U.S. patent application number 09/850258 was filed with the patent office on 2002-10-24 for genetic polymorphisms which are associated with autism spectrum disorders.
Invention is credited to Figlewicz, Denise A., Hyman, Susan L., Ingram, Jennifer L., Rodier, Patricia M., Stodgell, Christopher J..
Application Number | 20020155450 09/850258 |
Document ID | / |
Family ID | 21961832 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155450 |
Kind Code |
A1 |
Rodier, Patricia M. ; et
al. |
October 24, 2002 |
Genetic polymorphisms which are associated with autism spectrum
Disorders
Abstract
A method is provided for screening subjects for genetic markers
associated with autism. The method involves isolating a biological
sample from a mammal and then testing for the presence of a mutated
gene or a product thereof which is associated with autism. Also
disclosed are isolated nucleic acids encoding HoxA1 and HoxB1, both
of which have a polymorphism that is associated with autism
spectrum disorders.
Inventors: |
Rodier, Patricia M.;
(Rochester, NY) ; Ingram, Jennifer L.; (Rochester,
NY) ; Figlewicz, Denise A.; (Rochester, NY) ;
Hyman, Susan L.; (Rochester, NY) ; Stodgell,
Christopher J.; (Rochester, NY) |
Correspondence
Address: |
Michael L. Goldman, Esq.
NIXON PEABODY LLP
Clinton Square, P. O. 31051
Rochester
NY
14603
US
|
Family ID: |
21961832 |
Appl. No.: |
09/850258 |
Filed: |
May 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09850258 |
May 7, 2001 |
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09095117 |
Jun 10, 1998 |
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6228582 |
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60049803 |
Jun 17, 1997 |
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Current U.S.
Class: |
435/6.16 ;
434/433 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C07K 14/47 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 ;
434/433 |
International
Class: |
C12Q 001/68; A47B
041/00 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the United States Government under Grants No. RO1AA08666, RO1
NS 24287, RO1HD34295, RO1HD34969, and 2P30 ES01247 from the
National Institutes of Health and Grant No. R824758 from the
Environmental Protection Agency. The United States Government may
retain certain rights.
Claims
What is claimed:
1. A method for screening subjects for genetic markers associated
with autism, comprising: isolating a biological sample from a
mammal; and testing the sample or genetic material isolated from
the sample, for either a gene having a polymorphism or product
thereof, which is a genetic marker for autism.
2. The method according to claim 1, wherein the biological sample
is selected from the group consisting of blood, saliva, amniotic
fluid, and tissue.
3. The method according to claim 2, wherein the biological sample
is blood.
4. The method according to claim 1, wherein the mammal is a
human.
5. The method according to claim 4, wherein the biological sample
is isolated from developmentally disabled children.
6. The method according to claim 4, wherein the biological sample
is isolated from parents or relatives of developmentally disabled
children.
7. The method according to claim 4, wherein the biological sample
is isolated from children and said method further comprises: early
behavior training for children having genetic markers associated
with autism.
8. The method according to claim 1, wherein the gene is selected
from the group consisting of HoxA1, HoxB1, and HoxD1.
9. The method according to claim 8, wherein the polymorphism is
located in the homeobox.
10. The method according to claim 8, wherein the gene is HoxA1.
11. The method according to claim 10, wherein the gene has a single
base substitution resulting in an amino acid substitution.
12. The method according to claim 11, wherein the amino acid
substitution is an arginine for a histidine.
13. The method according to claim 8, wherein the gene is HoxB1.
14. The method according to claim 12, wherein the gene has an
insertion.
15. The method according to claim 14, wherein the insertion is
5'ACAGCGCCC-3'.
16. The method according to claim 8, wherein the mutated gene is
HoxD1.
17. The method according to claim 1, wherein the gene has a
polymorphism selected from the group consisting of a single base
substitution resulting in an amino acid substitution, a single base
substitution resulting in a translational stop, an insertion, a
deletion, and a rearrangement.
18. The method according to claim 1, wherein the gene has a
mutation in an exon.
19. The method according to claim 18, wherein the polymorphism
alters the sequence of the polypeptide encoded by the gene.
20. The method according to claim 1, wherein the gene has a
mutation in an intron.
21. The method according to claim 1, wherein the gene has a
mutation in a promotor or regulatory region.
22. The method according to claim 1, wherein said testing is
carried out by screening for a gene having a polymorphism.
23. The method according to claim 22, wherein said screening for
mutated nucleic acids is carried out by a method selected from the
group consisting of direct sequencing of nucleic acids, single
strand polymorphism assay, restriction fragment length polymorphism
assay, ligase chain reaction, enzymatic cleavage and southern
hybridization.
24. The method according to claim 23, wherein said screening is
carried out by direct sequencing of nucleic acids.
25. The method according to claim 23, wherein said screening is
carried out by single strand polymorphism assay.
26. The method according to claim 23, wherein said screening is
carried out by restriction fragment length polymorphism assay.
27. The method according to claim 23, wherein said screening is
carried out by ligase chain reaction.
28. The method according to claim 23, wherein said screening is
carried out by enzymatic cleavage.
29. The method according to claim 23, wherein said screening is
carried out by southern hybridization.
30. The method according to claim 23, wherein the nucleic acid is a
deoxyribonucleic acid.
31. The method according to claim 23, wherein the nucleic acid is a
messenger ribonucleic acid.
32. The method according to claim 1, wherein said testing is
carried out by screening for polypeptides resulting from said gene
having a polymorphism.
33. The method according to claim 32, wherein said screening for
the polypeptide resulting from said gene having a polymorphism is
carried out by a method selected from the group consisting of
probing with antibodies specific to said polypeptide, measurement
of the concentration of said polypeptide, and measuring the size of
said polypeptide.
34. The method according to claim 33, wherein said screening is
carried out by probing with antibodies specific to said
polypeptide.
35. The method according to claim 33, wherein said screening is
carried out by measuring the size of the polypeptides.
36. An isolated nucleic acid molecule comprising a single base
substitution at nucleotide 218 in SEQ. ID. No. 1, or a fragment
having at least 15 nucleotides encompassing said single base
substitution.
37. An isolated polypeptide encoded by the nucleic acid of claim
36.
38. An antibody which binds to the isolated polypeptide according
to claim 37 and which does not bind to the wild-type HoxA1 protein
of SEQ. ID. No. 2.
39. An isolated nucleic acid molecule comprising an insertion
between positions nucleotides 88 and 89 in SEQ. ID. No. 5, or a
fragment having at least 15 nucleotides encompassing said
insertion.
40. The isolated nucleic acid molecule according to claim 39,
wherein the insertion is 5'-ACAGCGCCC-3'.
41. An isolated polypeptide encoded by the nucleic acid of claim
39.
42. An antibody which binds to the isolated polypeptide according
to claim 41 and which does not bind to the wild-type HoxB1 protein
of SEQ. ID. No. 6.
Description
[0001] The present application claims the benefit of U.S.
Provisional Patent Application Ser. No. 60/049,803, filed Jun. 17,
1997.
FIELD OF THE INVENTION
[0003] The present invention relates to a method of screening
subjects for genetic markers associated with autism. The invention
further relates to isolated nucleic acids having polymorphisms
associated with autism, the polypeptide products of those nucleic
acids, and antibodies specific to the polypeptides produced by the
mutated genes.
BACKGROUND OF THE INVENTION
[0004] Autism is a behaviorally defined syndrome characterized by
impairment of social interaction, deficiency or abnormality of
speech development, and limited activities and interest (American
Psychiatric Association, 1994). The last category includes such
abnormal behaviors as fascination with spinning objects, repetitive
stereotypic movements, obsessive interests, and abnormal aversion
to change in the environment. Symptoms are present by 30 months of
age. The prevalence rate in recent Canadian studies using total
ascertainment is over {fraction (1/1,000)} (Bryson, S.E. et al., J.
Child Psychol. Psychiat., 29, 433 (1988)).
[0005] Attempts to identify the cause of the disease have been
difficult, in part, because the symptoms do not suggest a brain
region or system where injury would result in the diagnostic set of
behaviors. Further, the nature of the behaviors included in the
criteria preclude an animal model of the diagnostic symptoms and
make it difficult to relate much of the experimental literature on
brain injuries to the symptoms of autism.
[0006] Several quantitative changes have been observed in autistic
brains at autopsy. An elevation of about 100 g in brain weight has
been reported (Bauman, M. L. and Kemper, T. L., Neurology 35, 866
(1985)). While attempts to find anatomical changes in the cerebral
cortex have been unsuccessful (Williams, R. S. et al., Arch.
Neurol., 37, 749 (1980); Coleman P. D., et al., J. Autism Dev.
Disord., 15, 245 (1985)), several brains have been found to have
elevated neuron packing density in structures of the limbic system
(Bauman, M. L. and Kemper, T. L., Neurology 35, 866 (1985)),
including the amygdala, hippocampus, septal nuclei and mammillary
body. Multiple cases in multiple labs have been found to have
abnormalities of the cerebellum. A deficiency of Purkinje cell and
granule cell number, as well as reduced cell counts in the deep
nuclei of the cerebellum and neuron shrinkage in the inferior
olive, have been reported (Bauman, M. L. and Kemper, T. L.,
Neurology, 35, 866 (1985); Bauman, M. L. and Kemper, T. L.,
Neurology, 36 (suppl. 1), 190 (1986); Bauman, M. L. and Kemper, T.
L., The Neurobiology of Autism, Johns Hopkins University Press, 119
(1994); Ritvo, E. R. et al., Am. J. Psychiat., 143, 862 (1986);
Kemper, T. L. and Bauman M. L., Neurobiology of Infantile Autism,
Elsevier Science Publishers, 43 (1992)).
[0007] Imaging studies have allowed examination of some anatomical
characteristics in living autistic patients, providing larger
samples than those available for histologic evaluation. In general,
these confirm that the size of the brain in autistic individuals is
not reduced and that most regions are also normal in size (Piven,
J. et al., Biol. Psychiat. 31, 491 (1992)). Reports of size
reductions in the brainstem have been inconsistent (Gaffney, G. R.
et al., Biol. Psychiat., 24, 578 (1988); Hsu, M. et al., Arch.
Neurol. 48, 1160 (1991)), but a new, larger study suggests that the
midbrain, pons, and medulla are smaller in autistic cases than in
controls (Hashimoto, T. et al., J. Aut. Dev. Disord., 25, 1
(1995)). In light of the histological effects reported for the
cerebellum, it is interesting that the one region repeatedly
identified as abnormal in imaging studies is the neocerebellar
vermis (lobules VI and VII; Gaffney, G. R. et al., Am. J. Dis.
Child., 141, 1330 (1987); Courchesne E., et al., N. Engl. J. Med.,
318, 1349 (1988); Hashimoto, T. et al., J. Aut. Dev. Disord., 25, 1
(1995)). Not all comparisons have found a difference in
neocerebellar size (Piven, J. et al., Biol. Psychiat., 31, 491
(1992); Kleiman, M. D. et al., Neurology, 42, 753 (1992)), but a
recent reevaluation of positive and negative studies (Courchesne,
E. et al, Neurology, 44, 214 (1994)) indicates that a few autistic
cases have hyperplasia of the neocerebellar vermis, while many have
hypoplasia. Small samples of this heterogeneous population could
explain disparate results regarding the size of the neocerebellum
in autism. The proposal that the cerebellum in autistic cases can
be either large or small is reasonable from an embryological
standpoint, because injuries to the developing brain are sometimes
followed by rebounds of neurogenesis (e.g., Andreoli, J. et al.,
Am. J. Anat. 137, 87 (1973); Bohn, M. C. and Lauder, J. M., Dev.
Neurosci., 1, 250 (1978); Bohn, M. C., Neuroscience, 5, 2003
(1980)), and it is possible that such rebounds could overshoot the
normal cell number. Further, because increased cell density has
been observed in the limbic system, the cerebellum is not the only
brain region in which some form of overgrowth might account for the
neuro-anatomy of autistic cases. It may well be that some
autism-inducing injuries occur just prior to a period of rapid
growth for the cerebellar lobules in question or the limbic system,
leading to excess growth, while other injuries continue to be
damaging during the period of rapid growth, leading to hypoplasia.
However, the hypothesis that autism occurs with both hypoplastic
and hyperplastic cerebella calls into question whether cerebellar
anomalies play a major role in autistic symptoms.
[0008] A particularly instructive result has appeared in an MRI
study on the cerebral cortex (Piven, J. et al., Am. J. Psychiat.,
14, 734 (1992)). Of a small sample of autistic cases, the majority
showed gyral anomalies (e.g., patches of pachygyria). However, the
abnormal areas were not located in the same regions from case to
case. That is, while the functional symptoms were similar in all
the subjects, the brain damage observed was not. The investigators
argue convincingly that the cortical anomalies were not responsible
for the functional abnormalities. This is a central problem in all
attempts to screen for pathology in living patients or in autopsy
cases. While abnormalities may be present, it is not necessarily
true that they are related to the symptoms of autism.
[0009] To teratologists, the physical anomalies of a neonate,
child, or adult can serve as a guide to when the embryo was
injured. Years of research have amplified the details of that
timetable for the nervous system (Rodier, P. M., Dev. Med. Child
Neurol., 22, 525 (1980); Bayer, S. A. et al., Neurotoxicology, 14,
83 (1993)). In the case of autism, lack of specific information on
the neuroanatomy associated with the disease has made it difficult
to estimate the stage of development when the disorder arises.
However, in 1993, Miller and Stromland reported a finding that
conclusively identified the time of origin for some cases. They
observed that the rate of autism was 33% in people exposed to
thalidomide between the 20th and 24th days of gestation, and 0% in
cases exposed at other times (Stromland, K. et al., Devel. Med.
Child. Neurol., 36, 351 (1994)). Their deduction regarding the time
of injury was not based on neuroanatomy, which was not known in
their living subjects. Instead, it was based on the external
stigmata of the cases.
[0010] Because thousands of thalidomide-exposed offspring have been
evaluated for somatic malformations, the array of injuries
associated with the drug is well-known, and the time when each
arises has been carefully defined (Miller, M. T., Trans. Am.
Ophthalmol. Soc., 89, 623 (1991)). Of five cases of
thalidomide-induced autism, four had malformations of the ears,
without limb malformation, and the fifth had malformation of the
ears, forelimb, and hindlimb. Thalidomide is not teratogenic before
the 20th day of gestation. Starting on day 20 exposure causes ear
malformation and abnormalities of the thumb. Limb malformations
(other than those of the thumb) first appear with exposure on the
25th day, with effects moving from the forelimb to the hindlimb as
exposure occurs at later stages. After the 35th day, thalidomide
produces no malformations. Thus, the cases with malformations
restricted to the ear must have been exposed before day 25, and the
one patient with multiple malformations can only be explained as a
case of repeated injuries at several stages of development.
[0011] In fact, the idea that autism might arise very early in
gestation was suggested long ago. Steg and Rapoport (J. Aut. Child.
Schiz., 5, 299 (1975)) noted the significant increase in minor
physical anomalies among children with autism, and realized that
they indicated an injury in the first trimester. Several studies of
minor malformations have found ear effects to be the most common
anomalies in autism (Walker, H. A., J. Aut. Child. Schiz., 7, 165
(1977); Campbell, M. et al., Am. J. Psychiat., 135, 573 (1978)),
and the most recent study shows that they are not only the best
discriminator between people with autism and normal controls, but
also the only anomaly that discriminates autism from other
developmental disabilities (Rodier, P. M. et al., Teratology 55,
319 (1997)). Ear anomalies are among the earliest of all minor
physical malformations in their time of origin.
[0012] External malformations are not the only evidence which puts
the time of injury in autism at the time of neural tube closure.
The cranial nerve dysfunctions observed in the patients with autism
secondary to thalidomide exposure--facial nerve palsy, Duane
syndrome (lack of abducens innervation with reinnervation of the
lateral rectus by the oculomotor nerve), abnormal lacrimation, gaze
paresis, and hearing deficits (Stromland, K. et al., Devel. Med.
Child. Neurol., 36, 351 (1994))--suggest that the earliest-forming
structures of the brain stem were damaged, and it is now known that
these form during neural tube closure (Bayer, S. A. et al.,
Neurotoxicology, 14, 83 (1993)). Subsequent studies have shown that
a human brain from a patient with autism has the same pattern of
brain stem injury predicted by the thalidomide cases (Rodier, P. M.
et al., J. Comp. Neurol., 370, 247 (1996)). Perhaps even more
importantly, the autopsied brain has a shortening of the brain stem
in the region of the fifth rhombomere, and is missing two of the
nuclei known to form from that embryological structure. The
rhombomeres exist so briefly (Streeter, G. L., Contr. Embryol.
Carneg. Instn., 30,213 (1948)) that the evidence that one failed to
form is conclusive in pinpointing the time of injury. Like the
thalidomide cases, the autopsy case could have been injured only at
the time of neural tube closure.
[0013] The effect of injury around neural tube closure has been
tested experimentally, to see whether it can produce anatomical
results like those suspected in the thalidomide cases and observed
in human brain. Animals exposed during the critical period to
valproic acid, a teratogen with effects similar to thalidomide,
which has also been associated with autism (Christianson, A. L. et
al., Devel. Med. Child. Neurol., 36, 357 (1994); Williams, P. G. et
al., Dev. Med. Child. Neurol., 39, 632 (1997)) exhibit reductions
in the number of cranial nerve motor neurons (Rodier, P. M. et al.,
J. Comp. Neurol., 370, 247 (1996)). They are distinguished from
controls by shortening of the hindbrain in the region which forms
from the fifth rhombomere, just as the autopsied brain was (Rodier,
P. M., et al., Teratology 55, 319 (1997)). Additional data suggests
that the animal model has secondary changes in the cerebellum like
those reported in some human cases of autism (Ingram, J. L. et al.,
Teratology, 53, 86 (1996)).
[0014] It has long been known that heritable factors play an
important role in the etiology of autism. This was demonstrated by
the original twin studies of Folstein and Rutter (J. Child Psychol.
Psychiat., 18, 297 (1977)) and the subsequent addition of more twin
pairs to the sample has only increased the estimate of the
proportion of cases suspected to have a genetic basis (e.g. Bailey,
A. et al., Psychol. Med., 25, 63 (1995); LeCouteur, A. et al., J.
Child Psychol. Psychiat., 37, 785 (1996)). Family studies of
siblings (Smalley, S. L. et al., Arch. Gen. Psychiat., 45, 953
(1988)) and parents (Landa, R. et al., J. Speech Hear. Res., 34,
1339 (1991); Landa, R. et al., Psych. Med., 22, 245 (1992)) also
support the conclusion that an inherited risk is involved in many,
perhaps all, cases of autism spectrum disorders. While the rate of
autism is elevated in close relatives of cases, the rate of
symptoms short of the diagnosis is increased much more. That is,
individuals known to share genetic factors seem to vary in the
degree to which symptoms are expressed. This non-Mendelian pattern
(Jorde, L. B. et al., Am. J. Hum. Genet., 49, 932 (1991)) suggests
a complex disorder with major contributions from predisposing
genetic factors, which interact with the overall genetic background
and/or environmental insults to determine the phenotype.
[0015] The ability to identify the genetic factors that increase
the risk for autism would be a breakthrough for genetic counselling
for prevention of the disorder. In addition, it would allow the
creation of genetically-engineered animals in which to study the
environmental factors that interact with the inherited
predispositions. Tests for genetic factors would also serve as
biomarkers, valuable for diagnosis, and useful in research on all
aspects of the autism spectrum. Unfortunately, neither linkage nor
association studies have revealed any chromosomal regions strongly
related to autism (e.g. Spence, M. A. et al., Behav. Genet., 15, 1
(1985); Smalley, S. L. et al., Arch. Gen. Psychiat., 45, 953
(1988); Cook, E. H. et al., Molec. Psychiat., 2, 247 (1997);
Klauck, S. M. et al., Hum. Molec. Genet., 6, 2233 (1997); Cook, E.
H. et al., Am. J. Hum. Genet., 62, 1077 (1998)).
[0016] Furthermore, while there is no known medical treatment for
autism, some success has been reported for early intervention with
behavioral therapies. A biomarker would allow identification of the
disease, now typically diagnosed between ages three and five, in
infancy or prenatal life. Thus, there is an urgent need for a
method of reliably identifying subjects with autism. In particular
there is need for a blood test for polymorphisms causing autism
spectrum disorders. Families with affected members need to know
whether they carry a mutation which could affect future
pregnancies. Clinicians need a test as an aid in diagnosis, and
researchers would use the test to classify subjects according to
the etiology of their disease.
SUMMARY OF THE INVENTION
[0017] The present invention relates to a method for screening
subjects for genetic markers associated with autism. A biological
sample is isolated from a mammal and then tested for the presence
of a mutated gene or a product thereof which is associated with
autism.
[0018] Another aspect of the invention is an isolated nucleic acid
encoding a HoxA1 allele having a polymorphism which is associated
with autism spectrum disorders.
[0019] Yet another aspect of the invention is an isolated nucleic
acid encoding a HoxB1 allele having a polymorphism which is
associated with autism spectrum disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows two different alleles of HoxA1 from a case of
autism spectrum disorder. FIG. 1A shows the previously published
sequence of wild-type HoxA1. FIG. 1B shows a previously unknown
polymorphism having a single base substitution at position 218,
where an A is changed to a G.
[0021] FIG. 2 shows a second polymorphism was identified in the
first exon of HoxB1. The published sequence of wild-type HoxB1
(FIG. 2A) is compared to the previously unknown polymorphism in
this paralog of HoxB1 (FIG. 2B). In this case, the anomaly is a
nine-base insertion that adds a third repeat where two are normally
present. The result is three extra amino acids,
(serine-alanine-histidine). For each of the polymorphisms, it was
possible to test for the presence of the allele different from the
known sequence by digesting PCR product with a restriction enzyme
(Hph-I for HoxA1 and Msp-I for HoxB1). Sequencing reactions were
carried out on 30-40 subjects to be certain that the digestion
results match the sequencing results, demonstrating that the
digestion procedure detects the deviant sequence described and no
other.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention provides a method for screening
subjects for genetic markers associated with autism. A biological
sample is isolated from a mammal and then tested for the presence
of a mutated gene or a product thereof which is associated with
autism.
[0023] Polymorphisms in Hox genes are shown to be associated with
autism spectrum disorders. The Hox genes are a family of genes that
function in the patterning of body structures that develop along an
anteroposterior axis, such as the limbs, skeleton, and nervous
system; they are expressed during embryonic development at specific
times in limited regions of the embryo. In the mouse, for example,
Hox-a1 is expressed in rhombomeres 4 through 8 of the developing
hindbrain on days 8 to 8.5 of gestation. The Hox genes control the
pattern formation of the hindbrain. Similar abnormalities have been
observed in the brains of autistic individuals (Rodier et al., J.
Comp. Neuro. 370, 247 (1996), which is hereby incorporated by
reference).
[0024] The DNA and amino acid sequences for HoxA-1 have previously
been reported (Acampora, D. et al., Nucleic Acids Res., 17, 10385
(1989); Hong, Y. et al., Gene, 159, 209 (1995) which are hereby
incorporated by reference). Exon 1 stretches from base 1 to base
357. Exon 2 stretches from base 358 to the end (1008). The wildtype
gene sequences for HoxA1 is provided in SEQ. ID. No. 1 as
follows:
1 ATGGACAATG CAAGAATGAA CTCCTTCCTG GAATACCCCA TACTTAGCAG TGGCGACTCG
60 GGGACCTGCT CAGCCCGAGC CTACCCCTCG GACCATAGGA TTACAACTTT
CCAGTCGTGC 120 GCGGTCAGCG CCAACAGTTG CGGCGGCGAC GACCGCTTCC
TAGTGGGCAG GGGGGTGCAG 180 ATCGGTTCGC CCCACCACCA CCACCACCAC
CACCATCACC ACCCCCAGCC GGCTACCTAC 240 CAGACTTCCG GGAACCTGGG
GGTGTCCTAC TCCCACTCAA GTTGTGGTCC AAGCTATGGC 300 TCACAGAACT
TCAGTGCGCC TTACAGCCCC TACGCGTTAA ATCAGGAAGC AGACGTAAGT 360
GGTGGGTACC CCCAGTGCGC TCCCGCTGTT TACTCTGGAA ATCTCTCATC TCCCATGGTC
420 CAGCATCACC ACCACCACCA GGGTTATGCT GGGGGCGCGG TGGGCTCGCC
TCAATACATT 480 CACCACTCAT ATGGACAGGA GCACCAGAGC CTGGCCCTGG
CTACGTATAA TAACTCCTTG 540 TCCCCTCTCC ACGCCAGCCA CCAAGAAGCC
TGTCGCTCCC CCGCATCGGA GACATCTTCT 600 CCAGCGCAGA CTTTTGACTG
GATGAAAGTC AAAAGAAACC CTCCCAAAAC AGGGAAAGTT 660 GGAGAGTACG
GCTACCTGGG TCAACCCAAC GCGGTGCGCA CCAACTTCAC TACCAAGCAG 720
CTCACGGAAC TGGAGAAGGA GTTCCACTTC AACAAGTACC TGACGCGCGC CCGCAGGGTG
780 GAGATCGCTG CATCCCTGCA GCTCAACGAG ACCCAAGTGA AGATCTGGTT
CCAGAACCGC 840 CGAATGAAGC AAAAGAAACG TGAGAAGGAG GGTCTCTTGC
CCATCTCTCC GGCCACCCCG 900 CCAGGAAACG ACGAGAAGGC CGAGGAATCC
TCAGAGAAGT CCAGCTCTTC GCCCTGCGTT 960 CCTTCCCCGG GGTCTTCTAC
CTCAGACACT CTGACTACCT CCCACTGA 1008
[0025] The nucleic acid molecule of SEQ. ID. No. 1 encodes a
polypeptide having the amino acid sequence of SEQ. ID. No. 2, as
follows:
2 M D N A R M N S E L F Y P I L 15 S S G D S G T C S A R A Y P S 30
D H R I T T F Q S C A V S A N 45 S C G G D D R F L V G R G V Q 60 I
G S P H H H H H H H H H H P 75 Q P A T Y Q T S G N L G V S Y 90 S H
S S C C P S Y G S Q N F S 105 A P Y S P Y A L N Q F A D V S 120 G G
Y P Q C A P A V Y S G N L 135 S S P N V Q H H H H H Q G Y A 150 G C
A V G S P Q Y I H H S Y G 165 Q E H Q S L A L A T Y N N S L 180 S F
L H A S H Q E A C F S P A 195 S E T S S P A Q T F D N N K V 210 K F
N P P K T C K V G F Y G Y 225 L G Q P N A V F I N F T T K Q 240 L T
E L F K H F H F N K Y L T 255 R A R R V F I A A S L Q L N F 270 T Q
V K I W F Q N R R N K Q K 285 K R E K E G L L P I S P A T P 300 P G
N D E K A E E S S E K S S 315 S S P C V P S P G S S T S D T 330 L T
T S H 335
[0026] A polymorphism in the HoxA1 gene has been isolated and
sequenced. This polymorphism is associated with autism spectrum
disorders. A single base substitution is located at position 218
(underlined) of SEQ. ID. No. 3, where an A is changed to a G, as
follows:
3 ATGGACAATG CAAGAATGAA CTCCTTCCTG GAATACCCCA TACTTAGCAG TGGCGACTCG
60 GGGACCTGCT CAGCCCGAGC CTACCCCTCG GACCATAGGA TTACAACTTT
CCAGTCGTGC 120 GCGGTCAGCG CCAACAGTTG CGGCGGCGAC GACCGCTTCC
TAGTGGGCAG GGGGGTGCAG 180 ATCGGTTCGC CCCACCACCA CCACCACCAC
CACCATCGCC ACCCCCAGCC GGCTACCTAC 240 CAGACTTCCG GGAACCTGGG
GGTGTCCTAC TCCCACTCAA GTTGTGGTCC AAGCTATGGC 300 TCACAGAACT
TCAGTGCGCC TTACAGCCCC TACGCGTTAA ATCAGGAAGC AGACGTAAGT 360
GGTGGGTACC CCCAGTGCGC TCCCGCTGTT TACTCTGGAA ATCTCTCATC TCCCATGGTC
420 CAGCATCACC ACCACCACCA GGGTTATGCT GGGGGCGCGG TGGGCTCGCC
TCAATACATT 480 CACCACTCAT ATGGACAGGA GCACCAGAGC CTGGCCCTGG
CTACGTATAA TAACTCCTTG 540 TCCCCTCTCC ACGCCAGCCA CCAAGAAGCC
TGTCGCTCCC CCGCATCGGA GACATCTTCT 600 CCAGCGCAGA CTTTTGACTG
GATGAAAGTC AAAAGAAACC CTCCCAAAAC AGGGAAAGTT 660 GGAGAGTACG
GCTACCTGGG TCAACCCAAC GCGGTGCGCA CCAACTTCAC TACCAAGCAG 720
CTCACGGAAC TGGAGAACGA GTTCCACTTC AACAAGTACC TGACCCGCGC CCGCAGGGTG
780 GAGATCGCTG CATCCCTGCA GCTCAACGAG ACCCAAGTGA AGATCTGGTT
CCAGAACCGC 840 CGAATGAAGC AAAAGAAACG TGAGAAGGAG GGTCTCTTGC
CCATCTCTCC GGCCACCCCG 900 CCAGGAAACG ACGAGAAGGC CGAGGAATCC
TCAGAGAAGT CCAGCTCTTC GCCCTGCGTT 960 CCTTCCCCGG GGTCTTCTAC
CTCAGACACT CTGACTACCT CCCACTGA 1008
[0027] The single base substitution at position 218 results in the
replacement of histidine with arginine (underlined). The resulting
protein has the amino acid sequence (SEQ. ID. No. 4) as
follows:
4 M D N A R M N S F L E Y P I L 15 S S G D S G T C S A R A Y P S 30
D H R I T T F Q S C A V S A N 45 S C G G D D R F L V G R G V Q 60 I
G S P H H H H H H H H R H P 75 Q P A T Y Q T S G N L G V S Y 90 S H
S S C G P S Y G S Q N F S 105 A P Y S P Y A L N Q E A D V S 120 G G
Y P Q C A P A V Y S G N L 135 S S P M V Q H H H H H Q G Y A 150 G G
A V G S P Q Y I H H S Y G 165 Q E H Q S L A L A T Y N N S L 180 S P
L H A S H Q H A C R S P A 195 S H T S S P A Q T F D W M K V 210 K R
N P P K T C K V C H Y C Y 225 L C Q P N A V R T N F T T K Q 240 L T
H L H K F F H F N K Y L T 255 K A K R V F I A A S L Q L N F 270 T Q
V K I W F Q N R R M K Q K 285 K R H K H C L L P I S P A T P 300 P G
N D H K A H H S S E K S S 315 S S P C V P S P C S S T S D T 330 L T
T S H 335
[0028] In addition to the polymorphism in HoxA1, a polymorphism
associated with autism spectrum disorders has been isolated and
sequenced from the HoxB1 gene. The HoxB1 gene has not been studied
as comprehensively as HoxA1 in transgenic knockouts, but is
expressed at the same stage (Murphy, P et al., Development, 111, 61
(1991), which is hereby incorporated by reference). Its null
mutation produces similar malformations, including severe
diminution of the facial nucleus (Goddard, J. M. et al.,
Development, 122, 3217 (1996), which is hereby incorporated by
reference). The similarity of expression and function of these two
genes is due to the fact that they were originally a single gene in
invertebrates (Ruddle, F. H. et al., Annu. Rev. Genet., 28, 423
(1993), which is hereby incorporated by reference). In mammals, the
two appear on separate chromosomes (human 7 and 17), but the
sequence of each of the mammalian genes is similar to the others,
and similar to the original single gene from which the two
mammalian loci arose. The sequence of the wildtype hoxB1 gene (SEQ.
ID. No. 5) follows:
5 TGACGCATGG ACTATAATAG GATGAACTCC TTCTTAGAGT ACCCACTCTG TAACCGGGGA
60 CCCAGCGCCT ACAGCGCCCA CAGCGCCCCA ACCTCCTTTC CCCCAAGCTC
GGCTCAGGCG 120 GTTGACAGCT ATGCAAGCGA GGGCCGCTAC GCTGGCGGGC
TGTCCAGCCC TGCGTTTCAG 180 CAGAACTCCG GCTATCCCGC CCAGCAGCCG
CCTTCGACCC TGGGGGTGCC CTTCCCCAGC 240 TCCGCGCCCT CGGGGTATGC
TCCTGCCGCC TGCAGCCCCA GCTACGGGCC TTCTCAGTAC 300 TACCCTCTGG
GTCAATCAGA AGGAGACGGA GGCTATTTTC ATCCCTCGAG CTACGGGGCC 360
CAGCTAGGGG GCTTGTCCGA TGGCTACGGA GCAGGTGGAG CCGGTCCGGG GCCATATCCT
420 CCGCAGCATC CCCCTTATGG GAACGAGCAG ACCGCGAGCT TTGCACCGGC
CTATGCTGAT 480 CTCCTCTCCG AGGACAAGGA AACACCCTGC CCTTCAGAAC
CTAACACCCC CACGGCCCGG 540 ACCTTCGACT GGATGAAGGT TAAGAGAAAC
CCACCCAAGA CAGCGAAGGT GTCAGAGCCA 600 GGCCTGGGCT CGCCCAGTGG
CCTCCGCACC AACTTCACCA CAAGGCAGCT GACAGAACTG 660 GAAAAGGAGT
TCCATTTCAA CAAGTACCTG AGCCGGGCCC GGAGGGTGGA GATTGCCGCC 720
ACCCTGGAGC TCAATGAAAC ACAGGTCAAG ATTTGGTTCC AGAACCCACG AATGAAGCAG
780 AAGAAGCGCG AGCGAGAGGG AGGTCGGGTC CCCCCAGCCC CACCAGGCTG
CCCCAAGGAG 840 GCAGCTGGAG ATGCCTCAGA CCAGTCGACA TGCACCTCCC
CGGAAGCCTC ACCCAGCTCT 900 GTCACCTCCT GAACTGAACC TAGCCACCAA
TGGGGCTTCC AGGCACTGGA GCGCCCCAGT 960 CCAGCCCTAT CCCAGGCTCT
CCCAACCCAG GCCTGGCTTC ACTGCCTGGG ATCTCTAGGC 1020 T 1021
[0029] The protein encoded by nucleotides 7 to 909 of the wild-type
HoxB1 gene (SEQ. ID. No. 6) is as follows:
6 M D Y N R M N S F L H Y P L C 15 N R G P S A Y S A H S A P T S 30
F P P S S A Q A V D S Y A S H 45 G R Y G G G L S S P A F Q Q N 60 S
G Y P A Q Q P P S T L G V P 75 F P S S A P S C Y A P A A C S 90 P S
Y C P S Q Y Y P L G Q S F 105 C D G G Y F H P S S Y G A Q L 120 C C
L S D G Y C A C C A G P G 135 P Y P P Q H P P Y C N E Q T A 150 S F
A P A Y A D L L S F D K E 165 T P C P S F P N T P T A H T F 180 D W
M K V K R N P P K T A K V 195 S F P G L G S P S C L R T N F 210 T T
R Q L T E L E K F F H F N 225 K Y L S K A F R V F I A A T L 240 E L
N F T Q V K I K F Q N R R 255 M K Q K K K F R F C G K V P P 270 A P
P C C P K F A A G D A S D 285 Q S T C T S P F A S P 5 S V T 300 S
301
[0030] As with the HoxA1 gene, polymorphisms associated with autism
spectrum disorders were found with HoxB1. The HoxB1 mutation occurs
after base 88 (C) with the insertion of nine nucleotides
(ACAGCGCCC). The location of this insertion is such that the amino
acid sequence also changes. The normal sequence reads . . .
serine-alanine-histidine-serine-- alanine-proline. The mutant
sequence has an extra serine-alanine-histidine -sequence and then
the sequence resumes normally. The insertion and altered amino acid
sequence are underlined below. A mutated form of HoxB1 (SEQ. ID.
No. 7) is depicted as follows:
7 TGACGCATGG ACTATAATAG GATGAACTCC TTCTTAGAGT ACCCACTCTG TAACCGGGGA
60 CCCAGCGCCT ACAGCGCCCA CAGCGCCCAC AGCGCCCCAA CCTCCTTTCC
CCCAAGCTCG 120 GCTCAGGCGG TTGACAGCTA TGCAAGCGAG GGCCGCTACG
GTGGGGGGCT GTCCAGCCCT 180 GCGTTTCAGC AGAACTCCGG CTATCCCGCC
CAGCAGCCGC CTTCGACCCT GGGGGTGCCC 240 TTCCCCAGCT CCGCGCCCTC
GGGGTATGCT CCTGCCGCCT GCAGCCCCAG CTACGGGCCT 300 TCTCAGTACT
ACCCTCTGGG TCAATCAGAA GGAGACGGAG GCTATTTTCA TCCCTCGAGC 360
TACGGGGCCC AGCTAGGGGG CTTGTCCGAT GGCTACGGAG CAGGTGGAGC CGGTCCGCGG
420 CCATATCCTC CGCAGCATCC CCCTTATGGG AACGAGCAGA CCGCGAGCTT
TGCACCGGCC 480 TATGCTGATC TCCTCTCCGA GGACAAGGAA ACACCCTGCC
CTTCAGAACC TAACACCCCC 540 ACGGCCCGGA CCTTCGACTG GATGAAGGTT
AAGAGAAACC CACCCAAGAC AGCGAAGGTG 600 TCAGAGCCAG GCCTGGGCTC
GCCCAGTGGC CTCCGCACCA ACTTCACCAC AAGGCAGCTG 660 ACAGAACTGG
AAkAGGAGTT CCATTTCAAC AAGTACCTGA GCCGGGCCCG GAGGGTGGAG 720
ATTGCCGCCA CCCTGGAGCT CAATGAAACA CAGGTCAAGA TTTGGTTCCA GAACCGACGA
780 ATGAAGCAGA AGAAGCGCGA GCGAGAGGGA GGTCGGGTCC CCCCAGCCCC
ACCAGGCTGC 840 CCCAAGGAGG CAGCTGGAGA TGCCTCAGAC CAGTCGACAT
GCACCTCCCC GGAACCCTCA 900 CCCAGCTCTG TCACCTCCTG AACTGAACCT
AGCCACCAAT GGGGCTTCCA GGCACTGGAG 960 CGCCCCAGTC CAGCCCTATC
CCAGGCTCTC CCAACCCAGG CCTGGCTTCA CTGCCTGGGA 1020 TCTCTAGGCT
1030
[0031] The protein encoded by SEQ. ID. No. 8 is as follows:
8 M D Y N R N N S F L F Y P L C 15 N R C P S A Y S A H S A H S A 30
P T S F P P S S A Q A V D S Y 45 A S E C F Y C G C L S S P A F 60 Q
Q N S G Y P A Q Q P P S T L 75 G V P F P S S A P S C Y A P A 90 A C
S P S Y G P S Q Y Y P L C 105 Q S E C D C C Y F H P S S Y G 120 A Q
L C G L S D C Y C A G G A 135 G P G P Y P P Q H P P Y C N E 150 Q T
A S F A P A Y A D L L S S 165 D K S T P C P S S P N T P T A 180 R T
F D W M K V K R N P P K T 195 A K V S S P G L C S P S C L R 210 T N
F T T R Q L T F L F K F F 225 H F N K Y L S R A R R V S I A 240 A T
L F L N F T Q V K I W F Q 255 N R R N K Q K K R E R F C C R 270 V P
P A P P G C P K F A A C C 285 A S D Q S T C T S P F A S P S 300 S V
T S 304
[0032] Genes which have been duplicated and then maintained similar
functions over the course of evolution are called "paralogs." A
third paralog derived from the same invertebrate gene is known as
HoxD1. This gene has not yet been studied in knockouts, but is
known to have evolved to be expressed in somewhat different
embryonic tissues (mesoderm vs. ectoderm) in the hindbrain region
at the same stage of development as Hoxa1 and Hoxb1. Thus preferred
hox genes include HoxA1, HoxB1,and HoxD1.
[0033] Biological samples suitable for testing include blood,
saliva, amniotic fluid, and tissue. The most preferred biological
sample is blood. However, any biological sample from which genetic
material or the products of the marker genes can be isolated is
suitable.
[0034] Because the Hox genes are highly conserved among species,
the present invention is applicable for screening for autism
related polymorphisms in mammals. The screening method can be
utilized to identify animals carrying defects in genes like those
which give rise to autism in humans in order to study the
progression of the disease and test treatments. However, the
preferred mammal to be screened is humans. In particular, the
biological samples are isolated from developmentally disabled
children or adults in order to determine whether they carry the
marker associated with autism to assist in diagnosing the disease.
Similarly, the parents or relatives of disabled children may be
screened to determine whether they are carriers of the mutated
gene. Samples may also be tested from children including infants to
identify those children who have genetic markers associated with
autism in order to provide them with early behavior training.
[0035] As discussed more fully in the examples, polymorphisms in
the HoxA1 gene are associated with autism spectrum disorders. In
addition to HoxA1, the HoxB1 and HoxD1 genes are also involved in
the same stages of early brain development. Hoxb1 and Hoxd1 are
related developmental genes which are expressed at the same time
and in approximately the same region of the embryo as HoxA1. The
Hox genes are closely related and may perform similar functions in
development. Evolutionarily the various Hox genes were probably
derived from a common ancestral gene. Thus, the preferred genes to
be screened include Hoxa1, Hoxb1, and Hoxd1.
[0036] The mutation in the mutated gene may be a single base
substitution mutation resulting in an amino acid substitution, a
single base substitution mutation resulting in a translational
stop, an insertion mutation, a deletion mutation, or a gene
rearrangement. As demonstrated from the identified polymorphisms in
HoxA1 and HoxB1, polymorphisms which disrupt the gene or result in
an altered peptide are associated with autism spectrum
disorders.
[0037] The mutation may be located in an intron, an exon of the
gene, or a promotor or other regulatory region which affects the
expression of the gene.
[0038] Methods for screening for mutated nucleic acids include
direct sequencing of nucleic acids, single strand polymorphism
assay, ligase chain reaction, enzymatic cleavage, and southern
hybridization.
[0039] Screening for mutated nucleic acids can be accomplished by
direct sequencing of nucleic acids. In fact, putative mutants
identified by other methods may be sequenced to determine the exact
nature of the mutation. Nucleic acid sequences can be determined
through a number of different techniques which are well known to
those skilled in the art. In order to sequence the nucleic acid,
sufficient copies of the material must first be amplified.
[0040] Amplification of a selected, or target, nucleic acid
sequence may be carried out by any suitable means. (See generally
Kwoh, D. and Kwoh, T., Am Biotechnol Lab, 8, 14 (1990), which is
hereby incorporated by reference.) Examples of suitable
amplification techniques include, but are not limited to,
polymerase chain reaction, ligase chain reaction (see Barany, Proc
Natl Acad Sci USA 88, 189 (1991), which is hereby incorporated by
reference), strand displacement amplification (see generally
Walker, G. et al., Nucleic Acids Res. 20, 1691 (1992); Walker. G.
et al., Proc Natl Acad Sci USA 89, 392 (1992), which are hereby
incorporated by reference), transcription-based amplification (see
Kwoh, D. et al., Proc Natl Acad Sci USA, 86, 1173 (1989), which is
hereby incorporated by reference), self-sustained sequence
replication (or "3SR") (see Guatelli, J. et al., Proc Natl Acad Sci
USA, 87, 1874 (1990), which is hereby incorporated by reference),
the Q.beta. replicase system (see Lizardi, P. et al.,
Biotechnology, 6, 1197 (1988), which is hereby incorporated by
reference), nucleic acid sequence-based amplification (or "NASBA")
(see Lewis, R., Genetic Engineering News, 12(9), 1 (1992), which is
hereby incorporated by reference), the repair chain reaction (or
"RCR") (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992),
which is hereby incorporated by reference), and boomerang DNA
amplification (or "BDA") (see Lewis, R., Genetic Engineering News,
12(9), 1 (1992), which is hereby incorporated by reference).
Polymerase chain reaction is currently preferred.
[0041] In general, DNA amplification techniques such as the
foregoing involve the use of a probe, a pair of probes, or two
pairs of probes which specifically bind to DNA encoding the gene of
interest, but do not bind to DNA which does not encode the gene,
under the same hybridization conditions, and which serve as the
primer or primers for the amplification of the gene of interest or
a portion thereof in the amplification reaction.
[0042] Nucleic acid sequencing can be performed by chemical or
enzymatic methods. The enzymatic method relies on the ability of
DNA polymerase to extend a primer, hybridized to the template to be
sequenced, until a chain-terminating nucleotide is incorporated.
The most common methods utilize didoexynucleotides. Primers may be
labelled with radioactive or fluorescent labels. Various DNA
polymerases are available including Klenow fragment, AMV reverse
transcriptase, Thermus aquaticus DNA polymerase, and modified T7
polymerase.
[0043] Although DNA sequencing is clearly the most sensitive and
informative method, it is too cumbersome for routine use in
searching for polymorphisms, especially when the DNA segment of
interest is large. Several other methods are available for a rapid
search for changes in autism associated genes.
[0044] Recently, single strand polymorphism assay ("SSPA") analysis
and the closely related heteroduplex analysis methods have come
into use as effective methods for screening for single-base
polymorphisms (Orita, M. et al., Proc Natl Acad Sci USA, 86, 2766
(1989), which is hereby incorporated by reference). In these
methods, the mobility of PCR-amplified test DNA from clinical
specimens is compared with the mobility of DNA amplified from
normal sources by direct electrophoresis of samples in adjacent
lanes of native polyacrylamide or other types of matrix gels.
Single-base changes often alter the secondary structure of the
molecule sufficiently to cause slight mobility differences between
the normal and mutant PCR products after prolonged
electrophoresis.
[0045] Ligase chain reaction is yet another recently developed
method of screening for mutated nucleic acids. Ligase chain
reaction (LCR) is also carried out in accordance with known
techniques. LCR is especially useful to amplify, and thereby
detect, single nucleotide differences between two DNA samples. In
general, the reaction is carried out with two pairs of
oligonucleotide probes: one pair binds to one strand of the
sequence to be detected; the other pair binds to the other strand
of the sequence to be detected. The reaction is carried out by,
first, denaturing (e.g., separating) the strands of the sequence to
be detected, then reacting the strands with the two pairs of
oligonucleotide probes in the presence of a heat stable ligase so
that each pair of oligonucleotide probes hybridize to target DNA
and, if there is perfect complementarity at their junction,
adjacent probes are ligated together. The hybridized molecules are
then separated under denaturation conditions. The process is
cyclically repeated until the sequence has been amplified to the
desired degree. Detection may then be carried out in a manner like
that described above with respect to PCR.
[0046] Southern hybridization is also an effective method of
identifying differences in sequences. Hybridization conditions,
such as salt concentration and temperature can be adjusted for the
sequence to be screened. Southern blotting and hybridizations
protocols are described in Current Protocols in Molecular Biology
(Greene Publishing Associates and Wiley-Interscience), pages
2.9.1-2.9.10. Probes can be labelled for hybridization with random
oligomers (primarily 9-mers) and the Klenow fragment of DNA
polymerase. Very high specific activity probe can be obtained using
commercially available kits such as the Ready-To-Go DNA Labelling
Beads (Pharmacia Biotech), following the manufacturer's protocol.
Briefly, 25 ng of DNA (probe) is labelled with .sup.32P-dCTP in a
15 minute incubation at 37.degree. C. Labelled probe is then
purified over a ChromaSpin (Clontech) nucleic acid purification
column. Possible competition of probes having high repeat sequence
content, and stringency of hybridization and washdown will be
determined individually for each probe used. Alternatively,
fragments of a candidate gene may be generated by PCR, the
specificity may be verified using a rodent-human somatic cell
hybrid panel, and subcloning the fragment. This allows for a large
prep for sequencing and use as a probe. Once a given gene fragment
has been characterized, small probe preps can be done by gel- or
column-purifying the PCR product.
[0047] These mismatch detection protocols use samples generated by
PCR and thus require use of very little genomic template. All of
these methods can provide very good clues regarding the location of
the sequence change which leads to the appearance of anomalous
bands, hence facilitating subsequent cloning and sequencing
strategies.
[0048] Methods of screening for mutated nucleic acids can be
carried out using either deoxyribonucleic acids ("DNA") or
messenger ribonucleic acids ("mRNA") isolated from the biological
sample. During periods when the gene is expressed, mRNA may be
abundant and more readily detected. However, these genes are
temporally controlled and, at most stages of development, the
preferred material for screening is DNA.
[0049] Alternatively, the detection of a mutated gene associated
with autism can be carried out by collecting a biological sample
and testing for the presence or form of the protein produced by the
gene. The mutation in the gene may result in the production of a
mutated form of the peptide or the lack of production of the gene
product. In this embodiment, the determination of the presence of
the polymorphic form of the protein can be carried out, for
example, by isoelectric focusing, protein sizing, or immunoassay.
In an immunoassay, an antibody that selectively binds to the
mutated protein can be utilized (for example, an antibody that
selectively binds to the mutated form of HoxA1 encoded protein).
Such methods for isoelectric focusing and immunoassay are well
known in the art, and are discussed in further detail below.
[0050] Changes in the size or charge of the polypeptide can be
identified by isoelectric focusing or protein sizing techniques.
Changes resulting in amino acid substitutions, where the
substituted amino acid has a different charge than the original
amino acid, can be detected by isoelectric focusing. Isoelectric
focusing of the polypeptide through a gel having an ampholine
gradient at high voltages separates proteins by their pI. The pH
gradient gel can be compared to a simultaneously run gel containing
the wild-type protein. Protein sizing techniques such as protein
electrophoresis and sizing chromatography can also be used to
detect changes in the size of the product.
[0051] As an alternative to isoelectric focusing or protein sizing,
the step of determining the presence of the mutated polypeptides in
a sample may be carried out by an antibody assay with an antibody
which selectively binds to the mutated polypeptides (i.e., an
antibody which binds to the mutated polypeptides but exhibits
essentially no binding to the wild-type polypeptide without the
polymorphism in the same binding conditions).
[0052] Antibodies used to bind selectively the products of the
mutated genes can be produced by any suitable technique. For
example, monoclonal antibodies may be produced in a hybridoma cell
line according to the techniques of Kohler and Milstein, Nature,
265, 495 (1975), which is hereby incorporated by reference. A
hybridoma is an immortalized cell line which is capable of
secreting a specific monoclonal antibody. The mutated products of
genes which are associated with autism may be obtained from a human
patient, purified, and used as the immunogen for the production of
monoclonal or polyclonal antibodies. Purified polypeptides may be
produced by recombinant means to express a biologically active
isoform, or even an immunogenic fragment thereof may be used as an
immunogen. Monoclonal Fab fragments may be produced in Escherichia
coli from the known sequences by recombinant techniques known to
those skilled in the art. (See, e.g, Huse, W., Science 246, 1275
(1989), which is hereby incorporated by reference) (recombinant Fab
techniques).
[0053] The term "antibodies" as used herein refers to all types of
immunoglobulin, including IgG, IgM, IgA, IgD, and IgE. The
antibodies may be monoclonal or polyclonal and may be of any
species of origin, including (for example) mouse, rat, rabbit,
horse, or human, or may be chimeric antibodies, and include
antibody fragments such as, for example, Fab, F(ab').sub.2'and Fv
fragments, and the corresponding fragments obtained from antibodies
other than IgG.
[0054] Antibody assays may, in general, be homogeneous assays or
heterogeneous. In a homogeneous assay the immunological reaction
usually involves the specific antibody, a labeled analyte, and the
sample of interest. The signal arising from the label is modified,
directly or indirectly, upon the binding of the antibody to the
labeled analyte. Both the immunological reaction and detection of
the extent thereof are carried out in a homogeneous solution.
Immunochemical labels which may be employed include free radicals,
radioisotopes, fluorescent dyes, enzymes, bacteriophages,
coenzymes, and so forth.
[0055] In a heterogeneous assay approach, the reagents are usually
the specimen, the antibody of the invention and means for producing
a detectable signal. Similar specimens as described above may be
used. The antibody is generally immobilized on a support, such as a
bead, plate, or slide, and contacted with the specimen suspected of
containing the antigen in a liquid phase. The support is then
separated from the liquid phase and either the support phase or the
liquid phase is examined for a detectable signal employing means
for producing such signal. The signal is related to the presence of
the analyte in the specimen. Means for producing a detectable
signal include the use of radioactive labels, fluorescent labels,
enzyme labels, and so forth. For example, if the antigen to be
detected contains a second binding site, an antibody which binds to
that site can be conjugated to a detectable group and added to the
liquid phase reaction solution before the separation step. The
presence of the detectable group on the solid support indicates the
presence of the antigen in the test sample. Examples of suitable
immunoassays are the radioimmunoassay, immunofluorescence methods,
enzyme-linked immunoassays, and the like.
[0056] Those skilled in the art will be familiar with numerous
specific immunoassay formats and variations thereof which may be
useful for carrying out the method disclosed herein. See U.S. Pat.
Nos. 4,727,022, 4,659,678, 4,376,110, 4,275,149, 4,233,402 and
4,230,767.
[0057] Antibodies which selectively bind a polymorphic DLST isoform
may be conjugated to a solid support suitable for a diagnostic
assay (e.g., beads, plates, slides or wells formed from materials
such as latex or polystyrene) in accordance with known techniques,
such as precipitation. Antibodies which bind a polymorphic DLST
isoform may likewise be conjugated to detectable groups such as
radiolabels e.g,, .sup.35S, .sup.125I, .sup.131I) enzyme labels
(e.g., horseradish peroxidase, alkaline phosphatase), and
fluorescent labels (e.g., fluorescein) in accordance with known
techniques.
[0058] The invention further provides an isolated nucleic acid
molecule which encodes a HoxA1 gene having a single base
substitution at nucleotide 218 in SEQ. ID. No. 1. In another
embodiment, the invention provides an isolated nucleic acid
molecule which encodes a HoxB1 gene having an insertion between
positions nucleotides 88 and 89 in SEQ. ID. No. 5. In addition, the
invention provides fragments of the HoxA1 and HoxB1 genes having
the polymorphism, where the fragment has at least 15 nucleotides
and encompasses the polymorphism, i.e., the single base
substitution. Fragments longer than 15 nucleotides can be used to
probe for nucleic acid molecules containing the polymorphism.
Longer fragments may be used at higher stringency conditions.
[0059] The invention also provides isolated polypeptides that are
encoded by the genes having the polymorphisms. Either the whole
protein or fragments thereof may be used to induce the production
of antibodies specific to the portion of the protein which is
effected by the polymorphism. Such antibodies may then be used to
detect the presence of a polymorphism. Preferred antibodies bind
specifically to the protein or polypeptide effected by the
polymorphism but with less affinity to the wild-type Hox
protein.
[0060] In one embodiment, the antibody is a monoclonal antibody.
For use in an immunoassay, the antibody can be bound to a solid
support or bound to a detectable label.
EXAMPLES
Example 1
[0061] Collection of Blood Samples from Autistic Individuals
[0062] Blood was collected from patients with autism and their
immediate family members in order to determine whether any
polymorphisms in HoxA1 are present among this population. All blood
samples were procured following written consent by the patients or
their guardians. Among the samples collected were those of the
members of a family of four in which one child has autism and the
other has Asperger's syndrome; both children have malformed ears.
The first son is retarded and the second has normal intelligence.
The parents have no obvious symptoms. DNA was extracted from the
blood by phenolchloroform extraction following isolation and lysis
of the white blood cells. Control DNA was also used for these
excrements; this DNA was obtained from neurologically normal
donors.
[0063] The 20 cc blood samples were left for three-four days at
room temperature to allow continued proliferation of white blood
cells. White cells were pelleted, followed by isolation of the
nuclei. The nuclei were then incubated overnight at 37.degree. C.
in a lysis buffer consisting of EDTA, TNE-SDS, and proteinase K.
Protein contaminants were extracted by additions of buffered phenol
followed by chloroform, then DNA was precipitated by the addition
of ice-cold ethanol. The DNA was re-suspended in TE buffer for
storage at 4.degree. C. Extraction of genomic DNA from fixed tissue
was carried out using the protocol of Volkenandt et al., Methods in
Molecular Biology, 15, 81, Humana Press, (1993), which is hereby
incorporated by reference).
Example 2
[0064] Sequencing the Hoxa1 Gene
[0065] The HoxA1 gene was amplified by PCR from DNA samples to
provide sufficient material for sequencing. Two sets of
oligonucleotide primers were selected after examination of the
human HoxA1 nucleic acid sequence and comparison of the sequence to
those of human and mouse Hox genes. The first set was designed to
amplify residues 10-647, the second to amplify from residue 656 to
the stop codon at residue 1008, exons 1 and 2 of HoxA1,
respectively. The primers were used in polymerase chain reaction to
amplify the target gene in several control blood samples, in order
to determine the appropriate PCR conditions. Both exons were
amplified by 94.degree. C. denaturation for 1 min, 62.degree. C.
annealing for 30 sec, and 72.degree. C. extension for 2 min, for 35
cycles. The products were visualized with ethidium bromide staining
on a 1-2% agarose gel. PhiX174 RF DNA/Hae III fragments (Gibco)
were used as a molecular weight marker. The products were tested
for chromosome origin by using human-rodent monochromosomal somatic
cell hybrids. Both exons amplified by the HoxA1 primers amplified
the hybrid containing human chromosome 7 and do not amplify from
any other hybrids. Establishing that the product amplified by the
primers is from the correct chromosome rules out the possibility
that pseudogenes with the same sequence occur at other sites or
that the amplified product is another homologous homeobox gene. It
verifies that the PCR product represents only the targeted
gene.
[0066] The polymerase chain reaction (PCR) was performed with
various samples of control DNA in order to determine the
appropriate conditions. Once the optimal conditions were
ascertained, the gene was amplified from the patient samples.
[0067] Following PCR, an aliquot of the product was used for DNA
sequencing using the Sequenase system version 2.0 (United States
Biochemical), which is a chain-termination method of DNA
sequencing. The following procedure was used to read the nucleic
acid sequence of the amplified products. 7 .mu.l of PCR product was
mixed with 2 .mu.l shrimp alkaline phosphatase and 0.5 .mu.l
exonuclease I. The mixture was incubated at 37.degree. C. for 15
min and then at 80.degree. C. for 15 min. After addition of 1 .mu.l
of primer, the mixture was incubated at 100.degree. C. for 3 min
and then chilled on ice for 5 min. Next, the sample was incubated
for 5 min at room temperature with the following additions: 2 .mu.l
5.times.buffer, 1 .mu.l DTT, 2 .mu.l diluted dGTP, 0.5
.mu.l.sup.35S-dATP, and 2 .mu.l diluted Sequenase buffer. A 3.5
.mu.l aliquot of the mixture was then added to 1 .mu.l of one
dideoxyNTP. After 5 min at 37.degree. C., 4 .mu.l of stop solution
was added to the tube. The products were run on a 6% polyacrylamide
sequencing gel for 2-4 hr. Following this, the gel was dried on a
BioRad gel dryer and exposed to film overnight. Film was developed
on a Kodak M35A X-OMAT Processor. The method has been used
successfully to duplicate the published sequence of the Hoxa1 exons
in samples from a number of controls. The film was developed the
next afternoon, and the DNA sequence was read manually for
comparison to the published Hox A1 sequence.
[0068] The nucleotide sequence from some patients, including the
members of the family mentioned previously, showed the presence of
two discrete bands at the same levels on the gel.
Example 3
[0069] Sequencing the PCR Products
[0070] Since sequencing PCR products allows the DNA sequence to be
read from both alleles, a sequence with double bands suggests
heterozygosity--that the two alleles are not the same and that two
different sequences superimposed on one another are being read.
Based on these results, the PCR products were cloned in order to
get a cleaner sequence. Cloning separates the two alleles and
allowed each to be individually sequenced to determine whether one
or both alleles are abnormal.
[0071] The PCR products were cloned using Invitrogen's Zero Blunt
PCR Cloning Kit. This kit is designed to clone blunt-ended PCR
fragments, which can be generated by using a thermostable DNA
polymerase with proofreading activity. Once the products were
cloned, the clonal DNA was sequenced using the Sequenase version
2.0 chain-termination sequencing system. Each clone was sequenced
in both 5' and 3' directions, and the reactions were run out for 6
hours on a 6% polyacrylamide sequencing gel.
[0072] Cloning allowed the determination that three out of four
members of this family are indeed heterozygous for Hox A1. The
father and both children contain an identical mutation in the gene:
a single base-pair change of A to G in the first exon of the gene;
the mother's gene is normal. This mutation is dominant with
variable penetrance. Sequences showing the mutation can be seen in
FIG. 1. FIG. 1A shows the wild-type sequence. Substitution of
guanine for adenine at this single location as shown in FIG. 1B
causes an alteration in the resulting amino acid sequence, changing
an arginine to a histidine.
Example 4
[0073] Restriction Analysis of PCR Products
[0074] The PCR products from this family were also subjected to
restriction enzyme digestion to confirm the mutation. The enzyme
Hph I recognizes the specific sequence 3' . . . CCACT(N.sub.7). . .
5'. When normal HoxA1 is digested with this enzyme, it will be cut;
however, when mutated HoxA1 is digested, it will not be cut,
because the recognition site has been changed by the mutation. This
enzyme has been used to digest PCR products from this family and
confirm that the mutation does indeed exist in the father and the
children but not in the mother. This enzyme has been used to digest
PCR products from approximately 100 controls, 36 parent pairs, 26
affected relatives, and 46 probands. In forty cases, the results of
the restriction analysis has been compared to that from the
sequencing reactions. The two methods gave identical results in
every case.
Example 5
[0075] Sequencing of a Polymorphism in HoxB1
[0076] The sequence for the HoxB1 gene (accession number X16666)
was obtained from the Entrez data base. From this sequence primers
for the amplification of a 575 bp product of exon 1 by PCR were
designed (Sense: 5'-GCATGGACTATAATAGGATG-3' (SEQ. ID. No. 9);
Antisense: 5'-TCTTGGGTGGGTTTCTCTTA-3' (SEQ. ID. No. 10)). The final
concentration of the following components were used in the
amplification reaction: 1.5 U Taq polymerase; 200 .mu.M each of
dATP, dCTP, dGTP, dTTP; 1.5 mM MgCl; 0.4 mM of each sense and
antisense primer; 50-100 ng DNA template; and distilled H.sub.20 to
a final volume of 25 .mu.l. The Taq, dNTPs and MgCl are supplied in
a Ready-To-Go PCR Bead (Pharmacia 27-9555-01) and were used
according to manufacturer's directions. The PCR reaction was
carried out in a Perkin-Elmer 480 GeneAmp or a Perkin-Elmer 2400
thermocycler. Reaction conditions were: denaturing for 1 minute at
94 C., and then 35 cycles of denaturing at 94 C. for 45 sec,
annealing at 57 C. for 45 sec, and elongation at 72 C. for 45 sec.
Resulting PCR product was analyzed on a 1% agarose gel and compare
to a 100 bp ladder to determine the size of the product. Since the
size of the product was as expected (575 bp) and somatic cell
hybrid results indicated that the product is specific for
chromosome 17 DNA samples from probands, family members and
controls were amplified and sequenced using a radiolabeled
terminator cycle sequencing kit (Amersham Life Science US79750).
The sequencing reaction was ran on a 6% acrylamide sequencing gel
(National Diagnostics) and exposed to Kodak Biomax MS X ray film
for 24-48 hours. After developing the film, the resulting sequence
was compared to the published sequence found in the Entrez data
base.
Example 6
[0077] Association of the Newly-discovered Alleles with Autism
Spectrum Disorders.
[0078] Forty-six probands with autistic spectrum disorders and
evidence of genetic causation were selected for analysis.
Forty-three had one or more other affected family members and
thirty-five had ear anomalies or neurological deficits consistent
with malfunction of HoxA1 or its paralogs. For comparison, three
other groups were tested:
[0079] 1) An unstructured control group consisting of adults with
no evidence of neurological abnormality collected from many
different medical centers. These were mostly spouses of patients
with late onset degenerative diseases of the nervous system. The
purpose of this group was to determine the frequency of the alleles
in the general population.
[0080] 2) Parent controls--While each of the parents of a proband
obviously transmits half of his or her genetic material to the
proband, imaginary individuals with two alleles constructed from
the untransmitted allele of each parent pair should give an
accurate estimate of the frequency of the alleles in the study
population, aside from those transmitted to the probands. Thus, the
untransmitted alleles of the parent pair make a more stringent
control, taking into account known and unknown structure in the
local population.
[0081] 3) Affected family members of probands--When they were
available, the siblings, cousins, parents, or aunts and uncles of
probands diagnosed with autism spectrum disorders or related
symptoms (e.g. learning disabilities, language delays, neurological
anomalies of the cranial nerves) were tested. If an allele is
associated with autism, it should be more frequent in probands and
affected family members than in historic or parent controls.
9TABLE 1 Percent of individuals with polymorphic forms of HoxA1
and/or B1 HOXA1 HOXB1 HOXA1 or HOXB1 Historic controls (N = 101) 16
34 47 Parent controls (N = 36) 22 39 55.sup..dagger. Probands with
ASD (N = 46) 35** 52* 80*** Other affected relatives (N = 24) 38*
42 75* different from historical controls: *= p < .05, **= p
< .01, ***= p < .001 different from probands: .sup..dagger.=
<.05
[0082] Table 1 demonstrates that parent controls are, indeed,
similar to historic controls in their rates of the polymorphisms
under study, while affected family members are similar to probands.
This is especially true when the two functionally-related genes are
combined. Eighty percent of probands have one deviation from the
normal sequence or the other, while only 47% of historical controls
have an anomaly. Parent controls (untranslated alleles) match the
historical controls in their rate of abnormal alleles, indicating
that the local population is not structured differently from the
general population in its rate of these alleles. In contrast, both
probands (X.sup.2=14.83, p<0.001) and other affected family
members (X.sup.2=6.30, p<0.02)differ significantly from
historical controls. The probands differ significantly from the
parent controls, as well (X.sup.2=4.08 =, p<0.05)The probands
with genetic anomalies of HoxA1 or HoxB1 are concordant with the
other affected members of the family in 18/22 cases (X.sup.2=17.82,
p<0.001)Finally, both the HoxA1 and HoxB1 polymorphisms are
significantly associated with autism as judged by the Transmission
Disequilibrium Test for Association (Spielman and Ewens, 1996),
which compares the rate of transmission "into the disease" to the
50% rate one would expect in offspring of parents with the allele
of interest. The x.sup.2 s for this test are: HoxA1=5.16,
p<0.05; HoxB1=4.67, p<0.05.
[0083] In addition to the living probands, it was of interest to
determine the genotype of the patient whose brain anatomy first
suggested the involvement of the Hox genes in autism (Rodier et
al., 1996). Genomic DNA was extracted from the autopsy tissue, and
the patient was determined to have the B1 polymorphism (Stodgell et
al., 1998).
[0084] One proband is homozygous for the less common allele of
HoxA1, and he is severely affected. He was diagnosed early, at 21
months. None of the historic controls, and no parents, were
homozygous for the polymorphism. Homozygosity of the HoxB1
polymorphism occurred in two historic controls, one affected
parent, and in two severely-affected probands. Larger samples are
needed to determine whether either polymorphism reduces viability.
Three probands have both polymorphisms, and are severely disabled.
The detection and description of the polymorphisms in the first
exons of HoxA1 and HoxB1 and the progress of the association
studies have been described in a book chapter and two abstracts
(Rodier, 1998; Ingram et al., 1997; Stodgell et al., 1998).
Example 7
[0085] Identification of a Second Polymorphism in HoxA1
[0086] A third polymorphism has been detected in the homeobox
region of HoxA1 in the second exon. The second exon cannot be
amplified by PCR from the DNA of four probands indicating that an
anomaly exists. This indicates that they are homozygous for a
deviation from the published sequence on which the primers for the
exon were based. PCR amplification yields suggest that about ten
other probands are heterozygotes for this polymorphism of the
second exon of HoxA1.
[0087] Additional primers have been developed that will allow
complete sequencing of the altered region, which appears to be at
the 3 ' end of the homeobox. Once the sequence is established, a
test (such as the use of restriction length polymorphisms) can be
developed to allow rapid evaluation of DNA samples. The degree of
association of this polymorphism with autism spectrum disorders
will then be studied in the same groups already evaluated for the
others. Other studies in progress are designed to examine the
second exon of HoxB1 and the non-coding regions of both genes.
Example 8
[0088] Identification of Additional Polymorphisms in HoxB1 and
HoxD1 Associated with Autism
[0089] The procedures for evaluating the candidate gene HoxD1, as
well as for finding additional polymorphisms in HoxA1 and HoxB1,
will be the same as for those already identified in HoxA1 and
HoxB1. Mutation detection in the coding sequence of these genes
will consist of PCR amplification, cloning and sequencing. Mutation
detection for the entire genes will include large
deletion/insertion analysis by Southern blotting, analysis of
200-400 bp fragments by SSCP or heteroduplex analysis, and of
course cloning and sequencing when heterozygosity becomes apparent
for any region of the genes. Current Protocols in Human Genetics
(John Wiley & Sons, Inc.), Chapter 7, "Searching Candidate
Genes for Mutations."
[0090] Biological samples already isolated from patients with
autism which did not show any abnormalities in HoxA1 or HoxB1 will
be screened for polymorphisms in HoxD1 .
[0091] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these therefore are considered within
the scope of the invention as defined in the claims which follow.
Sequence CWU 1
1
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