U.S. patent application number 15/343323 was filed with the patent office on 2017-06-29 for mutations of the parkin gene, compositions, methods and uses.
The applicant listed for this patent is Aventis Pharma S.A., Institut National de la Sante et de la Recherche Medicale. Invention is credited to Nacer Eddine Abbas, Sandrine Bouley, Alexis Brice, Patrice Denefle, Christophe Lucking, Sylvain Ricard.
Application Number | 20170183736 15/343323 |
Document ID | / |
Family ID | 27253486 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170183736 |
Kind Code |
A1 |
Brice; Alexis ; et
al. |
June 29, 2017 |
MUTATIONS OF THE PARKIN GENE, COMPOSITIONS, METHODS AND USES
Abstract
The invention concerns nucleic acids coding for mutated or
truncated forms of the human parkin gene, or forms comprising
multiplication of exons, and the corresponding proteins and
antibodies. The invention also concerns methods and kits for
identifying mutations of the parkin gene, and for studying
compounds for therapeutic purposes.
Inventors: |
Brice; Alexis; (Paris,
FR) ; Lucking; Christophe; (Paris, FR) ;
Denefle; Patrice; (Saint Maur, FR) ; Ricard;
Sylvain; (Paris, FR) ; Abbas; Nacer Eddine;
(Paris, FR) ; Bouley; Sandrine; (Bletterans,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aventis Pharma S.A.
Institut National de la Sante et de la Recherche Medicale |
Antony Cedex
Paris |
|
FR
FR |
|
|
Family ID: |
27253486 |
Appl. No.: |
15/343323 |
Filed: |
November 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14454026 |
Aug 7, 2014 |
9540693 |
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15343323 |
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13209495 |
Aug 15, 2011 |
8835618 |
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14454026 |
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09856290 |
Aug 13, 2001 |
7998667 |
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PCT/FR99/02833 |
Nov 18, 1999 |
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13209495 |
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60124239 |
Mar 12, 1999 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/104 20130101;
C12Q 1/6883 20130101; C12Q 2600/16 20130101; C12Q 2600/158
20130101; A61P 25/16 20180101; C12N 9/93 20130101; C07K 16/40
20130101; C12Y 603/02019 20130101; A61P 25/06 20180101; C12Q
2600/156 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 9/10 20060101 C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 19, 1998 |
FR |
98/14524 |
Aug 4, 1999 |
FR |
99/10140 |
Claims
1-40. (canceled)
41. An oligonucleotide comprising a fragment, or the complementary
sequence thereof, of a parkin gene that comprises a genetic
alteration selected from the group consisting of: a) a deletion of
one or more exons selected from the group consisting of: exon 2,
exons 2-3, exons 2-4, exons 3-4, exons 3-6, exons 3-9, exon 5,
exons 5-6, exon 6, exons 6-7, exons 7-9, and exon 8; b) a
multiplication of exons selected from the group consisting of: a
triplication of exon 2, a duplication of exon 3, a duplication of
exon 6, a duplication of exon 7, and a duplication of exon 11; c) a
point mutation selected from the group consisting of: a mutation
from adenine to thymine at a position corresponding to position 584
of SEQ ID NO: 1, a mutation from guanine to adenine at a position
corresponding to position 601 of SEQ ID NO: 1, a mutation from
adenine to thymine at a position corresponding to position 734 of
SEQ ID NO: 1, a mutation from cytosine to thymine at a position
corresponding to position 867 of SEQ ID NO:1, a mutation from
thymine to adenine at a position corresponding to position 905 of
SEQ ID NO: 1, a mutation from cytosine to thymine at a position
corresponding to position 924 of SEQ ID NO: 1, a mutation from
guanine to adenine at a position corresponding to position 939 of
SEQ ID NO: 1, a mutation from thymine to guanine at a position
corresponding to position 966 of SEQ ID NO: 1, a mutation from
guanine to adenine at a position corresponding to position 1084 of
SEQ ID NO: 1, a mutation from cytosine to thymine at a position
corresponding to position 1101 of SEQ ID NO:1, a mutation from
guanine to cytosine at a position corresponding to position 1239 of
SEQ ID NO:1, a mutation from guanine to adenine at a position
corresponding to position 1281 of SEQ ID NO: 1, a mutation from
cytosine to adenine at a position corresponding to position 1345 of
SEQ ID NO: 1, a mutation from guanine to adenine at a position
corresponding to position 1390 of SEQ ID NO: 1, and a mutation from
guanine to adenine at a position corresponding to position 1459 of
SEQ ID NO:1; d) a deletion of 1 or more contiguous base pairs
selected from the group consisting of: a deletion of nucleotides
adenine and guanine at positions corresponding to positions 202-203
of SEQ ID NO:1, a deletion of adenine at a position corresponding
to position 255 of SEQ ID NO: 1, and a deletion of nucleotides
guanine and adenine at positions corresponding to positions
1142-1143 of SEQ ID NO:1; and e) an insertion of 1 or more
contiguous base pairs selected from the group consisting of: an
insertion of guanine and thymine at positions corresponding to
321-322 of SEQ ID NO:1; wherein the oligonucleotide is detectably
labeled.
42. The oligonucleotide of claim 41, wherein the detectable label
is selected from the group consisting of a radioactive label, a
fluorescent label, an enzymatic label, a chemical label.
43. The oligonucleotide of claim 41, wherein the oligonucleotide
has less than 500 bp.
44. The oligonucleotide of claim 41, wherein the oligonucleotide
has less than 300 bp.
45. The oligonucleotide of claim 41, wherein the oligonucleotide
has about 5 to about 100 bp.
46. The oligonucleotide of claim 41, wherein the oligonucleotide
has about 5 to about 50 bp.
47. The oligonucleotide of claim 41, wherein the oligonucleotide
specifically hybridizes to the parkin gene comprises a genetic
alteration under stringent conditions, and does not hybridize to
the wild type parkin gene under stringent conditions.
48. The oligonucleotide of claim 41, wherein the genetic alteration
comprises a point mutation that results in a nonsense mutation.
49. The oligonucleotide of claim 48, wherein the nonsense mutation
affects exon 7, exon 12, or a combination thereof.
50. The oligonucleotide of claim 48, wherein the point mutation
comprises the point mutation 905T>A that introduces a stop codon
at a position corresponding to the Cys268 residue encoded by the
sequence of SEQ ID NO: 1, or the point mutation 1459G>A that
introduces a stop codon at a position corresponding to the Trp453
residue encoded by the sequence of SEQ ID NO: 1.
51. The oligonucleotide of claim 41, wherein the genetic alteration
is a point mutation that results in a missense mutation.
52. The oligonucleotide of claim 51, wherein the missense mutation
causes a nonconservative change in the amino acid sequence encoded
by the sequence of SEQ ID NO: 1.
53. The oligonucleotide of claim 51, wherein the missense mutation
comprises a point mutation selected from the group consisting of
584A>T at a position corresponding to SEQ ID NO: 1 (Lys161Asn),
601G>A T at a position corresponding to SEQ ID NO:1 (Ser167Asn),
734A>T at a position corresponding to SEQ ID NO:1 (Lys211Asn),
867C>T T at a position corresponding to SEQ ID NO: 1
(Arg256Cys), 924C>T at a position corresponding to SEQ ID NO: 1
(Arg275Trp), 939G>A at a position corresponding to SEQ ID NO: 1
(Asp280Asn), 966T>G (Cys289Gly), 1084G>A at a position
corresponding to SEQ ID NO: 1 (Gly328Glu), 1101C>T at a position
corresponding to SEQ ID NO: 1 (Arg334Cys), 1281G>A T at a
position corresponding to SEQ ID NO:1 (Asp394Asn), and 1390G>A T
at a position corresponding to SEQ ID NO:1 (Gly430Asp).
54. The oligonucleotide of claim 51, wherein the missense mutation
causes a conservative change in the amino acid sequence encoded by
the sequence of SEQ ID NO: 1.
55. The oligonucleotide of claim 54, wherein the missense mutation
comprises a point mutation selected from the group consisting of
1239G>C T at a position corresponding to SEQ ID NO: 1
(Val380Leu) and 1345C>A T at a position corresponding to SEQ ID
NO:1 (Thr415Asn).
56. The oligonucleotide of claim 54, wherein the missense mutation
affects a potential phosphorylation site of a polypeptide encoded
by said isolated nucleic acid molecule.
57. The oligonucleotide of claim 56, wherein the point mutation is
Thr415Asn.
58. The oligonucleotide of claim 41, wherein the genetic alteration
comprises a deletion of one or more contiguous base pair(s) that
causes a reading frame shift.
59. The oligonucleotide of claim 58, wherein the deletion is
selected from the group consisting of: a deletion of the
nucleotides adenine and guanine at positions corresponding to
positions 202-203 of SEQ ID NO: 1, a deletion of the nucleotide
adenine at a position corresponding to position 255 of SEQ ID NO:1,
and a deletion of the nucleotides guanine and adenine at positions
corresponding to positions 1142-1143 of SEQ ID NO:1.
60. The oligonucleotide of claim 58, wherein the genetic alteration
comprises an insertion of one or more contiguous base pair(s) that
causes a reading frame shift.
61. The oligonucleotide of claim 60, wherein the insertion is an
insertion of nucleotides guanine and thymine at positions
corresponding to positions 321-322 of SEQ ID NO:1.
62. A kit for detection of a genetic alteration of a parkin gene,
comprising at least one of the oligonucleotides of claim 41.
Description
METHODS AND USES
[0001] The present invention relates to the field of genetics and,
more particularly, to the identification of mutations in the parkin
gene. It also relates to compositions and methods for the
identification of these mutations in samples, mutated or truncated
forms of parkin or forms comprising exon multiplications, and their
uses for diagnostic, screening or therapeutic purposes, for
example.
[0002] Parkinson's disease is a frequent neurodegenerative
condition whose prevalence is close to 2% after the age of 65 [de
Rijk et al., 1997]. The cardinal signs of the disease are rigidity,
bradykinesia, rest tremor and good reactivity, at least initially,
to levodopa. The disorders are due to a massive loss of
dopaminergic neurons from the substantia nigra. The causes of the
disease remain unknown, but the involvement of factors for genetic
susceptibility is strongly suspected [Wood, 1997]. Many familial
forms with dominant transmission have been reported. Mutations in
the gene encoding alpha-synuclein, located at 4q21-q23, have been
described in a small number of families with an early onset and a
rapid deterioration [Polymeropoulos et al., 1997; Kruger et al.,
1998]. A second locus is situated at 2p13 [Gasser et al., 1998]. A
parkinsonian syndrome with autosomal-recessive transmission (AR-JP)
has been described in Japan [Yamamura et al., 1973; Ishikawa and
Tsuji, 1996]. It manifests itself with the cardinal signs of
Parkinson's disease with certain specific features: i) early onset,
as a rule before the age of 40; ii) presence of a dystonia, often
at the lower limbs; iii) fluctuations during the day; and iv) slow
progressive evolution but always associated with dyskinesias under
levodopa. Neuropathological examination reveals a massive loss of
neurons from the substantia nigra pars compacta but without Lewy
bodies, a histopathological stigma of idiopathic Parkinson's
disease [Yamamura et al., 1973; Takahashi et al., 1994]. A genetic
linkage between the disease in Japanese families and 6q25.2-27
markers has been demonstrated which defines the PARK2 locus
[Matsumine et al., 1997]. Two teams then described PARK2 families
outside Japan, in particular in the United States, in Europe and in
the Middle East [Jones et al., 1998; Tassin et al., 1998]. Very
recently, Kitada et al. [Kitada et al., 1998] have identified
deletions of exons (3-7) or of exon 4 in a new gene, called parkin,
in 4 Japanese families.
[0003] The present application now describes the demonstration and
characterization, in 77 families and 102 isolated cases, mainly
European, having an early-onset parkinsonian syndrome, of the
presence of new genetic alterations affecting the parkin gene. In
addition, the present application shows that these genetic
alterations are present not only in early-onset parkinsonian
syndromes, but also in more tardive or atypical parkinsonian
syndromes. These new alterations therefore offer new tools both for
the diagnosis and the treatment of Parkinson's disease.
[0004] A more particular subject of the invention relates to a
nucleic acid encoding human parkin, characterized in that it
contains one or more genetic alterations chosen from: [0005] a) a
deletion of one or more exons, in combination or otherwise, [0006]
b) a multiplication (e.g. duplication, triplication) of exons,
[0007] c) a point mutation, [0008] d) a deletion of 1 or more
contiguous base pairs causing a reading frame shift, and [0009] e)
an insertion of 1 or more contiguous base pairs.
[0010] The term nucleic acid as defined in the present application
designates deoxyribonucleic (DNA) and ribonucleic (RNA) acids. In
addition, among the DNAs, they may correspond to genomic DNA (gDNA)
or complementary DNA (cDNA). The nucleic acids of the invention may
be of a natural or synthetic origin. They are generally prepared by
conventional molecular biology techniques, including the screening
of libraries, artificial synthesis, ligation, restriction, and the
like. The positions given in the present application are calculated
relative to the sequence of human parkin represented in FIG. 1 (SEQ
ID No: 1). This sequence represents the sequence of the cDNA
encoding human parkin. Compared with the sequence described by
Kitada et al., it contains a modification at the level of
nucleotide 768 (C->T).
[0011] The genetic alterations a) to e) defined above are mainly
exon alterations, that is to say which affect the coding region of
the human parkin gene. However, intron alterations, that is to say
alterations which affect the non-coding part of the gene, have also
been demonstrated.
[0012] The human parkin gene comprises 12 exons, the nucleotide
positions of which are given below:
TABLE-US-00001 Exon 1: nucleotides 1 to 108 Exon 2: nucleotides 109
to 272 Exon 3: nucleotides 273 to 513 Exon 4: nucleotides 514 to
635 Exon 5: nucleotides 636 to 719 Exon 6: nucleotides 720 to 835
Exon 7: nucleotides 836 to 972 Exon 8: nucleotides 973 to 1034 Exon
9: nucleotides 1035 to 1184 Exon 10: nucleotides 1185 to 1268 Exon
11: nucleotides 1269 to 1386 Exon 12: nucleotides 1387 to 2960
[0013] In a first embodiment, the invention relates to a nucleic
acid encoding parkin, comprising deletions of one or more exons, in
particular combinations of deletions of exons. More particularly,
these deletions affect exons 2 to 9 separately or in combination.
Particular examples of these deletions and combinations of
deletions of exons of the parkin gene are illustrated in Tables 2
and 4. In addition, these deletions may be homozygous, that is to
say may affect both chromosomes simultaneously, or heterozygous
(incidence on only one chromosome).
[0014] The applicant has in particular demonstrated the
heterozygous deletion of exon 2 as well as combinations of
deletions of this exon with other exons including in particular
exon 3 and exon 4. Thus, nine deletions or combinations of
deletions have for the first time been demonstrated and in
particular the following deletions: exons 2, 2+3, 2+3+4, 3-6, 3-9,
6, 6+7, 7+8+9 and 8. Moreover, these deletions or combinations of
deletions may be combined with each other in the case of composite
heterozygotes.
[0015] The applicant has also identified a number of homozygous
deletions, alone or in combinations, such as the deletions of exons
5 and 6.
[0016] The particular positions of the deletions described above
may be defined with reference to the numbering of the nucleotides
in FIG. 1.
[0017] In another specific embodiment, the invention relates to a
nucleic acid encoding parkin, containing a deletion of exon 3. More
particularly, this deletion affects nucleotides 273 to 513 in FIG.
1.
[0018] In another specific embodiment, the invention relates to a
nucleic acid encoding parkin, comprising a deletion of exons 8 and
9. More particularly, this deletion affects nucleotides 973 to 1184
in FIG. 1.
[0019] The consequences of these deletions or combinations of
deletions often remain a shift in the reading frame. Table 4
summarizes the consequences which appeared opposite the deletions
or combinations of deletions recorded.
[0020] In another particular embodiment, the invention relates to a
nucleic acid encoding parkin, comprising a multiplication of exons,
that is to say the repetition of one or more exons in the gene. The
present application shows for the first time, either a homozygous
and heterozygous duplication as is illustrated in the examples by
the duplication of exon 3, or a duplication of the heterozygous
type as illustrated in the duplication of exon 6, 7 or 11. The
present application also shows for the first time a triplication of
exons: either a homozygous triplication, or a heterozygous
triplication as is illustrated in the examples by the triplication
of exon 2.
[0021] Preferably, the term multiplication of exons indicates the
presence of 2 to 5 copies of the exon(s) considered, preferably 2
to 3 copies. Generally each copy of an exon is positioned in the
sequence beside the original exon.
[0022] In another specific embodiment, the invention relates to a
nucleic acid encoding parkin, comprising a point mutation, that is
to say the replacement of one base pair with another. The present
application indeed shows the existence of point mutants of parkin
and the causal character of some of them in the appearance and the
development of a parkinsonian syndrome. More particularly, the
point mutation(s) according to the invention are nonsense or
missense point mutations or mutations causing a reading frame
shift.
[0023] A nonsense mutation is a mutation which introduces a stop
codon into the sequence. Such a mutation leads to the premature
termination of translation, and therefore to the synthesis of a
truncated protein. Such a mutation is therefore also designated in
the text which follows by the term "truncating". More preferably,
according to the present invention, the nucleic acid comprises a
nonsense mutation located in a region corresponding to the N- or
C-terminal domain of human parkin. The present invention indeed
shows that this type of mutation occurs in patients more frequently
in the terminal regions of the gene (and therefore of the protein).
Still more preferably, the present invention relates to a nucleic
acid encoding human parkin, comprising a truncating point mutation
in a region corresponding to exons 2, 3, 11 or 12. It is more
preferably a mutation in exon 12 of the parkin gene, preferably
leading to the inactivation of the myristoylation site (residues
450-455 of the protein). By way of illustration, there may be
mentioned the point mutation G->A on nucleotide 1459,
introducing a stop codon in place of the residue Trp453. This
mutant therefore encodes a truncated protein comprising the first
452 amino acids of the wild-type protein. Surprisingly, the
applicant has shown that this parkin 1-452 mutant, which lacks only
the last 12 residues of the wild-type protein, causes Parkinson's
disease.
[0024] According to another embodiment, the present invention
relates to a nucleic acid encoding human parkin, comprising a
truncating point mutation in exon 7. By way of illustration, there
may be mentioned the T->A point mutation on nucleotide 905,
introducing a stop codon in place of the Cysteine 268 residue.
[0025] Another type of point mutation according to the invention is
a missense mutation. A missense mutation comprises the replacement
of a base pair in a codon, leading to a codon encoding an amino
acid different from the natural amino acid, without interruption of
the sequence. Such an isolated mutation therefore leads to a
protein having an unchanged number of residues, but in which one of
the residues differs from the wild-type protein. The present
application has now shown that missense point mutants of parkin
exist in subjects suffering from parkinsonian syndromes, and that
these mutants may have a causal character. More preferably, the
invention relates to a nucleic acid encoding human parkin,
comprising at least one missense point mutation located especially
in a region corresponding to exons 4 to 12 and preferably to exons
4, 6, 7, 9, 11 and 12.
[0026] A first type of more specific missense point mutations for
the purposes of the invention comprises the mutations which cause a
nonconservative change of amino acid in the encoded protein. Such
mutations are indeed more specifically associated with the
Parkinson's disease phenotype. Nonconservative change is understood
to mean the replacement of an amino acid with another amino acid
having structural, physicochemical and/or biological properties
which are different from the first. Thus, the change of one basic
amino acid with a nonbasic amino acid is said to be
nonconservative. This type of change comprises, more particularly,
the change of the amino acids of one or other of the following
categories: acidic, basic, polar neutral, nonpolar neutral.
Specific examples of mutants of this type according to the
invention are in particular the nucleic acids comprising the
following genetic alteration, alone or in combination: [0027] an
A->T mutation on nucleotide 584 (Lys161Asp) in exon 4, [0028] an
A->T mutation on nucleotide 734 (Lys211Asn) in exon 6, [0029] a
C->T mutation on nucleotide 867 (Arg256Cys) in exon 7, [0030] a
C->T mutation on nucleotide 924 (Arg275Trp) in exon 7, [0031] a
G->A mutation on nucleotide 939 (Asp280Asn) in exon 7, [0032] a
G->A mutation on nucleotide 1084 (Gly328Glu) in exon 9, [0033] a
C->T mutation on nucleotide 1101 (Arg334Cys) in exon 9, and/or
[0034] a G->A mutation on nucleotide 1390 (Gly430Asp) in exon
12.
[0035] A second type of more specific missense point mutations for
the purposes of the invention comprises conservative mutations.
Such mutations cover any replacement of a codon encoding an amino
acid with a codon encoding an amino acid of the same group. Amino
acid group is understood to mean the amino acids whose structural,
physicochemical and/or biological properties are very similar and
are defined according to the following categories: acidic, basic,
polar neutral, nonpolar neutral. As an example of a conservative
mutation, there may be mentioned in general the replacement of the
AAA codon (Lys) with the AGA codon (Arg), Lys and Arg forming part
of the same group of basic amino acids. A specific example of this
type of mutation according to the invention is in particular the
nucleic acid comprising the following genetic alteration, alone or
in combination: [0036] a T->G mutation on nucleotide 966
(Cys289Gly) in exon 7.
[0037] A third type of more specific missense point mutations for
the purposes of the invention comprises the mutations which affect
a potential phosphorylation site in the encoded protein. The
present application indeed shows that mutants of this type appear
in some subjects suffering from Parkinson's disease, and therefore
constitute events which participate in the development of the
pathology, because of the known biological functions of
phosphorylation events. Specific mutations are therefore those
which modify a phosphorylatable amino acid to a nonphosphorylatable
residue. In this regard, the residues capable of being
phosphorylated are, for example, the serine, threonine and tyrosine
residues.
[0038] Specific examples of mutants of this type according to the
invention are in particular the nucleic acids comprising the
following genetic alteration, alone or in combination: [0039]
C->A mutation on nucleotide 1345 (Thr415Asn), exon 11.
[0040] Moreover, as indicated above, the present invention also
relates to any nucleic acid comprising a deletion of one or more
contiguous base pairs and causing a reading frame shift (see d).
The present application demonstrates for the first time the
existence of restricted deletion mutants of parkin, and their
involvement in the appearance and the development of a parkinsonian
syndrome. More preferably, the restricted deletions according to
the invention lead, because of the reading frame shift, to the
synthesis of proteins (i) which are truncated and (ii) whose
C-terminal sequence is different from the wild-type protein. In the
case of intron deletions, the reading frame shift may lead either
to the nonrecognition of the intron if the mutation takes place at
the exon-intron junction and the production of an aberrant protein,
or to incorrect folding of the intron, thus preventing its
excision-splicing, when the mutation takes place inside the intron.
As indicated above, this protein (or the novel domain) constitutes
another subject of the present application.
[0041] More preferably, the invention relates to the nucleic acids
comprising a deletion of 1 to 10 and preferably 1 to 5 contiguous
base pairs. Still more particularly, the deletions according to the
invention are located in a region of the nucleic acid corresponding
to intron 8 or to exon 9 or to a terminal region of the protein,
especially in exons 2 or 3, for example to exon 2. By way of a
specific example, there may be mentioned a nucleic acid containing
the following alterations, alone or in combination: [0042] a
deletion of the AG nucleotides at positions 202 and 203. This
deletion introduces a change in the reading frame starting at the
level of amino acid residue 34 (Gln->Arg), and ending with a
stop codon (37). This protein is therefore truncated and comprises
a novel C-terminal region of 4 amino acids. [0043] a deletion of
the A nucleotide at position 255. This deletion introduces a change
in the reading frame starting at the level of amino acid residue 52
(Asn->Met), and ending with a stop codon (81). This protein is
therefore truncated and comprises a novel C-terminal region of 30
amino acids. [0044] a deletion of the five base pairs TCTGC in
intron 8, at position -21-17 relative to exon 9 and capable of
causing nonrecognition of the splicing site. [0045] a deletion of
two base pairs in exon 9 at position 1142-1143delGA which changes
Arg348 to Glu. The consequence of this deletion is the introduction
of a change in the reading frame, thus creating a stop codon at
position 368 in exon 10.
[0046] As indicated above, the present invention also relates to a
nucleic acid comprising one or more genetic alterations, such as,
in particular, an insertion of one or more contiguous base pairs
(see d)). The present application indeed demonstrates for the first
time the existence of insertion mutants of parkin, and their
involvement in the appearance and the development of Parkinson's
disease. More preferably, the insertion according to the invention
is such that it causes a reading frame shift. Because of this, the
insertion causes a change in the residues situated downstream
(C-terminal side) of the mutation. In addition, this insertion
leads more generally to the creation of a premature stop codon, and
therefore to the synthesis of a protein (i) which is truncated and
(ii) whose C-terminal sequence is different from the wild-type
protein. As indicated later, this protein (or the original domain)
constitutes another subject of the present application, and may be
used as a diagnostic or therapeutic tool.
[0047] More preferably, the invention relates to the nucleic acids
comprising an insertion of 1 to 5 contiguous base pairs, preferably
1 or 2. By way of a specific example, there may be mentioned a
nucleic acid comprising a GT insertion between nucleotides 321 and
322. This insertion introduces a change in the reading frame
starting at the level of the amino acid residue 74 (Trp->Cys),
and ending with a stop codon (81). This protein is therefore
truncated and comprises a novel C-terminal region of 8 amino
acids.
[0048] Of course the genetic alterations a) to e) described above
may be isolated or combined with each other, such as, in
particular, a missense mutation and a deletion or two cumulative
missence mutations. By way of examples illustrating this type of
combination, there may be mentioned in particular the combination
of the following modifications: [0049] a C->T missense mutation
at position 1101 (Arg334Cys) in exon 9 with a deletion of 5 base
pairs at position -21-17 relative to exon 9, in intron 8 [0050] a
C->T missense mutation at position 924 (Arg275Trp) in exon 7
with a G->A missense mutation at position 1390 (Gly430Asp) in
exon 12.
[0051] The subject of the invention is also a nucleic acid encoding
human parkin, characterized in that it comprises the sequence
presented in FIG. 1 (SEQ ID No: 1). This sequence contains,
compared with the sequence isolated by Kitada et al., a T at
position 768, in place of a C, resulting, in the encoded protein,
in an amino acid serine at position 223, in place of a proline.
This nucleic acid encodes the wild-type parkin found in European
populations.
[0052] The subject of the invention is also the polymorphic
variants of the nucleic acid presented in FIG. 1. The present
application indeed shows that the human parkin gene exhibits some
polymorphism, and describes more particularly certain variants
having more specifically one of the following sequence
modifications: [0053] G->A mutation on nucleotide 601 of exon 4
(Ser167Asn) [0054] G->C mutation on nucleotide 1239 of exon 10
(Val380Leu) [0055] G->A mutation on nucleotide 1281 of exon 11
(Asp394Asn).
[0056] The invention also relates to any vector comprising a
nucleic acid as defined above. It may be a plasmid vector, a
cosmid, viral vector, episome, artificial chromosome, and the like.
In a specific embodiment, such a vector also comprises a promoter
region allowing the expression of said nucleic acid.
[0057] Such vectors may be used to produce in large quantities the
nucleic acids of the invention, or to produce the corresponding
polypeptides, in an appropriate cellular host (prokaryotic,
eukaryotic, animal or plant cell, for example). Preferred cellular
hosts are in particular bacterial cells (E. coli for example) or
yeast cells, or alternatively mammalian, animal or human cells.
[0058] In this regard, the invention also relates to any
recombinant cell containing a nucleic acid or a vector as defined
above.
[0059] The invention also relates to any mammalian, in particular
human, cell containing a nucleic acid or a vector as defined above,
as a replacement for the wild-type gene for parkin.
[0060] The cells of the invention may be used in particular for
studying the properties of parkin, and also as models for the
search for compounds capable of compensating for the genetic
alterations of the parkin gene.
[0061] The invention relates, in addition, to any nonhuman mammal
comprising a nucleic acid as defined above in its cells.
Advantageously, these mammals are obtained by "knock-in" of the
alterations defined above, by homologous recombination, or also by
"knock-out" of the wild-type gene, which is replaced by the altered
version of the invention.
[0062] Such mammals (rodents, canines, rabbits and the like) can in
particular be used for studying the properties of parkin and the
identification of compounds for therapeutic purposes, for
example.
[0063] The invention also relates to any polypeptide encoded by a
nucleic acid as defined above. These polypeptides are therefore
human parkin, its polymorphic variants, and mutated and/or
truncated variants and/or variants comprising a multiplication of
exons, involved in the appearance and/or development of a
parkinsonian syndrome. The invention relates in particular to the
truncated or aberrant variants of parkin as described above, or a
portion thereof corresponding to the sequence created by the
reading frame shift. Such polypeptides or fragments, or the
corresponding nucleic acids, can be used for identifying and/or
studying compounds capable of restoring a normal phenotype to cells
expressing them. In particular, the nucleic acids described above
may be transferred into appropriate host cells, preferably
eukaryotic cells (mammal, yeast for example), to be used in a test
for screening compounds capable of counteracting their activity,
whether they are chemical, biochemical or genetic compounds. Such
polypeptides or fragments can also be used as antigens, for the
preparation of specific antibodies, which can be used for the
detection of the variants. In particular, the specific polypeptide
regions of the truncated forms (in particular the ends) may be used
for the preparation of antibodies, according to conventional
immunological techniques, which antibodies then constitute tools
for detecting the presence of these forms in biological samples
obtained from subjects. Such antibodies may be polyclonal or
monoclonal (prepared for example by fusion of spleen cells of
animals immunized with the antigens, with myeloma cells, followed
by selection of clones producing monoclonal antibodies).
[0064] In this regard, the invention also relates to any antibody
specific for a polypeptide as described above, or for a specific
region of such a polypeptide. The term "specific antibody"
designates an antibody having a particularly high affinity for the
given antigen, compared with any other antigen.
[0065] The invention relates, in addition, to any composition
comprising a polypeptide or an antibody or alternatively a vector
or a host cell transformed by the nucleic acid of the invention, as
described above. These compositions may be packaged in various
types of media (isotonic, saline solutions, and the like) in the
presence of stabilizers or preservatives, for example. These
compositions may be stored at cold temperature or frozen, in any
appropriate device (tube, box, bottle, flask, bag and the
like).
[0066] Moreover, in addition to the genetic exon alterations
described above, the invention also describes genetic intron
alterations of the parkin gene. These alterations do not induce any
change in the sequence of the encoded protein, and essentially
constitute polymorphic variants. These variants are more
particularly described in Table 2.
[0067] One of the applications of the invention consists in the
detection of the presence of mutations in the parkin gene, and
their correlation with the susceptibility to Parkinson's disease,
for example. In this regard, the invention also relates to various
tools (probes, primers, antibodies and the like), which are useful
for carrying out such detection methods.
[0068] In particular, the invention relates to any probe or
oligonucleotide which hybridizes specifically with a nucleic acid
as defined above.
[0069] The specific probes or oligonucleotides of the invention
generally comprise less than 500 bp, more preferably less than 300
bp. Typically, a specific oligonucleotide of the invention
comprises from 5 to 100 bp, advantageously from 5 to 50 bp. The
length of the oligonucleotide may of course be adjusted by persons
skilled in the art. The probes or oligonucleotides of the invention
are moreover generally labeled, so as to allow their detection.
Various types of labelings known to persons skilled in the art may
be used (radioactive, fluorescent, enzymatic, chemical, end or
internal labeling, and the like). Finally, the probes or
oligonucleotides of the invention have the capacity to specifically
hybridize with the nucleic acids as defined above, that is to say
with the various altered forms of the parkin gene. The
hybridization is said to be specific when the probe or
oligonucleotide hybridize, under conditions of high stringency,
with the nucleic acid carrying the desired alteration, and not or
not to any great extent with the same nucleic acid not carrying
said alteration. The hybridization is therefore said to be specific
when the specific signal/background noise differential is
sufficiently high to be detected.
[0070] The probes or oligonucleotides of the invention are
therefore generally complementary to at least one region of the
parkin gene carrying the genetic alterations a) to d) described
above. The complementarity is generally perfect, so as to ensure a
better hybridization selectivity. These probes or oligonucleotides
may be synthesized by any technique known to persons skilled in the
art, for example by cleavage from the nucleic acids described
above, or by artificial synthesis, or by combining these
techniques. These probes or oligonucleotides can be used for the
identification, on biological samples, of the presence of genetic
alterations of the parkin gene.
[0071] The invention also relates to a pair of primers for the
amplification of all or part of a nucleic acid as described above,
characterized in that it comprises: [0072] a first primer
complementary to a region of the parkin gene situated in 5' of a
genetic alteration, and [0073] a second primer complementary to a
region of the parkin gene situated in 3' of said genetic
alteration.
[0074] The primers of the invention are generally complementary to
a region of the parkin gene, and advantageously comprise less than
30 bp.
[0075] The invention further relates to a method for the
identification of a genetic alteration in the parkin gene, and in
particular the detection of deletion(s) and/or multiplication (e.g.
duplication, triplication) of exons in the homozygous and
heterozygous state.
[0076] This method according to the invention comprises:
[0077] i) the provision of a sample comprising the parkin gene,
[0078] ii) the amplification (semi-quantitative) of at least a
portion of said gene, said portion comprising a genetic alteration
as defined above, and
[0079] iii) the detection of the presence of the genetic
alteration.
[0080] Advantageously, in the method of the invention, the sample
is a sample of blood, tissue, plasma or a cell culture, obtained
from a subject, in particular from a mammal, in particular from a
human. In a preferred embodiment, the sample is pretreated so as to
make the parkin gene, or a portion thereof, accessible for the
amplification. This pretreatment may comprise the lysis of the
cells, an enzymatic treatment, a denaturation, and the like.
[0081] Advantageously, the amplification is carried out by means of
a pair of primers as described above or those described by Kitada
et al. and included by way of reference.
[0082] By way of a specific example of a pair of primers according
to the invention, there may be mentioned the primers serving for
the detection of alterations in exon 3 or of point mutations. Thus,
the following pair of primers was used in the context of the
invention:
TABLE-US-00002 (SEQ ID No: 2) For: 5'-(Hex)AATTGTGACCTGGATCAGC-3'
and (SEQ ID No: 3) Rev: 5'-CTGGACTTCCAGCTGGTGGTGAG-3'
[0083] The following primers were also used for the detection of
the following point mutations:
TABLE-US-00003 Asp280Asn: (SEQ ID No: 4)
5'-GGCAGGGAGTAGCCAAGTTGAGGAT-3' wild-type sequence G Arg334Cys:
(SEQ ID No: 5) 5'-AGCCCCGCTCCACAGCCAGCGC-3' wild-type sequence
G
[0084] The detection of a genetic alteration as described above may
be carried out by various techniques, and in particular by
sequencing, PCR/restriction, ASO, PAGE or by semi-quantitative
multiplex PCR, as detailed in the experimental part. Briefly, this
method is based on semi-quantitative PCR amplification and in the
exponential phase of template DNA. According to this method,
comparison of the relative level of template DNA is sufficient to
demonstrate a loss of the quantity of DNA (deletion of exon(s)) or
on the contrary an increase in the quantity of DNA (multiplication
of exon(s)).
[0085] The invention relates, in addition, to a kit for carrying
out the methods of the invention, comprising a probe or an
oligonucleotide or a pair of primers as described above. The kits
of the invention advantageously comprise the appropriate reagents
for an amplification and/or hybridization reaction, and,
optionally, a support for such reactions (filters, membranes, chips
and the like).
[0086] The present invention is particularly appropriate for the
diagnosis of a susceptibility to Parkinson's disease, by the search
for a genetic alteration as described above in the parkin gene.
[0087] The present invention also relates to the use of the tools
described above (nucleic acids, probes, polypeptides, antibodies,
cells, animals) for the identification of compounds capable of
counteracting, at least in part, the effects of a genetic
alteration in the parkin gene, in particular with a therapeutic
objective. Thus, such compounds may be detected by bringing into
contact with a test composition (or product) in the presence of a
cell or an animal as described above, and detecting a phenotypic or
genotypic effect.
[0088] The method of the invention may in particular allow the
identification of compounds which can be used, alone or in
combination with other products or treatments, for treating (i.e.
reducing) Parkinson's disease. Such compounds constitute another
subject of the present invention.
[0089] Other advantages and applications of the present invention
will emerge on reading the following examples which should be
considered as illustrative and nonlimiting.
EXAMPLES
A--Legend to the Figures
[0090] FIG. 1: cDNA sequence encoding human parkin. The junctions
between the exons are indicated. The initiator codon (atg) and the
stop codon (tag) are in bold. The C>T change at position 768 is
in bold and underlined.
[0091] FIG. 2: Families having point mutations in the parkin gene.
The complete cosegregation of the mutation with the disease is
represented. The black squares (men) and circles (women) represent
the individuals affected with the age of appearance (in years)
indicated under the symbol for the patient. The crossed symbols
indicate deceased patients. The number of nonaffected and
nonanalyzed brothers and sisters is given as a diamond. For each
mutation (change in amino acid), the genotype of the family member
is indicated (+/+ wild-type homozygote, +/- heterozygous for the
mutation; -/- homozygous for the mutation). Under each genotype,
the detection results are given. PAGE: electrophoretogram with the
size of the allele in bp; ASO: autoradiograms of the mutated and
wild-type alleles; PCR/restriction: PCR products after digestion
with the appropriate restriction enzymes. The length of the
fragments in bp is given. Mut: mutated; nd: age of appearance not
determined, since the patient is not conscious of the symptoms.
[0092] FIG. 3: Representation and location of the point mutations
in the parkin gene. The coding sequence of the gene, with its 12
exons, is represented (bar). The exons are numbered 1 to 12. The 8
causal point mutations are positioned according to their effect on
the protein (truncation vs missense). The ubiquitin-like domain and
the ring motif ("Ring Finger") are hatched. For the Thr415Asn and
Trp453Stop mutations, the phosphorylation (P) and myristoylation
(M) sites are indicated. UTR; untranslation region.
[0093] FIG. 4: Results of the detection of deletions of
heterozygous exons in a family with early onset parkinsonian
syndrome (FPD-GRE-WAG-155) according to the semi-quantitative
multiplex PCR method of the invention. The black squares (men) and
circles (women) represent the individuals affected.
[0094] The peaks represent the exons produced by semi-quantitative
multiplex PCR. The encircled figures indicate the height of the
peaks. The graduated ruler above the electrophoretograms indicates
the size of the PCR products in base pair.
[0095] Table 1: Oligonucleotides used for the ASO technique. The
nucleotide changes in the sequence of the oligonucleotides are
represented in bold and underlined. WT=wild type; V=variant.
[0096] Table 2: Summary of the mutations identified. The positions
of the nucleotides are given according to the cDNA sequence
published in the DNA Data Bank of Japan (DDBJ; accession number
AB009973) and are illustrated in FIG. 1. PAGE=polyacrylamide gel
electrophoresis; ASO=technique for the detection of mutations using
an allele specific oligonucleotide.
[0097] Table 3: Clinical characteristics of patients as a function
of the type of genetic alteration. The patients of the IT-020
family who are composite heterozygous for a missense mutation and a
truncating mutation do not appear in the table. a: p<0.05 for
the comparison between the patients with a homozygous deletion and
the patients with truncating mutations.
[0098] Table 4: Frequency and consequences of the
deletions/multiplications of exons
del=deletion, het=heterozygote; hom=homozygote
[0099] Table 5: Ratio of the results obtained in FIG. 4. The height
of the peaks is given for each exon in the left hand part of the
table, the values for the double peaks having been added. The right
hand part of the table provides the calculation of the ratios of
the values of the peaks. Italics=normal value;
underlined=pathological value compared with the control. In the
second line of the table, for each case, the ratio of the control
is divided by the ratio of the case. The pathological values are
either .ltoreq.0.625 or .gtoreq.1.6 (=1/0.625). Deletions of exons
were detected for the subjects FR 155 5 (exon 3), FR 155 6 (exon
2), FR 155 8 and FR 155 9 (exons 2+3). For the latter two affected
subjects the value of the exon 3/2 ratio is normal given that the
two exons were heterozygously deleted.
B--Materials and Methods
[0100] 1. Families and Patients
[0101] In a first series of experiments, 38 families were selected
according to the following criteria: parkinsonian syndrome reactive
to levodopa, ii) starting age at most 45 years for at least one of
the affected members, and iii) transmission compatible with an
autosomal-recessive heredity.
[0102] In another series of experiments, 77 families were selected
according to the following criteria (as indicated in Lucking et al,
1998; Abbas et al, 1999): i) presence of a parkinsonian syndrome
with a good response to levodopa (.gtoreq.30% improvement) in at
least two members of a phratry (or only one if there is a notion of
consanguinity); ii) absence of exclusion criteria such as
Babinski's syndrome, ophthalmoplegia, dementia or dysautonomia
occurring before two years of progression; iii) beginning 45 years
in at least one of those affected; iv) heredity compatible with
recessive autosomal transmission (several patients in a single
generation with or without a notion of consanguinity). The families
were from France (n=20), Italy (n=19), Great Britain (n=14), the
Netherlands (n=9), Germany (n=9), Lebanon (n=2), Algeria (n=1),
Morocco (n=1), Portugal (n=1), Vietnam (n=1).
[0103] Furthermore, 102 isolated cases, with no known
consanguinity, were selected with the same clinical criteria. They
were from France (n=31), Italy (n=23) and Great Britain (n=26),
Germany (n=21)n the Netherlands (n=1).
All the patients were evaluated according to a standard protocol.
The informed consent of all the participants was obtained in
writing.
[0104] 2. Analysis of the Parkin Gene
[0105] The DNA of the 12 exons encoding the parkin gene was
amplified by PCR from peripheral blood leukocytes, for each index
case, according to the conditions described in Kitada et al.
Briefly, the amplification was carried out on 100 ng of DNA, in the
presence of 350 .mu.M of each dNTP, 350 .mu.M of each primer, and
Taq polymerase. The amplification conditions are cycles at
94.degree. C. for 30 sec, at 55-61.degree. C. for 30 sec, and then
at 68.degree. C. for 30 sec. For exons 4 and 7, only the pair of
intron primers was used. The sequence of the 12 exons was prepared
on two strands with the primers used for the PCR amplification,
with the sequencing kit "Big Dye Terminator Cycle Sequencing Ready
Reaction" (ABI PRISM) and analyzed after electrophoresis on the ABI
377 sequencer with the "sequence analysis 3.0" software (ABI
PRISM).
[0106] The detection of the mutations in the samples and the
analysis of a population of 45 control individuals was carried out
by three techniques, which may be used alone or in combination(s):
PCR/restriction with the appropriate restriction enzyme; ASO
technique ("Allele Specific Oligonucleotide"), and polyacrylamide
gel electrophoresis ("PAGE") as illustrated in Table 2. More
particularly, these techniques were carried out as described
below.
[0107] The ASO technique: this approach consists in hybridizing two
oligonucleotide probes with an amplified sample (for example by
PCR), the first specific for and covering a genetic alteration, the
second specific for and covering the corresponding wild-type
region. Thus, in the presence of a mutated gene, only the first
probe allows hybridization with the DNA fragment, whereas in the
presence of a nonmutated gene, only the second probe allows
hybridization with the DNA fragment. In the case of a heterozygous
gene, a hybridization is obtained with each of the probes. This
technique may also be carried out concomitantly with the
amplification, using two pairs of primers, the first comprising a
primer specific for and covering the corresponding wild-type
region. In this embodiment, in the presence of a mutated gene, only
the first pair allows the amplification of a DNA fragment, whereas
in the presence of a nonmutated gene, only the second pair of
primers allows the amplification of a DNA fragment. In the case of
a heterozygous gene, an amplification product is obtained with each
of the pairs of primers.
[0108] For carrying out this technique, 10 .mu.l of PCR product
were denatured at 95.degree. C. for 5 min, deposited on Hybond N+
nylon membranes (Amersham), and then microwave-fixed at 600 W for 2
min. The specific primers (or oligonucleotides) used for the
detection (or, where appropriate, for the amplification), are
described in the examples (see Table 1). For exon 3, the exon
primers Ex3iFor (forward) and Ex3iRev (back) were used. The
sequence of these primers is the following:
TABLE-US-00004 Ex3iFor: (SEQ ID No: 6) 5'-AATTGTGACCTGGATCAGC-3'
Ex3iRev: (SEQ ID No: 7) 5'-CTGGACTTCCAGCTGGTGGTGAG-3'
[0109] These oligonucleotides (including the primers, in the case
of a simultaneous amplification), labeled with dCTP32 by means of
the Terminal Transferase Kit (Boehringer Mannheim) were hybridized
with the membranes at 44.degree. C. overnight in a buffer
consisting of 5.times.SSC, 5.times.Denhardts and 0.1% SDS. The
membranes were then washed twice for 30 min in a 2.times.SSC medium
at 59.degree. C. and exposed to an MP film (Amersham) for 3-6
hours.
[0110] PCR/restriction technique: this technique is based on the
use of restriction enzymes whose digestion profile becomes modified
because of the genetic alteration. Preferably, this technique
therefore uses restriction enzymes whose site is modified
(destroyed or created) by the genetic alteration. Thus, depending
on the nucleic acid digestion profile (generally amplification
product), it is possible to distinguish the presence or otherwise
of the genetic alteration searched for. Of course, this technique
is most particularly appropriate for the search for straightforward
genetic alterations, causing a modification in an enzymatic
cleavage site. For its use, 15 .mu.l of amplification product is
digested in the presence of appropriate restriction enzyme(s),
according to the manufacturer's recommendations. The particular
enzymes used in the examples and the expected size of the
restriction fragments are given in Table 2.
[0111] Polyacrylamide gel electrophoresis ("PAGE") technique: this
technique makes it possible to detect the presence of mutations by
measuring the size of the amplification products. It is therefore
most particularly appropriate for the detection of genetic
alterations of the insertion or deletion type. For its use, a
labeled forward primer (5'-fluorescent, Hex) was used to amplify
exon 2 of the parkin gene. The presence of the 202-203delAG
alteration, resulting in a shorter PCR product (306 vs 308 bp) was
established by measuring the size of the amplified fragment using
an ABI377 automated sequencer equipped with "Genescan 2.0.2" and
"Genotyper 1.1.1" software (ABI PRISM).
[0112] The numbering of the nucleotides used in the present
application is given with reference to the sequence of the cDNA
which exists in the DNA Data Bank of Japan (DDBJ; accession number:
AB009973). The sequence is represented in FIG. 1. This sequence
differs from the sequence described by Kitada et al. at the level
of nucleotide 768. The sequence presented in FIG. 1 corresponds to
the wild-type protein found in European populations.
[0113] 3. Semi-Quantitative Multiplex PCR for the Detection of
Deletions/Multiplications of Exons in the Homozygous and
Heterozygous State
[0114] a) Principles
[0115] The detection of heterozygous deletions or multiplications
of exons in the Parkin gene cannot be carried out by
nonquantitative PCR. Thus, a semi-quantitative PCR which compares
the relative amount of template DNA is sufficient to know if 50% of
the template DNA is missing for one or more exons or, on the
contrary in the case of a heterozygous or homozygous
multiplication, if there is for example 50% (heterozygous
duplication), 100% (homozygous duplication or heterozygous
triplication) or 200% (homozygous triplication) of DNA in excess
for one or more exons. To carry out this comparison, several exons
from the same individual are simultaneously amplified, in a PCR
reaction (multiplex PCR), the coamplified exons serving as internal
standard for quantity. The PCR is carried out with fluorescent
primers, such that the quantity of PCR product can be measured by
the height of peaks on an automated sequencer (ABI Prism 377), as
applied for example in the Applied Biosystems LOH (Loss of
Heterozygosity) Assay. The quantity of PCR product (height of the
peak) is directly linked to the quantity of template DNA as long as
the PCR is in its exponential phase which means an absence of
limitation by the available substrates. Each multiplex PCR, for a
given combination of exons, produces a typical peak height
distribution for a control individual as well as defined ratios
between the different peaks.
[0116] A homozygous deletion of an exon will be demonstrated by the
absence of the corresponding peak. If an exon is deleted in the
heterozygous state, the corresponding peak will have half of its
normal height, which will change the ratio between the deleted and
nondeleted exons by a factor of 2 compared with a control
(comparing the high value with the low value; FIG. 4 and Table 4
relating to FIG. 4). For the duplications of exons, the ratios
change by a factor of 1.5 for the heterozygotes and by a factor of
2 for the homozygotes (still by comparing the high value with the
low value). Thus, the factors for a heterozygous or homozygous
triplication are 2 or 3, respectively (still by comparing the high
value with the low value). In order to be able to also detect a
deletion or a multiplication of the entire Parkin gene, a PCR
product of 328 base pairs (C328) of a gene situated at a distance
(gene for Transthyretin) is amplified and serves as external
standard in one of the multiplex PCRs. The fact that only the
ratios of the heights between the peaks are compared renders, by
first approximation, the method independent of the quantity and of
the quality of the DNA.
[0117] b) Establishment of the Appropriate Conditions for Multiplex
PCR
[0118] During preliminary experiments, it was noted that the exons
which exhibit the best amplification could negatively influence the
amplification of other exons, for which the efficiency was not as
good. Thus, the exons whose amplification efficiency was comparable
were grouped together. Furthermore, as the size of the PCR product
can influence the amplification yield (the short sequences being as
a rule better amplified than the long sequences), the PCR products
of comparable size were grouped together in the multiplex reaction.
Thus, three combinations of exons were chosen:
Ex 4o (261 bp)+7o (239 bp)+8 (206 bp)+11 (303 bp), Comb 1:
Ex 5 (227 bp)+6 (268 bp)+8 (206 bp)+(165 bp) and Comb 2:
Ex 2 (308 bp)+3i (243 bp)+9 (278 bp)+12 (255 bp)+C328 Comb 3:
[0119] (external control of 328 base pairs).
[0120] The primers used are those described by Kitada et al (1998).
For exon 3, a pair of exonic primers was used:
TABLE-US-00005 (SEQ ID No: 8) For: 5'-(Hex)AATTGTGACCTGGATCAGC-3'
and (SEQ ID No: 9) Rev: 5'-CTGGACTTCCAGCTGGTGGTGAG-3'.
[0121] The primers for C328 being:
TABLE-US-00006 The primers for C328 being: TTRForHex: (SEQ ID No:
10) 5'-(Hex)ACGTTCCTGATAATGGGATC-3' and TTR328Rev: (SEQ ID No: 11)
5'-CCTCTCTCTACCAAGTGAGG-3'.
[0122] In order to obtain peaks of comparable heights in each
multiplex PCR and to be situated in the exponential phase for each
exon, the PCR conditions were adjusted permanently (by partly
following the recommendations of Henegariu et al (Henegariu et al,
1997). In particular, the hybridization and extension temperatures
were reduced and the concentration of MgCl.sub.2 and the duration
of the extension were increased. Furthermore, the concentrations of
primers were adjusted from a standard concentration of 0.8 .mu.M,
according to the amplification efficiency (the concentrations of
primers being reduced for the exons which amplify well, and
increased for the others).
[0123] Each combination of exons was tested in order to verify that
the exponential phase was established, this being in two multiplex
PCRs in parallel for the 3 combinations of primers, on a control
individual with 22, 23 and 24 cycles. The peak heights were
corrected for the variations in loadings according to the internal
molecular weight marker (Applied Biosystems TAMRA 500 XL). The
corrected peak heights were compared to the number of cycles, and
represent, on a logarithmic scale, an ascending straight line which
demonstrates that the exponential phase was established for the
following conditions:
[0124] 5 minutes at 95.degree. C. for one cycle,
[0125] 30 seconds at 95.degree. C., 45 seconds at 53.degree. C. and
2.5 minutes at 68.degree. C. for 23 cycles,
[0126] 5 minutes at 68.degree. C. for one cycle.
[0127] The reaction was carried out with 40 ng of DNA in a volume
of 25 .mu.l of PCR solution, with 3 mM MgCl.sub.2, 0.2 mM dNTP and
1 U Taq/25 .mu.l. The concentration of each primer was:
Ex 2 (0.8 .mu.M), Ex 3 (0.4 .mu.M), Ex 4 (1.0 .mu.M), Ex 5 (0.6
.mu.M), Ex 6 (1.4 .mu.M), Ex 7 (0.44 .mu.M), Ex 8 (in comb 1:1.0
.mu.M and in comb 2:0.8 .mu.M), Ex 9 (0.4 .mu.M), Ex 10 (1.04
.mu.M), Ex 11 (0.8 .mu.M), Ex 12 (1.2 .mu.M) and C328 (1.92
.mu.M).
[0128] c) Applications of Multiplex PCR, Internal Controls and
Electrophoresis
[0129] As a general rule, the multiplex PCRs were carried out at
least in two parallel reactions for each individual. For each
series of patient, at least one positive control (with a
heterozygous deletion of known exons) and one negative control
(control individual) were treated in parallel in order to obtain
the normal and pathological values for each reaction premix, so as
to avoid erroneous results due to possible differences in the
premix (variation of pipetting). Two additional controls were
added, which did not contain template DNA. 1.5 to 2.5 .mu.l of the
PCR product were mixed with 4 .mu.l of loading buffer (comprising
0.3 .mu.l of the Applied Biosystems TAMRA 500 XL size marker). 1.5
.mu.l of this mixture was loaded onto a 4% denatured polyacrylamide
gel containing 96 wells on an ABI 377 automated sequencer. The gels
are analyzed by the GeneScan 3.1 and Genotyper 1.1.1 software
packages (Applied Biosystems). The peak heights are measured as
indicated in Genotyper. For the double peaks with one base pair
difference (caused by the fact that Taq polymerase inconstantly
adds an A to each end), the two peak heights are added. The ratios
of each combination of peaks are calculated for each reaction,
using the Excel 5.0 software (Table 5) and the mean values are
calculated for two reactions.
[0130] d) Interpretation
[0131] For the deletions, the results are interpreted as
pathological if the difference in ratio was a factor of at least
1.6 or .ltoreq.0.625 (=1/1.6) in all the respective ratios between
the control and the case (ratio of the subject/ratio of the
control--Table relating to FIG. 4). When the differences in ratios
between the parallel reactions are contradictory (for example
because of a weak amplification in one of the PCRs), the ratios
obtained with a satisfactory amplification are taken into account
on condition that they are normal.
[0132] For the duplications, a change in the ratios by a factor of
1.30-1.65 or >1.75 is interpreted as a heterozygous or
homozygous duplication respectively (by comparing the high value
with the low value).
[0133] For a triplication, a change in the ratios by a factor of
1.6-2.4 or >2.6 is interpreted as a heterozygous or homozygous
triplication respectively (by comparing the high value with the low
value).
[0134] However, as the conditions were continuously adjusted during
the development of the method, some of the results were obtained
under slightly different conditions. These results are taken into
account when they are clearly normal or pathological and
reproducible. In ambiguous situations, the experiment was repeated
under appropriate conditions.
[0135] 4. Analysis of Cosegregation and of a Control Population
[0136] a) Point Mutations
[0137] The variants of the Parkin sequence were tested for their
cosegregation in the families (according to the availability of
other samples) and for their presence in a population of controls
without Parkinsonian syndrome (61 to 73 individuals). Because of
the certainly pathogenic character of the 1142-1143delGA mutation,
controls were not tested for this mutation. The techniques used are
PCR and digestion with the appropriate restriction enzyme or
polyacrylamide gel electrophoresis (PAGE) (see Table 2). When the
variant did not cause any change in restriction site by itself, a
site was artificially created with the aid of a primer with a
mismatch. The primers were designed so as to introduce the change
of base near the position of the sequence variant, so as to create
a restriction site which includes this variant. The primers are
indicated in the table below.
[0138] Modified primers (not complementary to the wild-type
sequence for one base) for PCR:
TABLE-US-00007 Restriction Mutation enzyme Primer F Primer R
Asp280Asn AlwI Ex 70 For 5'-GGCAGGGAGTAGCCAAGTTGAGGAT-3' (SEQ ID
NO. 12) wild-type sequence G Arg334Cys BstUI Ex 9 For
5'-AGCCCCGCTCCACAGCCAGCGC-3' (SEQ ID NO. 13) wild-type sequence
The change in base pair introduced is underlined by comparison with
the wild-type sequence.
[0139] b) Deletions or Multiplications of Homozygous or
Heterozygous Exons
[0140] The cosegregation of a deletion or of a multiplication of
exons in the families was analyzed with the aid of the methods
described above. A control population was not tested because of the
highly probable pathogenic character of the mutations, which causes
an internal deletion of the protein, with or without a reading
frame shift.
[0141] 5. Linkage Analysis
[0142] To test the linkage to the PARK2 locus, four
microsatellite-type markers, situated near the locus, were tested
(D6S1579, D6S411, D6S1550 and D6S305) as described by Tassin et al
(1998).
C--Results
[0143] a) In a First Series of Experiments, the Analysis of the
Parkin Gene was Carried Out in the Index Case of 38 Families with
AR-JP which Contain 87 Patients.
[0144] 1. Detection of Deletions of Exons
[0145] The amplification of the exons revealed the presence of a
deletion in the homozygous state in three families: deletion of
exon 3 in a French family (SAL-024) and a Portuguese family
(SAL-711), and of exons 8 and 9 in an Algerian family (DEL-001).
These deletions are transmitted with the disease because they are
detected in each family in the homozygous state in all patients but
not in the healthy related ones sampled (FIG. 2).
[0146] 2. Detection of Point Mutations
[0147] The sequence analysis in the families without homozygous
deletion revealed the presence of 16 variants of the nucleic
sequence: 12 in the exons and 4 in the introns (Tables 2 and 3,
FIG. 3). Three variants cause a reading frame shift and the
synthesis of a truncated protein. They are mutations 202-203delAG
(Gln34Arg(Stop37)) and 225delA (Asn52Met(Stop81)) in exon 2 and
321-322insGT (Trp74Cys(Stop81)) in exon 3 which are found
respectively in the families IT-020 and UK-086, TOU-096, LYO-119.
These mutations, with the exception of 202-203delAG are in the
homozygous state. A nonsense mutation 1459G>A (Trp453Stop) in
exon 12 is present in the homozygous state in the IT-006 family.
Eight of the variants are of the missense type. In exon 4,
584A>T (Lys161Asp) and 601G>A (Ser167Asn) are in the
heterozygous state in patients of the IT-020 and SAL-730 families,
respectively. In exon 7, the variants 867C>T (Arg256Cys) and
924C>T (Arg275Trp) are found in the heterozygous state in the
DE-012 and IT-015 families, respectively. In exon 10, the variant
1239G>C (Val380Leu) is found in the heterozygous and homozygous
state in 11 families (IT-014, IT-020, IT-058, SAL-017, GRE-029,
SAL-038, TOU-096, SAL-431, UK-006, UK-086, DE-022). In exon 11, the
variant 1281G>A (Asp394Asn) is detected in the heterozygous
state in the UK-046 family and 1345C>A (Thr415Asn) in the
homozygous state in the IT-014 family. Finally, the variant
768C>T (Pro223Ser) is not pathogenic, because it is detected in
the homozygous state in all the individuals sequenced, suggesting
that it is a typographical error in the parkin sequence [Kitada et
al., 1998]. The search for these variants in the control population
reveals that three of them represent polymorphisms (Table 2):
Ser167Asn, Val380Leu and Asp394Asn. The other variants most
probably constitute causal mutations because they cause the
synthesis of truncated parkin or nonconservative substitutions or
substitutions affecting one of the amino acids capable of being
phosphorylated. Furthermore, they segregate with the disease in the
families and are not detected in 90 control chromosomes.
[0148] The variants identified in introns 2, 3, 6 and 7
(IVS2+25T>C (272+25T>C), IVS3-20C>T (514-20C>T),
IVS6+19T>C (835+19T>C) and IVS7-35A>G (973-35A>G))
constitute polymorphisms (Table 2). They are not located near
splicing sites and are detected in the control chromosomes.
[0149] 3. Functional Domains of Parkin
[0150] A study of the functional domains of parkin was undertaken
by analysis and comparison of sequences. This study shows that the
conservative change in amino acid Thr415Asn is located in the
consensus sequence of a cAMP- and cGMP-dependent protein kinase
(KKTT) and in the phosphorylation site of a protein kinase C (TTK).
This study shows, in addition, that the nonsense mutation
Trp453Stop is located in an N-terminal myristoylation site
(GCEWNR).
[0151] 4. Phenotype Genotype Correlations
[0152] The homozygous deletions and the point mutations were
detected in 12 families which contain 26 patients. The average age
at onset is 36.7 years with extremes of 7 to 56 years (Table 3).
The comparison between the families according to the functional
consequences of the mutations (homozygous deletion, truncating
mutation and missense mutation) does not reveal any significant
difference in the age at onset, in the severity or the frequency of
the associated signs, except for tremor which is significantly less
frequent in families with a homozygous deletion, compared with
families with truncating mutations (Table 3).
[0153] b) Detection of New Point Mutations
[0154] Eight new point mutations in exons were identified, of which
six are missense mutations one truncating and one nonsense:
734A>T (Lys211Asn) in exon 6, 905T>A (Cys268Stop), 939G>A
(Asp280Asn) and 966T>G (Cys289Gly) in exon 7, 1084G>A
(Gly328Glu), 1142-1143delGA and 1101C>T (Arg334Cys) in exon 9
and 1390G>A (Gly430Asp) in exon 12. Five of the missense
mutations lead to nonconservative amino acid changes and one to a
conservative change (Cys289Gly). Furthermore, a deletion of five
base pairs in intron 8, located at positions -21 to -17 relative to
exon 9 was detected. All these sequence variants were not detected
in 61 to 73 control individuals (the 1142-1143delGA mutation was
not tested) and do not therefore represent polymorphisms. The
results are detailed in Table 2.
[0155] c) Detection of New Homozygous Deletions of Exons
[0156] Homozygous deletions of exons were detected in 3 families in
addition to the deletions previously reported by Hattori et al
(1998a) and Lucking et al (1998) for exon 3 and by Hattori et al
(1998a) for exons 3+4. These deletions relate to exons 3
(FDP-ANG-GEO-141), 3+4 (IT-064) and 5+6 (SPD-LIB-HAG-076). The
consequences of the deletions of exons on the reading frame and
their relative frequency in the sample are indicated in Table
4.
[0157] d) Detection of Homozygous and Heterozygous
Duplications/Triplications
[0158] Five new types of duplications of exons were detected: a
duplication of exon 3 in the homozygous state (SPD-NIC-AIT-091) and
a duplication of exon 3 in the heterozygous state (SAL 399 213). In
addition, heterozygous duplications of exon 6 (FPD-LIL-CHA-171), of
exon 7 (DE 4001) and of exon 11 (SAL 399 213) were detected. Two
types of triplication were detected: a triplication of exon 2 in
the homozygous state (RM 347) and a the heterozygous state (RM
330).
[0159] e) Detection of New Heterozygous Deletions
[0160] Thirteen different combinations of heterozygous deletions of
exons were detected in 21 families. The following deletions were
observed: exons 2, 2+3, 2+3+4, 3, 3+4, 3-6, 3-9, 4, 5, 6, 6+7,
7+8+9 and 8. The deletions of exons 2, 2+3, 2+3+4, 3-6, 3-9, 6,
6+7, 7+8+9 and 8 are new.
[0161] For two families (Sal-Hab-436 and UK 12416), it was not
possible to establish with certainty if the heterozygous mutations
of exons 2+3 or 6-7, respectively, were situated on the same
chromosome or if they were composite heterozygous cases because of
the absence of DNA for other members of these families. The
consequences of the deletions and of the multiplications of exons
described on the reading frame and their relative frequency in our
sample are indicated in Table 4.
[0162] f) Recurring Point Mutations
[0163] Five point mutations were detected in more than one family.
These mutations are 202-203delAG (in the heterozygous or homozygous
state in 5 families), 255delA (in the homozygous or heterozygous
state in 6 families), Lys211Asn (in the heterozygous state in 2
families), and Arg275Trp (in the heterozygous state in
families).
[0164] g) Frequencies of the Different Types of Mutations and of
the Composite Heterozygotes
[0165] Among the families with Parkin mutations, homozygous
deletions of exons were detected in 8 families, point mutations in
the homozygous state in 10 families, a duplication of homozygous
exon in one family and a triplication of exon in one family. The
patients from 21 families were composite heterozygotes for two
different mutations (3 times for the different point mutations, 6
times for a point mutation and an exon deletion, twice for a point
mutation and a duplication, once for a triplication and an exon
deletion, once for two different duplications of exon and 6 times
for two different deletions of exons; see example FIG. 4). In two
cases, it was not possible to determine if heterozygotes with
deletions of several composite adjacent exons were involved, and in
13 cases, only one mutation in the heterozygous state (6 point
mutations and 7 exon mutations) was detected.
D--Discussion
[0166] The present invention relates to variants of the Parkin
gene, their diagnostic and/or therapeutic use, as well as
techniques for the detection of alterations (in particular of
deletions of heterozygous exons and of multiplications of exons) of
the Parkin gene.
[0167] The detection of different causal genetic alterations (in
particular of homozygous deletions, point mutations, insertions and
multiplications of exons) demonstrate that the abnormalities in the
parkin gene constitute a frequent cause of AR-JP.
1. FIRST STUDY ON 38 EUROPEAN FAMILIES
[0168] A first study made it possible to demonstrate the existence
of deletions, mutations and insertions in the parkin gene.
[0169] The pathogenic role of the homozygous deletions appears to
be easy to establish. In the 2 mutations described, deletions of
exon 3 and of exons 8-9, the loss of the exon is accompanied by a
reading frame shift leading to the appearance of a premature stop
codon. In the absence of alternative splicing, a truncated protein
results therefrom.
[0170] Eight of the exon variants constitute causal mutations.
First, these mutations segregate with the disease in the families.
Secondly, these variants are not detected by ASO, PAGE or
PCR/restriction in 90 control chromosomes. Thirdly, the functional
consequences of the mutations appear to be deleterious. It is easy
to understand that the 4 truncating point mutations
(Gln34Arg(Stop37), Asn52Met(Stop81), Trp74Cys(Stop81), Trp453Stop)
detected in the homozygous state in the patients of 3 of the 5
families will cause a loss in the parkin function in accordance
with the autosomal-recessive transmission of the disease. Three of
the four missense mutations cause nonconservative changes in amino
acids. One of them (Lys161Asp) is associated with a truncating
mutation on the other allele which reinforces the assumption of a
pathogenic role. A missense mutation is conservative (Thr415Asn),
but affects a potential phosphorylation site. Three of the missense
mutations are present in the heterozygous state in patients whose
other mutation has not been characterized. It is probable that
deletions of one or more exons in the heterozygous state are
involved which cannot be visualized with the techniques used for
this study.
[0171] The abnormalities detected in the parkin gene are varied and
there are no hot spot mutations. It should be noted that the
truncating point mutations preferably correspond to the N- and
C-terminal regions of parkin (comprising in particular the
ubiquitin-like and ring "RING-finger" units, respectively) whereas
the missense-type mutations affect the central region. Only two of
the 11 mutations described in this first study are found in several
families. The homozygous deletion of exon 3 is detected in the
French SAL-024 and Portuguese SAL-711 families. The mutation with a
reading frame shift 202-203delAG (Gln34Arg(Stop37)) is visualized
in the heterozygous state in the Italian IT-020 and English UK-086
families. The different 2.0 origin of the families is in favor of
the hypothesis for the independent occurrence of these
mutations.
[0172] The mutations described affect families from 6 countries:
Algeria, Germany, England, France, Italy and Portugal. The study of
the phenotype in the families with a mutation shows that the
clinical spectrum associated with the abnormalities of parkin is
broader than in the Japanese families [Kitada et al., 1998]. These
results confirm the observations made in the European and North
African families studied by genetic linkage [Tassin et al., 1998].
The age of onset is above 50 in several patients, ranging up to 56.
Certain clinical signs such as dystonia or pyramidal signs in the
lower limbs are not always present in the carriers of mutation even
after periods of evolution of several decades. Overall, the
phenotype remains very similar between the groups of patients
classified according to the functional consequences of the
mutations. However, the presence of painful dystonia episodes
appears to be encountered exclusively in patients carrying
homozygous deletions. The absence of a significant difference for
the age of onset, the severity and the frequency of the associated
signs between the truncating point mutations and the missense
mutations suggests that the modified amino acids in the latter play
an important role in the physiology of parkin.
[0173] In conclusion, this first study underlines the frequency of
the mutations in the parkin gene in early-onset familial
parkinsonian syndromes in Europe. Abnormalities in this gene are
also responsible for more tardive or atypical parkinsonian
syndromes. The role of mutations of parkin or of its polymorphisms
in the isolated cases remains to be determined. The mutations
detected are very diverse both by their nature and by their
location. The study of their location in parkin suggests that many
regions of the protein contribute to its as yet unknown
function.
2. METHOD FOR THE DETECTION OF DELETIONS OF EXONS AND
MULTIPLICATIONS OF EXONS
[0174] For the first time, the detection of deletions of exons in
the heterozygous state and of multiplications of exons (for example
duplication, triplication) in the homozygous and heterozygous state
in the Parkin gene is described. This aspect is advantageous
because exon deletions are relatively frequent (see later). As a
method of detection, a semiquantitative multiplex PCR protocol was
chosen and developed. This method had previously been validated for
gene assay, for example for the detection of deletions of the PMP22
gene (Poropat and Nicholson, 1998), provided that the PCR
amplification is in the exponential phase. In all these
experiments, the choice of coamplified controls which serve as a
standard for the quantification is critical (Prior, 1998). In the
experiment, the nondeleted exons serve as internal controls in the
multiplex PCR amplification in the same individual. Combinations of
4 or 5 exons were chosen so as not to contain more than 2 adjacent
exons, because such exons cannot serve as controls in the case of a
deletion of the two. The exon on a different gene (Transthyretin)
was coamplified in one of the three combinations in order to
identify heterozygous deletions of the entire Parkin gene. This
external control was indirectly represented in the other two
combinations, which include exons on either side of exon 9; the
latter being tested with Transthyretin.
[0175] The results obtained by this method were very reproducible
and the abnormal results show differences in the ratios of a
factor, which corresponds to that expected in theory. These results
show that this is a simple and validated method for rapid
screening. Furthermore, small deletions or insertions in the PCR
product, which are relatively frequent (see below) may be
simultaneously detected by this method.
3. DELETIONS OF EXONS AND MULTIPLICATIONS OF EXONS
[0176] It was possible to identify four duplications of exons and
one triplication of exons which had never been described in the
Parkin gene before, but whose relative frequency is low.
Furthermore, 10 combinations of new deletions of exons were
identified with, for the first time, the demonstration of the
deletions which carry exon 2. The relative frequency of the point
mutations and of the deletions of exons was estimated at about 50%.
Thus, the deletions of exons (heterozygous or homozygous) may
represent up to 50% of the Parkin mutations, emphasizing the
importance of the technique described here. In fact, this technique
has made it possible to detect mutations in 26 of the 53 families.
Thus, in the sample studied, the point mutations and the deletions
in the Parkin gene have the same frequency, whereas the deletions
of exons are predominant in the Japanese population (Hattori et al,
1998). The functional consequences of the deletions or of the
multiplications of exons described (reading frame shift or
deletion/multiplication in phase) were deduced from the published
cDNA sequences for Parkin (Kitada et al, 1998) and are speculative,
because the absence of a PCR product does not indicate that there
is necessarily a deletion of the exon in its entirety (see above).
However, the pathological role of the modifications detected is
highly probable, because they are transmitted with the disease in
the families which have been able to be tested, they are associated
with point mutations in composite heterozygotes and the deletions
are identified with a frequency similar to that of point mutations.
Likewise; in the isolated cases, the frequency of the heterozygous
deletions and of the point mutations is similar. In the case of
exon 3, exonic primers were used, which demonstrate the alteration
of this exon when there is no PCR product. Furthermore, in some of
the cases, several juxtaposed exons were simultaneously deleted,
which is an argument for a large genomic deletion.
[0177] Heterozygous deletions or multiplications of the entire
Parkin gene were not observed. This is probably a rare event given
the very large size (about 500 kb) of this gene (Kitada et al,
1998).
[0178] The exon deletions observed frequently effect exons 3 to 5.
This observation has been confirmed in European families.
Furthermore, it has been demonstrated that exon 2 alone or
associated with others is also frequently involved in European
families (Table 2).
4. NEW POINT MUTATIONS
[0179] The identification of 8 new point mutations (6 of the
missense type, 1 truncating and 1 of the nonsense type) increases
the diversity of the point mutations in the Parkin gene. The
mutations described are pathogenic, as the segregation with the
disease has shown, and are not detected in 122 to 147 control
chromosomes (mutation 1142-1143delGA not included). Even if the
Cys289Gly change is conservative, this change in amino acid may
have substantial deleterious consequences, if the cysteine at
position 289 is involved in a disulphide bridge, which is important
for the function of the protein.
[0180] Interestingly, 2 patients of the UK-040 family exhibit 3
different mutations (see Table 5): one Arg334Cys missense mutation
in exon 9 in the homozygous state, one homozygous deletion of 5
base pairs at position -17 to -21 of intron 8, and one
nonconservative Asp280Asn missense mutation in the heterozygous
state. It may be suspected that the Arg334Cys mutation in the
homozygous state is causal, but the deletion of five base pairs in
the homozygous state, near the acceptor splicing site of exon 9,
could also have functional consequences.
[0181] Five point mutations are present in several families
analyzed. The three most frequent are 255delA (detected in 6
families) and 202-203delAG (found in 5 families) and Arg275Trp
(detected in 5 families). A foundation effect could be suspected
for the 255delA mutation which affects 5 French families. However,
this hypothesis can only be verified by analysis of the
haplotypes.
5. EPIDEMIOLOGICAL GENETICS
[0182] The results obtained show that 34 of the 77 families with an
early-onset parkinsonian syndrome exhibit mutations of the Parkin
gene, emphasizing the importance of this gene in European families.
The detection of mutations in 18 of the 102 cases isolated and
analyzed is more difficult to interpret because the number is
smaller and the analysis of some cases is not complete. However, it
is striking to note that the age at onset of the 7 cases for which
it is known is particularly early (13 to 22 years) and that there
are very few cases with very early onset without mutation in the
Parkin gene (for example IT-NA-JMP-3). This result suggests that
the frequency of the mutations of the Parkin gene in isolated cases
increases when their age decreases, especially before the age of
25. The observation of mutations in the Parkin gene in isolated
cases is not surprising if it is considered that in small families,
an autosomal recessive disease has every chance of appearing as an
isolated case. Analyzing a larger sample will be useful for
determining precisely the frequency of the Parkin mutations in the
isolated cases, according to the age at onset.
[0183] Mutations were identified in families from a wide variety of
origins: France, Italy, Great Britain, Germany, The Netherlands,
Algeria, Portugal. These results show that the mutations in the
Parkin gene are detected in all the populations tested so far.
6. PATIENTS WITH AN ABNORMALITY IN THE PARKIN GENE IN THE
HETEROZYGOUS STATE
[0184] Although the technique for the detection of heterozygous
deletions of exons or of multiplications of exons allowed us to
identify composite heterozygous cases, in about 1/4 of the families
(13 out of 53), a single mutation was detected. This includes 6
cases with a point mutation in the heterozygous state and 7 with an
exon deletion in the heterozygous state. The pathogenic role of
these mutations is highly probable because they cause a
nonconservative change in amino acid or a truncated protein.
Furthermore, one of these mutations of the missense type
(Arg275Trp) is associated with another heterozygous point mutation
(Gly430Asp) and with heterozygous exon deletions (exon 3-6 or exon
5+6), carried by the other allele in three different families. The
absence of detection of a mutation on the other allele in 13
families suggests that a second undetected mutation affects another
region of the gene. This hypothesis is strengthened by the fact
that in 6 families probably linked to the PARK2 locus, because the
patients are haploidentical for 4 markers for the region, no
mutation was detected. Thus, other regions of the gene could be
affected, such as the promoter regions, the untranslated 5' and 3'
regions, or intron sequences.
7. GENETIC HETEROGENEITY OF THE EARLY ONSET AUTOSOMAL RECESSIVE
PARKINSONIAN SYNDROMES
[0185] In 5 of the 77 families, it has been possible to exclude a
genetic linkage at the Parkin locus. Furthermore, no mutation was
identified in 21 families for which this locus could not be
conclusively excluded. These results suggest that there may be at
least one other locus for families with an early onset autosomal
recessive parkinsonian syndrome in Europe. This hypothesis had been
proposed by Leroy et al (1998), which reports a family with two
branches, of which one exhibits deletions of the Parkin gene,
whereas the other does not exhibit either these deletions or the
same haplotype, which excludes a linkage to this locus.
8. CONCLUSIONS
[0186] A novel method for the detection of heterozygous deletions
of exons or multiplications of exons is reported. In particular,
the duplications/triplications of exons and deletions of exon 2 and
of other combinations of exons are novel. In combination with the
sequencing of exons, it has been possible to identify eight novel
point mutations and an intron deletion which could affect a
splicing site. Thus, 34 of the 77 families analyzed (about 50%)
exhibit mutations in the Parkin gene. Furthermore, the mutations in
this gene were detected in 19 isolated cases. In the European
population, the proportion of point mutations and deletions of
exons appear to be identical. Two mutation hot points which
correspond to deletions at exons 3 to 5 and to three point
mutations (202-203delAG, 255delA and Arg275Trp) were in addition
detected.
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Nussbaum R L. Mutation in the alpha-Synuclein Gene Identified in
Families with Parkinson's Disease. Science (1997), 276: 2045-2047
[0201] Poropat R A and Nicholson G A. Determination of gene dosage
at the PMP22 and androgen receptor loci by quantitative PCR.
Clinical Chemistry (1998), 44: 724-730 Prior T W. Determining Gene
Dosage (editorial). Clinical Chemistry (1998), 44: 703-704. [0202]
Sunada Y, Saito F, Matsumura K and Shimizu T. Differential
expression of the parkin gene in the human brain and peripheral
leukocytes. Neurosci Lett (1998), 254: 180-182 [0203] Takahashi H,
Ohama E, Suzuki S, Horikawa Y, Ishikawa A, Morita T, Tsuji S and
Ikuta F. Familial juvenile parkinsonism: clinical and pathologic
study in a family. Neurology (1994), 44: 437-41 [0204] Tassin J, D
rr A, de Broucker T, Abbas N, Bonifati V, De Michele G, Bonnet A M,
Broussolle E, Pollak P, Vidailhet M, De Mari M, Marconi R, Medjbeur
S, Filla A, Meco G, Agid Y and Brice A. Chromosome 6-Linked
Autosomal Recessive Early-Onset Parkinsonism: Linkage in European
and Algerian Families, Extension of the Clinical Spectrum, and
Evidence of a Small Homozygous Deletion in One Family. Am J Hum
Genet (1998), 63: 88-94 [0205] Wood N. Genes and parkinsonism
[editorial]. J Neurol Neurosurg Psychiatry (1997), 62: 305-9 [0206]
Yamamura Y, Sobue I, Ando K, Iida M and Yanagi T. Paralysis agitans
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TABLE-US-00008 [0206] TABLE 1 Posi- Nucleotide tion change
Oligonucleotide sequence Ex3 321-322insGT WT:
5'TGCAGAGACC-GTGGAGAAAA-3' (SEQ ID No: 14) V:
5'GCAGAGACCGTGTGGAGAAA-3' (SEQ ID No: 15) Ex4 584A > T WT:
5'-GCCGGGAAAACTCAGGGTA-3' (SEQ ID No: 16) V:
5'-GCCGGGAAATCTCAGGGTA-3' (SEQ ID No: 17) Ex7 867C > T WT:
5'-TGCAACTCCCGCCACGTGA-3' (SEQ ID No: 18) V:
5'-TGCAACTCCTGCCACGTGA-3' (SEQ ID No: 19) Ex10 1239G > C WT:
5'-TGCAGTGCCGTATTTGAAG-3' (SEQ ID No: 20) V:
5'-TGCAGTGCCCTATTTGAAG-3' (SEQ ID No: 21) Ex11 1345C > A WT:
5'-AGAAAACCACCAAGCCCTG-3' (SEQ ID No: 22) V:
5'-AGAAAACCAACAAGCCCTG-3' (SEQ ID No: 23)
TABLE-US-00009 TABLE 2 Amino acid changed Expected length
Nucleotide (position of the Detection of the fragment changed stop
codon) Type of mutation technique (bp) Exon Ex2 202-203delAG
Gln34Arg(Stop37) reading frame PAGE WT: 308 V: 306 Ex2 255delA
Asn52Met(Stop81) reading frame Fok 1 WT: 278 + 30 creation of the
V: 222 + 57 + 30 site Ex3 321-322insGT Trp74Cys(Stop81) reading
frame ASO Ex4 584A > T Lys161Asn missense ASO (nonconservative)
Ex4 601G > A Ser167Asn missense Alw NI WT: 164 + 97
(nonconservative) loss of the site V: 261 Ex6 734A > T Lys211Asn
missense Dra I WT: 171 + 98 (nonconservative) loss of the site V:
269 Ex7 867C > T Arg256Cys missense ASO (nonconservative) Ex7
905T > A Cys268Stop nonsense Dde I WT: 141 + 100 gain of the
site V: 117 + 100 + 24 Ex7 924C > T Arg275Trp missense Sau3A I
WT: 142 + 97 (nonconservative) loss of the site V: 239 Ex7 939G
> A Asp280Asn missense Alw I with WT: 153 + 30 (nonconservative)
mismatched primer V: 183 loss of the site Ex7 966T > G Cys289Gly
missense BstN I WT: 177 + 64 (nonconservative) gain of the site V:
118 + 64 + 59 Ex9 1142-1143delGA Arg348Glu(Stop368) reading frame
PAGE WT: 278 V: 276 Ex9 1084G > A Gly328Glu missense Mnl I WT:
124 + 80 + 74 (nonconservative) gain of the site v: 124 + 74 + 60 +
20 Ex9 1101C > T Arg334Cys missense BstU I with WT: 123 + 21
(nonconservative) mismatched primer V: 144 loss of the site Ex10
1239G > C Val380Leu missense ASO (conservative) Ex11 1281G >
A Asp394Asn missense Taq I WT: 221 + 84 (nonconservative) loss of
the site V: 303 Ex11 1345C > A Thr415Asn missense ASO
(conservative) Ex12 1390G > A Gly430Asp missense Mnl I WT: 191 +
65 (nonconservative) loss of the site V: 256 Ex12 1459G > A
Trp453Stop nonsense Nla IV WT: 142 + 17 + 35 + 61 loss of the site
V: 159 + 35 + 61 intron Intron 2 IVS2 + 25T > C BstN I WT: 308
bp (272 + 25T > C) creation of the V: 264 + 44 bp site Intron 3
IVS3 - 20C > T Mnl I WT: 201 + 60 (514 - 20C > T) loss of the
site V: 261 Intron 7 IVS7 - 35A > G Mae III WT: 206 (973 - 35A
> G) creation of the V: 159 + 47 site Intron 8 IVS8 - 21 - 17del
splice site PAGE (1035 - 21 - 17del) TCTGC
TABLE-US-00010 TABLE 3 CLINICAL CHARACTERISTICS OF FAMILIES WITH
MUTATIONS IN THE PARKIN GENE Homozygous Missense Truncating
deletions mutations mutations Total Families (patients) 3 (8) 3 (8)
4 (9) 10 (25) Average age at onset (extremes) 30 .+-. 16 (7-55) 44
.+-. 9 (30-56) 37 .+-. 6 (29-45) 37 .+-. 12 (7-56) Average duration
of evolution 13 .+-. 6 (3-20) 13 .+-. 7 (0.5-27) 16 .+-. 10 (3-29)
14 .+-. 8 (0.5-29) (extremes) Hoehn and Yahr scale 3.1 .+-. 1.2 2.6
.+-. 0.8 2.0 .+-. 0.6 2.5 .+-. 0.98 Akinesia 8/8 8/8 8/9 96%
Rigidity 8/8 8/8 9/9 100% Tremor 3/8 4/8 8/9 60% Dystonia 4/8 0/5
1/7 25% Good reaction to levodopa 7/7 (1) 6/6 (2) 7/7 100% (case de
novo) Dyskinesia 4/7 5/6 6/9 68% Fluctuations.sup.a 7/7 ND 2/6 62%
Sharp reflexes in the 2/8 3/4 0/6 28% lower limbs
TABLE-US-00011 TABLE 4 exon(s) deleted/multiplied Number of
families Consequences 2 del 3 .times. het Reading frame shift 2
triplication 1 .times. hom + 1 .times. het No reading frame shift 2
+ 3 del 1 .times. het No reading frame shift 2 + 3 + 4 del 1
.times. het Reading frame shift 3 del 3 .times. hom + 7 .times. het
Reading frame shift 3 duplication 1 .times. hom + 1 .times. het
Reading frame shift 3 + 4 del 1 .times. hom + 3 .times. het No
reading frame shift 3 - 6 del 1 .times. het Reading frame shift 3 -
9 del 1 .times. het No reading frame shift 4 del 1 .times. hom + 3
.times. het Reading frame shift 5 del 3 .times. het No reading
frame shift 5 + 6 del 2 .times. hom Reading frame shift 6 del 1
.times. het Reading frame shift 6 duplication 1 .times. het Reading
frame shift 6 + 7 del 1 .times. het Reading frame shift 7
duplication 1 .times. het Reading frame shift 7 + 8 + 9 del 1
.times. het Reading frame shift 8 del 1 .times. het Reading frame
shift 8 + 9 del 1 .times. hom Reading frame shift 11 duplication 1
.times. het Reading frame shift
TABLE-US-00012 TABLE 5 C328/ C328/ Ex3i/ Case 3i 12 9 2 C328 3i 12
C328/9 C328/2 12 Ex3i/9 Ex3i/2 Ex12/9 Ex12/2 Ex9/2 T2 743 838 1040
935 455 0.61 0.54 0.44 0.49 0.89 0.71 0.79 0.81 0.90 1.11 FR 155 5
608 1245 1588 1466 635 1.04 0.51 0.40 0.43 0.49 0.38 0.41 0.78 0.85
1.08 T2/FR 0.59 1.06 1.09 1.12 1.82 1.87 1.92 1.03 1.06 1.03 155 5
FR 155 6 759 861 1120 540 498 0.66 0.58 0.44 0.92 0.88 0.68 1.41
0.77 1.59 2.07 T2/FR 0.93 0.94 0.98 0.53 1.01 1.05 0.57 1.05 0.56
0.54 155 6 FR 155 8 597 1185 1467 766 623 1.04 0.53 0.42 0.81 0.50
0.41 0.78 0.81 1.55 1.92 T2/FR 0.59 1.03 1.03 0.60 1.76 1.76 1.02
1.00 0.58 0.58 155 8 FR 155 9 495 1200 1438 754 688 1.39 0.57 0.48
0.91 0.41 0.34 0.66 0.83 1.59 1.91 T2/FR 0.44 0.95 0.91 0.53 2.15
2.08 1.21 0.97 0.56 0.58 155 9
Sequence CWU 1
1
2412960DNAHomo sapiens 1tccgggagga ttacccagga gaccgctggt gggaggcgcg
gctggcgccg ctgcgcgcat 60gggcctgttc ctggcccgca gccgccacct acccagtgac
catgatagtg tttgtcaggt 120tcaactccag ccatggtttc ccagtggagg
tcgattctga caccagcatc ttccagctca 180aggaggtggt tgctaagcga
cagggggttc cggctgacca gttgcgtgtg attttcgcag 240ggaaggagct
gaggaatgac tggactgtgc agaattgtga cctggatcag cagagcattg
300ttcacattgt gcagagaccg tggagaaaag gtcaagaaat gaatgcaact
ggaggcgacg 360accccagaaa cgcggcggga ggctgtgagc gggagcccca
gagcttgact cgggtggacc 420tcagcagctc agtcctccca ggagactctg
tggggctggc tgtcattctg cacactgaca 480gcaggaagga ctcaccacca
gctggaagtc cagcaggtag atcaatctac aacagctttt 540atgtgtattg
caaaggcccc tgtcaaagag tgcagccggg aaaactcagg gtacagtgca
600gcacctgcag gcaggcaacg ctcaccttga cccagggtcc atcttgctgg
gatgatgttt 660taattccaaa ccggatgagt ggtgaatgcc aatccccaca
ctgccctggg actagtgcag 720aatttttctt taaatgtgga gcacacccca
cctctgacaa ggaaacatca gtagctttgc 780acctgatcgc aacaaatagt
cggaacatca cttgcattac gtgcacagac gtcaggagcc 840ccgtcctggt
tttccagtgc aactcccgcc acgtgatttg cttagactgt ttccacttat
900actgtgtgac aagactcaat gatcggcagt ttgttcacga ccctcaactt
ggctactccc 960tgccttgtgt ggctggctgt cccaactcct tgattaaaga
gctccatcac ttcaggattc 1020tgggagaaga gcagtacaac cggtaccagc
agtatggtgc agaggagtgt gtcctgcaga 1080tggggggcgt gttatgcccc
cgccctggct gtggagcggg gctgctgccg gagcctgacc 1140agaggaaagt
cacctgcgaa gggggcaatg gcctgggctg tgggtttgcc ttctgccggg
1200aatgtaaaga agcgtaccat gaaggggagt gcagtgccgt atttgaagcc
tcaggaacaa 1260ctactcaggc ctacagagtc gatgaaagag ccgccgagca
ggctcgttgg gaagcagcct 1320ccaaagaaac catcaagaaa accaccaagc
cctgtccccg ctgccatgta ccagtggaaa 1380aaaatggagg ctgcatgcac
atgaagtgtc cgcagcccca gtgcaggctc gagtggtgct 1440ggaactgtgg
ctgcgagtgg aaccgcgtct gcatggggga ccactggttc gacgtgtagc
1500cagggcggcc gggcgcccca tcgccacatc ctgggggagc atacccagtg
tctaccttca 1560ttttctaatt ctcttttcaa acacacacac acacgcgcgc
gcgcgcacac acactcttca 1620agtttttttc aaagtccaac tacagccaaa
ttgcagaaga aactcctgga tccctttcac 1680tatgtccatg aaaaacagca
gagtaaaatt acagaagaag ctcctgaatc cctttcagtt 1740tgtccacaca
agacagcaga gccatctgcg acaccaccaa caggcgttct cagcctccgg
1800atgacacaaa taccagagca cagattcaag tgcaatccat gtatctgtat
gggtcattct 1860cacctgaatt cgagacaggc agaatcagta gctggagaga
gagttctcac atttaatatc 1920ctgcctttta ccttcagtaa acaccatgaa
gatgccattg acaaggtgtt tctctgtaaa 1980atgaactgca gtgggttctc
caaactagat tcatggcttt aacagtaatg ttcttattta 2040aattttcaga
aagcatctat tcccaaagaa ccccaggcaa tagtcaaaaa catttgttta
2100tccttaagaa ttccatctat ataaatcgca ttaatcgaaa taccaactat
gtgtaaatca 2160acttgtcaca aagtgagaaa ttatgaaagt taatttgaat
gttgaatgtt tgaattacag 2220ggaagaaatc aagttaatgt actttcattc
cctttcatga tttgcaactt tagaaagaaa 2280ttgtttttct gaaagtatca
ccaaaaaatc tatagtttga ttctgagtat tcattttgca 2340acttggagat
tttgctaata catttggctc cactgtaaat ttaatagata aagtgcctat
2400aaaggaaaca cgtttagaaa tgatttcaaa atgatattca atcttaacaa
aagtgaacat 2460tattaaatca gaatctttaa agaggagcct ttccagaact
accaaaatga agacacgccc 2520gactctctcc atcagaaggg tttatacccc
tttggcacac cctctctgtc caatctgcaa 2580gtcccaggga gctctgcata
ccaggggttc cccaggagag accttctctt aggacagtaa 2640actcactaga
atattcctta tgttgacatg gattggattt cagttcaatc aaactttcag
2700cttttttttc agccattcac aacacaatca aaagattaac aacactgcat
gcggcaaacc 2760gcatgctctt acccacacta cgcagaagag aaagtacaac
cactatcttt tgttctacct 2820gtattgtctg acttctcagg aagatcgtga
acataactga gggcatgagt ctcactagca 2880catggaggcc cttttggatt
tagagactgt aaattattaa atcggcaaca gggcttctct 2940ttttagatgt
agcactgaaa 2960219DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 2aattgtgacc tggatcagc 19323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3ctggacttcc agctggtggt gag 23425DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 4ggcagggagt agccaagttg
aggat 25522DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5agccccgctc cacagccagc gc 22619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6aattgtgacc tggatcagc 19723DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7ctggacttcc agctggtggt gag
23819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8aattgtgacc tggatcagc 19923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9ctggacttcc agctggtggt gag 231020DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 10acgttcctga taatgggatc
201120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11cctctctcta ccaagtgagg 201225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12ggcagggagt agccaagttg aggat 251322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13agccccgctc cacagccagc gc 221420DNAHomo sapiens 14tgcagagacc
gtggagaaaa 201520DNAHomo sapiens 15gcagagaccg tgtggagaaa
201619DNAHomo sapiens 16gccgggaaaa ctcagggta 191719DNAHomo sapiens
17gccgggaaat ctcagggta 191819DNAHomo sapiens 18tgcaactccc gccacgtga
191919DNAHomo sapiens 19tgcaactcct gccacgtga 192019DNAHomo sapiens
20tgcagtgccg tatttgaag 192119DNAHomo sapiens 21tgcagtgccc tatttgaag
192219DNAHomo sapiens 22agaaaaccac caagccctg 192319DNAHomo sapiens
23agaaaaccaa caagccctg 19246PRTUnknownDescription of Unknown
Myristoylation site peptide 24Gly Cys Glu Trp Asn Arg 1 5
* * * * *