Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-23
2001-10-16
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06303307
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular genetics and diagnosis of genetic diseases. More specifically, the present invention relates to a large scale genotyping of diseases and diagnostic tests and kits for same.
2. Description of the Related Art
Expansion of repeat sequences involving the trinucleotides CAG, CTG, CGG or GAA has been shown to be the primary cause of several neurological disorders
1
. Among them, CAG repeat expansions have been associated with a group of neurodegenerative disorders including Huntington disease
2
, spinobulbar muscular atrophy
3
, spinocerebellar ataxia type 1 (SCA1)
4
, spinocerebellar ataxia type 2 (SCA2)
5-7
, spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD)
8
, and dentatorubral-pallidoluysian atrophy/Haw-River syndrome
9
. All these disorders are progressive diseases leading to degeneration of the neurons in central nervous system. The CAG repeats in the respective genes show length polymorphism in the human population, typically, not exceeding 40 repeats. In affected individuals, the expanded alleles contain 36-121 repeats
10
.
CAG repeat expansions are much smaller than t h e hundreds or thousands of repeats often seen in diseases with CGG, CTG, and GAA expansions
11-14
. The expanded CAG alleles show variable degrees of instability in both germline and somatic tissuesl
15,16
. Intergenerational changes of the CAG repeat size are often biased toward further expansion, particularly if paternally transmitted, providing the molecular basis for anticipation. The CAG repeat arrays in these diseases are located in the coding regions of the involved genes and are translated into polyglutamine tracts in the protein products
17
. It has been postulated that an expansion of the polyglutamine tract produces a gain of function in the protein product in each disease accounting for the dominant inheritance. Based on the relatively uniform characteristics of diseases caused by CAG repeat expansions, it has been speculated that other neurodegenerative diseases with similar clinical characteristics may have expansions of CAG repeats. Indeed, a study by Trottier and colleagues demonstrated that an antibody against a polyglutamine tract detects abnormally large proteins in tissues from patients with either SCA2 or spinocerebellar ataxia type 7 (SCA7), suggesting that the mutation responsible for SCA2 and SCA7 is an expansion of a polyglutamine repeat tract
18
.
The prior art is deficient in the lack of effective means for the large scale genotyping of genetic diseases and diagnostic tests and kits for diagnosing such diseases. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
A polymorphic CAG repeat was identified in the human &agr;
1A
voltage-dependent calcium channel subunit. To demonstrate that expansion of this CAG repeat could be the cause of an inherited progressive ataxia, a large number of unrelated controls and ataxia patients were genotyped. Eight unrelated patients with late onset ataxia had alleles with larger repeat numbers (21-27) compared to the number of repeats (4-16) in 475 non-ataxia individuals. Analysis of the repeat length in families of the affected individuals revealed that the expansion segregated with the phenotype in every patient. Six isoforms of the human &agr;
1A
calcium channel subunit were identified. The CAG repeat is within the open reading frame and is predicted to encode glutamine in three of the isoforms. Thus, a small polyglutamine expansion in the human &agr;
1A
calcium channel is most likely the cause of a newly classified autosomal dominant spinocerebellar ataxia, SCA6.
In one object of the present invention, there is provided a method of screening individuals at risk for developing diseases caused by trinucleotide repeat sequence instability, comprising the steps of: amplifying genomic DNA trinucleotide repeat sequences in a sample from an individual by polymerase chain reaction using one or more oligonucleotide primers; restricting said amplified genomic DNA trinucleotide repeat sequences with a restriction enzyme; separating said restricted amplified genomic DNA trinucleotide repeat sequences by electrophoresis to form a sample electrophoresis pattern; labeling a probe capable of detecting said amplified genomic DNA trinucleotide repeat sequences in said sample; hybridizing said sample of restricted, amplified genomic DNA trinucleotide repeat sequences with a first aliquot of said labeled probe under hybridizing conditions to produce a sample hybridization pattern for said sample genomic DNA trinucleotide repeat sequence; amplifying a control genomic DNA trinucleotide repeat sequence by polymerase chain reaction using said one or more oligonucleotide primers, wherein said control genomic DNA trinucleotide repeat sequence is from non-diseased source; restricting said control genomic DNA trinucleotide repeat sequence with a restriction enzyme; separating said restricted control genomic DNA trinucleotide repeat sequence b y electrophoresis to form a control electrophoresis pattern; combining said restricted control genomic DNA trinucleotide repeat sequence with a second aliquot of said probe under hybridizing conditions to form a control hybridization pattern for said genomic DNA trinucleotide repeat sequence; comparing said sample hybridization pattern for said sample genomic DNA trinucleotide repeat sequence to said control hybridization pattern for said control genomic DNA trinucleotide repeat sequence; and determining whether said individual to be tested may be at risk for developing diseases caused by trinucleotide repeat sequence instability, wherein if said sample genomic DNA trinucleotide repeat sequence is larger than said control genomic DNA trinucleotide repeat sequence, said individual may be at risk for developing diseases caused by trinucleotide repeat sequence instability.
In another object of the present invention, there is provided a method of identifying genes in which a disease-causing allele is due to trinucleotide repeat sequence instability, comprising the steps of: screening a library with an oligonucleotide having a triplet base repeat; identifying clones which have said triplet base repeat; sequencing said identified clones to determine sequences of nucleotides flanking said triplet base repeat; synthesizing primers complementary to said sequences of nucleotides flanking said triplet base repeat; isolating DNA from a large sampling of individuals, including diseased and non-diseased individuals; amplifying said isolated DNA with said primers to produce amplified triplet base repeat regions; determining a number of triplet base repeats in said triplet base repeat region for each of said individuals in said large sampling; determining whether triplet base repeat expansions are observed at a relatively high frequency in diseased individuals but are absent or occur at very low frequency in non-disease individuals, wherein if triplet base repeat expansions are observed at a relatively high frequency in diseased individuals but are absent or occur at very low frequency in non-disease individuals, it is likely that a disease-causing allele is due to trinucleotide repeat sequence instability.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
REFERENCES:
Ishikawa et al “Japanese Families with Autosomal Dominant Pure Cerebellar Ataxia Map to Chromosome 19p13.1-p13.2” Am. J. Human Genet. vol. 61, pp. 336-346, 1997.*
Shizuka et al “Molecular analysis of de novo mutation for SCA6 and (CAG)n repeat units in normal elder controls” J. of Neurological Sciences. Vol. 161, pp. 85-87, 1998.*
Yabe et al “SCA6 mutation analysis in a large cohort of the Japanese patients with late-onset pure cerebellar ataxia” J. of Neurological Sciences. Vol. 156, pp. 89-95, 1998.*
Nagai et al “Clinical and molec
Adler Benjamin Aaron
Arthur Lisa B.
Goldberg Jeanine
Research Development Foundation
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