucleotide signals in the sequences of the mtDNA control
region, PCR products were directly inserted into the pCR
2,1-TOPO.RTM. vector and transformed into competent E. coli (TOP10
cells) using the TOPO TA cloning kit (Invitrogen, Carlsbad,
Calif.). Recombinant plasmids isolated from 8 to 12 white colonies
were sequenced.
[0260] A Chi square test was used to determine statistical
differences in the frequency of heteroplasmy in adult bone marrow
and circulating blood. The one-way ANOVA (analysis of variance)
test was performed to examine whether total rate and unique
difference among cord blood CD34.sup.+ clones, adult BM CD34.sup.+
clones, circulating CD34.sup.+ clones and single granulocytes
produced significant statistical differences in mtDNA
heterogeneity. P<0.05 was considered significant.
[0261] In order to identify mtDNA heterogeneity in individual
CD34.sup.+ clones and single granulocytes, the aggregate (bulk)
cell genotype from total bone marrow cells from each donor was
first examined (see Example 1, above). There was marked variation
in the number of nucleotide changes among individual donors, with
ranges of 6 (donor 1) to 23 (donor 4) (11.3.+-.6.1, mean .+-.SD)
(Table 13A). A total of 68 mtDNA-sequence variants were found in
bulk cells from six normal donors. Among these, 66 variants were
already listed in the Mitomap polymorphism database and two new
nucleotide variants were classified as mutations (A478G and A517G
in donor 2 and 6, respectively). Donors 2 and 3 had length
variations of the poly C tract at nucleotide positions
16,183-16,193 (T16,189C, 12C) and 303-315 (Table 13A).
TABLE-US-00013 TABLE 13 Nucleotide sequence changes of mtDNA
control region (A) and coding region (B) from total (bulk) bone
marrow cells A. Control region Donors Affected mtDNA (Age/Sex)
Polymorphism gene 1 A73G HV2, 7S (47/F) C150T HV2, 7S, OH A263G
HV2, OH 8CT6C* HV2, OH, CSB2 C16,192T HV1, 7S C16,270T HV1, 7S 2
A73G HV2, 7S (38/F) G185A HV2, 7S, OH A263G HV2 + OH 7CT6C* HV2,
OH, CSB2 A478G.sup.# T16,093C HV1 A16,158G HV1, 7S, TAS T16,172C
HV1, 7S, TAS A16,183C HV1, 7S T16,189C (12C) HV1, 7S A16,219G HV1,
7S C16,278T HV1, 7S 3 A73G HV2, 7S (43/M) T146C HV2, 7S, OH T152C
HV2, 7S, OH T195C HV2, OH A263G HV2, OH 9CT6C*, 8CT6C* HV2 + OH +
CSB2 del 514 C del 515 A C16,223T HV1, 7S C16,278T HV1, 7S C16,294T
HV1, 7S G16,390A 7S 4 A93G HV2, 7S (34/M) A95C HV2, 7S G185A HV2,
7S, OH A189G HV2, 7S, OH T236C HV2, OH 8CT6C* HV2, OH, CSB2 G247A
HV2, OH, TFB1 A263G HV2, OH del C 514 del A 515 T16,093C HV1
G16,129A HV1, 7S C16,148T HV1, 7S C16,168T HV1, 7S, TAS T16,172C
HV1, 7S, TAS C16,187T HV1, 7S C16,188G HV1, 7S T16,189C HV1, 7S
C16,223T HV1, 7S A16,230G HV1, 7S C16,278T HV1, 7S A16,293G HV1, 7S
T16,311C HV1, 7S C16,320T HV1, 7S 5 A73G HV2, 7S (54/M) A263G HV2,
OH 7CT6C* HV2, OH, CSB2 del 514C del 515A T16,126C HV1, 7S C16,294T
HV1, OH C16,296T HV1, OH T16,519C 7S 6 A73G HV2, 7S (34/F) C150T
HV2, 7S, OH A263G HV2, OH 8CT6C* HV2, OH, CSB2 A517G.sup.# C16,270T
HV1, 7S C16,292T HV1, 7S T16,362C HV1, 7S B. coding region (CO1 and
Cytb) Donors MtDNA Nucleotide Amino acid Polymorphism (P)/
(Age/Sex) gene change change Mutation (M) 1 (47/F) CO1 C7028T No P
Cytb A15326G Thr-Ala P 3 (43/M) CO1 A6663G Ile-Val P CO1 C7028T No
P CO1 T7175C No P CO1 C7256T No P CO1 C7274T No P Cytb G15301A No P
Cytb A15326G Thr-Ala P Cytb T15784C.sup.# No M 5 (54/M) CO1
T7022C.sup.# No M CO1 C7028T No P Cytb C14766T No P Cytb G14905A No
P Cytb A15326G Thr-Ala P Cytb C15452A Leu-Ile P Cytb A15607G No P
Abbreviations: HV1, hypervariable segment 1; HV2, hypervariable
segment 2; 7S, 7S DNA; OH, H-strand origin; CSB2, conserved
sequence block II; TAS, termination-association sequence; TFB1,
mitochondrial transcription factor 1 binding site; *homopolymeric C
tract localized between nucleotide 303 and 315 (for example, 8CT6C
defined CCCCCCCCTCCCCCC); .sup.#new mtDNA polymorphisms (not listed
in accepted database); Ala, alanine; Ile, isoleucine; Leu, leucine;
Thr, threonine; Val, valine.
[0262] The bulk genotype of the mtDNA coding region (CO1 and Cytb
genes) from total bone marrow cells was also examined. A total of
17 mtDNA nucleotide changes (5.7.+-.3.7, mean .+-.SD) were noted in
the CO1 and Cytb genes among three donors (Table 13B): 15
nucleotide variants were already listed in the published Mitomap
polymorphism database and 2 new sequence variations were identified
that were not previously recorded (including among unpublished
mtDNA polymorphisms). These two mutations were not predicted to
produce amino acid change (Table 13B).
[0263] CD34.sup.+ cell-derived colonies cultured in individual
wells of 96-well plates in serum-free medium containing selected
hematopoietic growth factors were classified according to the cell
number per well (FIG. 3C). Although there was some variation of
plating efficiency of CD34.sup.+ cells among six normal, overall
average plating efficiency in PB and BM was 45% (45.3.+-.9.3, mean
.+-.SD) and 36% (35.7.+-.3.6) respectively (Table 13A). This
difference was not statistically significant (P=0.12).
[0264] The mtDNA control regions from a total of 4,704 single
granulocytes from six donors was amplified using a double nested
PCR. A summary of the amplification results is provided in Table
14A. From the 4,704 granulocytes, 355 (8%) (7.5.+-.4.2, mean
.+-.SD) produced a product of the correct size. TABLE-US-00014
TABLE 14 CD34.sup.+ clones from peripheral blood and bone marrow,
and single granulocyte for mtDNA analysis A. Plating efficiency and
grade of CD34.sup.+ clones after 5 day-suspension culture, and
nested mtDNA PCR efficiency from single granulocytes Samples PB
CD34.sup.+ clone BM CD34.sup.+ clone Grade Sub- Micro- Grade Donor
1 2 3 4 total plate* PEf (%) 1 2 3 4 1 (47/F) 13 54 128 85 280 5
58.3 33 21 30 38 2 (38/F) 33 61 64 34 192 4 50.0 -- -- -- -- 3
(43/M) 24 20 23 26 93 3 32.3 279 149 91 127 4 (34/M) 38 38 58 66
200 5 41.7 -- -- -- -- 5 (54/M) 35 50 61 36 182 5 37.9 306 177 119
140 6 (34/F) 37 46 52 92 227 5 47.3 -- -- -- -- Total 180 269 386
339 1174 27 45.3 618 347 240 305 Samples BM CD34.sup.+ clone Single
granulocyte mtDNA PCR Sub- Micro- Granulocyte PCR efficiency Donor
total plate PEf (%) No Positive No % 1 (47/F) 122 4 31.8 480 51
10.6 2 (38/F) -- -- -- 1440 37 2.6 3 (43/M) 646 20 33.6 480 50 10.4
4 (34/M) -- -- -- 768 111 14.5 5 (54/M) 742 20 38.6 768 42 5.5 6
(34/F) -- -- -- 768 64 8.3 Total 1510 44 35.27 4704 355 7.5 B.
Assay number MtDNA Control region BM CD34.sup.+ clone PB CD34.sup.+
clone Single Donor G1 G2 G3 G4 Subtotal G1 G2 G3 G4 Subtotal
granulocyte 1 (47/F) 28 20 30 37 115 13 30 47 30 120 51 2 (38/F) 23
3 4 6 36 23 24 23 24 94 37 3 (43/M) 30 30 30 30 120 24 20 23 26 93
50 4 (34/M) 26 28 28 29 111 24 24 24 24 96 111 5 (54/M) 26 28 30 30
114 24 23 24 24 95 42 6 (34/F) 25 30 30 30 115 24 24 24 24 96 64
Total 158 139 152 162 611 133 146 166 152 594 355 MtDNA Coding
region (CO1 and Cytb) BM CD34.sup.+ clone PB CD34.sup.+ clone Donor
G1 G2 G3 G4 Subtotal G1 G2 G3 G4 Subtotal 1 (47/F) 29 20 29 18 96
13 30 35 18 96 2 (38/F) -- -- -- -- -- -- -- -- -- -- 3 (43/M) 26
26 24 20 96 24 20 23 26 93 4 (34/M) -- -- -- -- -- -- -- -- -- -- 5
(54/M) 25 29 30 12 96 24 24 24 24 96 6 (34/F) -- -- -- -- -- -- --
-- -- -- Total 80 75 83 50 288 61 74 82 68 285 Abbreviations: PB,
peripheral blood; BM, bone marrow; suspension culture media
containing 100 ng/ml of each stem cell factor (SCF), Flt-3 and
thrombopoietin (TPO), free-serum media and 50 ng/ml of G-CSF; PEf,
plating efficiency; grade 1, less than 5 cells/well; grade 2, 6 to
10 cells/well; grade 3, 11 to 20 cells/well; grade 4, more than 21
cells/well; *No of 96 well microplates. G, grade; CO1, cytochrome c
oxidase 1; Cytb, cytochrome b.
[0265] To assess the neutral mutational spectra of the mtDNA
sequences and their clonal expansion among CD34.sup.+ clones and
single granulocytes, the 1,121 base pair mtDNA control region,
which contains multiple mutational hot spots (FIG. 3B) was
examined. On average, 100 CD34.sup.+ clones from each donor was
subjected to sequencing analysis. A total number of 594 circulating
(peripheral blood) CD34.sup.+ clones were examined from six adult
donors (Table 14B) and a total of 355 single granulocytes from the
same six donors were used directly for sequencing analysis to
investigate the evidence of clonal expansion of neutral mutations
in these cells (Table 14B).
[0266] As observed in Example 2, above, marked sequence
heterogeneity (mtDNA neutral mutations) was detected in cells from
all of the donors. Analysis of 594 circulating CD34.sup.+ clones
revealed that a total of 151 clones (25.4.+-.8.4%, mean .+-.SD)
displayed mtDNA heterogeneity that was distinct from the donor's
corresponding bulk mtDNA sequences (Tables 15 and 16). Common
patterns of mtDNA heterogeneity in the circulating CD34.sup.+
clones among the six donors included one or two nucleotide changes
(substitution, insertion or deletion) in addition to the
polymorphisms detected in the bulk mtDNA. Most differences were due
to single nucleotide substitutions at various nucleotide positions
and to length alterations in the homopolymeric C tract that is
localized between nucleotide 303 and nucleotide 315. The mtDNA
heterogeneity of CD34.sup.+ clones in six donors was classified
into several patterns according to nucleotide changes: donors 1
through 6 had 5, 3, 3, 5, 9 and 6 different types of nucleotide
change, respectively (Table 15). The mean proportion of unique
patterns of mtDNA heterogeneity among the CD34.sup.+ clones from
peripheral blood was 5.2% (5.2.+-.2.2) (Table 15).
[0267] One hundred and three of 355 single granulocytes (29%,
29.0.+-.9.1) from the same six donors showed mtDNA heterogeneity
that was distinct from mtDNA sequences of the donor's corresponding
bulk mtDNA and from other single granulocytes (Table 15). The mean
proportion of unique patterns of mtDNA heterogeneity among single
granulocytes was 15.2% (15.2.+-.5.2). TABLE-US-00015 TABLE 15
Mutational spectra of mtDNA control region in individual CD34
clones and single granulocytes A. BM CD34.sup.+ clones B. PB
CD34.sup.+ clones C. single granulocyte Heterogeneity Heterogeneity
Heterogeneity Clone Frequency Unique Clone Frequency Unique Clone
Frequency Unique Donor MtDNA sequence No No % No % MtDNA sequence
No No % No % MtDNA sequence No No % No % 1 `bulk` sequence 85
`bulk` sequence 90 `bulk` sequence 40 (47/F) +8CT6C*, 9CT6C* 22 30
26.1 8 7.0 +8CT6C*, 9CT6C* 23 30 25.0 5 4.2 11 21.6 5 9.8 +9CT6C* 2
+9CT6C*, 10CT6C* 4 +9CT6C*, 10CT6C* 7 +7CT6C* 1 +7CT6C* 1 +A189G/A
1 +G16129A/G 1 +10CT6C*, 11CT6C 1 +T204C 1 +G16129A 1 +C349T/C(M),
1 A368G(M) +C277T 1 +C16256T/C 1 +C369T (M) 1 +ins 514CA 1 +C16114T
1 Subtotal 115 Subtotal 120 Subtotal 51 2 `bulk` sequence 27 `bulk`
sequence 85 `bulk` sequence 24 (38/F) +C16184CC (11C) 5 9 25.0 5
13.9 +C16184CC (11C) 5 9 9.6 3 3.2 +C16184CC(11C) 11 13 35.1 3 8.1
+T16131C/T 1 +C16184CCCC (13C) 3 +C16184CCCC (13C) 1 +G16145A 1
+A478G/A 1 +C16459T (M) 1 +C16184CCCC (13C) 1 A73G, A263G, A191AA,
C194T, 1 T199C, G207A, 8CT6C*, T489C, C16147T, C16173T, C16245T,
T16362C Subtotal 36 Subtotal 94 Subtotal 37 3 `bulk` sequence 96
`bulk` sequence 66 `bulk` sequence 34 (43/M) +9CT6C*, 10CT6C* 11 24
20.0 7 5.8 +9CT6C*, 10CT6C* 26 27 29.0 3 3.2 +9CT6C*, 10CT6C* 4 16
32.0 10 20.0 +9CT6C* 6 +8CT6C* 5 +8CT6C* 2 +8CT6C*, 9CT6C*, 1
+C182T/C, 8CT6C*, 9CT6C* 2 T16124C/T +8CT6C*, 9CT6C*, C104T(M) 1
+del 71G, 9CT6C*, 10CT6C* 1 +8CT6C*, 9CT6C*, A181A/G(M) 1 +T279C/T
1 +8CT6C*, 9CT6C*, C296C/T(M) 1 +G16153A 1 +8CT6C*, 9CT6C*,
C16339T(M) 1 +8CT6C*, 9CT6C*, T16352T/C 1 +9CT6C*, 10CT6C*, 1
G225A, T16263C +8CT6C*, 9CT6C*, G16035A 1 Subtotal 120 Subtotal 93
Subtotal 50 4 `bulk` sequence 92 `bulk` sequence 70 `bulk` sequence
77 (34/M) +8CT6C*, 9CT6C* 11 19 17.4 6 5.4 +8CT6C*, 9CT6C* 16 25
26.3 5 5.3 +8CT6C*, 9CT6C* 6 34 30.6 16 14.4 +del514-515CA, ins
514CA.sup.# 3 +7CT6C*, 8CT6C* 3 +7CT6C*, 8CT6C* 3 +7CT6C*, 8CT6C*,
2 +T16093T/C 4 +9CT6C*, 10CT6C* 6 +9CT6C*, 10CT6C* 1 +8CT6C*,
9CT6C* 1 +9CT6C*, 10CT6C* 2 +T89C 1 T16044C/T (M) T16,093T/C
+8CT6C*, 9CT6C*, T16,093T 1 +8CT6C*, 8CT7C 1 +T16093T/C 3
+16093T.sup.s 4 +16093T.sup.s, G16208A (M) 1 +A28G/A (M) 1 +8CT6C*,
9CT6C, G185G/A 1 +T195T/C 1 +T195C, A16175C (M) 1 +C277T (M) 1
+C315C/T 1 +C411T (M) 1 +del 452T (M) 1 +C469T (M), T16093T/C 1
Subtotal 111 Subtotal 95 Subtotal 111 5 `bulk` sequence 57 `bulk`
sequence 63 `bulk` sequence 22 (54/M) +ins 514CA.sup.s 19 57 50.0
14 12.3 +ins 514CA.sup.s 11 33 34.4 9 9.4 +C264T 7 20 47.6 14 33.3
+C264T 18 +C264T 11 +T146C 1 +del514-515CA, ins 514CA.sup.# 5
+C264T/C 2 +7CT6C*, 8CT6C* 1 +C264T/C 3 +T146C/T, ins 514CA.sup.s 4
+8CT6C 1 +7CT6C*, 8CT6C* 2 +T146C, 514-515CA 2 +G94A/G 1 +T321T/C
(M) 1 +T146C, C264T/C 1 +C320C/A 1 +A376G (M) 1 +T146C/T, ins
514CA.sup.s 1 +T16189C 1 +T582T/C (M), T16075C/T 1 +T146C 1
+A16265G 1 +T1605C/T (M) 1 +A189G 1 +A357AA (M), 1 +A16098A/G 1
+C264T/C, 514-515CA 1 ins 514.sup.s, T16136C/T +C16259G 1 +T161C/T,
C264T, ins 514CA.sup.s 1 +G16274A, C16296T/C 1 +T16,189C 1 +C16292T
1 +C16,296C/T 1 +C16332T (M), C16366T 1 +T16422C 1 Subtotal 114
Subtotal 96 Subtotal 42 6 `bulk` sequence 102 `bulk` sequence 69
`bulk` sequence 55 (34/F) +8CT6C*, 9CT6C* 5 13 11.3 6 5.6 +8CT6C*,
9CT6C* 18 27 28.1 6 6.3 +8CT6C*, 9CT6C* 4 9 14.1 6 9.4 +A200G/A 3
+A200G/A 4 +A200G 2 +A200G 1 +A28G (M) 1 +A200G/A, 7CT6C*, 8CT6C* 1
+A200G/A. 2 +C571A (M) 1 +A200G/A. 8CT6C*, 9CT6C* 1 8CT6C*, 9CT6C*
+A16080G/A 1 +7CT6C*, 8CT6C* 1 +A16116G (M) 1 +A234G/A 1 +G16412G/A
1 +C16111T/C 1 Subtotal 115 Subtotal 96 Subtotal 64 Total 611 152
24.9 46 7.5 594 151 25.4 31 5.2 355 103 29.0 54 15.2 Abbreviations:
Different, different from bulk cell sequence; Unique, uniquely
different heterogeneity; +, mtDNA nucleotide changes in comparison
to bulk cell mtDNA sequence; BM, bone marrow; PB, peripheral blood;
s, the same as the Cambridge Reference Sequence but different from
the bulk sequence.
[0268] TABLE-US-00016 TABLE 16 Summary of mtDNA neutral mutations
in CD 34.sup.+ clones and single granulocytes Heterogeneity
(neutral mutation) MtDNA Assay No Total rate Substitution Poly C
tract genes Specimens (Donor No) (No) Unique (No) (No) np 303-315
Control CB CD34.sup.+# 580 (5) 1.6% (9) 1.2% (7) 0.0% (0) 0.0% (0)
region Adult BM CD34.sup.+# 611 (6) 24.9% (152) 7.5% (46) 10.5%
(64) 10.8% (66) Adult PB CD34.sup.+ 594 (6) 25.4% (151) 5.2% (31)
7.9% (47) 15.3% (91) Single granulocyte 355 (6) 29.0% (103) 15.2%
(54) 11.8% (42) 11.8% (42) CO1, Adult BM CD34.sup.+ 285 (3) 3.9%
(11) 3.9% (11) 3.9% (11) Cytb Adult PB CD34.sup.+ 284 (3) 6.7% (19)
5.6% (16) 6.7% (19) Subtotal 569 (6) 5.3% (30) 4.7% (27) 5.3% (30)
Heterogeneity (neutral mutation) MtDNA Poly C tract AA change
Interpretation genes np 16184-16189 NC (No) P (No) M (No) Control
0.5% (3) 0.7% (4) region 1.1% (7) 0.0% (0) 1.5% (9) 0.0% (0) 2.0%
(12) 0.0% (0) CO1, 61.5% (8/13*) 30.8% (4/13) 69.2% (9/13) Cytb
43.5% (10/23*) 56.5% (13/23) 43.5% (10/23) 50.0% (18/36*) 47.2%
(17/36) 52.8% (19/36) Abbreviations: CB, cord blood; np, nucleotide
position; AA, amino acid; NC, nucleotide change; P, polymorphism;
M, mutation; *total No of mutational events.
[0269] The mtDNA mutational frequency of the CO1 and Cytb genes,
located in the mtDNA coding region, were examined in order to
determine the functional significance of mtDNA heterogeneity among
bone marrow (BM) CD34.sup.+ and peripheral blood CD34.sup.+ clones.
CO1 and Cytb gene products are central components of respiratory
chain complex III and IV in mitochondria. Cytb mutations may not
only damage complex III, but by interfering with the flow of
electrons through the respiratory chain, they may also affect the
function of complex IV (cytochrome c oxidase) (DiMauro and Schon,
N. Eng J. Med. 348:2656-2668, 2003). Complex IV may be the site of
iron reduction for heme synthesis (Gattermann, Leuk. Res.,
24:141-151, 2000).
[0270] Analysis of 569 CD34.sup.+ clones from BM (285 clones) and
peripheral blood (PB) (284 clones) from 3 donors showed that 5.3%
(30 clones) had mutations distinct from the donor's corresponding
bulk sequences (Table 13B). All of the mtDNA heterogeneity (neutral
mutations) were nucleic acid base substitutions. The mean
proportion of unique mtDNA neutral mutations was 4.7% (27 clones)
(15.2.+-.5.2). Half of them led into amino acid changes (Table 17).
TABLE-US-00017 TABLE 17 Mutational spectra of mtDNA CO1 and Cytb
genes in individual CD34+ clones from BM and PB A. BM CD34.sup.+
clones Heterogeneity Fre- Amino acid Clone quency Unique Donor
MtDNA sequence Gene change Interpretation No No % No % 1 (47/F)
`bulk` sequence 91 +T7071C/T CO1 Met-Thr Mutation/TS 1 3 3.2 3 3.2
+G7207G/A CO1 Gly-Glu Mutation/TS 1 +T14924C Cytb Ser-Pro
Mutation/TS 1 Subtotal 94 3 (43/M) `bulk` sequence 92 +G6955G/A CO1
Gly-Asp Mutation/TS 1 3 3.2 3 3.2 +T6987C/T CO1 Ser-Pro Mutation/TS
1 +T7110T/C CO1 Tyr-His Mutation/TS 1 Subtotal 95 5 (54/M) `bulk`
sequence 91 +T6711G/T CO1 Tyr-Asp Mutation/TV 1 5 5.2 5 5.2
+G6899A/G CO1 No Mutation/TS 1 +7022T.sup.s CO1 1 +T7297C/T CO1
Val-Ala Mutation/TS 1 +15607A.sup.- Cytb 1 G14905G/A, No
Polymorphism/TS T15067C No Polymorphism/TS Subtotal 96 Total 285 11
3.9 11 3.9 B. PB CD34.sup.+clones Heterogeneity Fre- Amino acid
Clone quency Unique Donor MtDNA sequence Gene change Interpretation
No No % No % 1 (47/F) `bulk` sequence 91 +T6952C CO1 Val-Ala
Mutation/TS 1 4 4.2 4 4.2 +T7312C CO1 Phe-Ser Mutation/TS 1
+C14832C/G Cytb Ala-Gly Mutation/TV 1 +A15098A/G Cytb Ile-Val
Mutation/TS 1 Subtotal 95 3 (43/M) `bulk` sequence 89 +G6955G/A CO1
Gly-Asp Mutation/TS 1 4 4.3 4 4.3 +T14935C Cytb No Mutation/TS 1
+T15067T/C Cytb No Polymorphism/TS 1 +A15724G/A Cytb No
Polymorphism/TS 1 Subtotal 93 5 (54/M) `bulk` sequence 85 +T7022T/C
CO1 No Polymorphism/TS 4 11 11.5 8 8.3 +G7075A/G CO1 No Mutation/TS
1 +T14864C Cytb Cys-Arg Mutation/TS 1 +T15067T/C Cytb No
Polymorphism/TS 1 +T15132C Cytb Met-Thr Mutation/TS 1 +T15818C Cytb
Tyr-His Mutation/TS 1 +C15452C/A, Cytb Leu-Ile Polymorphism/TS 1
A15607A/G No Polymorphism/TS +G14905G/A, Cytb No Polymorphism/TS 1
+T15067T/C, No Polymorphism/TS C15452C/A, Leu-Ile Polymorphism/TS
A15607A/G No Polymorphism/TS Subtotal 96 Total 284 19 6.7 16 5.6
Heterogeneity of mtDNA CO1 and Cytb genes in 569 30 5.3 27 4.7
total CD34.sup.+ clones from BM and PB
Abbreviations and symbols used in Table 17 include: CO1, cytochrome
c oxidase 1; Cytb, cytochrome b; BM, bone marrow; PB, peripheral
blood; TS, transition; TV, transversion; s, the same as the
Cambridge Reference Sequence but different from the bulk
(aggregate) sequence.
[0271] Analysis of 355 single granulocytes from six donors revealed
that 63 cells (17.7%, 63/355) had the same mutations found in their
progenitor cells (BM CD34.sup.+ and/or circulating CD34.sup.+). In
most cases, there were one or two mutations in each cell. The
majority of clonally expanded mutations in hematopoietic tissue are
length changes in the homopolymeric C tract between nucleotide 303
and nucleotide 315 at hypervariable segment 2. Clonal expansion
among substitution mutations, such as T16093T/C and C264T, were
also observed in donors 4 and 5 (Table 15). These data indicate
that each of these mutations expanded from a single initial
mutational event.
[0272] The characteristics of mtDNA mutations in cells obtained
from different hematopoietic tissues are summarized in Table 16. On
average, approximately 25% of single granulocytes and CD34.sup.+
cell clones from adult BM and PB differed from the bulk mtDNA
sequence of each specific donor. In contrast, less than 2% of the
cord blood (CB) CD34.sup.+ cell clones were different from their
respective individual bulk sequence (see also Example 3). The
proportion of unique differences (different from the bulk sequence
and other CD34.sup.+ clones and granulocytes) was 7.5%, 5.2% and
15.2% in BM CD34.sup.+ cells, circulating CD34.sup.+ cells, and
single granulocytes, respectively. The proportion of solitary
nucleotide substitutions in BM CD34.sup.+ cells, circulating
CD34.sup.+ cells, and single granulocytes was similar and these
substitutions were not observed in CB CD34.sup.+ clones.
[0273] As discussed in Example 2, above, a high incidence of
nucleotide variations was also observed in both HV2 and HV1
segments among circulating CD34.sup.+ clones and single
granulocytes. Most mutations from circulating CD34.sup.+ clones and
single granulocytes were localized in the HV2 homopolymeric C
tracts between nucleotide 303 and nucleotide 315 (60.3% or 91/151
in PB CD34.sup.+ clones; 40.8% or 42/103 in single granulocytes)
(Table 16).
EXAMPLE 5
MtDNA Heterogeneity in Single CD34.sup.+ Cell Clones from Normal
Bone Marrow without Amplification of the MtDNA
[0274] This example provides a description of one embodiment in
which the mutational frequency in mtDNA is measured in clonal
populations of CD34.sup.+ cells but where the mtDNA is sequenced
without prior amplification.
[0275] To determine the heterogeneity of mtDNA sequences in
CD34.sup.+ cells from umbilical cord blood, normal umbilical cord
blood is collected from five normal (do not have any apparent
hematologic disease or symptom) donors. Alternatively, peripheral
blood, or bone marrow samples are collected from the donors either
instead of, or in addition to, the umbilical cord blood
samples.
[0276] In order to determine the heterogeneity of single CD34.sup.+
clones from umbilical cord blood, mononuclear cells are separated
from other hematopoietic cells by standard Ficoll separation and
washed twice in phosphate-buffered saline (PBS). Cells suspended in
PBS are adjusted to 2.times.10.sup.7 cells/ml. To each 12.times.75
mm tube containing 100 .mu.l of the cell suspension, 10 .mu.l of
phycoerythrin (PE)-conjugated anti-CD34, or 10 .mu.l of
PE-conjugated IgG1 (BD Bioscience, Franklin Lakes, N.J.) are added.
Following a 30 minute incubation at 4.degree. C., cells are washed
using cold PBS and resuspended in 0.5 ml PBS.
[0277] Human CD34.sup.+ cells are sorted using a MoFlo cytometer
(Dako-Cytomation, Fort Collins, Colo.) and an 1-90 argon laser
(emitting at 488 nm, Coherent Inc., Palo Alto, Calif.) for
excitation. Forward scatter is used as the triggering parameter. PE
fluorescence is detected using a 580/30 bandpass filter. Single
cell deposition is performed using a CyClone automated cloner
(Dako-Cytomation) in the 0.5 single drop mode. Gating of the cells
is based on forward scatter and PE fluorescence. Individual
CD34.sup.+ cells are plated into each well of a 96-well culture
plate with 100 .mu.l of serum-free medium containing 100 ng/ml of
stem cell factor (SCF), 100 ng/ml of Flt-3, 100 ng/ml of
thrombopoietin (TPO), in the presence or absence of 50 ng/ml of
granulocyte-colony stimulating factor (G-CSF).
[0278] After five days of culture, each well of the microtiter
plate is carefully examined with an inverted microscope (Olympus
IX50; Melville, N.Y.) in order to examine growth and plating
efficiency of single CD34.sup.+ cells. As described above in
Example 2, cells in each well are graded based on the number of
cells present in the well following the five-day culture. Cloning
(plating) efficiency is defined as the number of positive wells
(any cells present)/total wells.times.100.
[0279] To assess heterogeneity of the mtDNA sequences among
CD34.sup.+ cells from each of the normal umbilical cord blood
donors, the 1,121 base pair mtDNA control region in a set of
CD34.sup.+ clones (clonal populations) is subjected to sequencing
analysis.
[0280] To prepare for sequencing analysis, each CD34.sup.+ clone is
harvested from the well into a 1.5 ml microcentrifuge tube by
vigorous pipetting and dispensing followed by rinse of the well
with 200 .mu.l of PBS. Cells are collected after centrifugation at
300.times.g for 5 minutes, and then washed with PBS. Cell pellets
are stored at -80.degree. C. In order to extract DNA from
individual CD34.sup.+ clones, each cell pellet is covered with 30
.mu.l of 1.times.TE buffer and lysed by incubating the cells at
95.degree. C. for 10 minutes, with occasional shaking. The lysate
is briefly microfuged and stored at -20.degree. C.
[0281] The mtDNA is sequenced with the appropriate primers using
the BigDye Terminator v3.0 Ready Reaction kit (Applied Biosystems,
Foster City, Calif.) according to the manufacturer's protocol, and
then applied to the ABI Prism 3100 Genetic Analyzer (Applied
Biosystems). The following oligonucleotide primers are used for
sequencing: 5'CAGTGTATTGCTTTGAGGAGG3' (SEQ ID NO: 5),
5'CATCTGGTTCCTACTTCAGGGTC3' (SEQ ID NO: 6),
5'TTAACTCCACCATTAGCACC3' (SEQ ID NO: 7),
5'GCATGGAGAGCTCCCGTGAGTGG3' (SEQ ID NO: 8),
5'CACCCTATTAACCACTCACG3' (SEQ ID NO: 9) and
5'TACATTACTGCCAGCCACCATG3' (SEQ ID NO: 10). The mtDNA sequences
experimentally obtained are compared to the 2001 Revised Cambridge
Reference Sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998) using the
National Center for Biotechnology Information (NCBI) Blast2 program
and the database search tool, MitoAnalyzer (National Institute of
Standards and Technology, Gaithersburg, Md.), in order to identify
polymorphisms and mutations. All automated results are manually
confirmed.
[0282] Analysis of the mtDNA sequence from the set of CD34.sup.+
clonal populations from normal donors is used to determine the
number of CD34.sup.+ clonal populations of cells possessing at
least one mtDNA mutation within the sequenced region of the mtDNA.
The mutational frequency of the mtDNA can be determined by
calculating the proportion of clonal populations of cells, within
the set of clonal populations of cells, having at least one mtDNA
mutation.
EXAMPLE 6
MtDNA Heterogeneity in Single Hematopoietic Cells
[0283] This example provides a description of one embodiment of
measuring the mutational frequency of mtDNA, wherein individual
hematopoietic cells are substituted for clonal populations of
hematopoietic cells.
[0284] To determine the heterogeneity of mtDNA sequences in
individual hematopoietic cells (for example, CD34.sup.+ cells,
granulocytes, monocytes, or macrophages), bone marrow, peripheral
blood, or umbilical cord blood specimens are collected from normal
donors. Mononuclear cells are separated from other hematopoietic
cells by standard Ficoll separation and washed twice in
phosphate-buffered saline (PBS). In order to isolate granulocytes,
the cells suspended in PBS are adjusted to 2.times.10.sup.7
cells/ml. To each 12.times.75 mm tube containing 100 .mu.l of the
cell suspension, 10 .mu.l of phycoerythrin (PE)-conjugated
anti-CD33, or 10 .mu.l of PE-conjugated IgG1 (BD Bioscience,
Franklin Lakes, N.J.) are added. Following a 30 minute incubation
at 4.degree. C., cells are washed using cold PBS and resuspended in
0.5 ml PBS.
[0285] Human granulocytes are sorted using a MoFlo cytometer
(Dako-Cytomation, Fort Collins, Colo.) and an 1-90 argon laser
(emitting at 488 nm, Coherent Inc., Palo Alto, Calif.) for
excitation. Forward scatter is used as the triggering parameter. PE
fluorescence is detected using a 580/30 bandpass filter. Single
cell deposition is performed using a CyClone automated cloner
(Dako-Cytomation) in the 0.5 single drop mode. Gating of the cells
is based on forward scatter and PE fluorescence. Each individual
granulocyte is placed in separate 1.5 ml microcentrifuge tubes and
the cells are lysed by any means known to one of skill in the art
(for example sonication).
[0286] To assess heterogeneity of the mtDNA sequences among a set
of granulocytes from normal donors, the 1,121 base pair mtDNA
control region (a region known to contain multiple mutational
hotspots) in a set of granulocytes is subjected to PCR
amplification and sequencing analysis. Nested gene PCR
amplification is performed using the following nested primers:
outer primer pair 5'-CGCCTACACAATTCTCCGATC-3' (SEQ ID NO: 1) and
5'-ACTTGGGTTAATCGTGTGACC-3' (SEQ ID NO: 2), which amplify the
region between nucleotide 15,974 and nucleotide 921 of the human
mtDNA genome as represented by the revised Human mtDNA Cambridge
Reference sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998), and inner primer
pair 5'-TTAACTCCACCATTAGCACC-3' (SEQ ID NO: 3) and
5'-GAAAGGCTAGGACCAAACCTA-3' (SEQ ID NO: 4), which amplify the
region between nucleotide 15,971 and nucleotide 670 of the human
mtDNA genome as represented by the revised Human mtDNA Cambridge
Reference sequence (Andrews et al., Nat. Genet. 23:147, 1999;
Kogelnik et al., Nucleic Acids Res. 26:112, 1998). Amplification of
mtDNA is performed with the TaKaRa LA PCR kit (PanVera, Madison,
Wis.).
[0287] The primary and secondary PCR amplifications are carried out
in a DNA thermal cycler 480 (Perkin-Elmer, Foster City, Calif.)
under the following amplification conditions: one cycle at
96.degree. C. for 1 minute; 35 cycles at 94.degree. C. for 30
seconds, 52.degree. C. for 50 seconds and 72.degree. C. for 1
minute with a 5-second increase per cycle; one cycle of 72.degree.
C. for 5 minutes. Secondary PCR samples are electrophoresed on 1%
agarose gels and stained with ethidium bromide to assess the purity
and size of DNA fragments, and the samples are subsequently
purified using the QIAquick.TM. PCR purification kit (Qiagen,
Valencia, Calif.). As negative controls, reaction mixtures without
DNA templates are subjected to PCR amplification. To prevent DNA
cross-contamination, special precautions are taken for each
procedure of cell isolation, PCR amplification and DNA
sequencing.
[0288] Purified-PCR products are prepared for sequencing, using the
appropriate primers (see Example 3, above), with the BigDye
Terminator v3.0 Ready Reaction kit (Applied Biosystems, Foster
City, Calif.) according to the manufacturer's protocol, and then
applied to the ABI Prism 3100 Genetic Analyzer (Applied
Biosystems). Sequencing is performed on an ABI Prism 3100 Genetic
Analyzer in both orientations. The resultant mtDNA sequences are
compared to the 2001 Human mtDNA Revised Cambridge Reference
sequence (Andrews et al., Nat. Genet. 23:147, 1999; Kogelnik et
al., Nucleic Acids Res. 26:112, 1998) using the National Center for
Biotechnology Information (NCBI) Blast2 program and the database
search tool, MitoAnalyzer (National Institute of Standards and
Technology, Gaithersburg, Md.) in order to identify mutations
(mtDNA heterogeneity) and polymorphisms. All automated results are
manually confirmed.
[0289] Analysis of the mtDNA sequence from the set of granulocytes
from normal donors is used to determine the number of granulocytes
possessing at least one mtDNA mutation within the sequenced region
of the mtDNA. The mutational frequency of the mtDNA can be
determined by calculating the proportion of cells, within the set
of cells, having at least one mtDNA mutation.
EXAMPLE 7
Establishing a Correlation Between MtDNA Mutational Frequency and
Circumstances in which Genomic (Nuclear) Mutations are Increased In
Vitro
[0290] This example provides a description of one way in which the
effect of mutagenic agents, for example chemotherapeutic drugs and
X-rays, is measured in cells such as cultured cell lines and
primary cells.
[0291] To determine the correlation between mtDNA mutational
frequency and circumstances that increase genomic (nuclear)
mutations in vitro, hematopoietic cells are obtained from normal
(do not have any apparent hematologic disease or symptom) donors,
for example a healthy subject that has not been exposed to a
mutagen, and mononuclear cells are separated from other
hematopoietic cells in the sample, for instance as described in
Example 1. The isolated mononuclear cells, in addition to cells
from a variety of hematopoietic cell lines, are incubated in the
presence or absence of at least one mutagenic agent that is known
to cause genomic mutations in vitro. The treated and untreated
cells are sorted by single cell deposition into 96 well microtiter
plates, using a phycoerythrin anti-CD34 monoclonal antibody, a
MoFlo cytometer, and a CyClone automated cloner in the 0.5 single
drop mode. The cells are expanded into clonal populations of cells
during five days of culture in media containing stem cell factor,
Flt-3, thrombopoietin, in the presence or absence of
granulocyte-colony stimulating factor (G-CSF). DNA is extracted
from the treated and untreated clonal populations of cells, mtDNA
and/or genomic DNA are amplified, for instance using the primers
described in Example 1, and the amplified DNA is sequenced and
compared to the 2001 Human mtDNA Revised Cambridge Reference
sequence (Andrews et al., Nat. Genet. 23:147, 1999; Kogelnik et
al., Nucleic Acids Res. 26:112, 1998) using the National Center for
Biotechnology Information (NCBI) Blast2 program and the database
search tool, MitoAnalyzer (National Institute of Standards and
Technology, Gaithersburg, Md.), as described herein.
[0292] The mutational frequency of mtDNA or genomic DNA in treated
and untreated cells in vitro is examined. For example, the number
(or percentage or proportion) of clonal populations with new
mutations in mtDNA divided by the total number of clonal
populations studied (the set of clonal populations of cells),
multiplied by 100, is the mutational frequency of the mtDNA. The
mutational frequency of genomic DNA is determined in the same
manner. An increase in the mutational frequency of both mtDNA and
genomic DNA obtained from the cells treated with the mutagenic
agent, compared to the untreated cells, is an indication that the
increase in mutational frequency of mtDNA can be correlated to an
increase in the mutational frequency of genomic DNA. Thus,
mutagenic agents that increase the mutational frequency of mtDNA
are expected to increase the mutational frequency of genomic
DNA.
EXAMPLE 8
Establishing a Correlation Between an Increase in MtDNA Mutational
Frequency and Circumstances in which Genomic (Nuclear) Mutations
are Increased In Vivo
[0293] This example provides a description of one way in which the
effect of mutagenic agents on mutational frequency is measured in
cells expanded into clonal populations of cells from subjects of
different age and/or medical history. For example, bone marrow
specimens from children and umbilical cord blood from newborns are
collected from normal donors, for example a healthy subject that
has not been exposed to a mutagen, for which the prevalence of
mtDNA mutations is very low. These specimens are compared to bone
marrow specimens obtained from adults. Alternatively, bone marrow
is collected from subjects before and after they have undergone
cytotoxic chemotherapy or radiation. Bone marrow samples also are
collected from subjects with genetic syndromes that are known to
result in a high prevalence of mtDNA mutations, for example
Pearson's syndrome.
[0294] To determine the correlation between mtDNA mutational
frequency and circumstances which increase genomic mutations in
vivo, mononuclear cells in bone marrow specimens obtained from
subjects before and after chemotherapy treatment are separated from
other hematopoietic cells in the samples by density gradient
centrifugation. CD34.sup.+ cells are sorted by single cell
deposition into 96 well microtiter plates, using a phycoerythrin
anti-CD34 monoclonal antibody, a MoFlo cytometer, and a CyClone
automated cloner in the 0.5 single drop mode. After five days of
culture in media containing stem cell factor, Flt-3,
thrombopoietin, in the presence or absence of granulocyte-colony
stimulating factor (G-CSF), each well of the microtiter plate is
carefully examined microscopically. DNA is extracted, mtDNA and/or
genomic DNA are amplified, for instance using the primers described
in Example 1, and the amplified DNA is sequenced and compared to
the 2001 Human mtDNA Revised Cambridge Reference sequence (Andrews
et al., Nat. Genet. 23:147, 1999; Kogelnik et al., Nucleic Acids
Res. 26:112, 1998) using the National Center for Biotechnology
Information (NCBI) Blast2 program and the database search tool,
MitoAnalyzer (National Institute of Standards and Technology,
Gaithersburg, Md.), as described herein.
[0295] The mutational frequency of mtDNA or genomic DNA in
chemotherapy-treated and -untreated cells in vivo is examined. For
example, the number of clonal populations of cells with new
mutations in mtDNA divided by the total number of clonal
populations of cells studied (the set of clonal populations of
cells), multiplied by 100, is the mutational frequency of the
mtDNA. The mutational frequency of genomic DNA is determined in the
same manner. An increase in the mutational frequency of both mtDNA
and genomic DNA obtained from the chemotherapy-treated cells,
compared to the chemotherapy-untreated cells, is an indication that
the increase in mutational frequency of mtDNA can be correlated to
an increase in the mutational frequency of genomic DNA. Thus,
mutagenic agents that increase the mutational frequency of mtDNA in
vivo are believed to increase the mutational frequency of genomic
DNA in vivo.
EXAMPLE 9
Screening for the Mutagenic Effect of Agents
[0296] This example provides a description of one way of screening
for agents that have a mutagenic effect on a cell, or screening for
the mutagenic effect of agents.
[0297] Bone marrow or peripheral blood specimens are collected from
normal donors and mononuclear cells are separated from other
hematopoietic cells by density gradient centrifugation. CD34.sup.+
cells are isolated as described herein and the cells are then
cultured either in the presence or absence of a series of known or
putative mutagenic agents for various lengths of time. The treated
and untreated CD34.sup.+ cells are sorted by single cell deposition
into 96 well microtiter plates, using a phycoerythrin anti-CD34
monoclonal antibody, a MoFlo cytometer, and a CyClone automated
cloner in the 0.5 single drop mode. The cells also can be cultured
in media containing stem cell factor, Flt-3, thrombopoietin, in the
presence or absence of granulocyte-colony stimulating factor
(G-CSF). Following the culture period, each well of the microtiter
plate is carefully examined microscopically, as described above in
Example 1. DNA is then extracted from the treated and untreated
cells, mtDNA and genomic DNA are amplified, for instance using the
primers described in Example 1, and the amplified DNA is sequenced.
The mutational frequency of mtDNA or genomic DNA in treated cells,
compared to the untreated cells, is examined to screen for the
mutagenic effect of the agents. An increase in the mutational
frequency of mtDNA and/or genomic DNA obtained from treated cells,
compared to the mtDNA and/or genomic DNA obtained from untreated
cells, is an indication that the agent is a mutagen. In particular
examples, the mutational frequency of mtDNA obtained from treated
cells is compared to the mutational frequency of mtDNA from
untreated cells.
EXAMPLE 10
Tracking a Set of MtDNA Mutations in Subjects Suffering from
Disease
[0298] This example provides a description of one way to track a
set of mtDNA mutations (a profile) in a subject having a disease.
The subject's progress in overcoming the disease can be measured by
the presence or absence of cells with the mtDNA mutation pattern in
the subject following treatment.
[0299] To track the mtDNA mutation pattern in subjects undergoing
treatment for a disease, bone marrow or peripheral blood is
collected from subjects before and after they undergo treatment,
for example cytotoxic chemotherapy or radiation. Multiple samples
can be taken after or during treatment to track the subject's
progress. Blood or bone marrow samples can be obtained from the
subject at any time following treatment. Mononuclear cells are
separated from other hematopoietic cells in the samples by density
gradient centrifugation. CD34.sup.+ cells are sorted by single cell
deposition into 96 well microtiter plates, using a phycoerythrin
anti-CD34 monoclonal antibody, a MoFlo cytometer, and a CyClone
automated cloner in the 0.5 single drop mode. After five days of
culture in media containing stem cell factor, Flt-3,
thrombopoietin, in the presence or absence of G-CSF, each well of
the microtiter plate is carefully examined microscopically. DNA is
extracted, mtDNA is amplified, for instance using the primers
described in Example 1, and the amplified DNA is sequenced. The
mtDNA mutation pattern in cells obtained from the subject before
and after treatment is examined to determine the effect of the
treatment on eradicating the cells with the particular mtDNA
mutation profile.
[0300] This disclosure provides methods of determining the mutation
frequency in a cell, and more particularly in a hematopoietic cell,
such as a CD34.sup.+ cell or a cell in which the mutation frequency
can be correlated to that measured in a CD34.sup.+ cell. The
disclosure further provides methods of screening various agents for
their mutagenic effect on cells. It will be apparent that the
precise details of the methods described may be varied or modified
without departing from the spirit of the described invention. We
claim all such modifications and variations that fall within the
scope and spirit of the claims below.
Sequence CWU 1
1
23 1 21 DNA Artificial Sequence oligonucleotide primer 1 cgcctacaca
attctccgat c 21 2 21 DNA Artificial Sequence oligonucleotide primer
2 acttgggtta atcgtgtgac c 21 3 20 DNA Artificial Sequence
oligonucleotide primer 3 ttaactccac cattagcacc 20 4 21 DNA
Artificial Sequence oligonucleotide primer 4 gaaaggctag gaccaaacct
a 21 5 21 DNA Artificial Sequence oligonucleotide primer 5
cagtgtattg ctttgaggag g 21 6 23 DNA Artificial Sequence
oligonucleotide primer 6 catctggttc ctacttcagg gtc 23 7 20 DNA
Artificial Sequence oligonucleotide primer 7 ttaactccac cattagcacc
20 8 23 DNA Artificial Sequence oligonucleotide primer 8 gcatggagag
ctcccgtgag tgg 23 9 20 DNA Artificial Sequence oligonucleotide
primer 9 caccctatta accactcacg 20 10 22 DNA Artificial Sequence
oligonucleotide primer 10 tacattactg ccagccacca tg 22 11 25 DNA
Artificial Sequence oligonucleotide primer 11 ccacaaaccc cattactaaa
cccac 25 12 20 DNA Artificial Sequence oligonucleotide primer 12
cggttgttga tgggtgagtc 20 13 24 DNA Artificial Sequence
oligonucleotide primer 13 gcatacatca ttattctcgc acgg 24 14 23 DNA
Artificial Sequence oligonucleotide primer 14 gggtggtacc caaatctgct
tcc 23 15 24 DNA Artificial Sequence oligonucleotide primer 15
gcaacctcaa caccaccttc ttcg 24 16 25 DNA Artificial Sequence
oligonucleotide primer 16 gtagacctac ttgcgctgca tgtgc 25 17 25 DNA
Artificial Sequence oligonucleotide primer 17 ccattctata ccaacaccta
ttctg 25 18 24 DNA Artificial Sequence oligonucleotide primer 18
ccatggggtt ggcttgaaac cagc 24 19 21 DNA Artificial Sequence
oligonucleotide primer 19 cagtgtattg ctttgaggag g 21 20 23 DNA
Artificial Sequence oligonucleotide primer 20 ctacaaccac gaccaatgat
atg 23 21 22 DNA Artificial Sequence oligonucleotide primer 21
gctttgggtg ctaatggtgg ag 22 22 25 DNA Artificial Sequence
oligonucleotide primer 22 ctaccaggct tcggaataat ctccc 25 23 24 DNA
Artificial Sequence oligonucleotide primer 23 gttcttcgaa tgtgtggtag
ggtg 24
* * * * *