Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-25
2002-01-01
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06335165
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to methods and kits for characterizing GC rich nucleic acid sequences. More particularly, the present invention relates to methods and kits for amplification, size determination and sequencing of GC rich nucleic acid sequences. Most particularly, the present invention relates to methods and kits for amplification, size determination and sequencing of GC rich nucleic acid sequences, such as the trinucleotide repeats in the FMRI gene causing the Fragile X syndrome, and other genes.
Triple Repeat Mutations
Trinucleotide repeats are the sites of mutation in several heritable human disorders. These repeats are usually GC rich (e.g., over 65% GC) and are highly polymorphic in the normal population. Fragile X syndrome and myotonic dystrophy (DM) are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of penetration has so far been identified in the following diseases: Fragile X syndrome (FRAXA); spinal and bulbar muscular atrophy (SMBA); myotonic dystrophy (DM); Huntington's disease (HD); spinocerebrellar ataxia type 1 (SCA1) fragile XE (E site) mental retardation (FRAXE-MR) and dentatorubral pallidoluysian atrophy (DRAPLA). Triplet repeats are found both near to, and within additional genes, as one can learn from screening data bases of gene sequences. It is probable that in the future it will be found that the same penetration mechanism is responsible for the existence of additional genetic diseases.
Of the seven diseases listed hereinabove, Fragile XA is the most common. It is a recessive X-linked genetic disorder (therefore affecting mostly males) with an incomplete penetrance. The syndrome is difficult to diagnose in newborns and the disease features accumulate slowly with age. Developmental delay and mental retardation are the predominant clinical features of Fragile X syndrome. Mental retardation varies from extreme to borderline with the average IQ in the moderately retarded range. Female patients are more mildly affected, with few somatic signs and generally the retardation falls in to the mild—borderline category. Fragile-XA is one of the most common forms of mental retardation. It is the most common cause (one in 1500 males and one in 2500 females) of mental retardation from a single gene defect. It is also one of the commonest heritable disorders and the most common familial, heritable mental retardation. Furthermore, as the Fragile XA disease appears in all ethnic groups studied so far, it may be considered one of the most common single-gene disorders found in humans.
Fragile XA is characterized by an incomplete penetrance, consequently (i) some males are normal transmitting males' (NTMs); They are clinically normal, but their positions in the genetic pedigree makes them obligate carriers of the mutated allele. (ii) About a third of the carrier women (heterozygotes) exhibit slight symptoms of mental disturbances. The gene responsible for the syndrome was located to chromosomal position Xq27.3. Once it was cloned, it was found that within the gene there is a (CGG)n repeat that is highly polymorphic in the number (n) of repeats. This sequence is located in the 5′ non-translated region of the gene. A survey conducted among healthy individuals and among individuals who suffer from the syndrome has shown that the number of triplet repeats of the sequence CGG in said polymorphic locus in the first group (normal individuals) is lower than the number of such repeats in the second group (that suffers from the syndrome). While the number of CGG repeats that characterizes X chromosomes derived from healthy individuals is low, e.g., 6-52; the number of repeats in carriers is medium, e.g., 50-200; and the number of triplet repeats in individuals who suffer from the syndrome is high, e.g., 230-1000.
It was also found that when the number of CGG repeats in the FMR1 gene increases over 230, the DNA in the 5′ region of the gene is characterized by an abnormal number of methylated cytosine residues. This methylation covers also the promoter region of the gene and therefore causes its failure to replicate and the lack of expression of the FMR1 protein. This lack of expression and the changes in structure and organization of the DNA are most probably the direct molecular cause for the phenotype associated with the fragile XA syndrome (Caskey et al., Science, 1992, 256(5058):784-9; Pieretti et al., Cell, 1991, 66(4):817-22; and Annemieke, Cell, 1991, 65:905-914.
NTMs carry numbers of CGG repeats outside the range of normal and below those found in affected males. Such males transmit the repeats to their progeny with relatively small changes in the number of repeats. On the other hand, females who carry similar premutation alleles are prone to bear progeny (male or female) with large expansion of the repeats region. Thus, large CGG amplification associated with fragile XA syndrome appears to be predominantly a female meiotic event. See, Caskey et al. Science, 1992, 256:784-789.
Many fragile XA diseased individuals were found to be mosaic with respect to the number of the CGG trinucleotide repeats characterizing different cells in their body, a phenomenon indicating somatic instability of expanded repeats.
Instability, characterized by expansion of trinucleotide repeats, is observed also in DM, HD, FRAXE, DRPLA and SCA1 pedigrees. As opposed to FRAXA, DM and FRAXE high risk alleles can expand to similar extent through both male and female meioses and, to the best of our knowledge, somatic mosaicism has not yet been observed in DM and FRAXE patients. High risk alleles have yet to be found for HD and DRPLA, that is, alleles of these diseases either cause or do not cause the disease. Nevertheless, HD repeats are also unstable in more than 80% of meiotic transmissions; on the other hand, they are characterized by increasing, or alternatively, decreasing number of repeats with the largest increase occurring in paternal transmission (Duyao. et al. Nature Genetics, 1993, 4:387-392), whereas DRPLA alleles have a tendency to increase in size along generations. See, Nagafuchi et al. Nature Genetics, 1994, 6:14-18; Koide et al. Nature Genetics, 1994, 6:9-13.
Attempts to correlate the size of trinucleotide repeat mutations and the severity of the associated genetic diseases were made for Fragile XA syndrome, Myotonic Dystrophy, Dentatorubral Pallidoluysian Atrophy and Spinocerebellar Ataxia Type 1.
For Fragile XA, as expected, median IQ score was significantly lower for females carrying a fully expanded mutation (above 230 repeats) than for females carrying a premutation (50-200 repeats) on one of their X chromosomes. On the other hand, no significant relationship was found between IQ score and number of CGG repeats, see, Taylor et al. JAMA, 1994, 271:507-514. Nevertheless, it was found that prenatal DNA studies of the number of trinucleotide repeats characterizing Myotonic Dystrophy alleles can improve the estimation of clinical severity; and that the number of CAG trinucleotide repeats in Spinocerebellar Ataxia Type 1 and Dentatorubral Pallidoluysian atrophy is correlated with increased progression of the disease (Nagafuchi et al. Nature Genetics, 1994, 6:14-18; Koide et al. Nature Genetics, 1994, 6:9-13; Orr et al. Nature Genetics, 1993, 4:221-226).
Attempts to correlate between the size of trinucleotide repeat mutations and the age of onset of Huntington's Disease resulted in finding a reverse correlation confined to the upper range of trinucleotide repeat numbers (ca. 60-100 repeats), see Andrew S. E. et al. (1993) Nature Genetics, 4:398-403.
Furthermore, for Spinocerebellar Ataxia Type 1 and Dentatorubral Pallidoluysian Atrophy (Nagafuchi S. et al. (1994) Nature Genetics, 6:14-18; Koide R. et al. (1994) Nature Genetics, 6:9-13), a direct correlation between the number of the (CAG)n trinucleotide repeats expansion and earlier ages of onset was found.
Amplificatio
Lederkremer Martine
Navot Nir
Gamidagen Ltd.
Horlick Kenneth R.
Tung Joyce
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