A novel PCR-based approach for the detection of the Huntington disease associated trinucleotide repeat expansion

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder associated with expansions of an unstable CAG trinucleotide repeat in exon 1 of the IT15 gene. In normal individuals, IT15 contains up to 35 CAG repeats, while in affected the repeat length is >36. Polymerase chain reaction (PCR) is used to estimate the number of CAG repeats but may be inefficient in long repeats because of the high C+G content of the HD locus. We present a novel PCR approach for the diagnosis of HD, which permits direct visualization of the amplified products on agarose gel, using ethidium bromide. It is based on the methylation-sensitive conversion of C residues to U by bisulfite treatment of single-stranded DNA and subsequent amplification of the sense strand with specific primers. The bisulfite treatment dramatically reduces the C + G content of the region; thus, the high Tm and stable secondary structures are no longer obstacles to PCR. In both normal and affected individuals, UAG repeats (5'- CAG-3', before bisulfite treatment) in the sense strand can easily be amplified and visualized on a gel by ethidium bromide staining. The method has considerable advantages compared with other described PCR-based diagnostic tests for HD.     

Trinucleotide repeat length and progression of illness in Huntington''s disease.

The genetic defect causing Huntington's disease (HD) has been identified as an unstable expansion of a trinucleotide (CAG) repeat sequence within the coding region of the IT15 gene on chromosome 4. In 50 patients with manifest HD who were evaluated prospectively and uniformly, we examined the relationship between the extent of the DNA expansion and the rate of illness progression. Although the length of CAG repeats showed a strong inverse correlation with the age at onset of HD, there was no such relationship between the number of CAG repeats and the rate of clinical decline. These findings suggest that the CAG repeat length may influence or trigger the onset of HD, but other genetic, neurobiological, or environmental factors contribute to the progression of illness and the underlying pace of neuronal degeneration.     

Parental factors associated with rigidity in Huntington''s disease.

The discriminatory power of ten factors has been explored in relation to the presence or absence of muscular rigidity in patients with Huntington's disease. The sex and neurological sign of an affected parent were the only two significant determinants of rigidity or choreoathetosis in offspring. It was shown, using the Mantel-Haenszel method of adjusting for confounding variables that the risk of a patient displaying rigidity (and thereby having a graver prognosis) is five times as great for those with rigid parents as it is for those with non-rigid parents. Additionally, the risk of a patient displaying rigidity is more than three times as great for those with affected fathers as it is for those with affected mothers. Some implications of these findings are discussed.     

Gametic but not somatic instability of CAG repeat length in Huntington''s disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntington's disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.     

Meiotic instability associated with the CAGR1 trinucleotide repeat at 13q13.

CAGR1 is a recently characterised polymorphic trinucleotide repeat localised to 13q13, which has been suggested as a possible candidate gene for neurological disorders that manifest genetic anticipation. To provide evidence in support of this hypothesis, a large number of chromosomes (n = 928) from patients with a wide variety of neurological diseases were screened for evidence of repeat expansion and meiotic instability. One person with a CAGR1 repeat number of 50 was identified (normal range 9-29). Subsequent molecular analyses of CAGR1 repeat number in additional family members showed meiotic instability of a (CAG)45 allele through three generations. While CAGR1 repeat number did not correlate with a readily discernible phenotype in this family, the finding of meiotic stability and mendelian inheritance of normal CAG alleles and meiotic instability of larger repeats fulfil several criteria thought essential for pathologically relevant mutations of this type. Thus, these data strengthen the hypothesis for a role of CAGR1 in the development of an as yet molecularly uncharacterised human neurological disease.     

Huntington''s disease in Tanzania.

Huntington's disease was studied in a Bantu community in northern Tanzania. Although there is evidence to suggest that the disease has been present here for over one hundred years, this is the first report of the condition in Tanzania. A survey of published reports indicates that the disease is infrequently reported in persons of Negro ancestry.     

An association study of Huntington''s disease and HLA.

HLA antigens were determined in a sample of 47 patients with the diagnosis and family history of Huntington's disease (HD). Two groups consisting of 20 and 11 unrelated patients respectively were investigated. In the first group an increased frequency of HLA-Bw44 (p less than 0.05) and of HLA-A11 (p less than 0.05) was found, but after correcting for multiple inferences the differences were no longer statistically significant. No correlation was found between sex, age of onset, initial symptom, and HLA type. In order to find out if the increased frequency of HLA-Bw44 was real or due to chance, the second group of patients was investigated. In this group the frequency of HLA-Bw44 did not differ from the normal population and a strong association between HLA and HD could be excluded.     

Analysis of the trinucleotide repeat expansion in Italian families affected with Huntington disease

150 subjects affected with HD and 45 at high risk for the diseasewere typed for the CAG trinucleotide repeat at the 5‘end of IT15. In all of them we find expanded segments showingmarked instability upon transmission. Their length distributionmatches those previously reported and inversely correlates (–0.686)with age at onset. Two at risk sibs from a large HD pedigreeshow expanded segments that overlap the normal distributionand can represent reductions from the HD to the normal range.A case of instability on a normal chromosome is also reported.Finally, an analysis of the CAG repeat as a function of threepolymorphic DNA markers at D4S127 and D4S95 loci shows no significantdifference in the average repeat length on HD chromosomes groupedaccording to the cosegregating allele of each marker or to thecorresponding haplotype. Despite the marked heterogeneity inrepeat length among HD families, haplotypes are not associatedwith different values around which the repeat length fluctuates.     

Huntington''s disease in Grampian region: correlation of the CAG repeat number and the age of onset of the disease.

The identification of an unstable trinucleotide repeat as the mutation responsible for Huntington's disease (HD) has given the hope that additional information can be provided about age of onset and mode of action of the mutated gene. We present in this paper results of a clinical and molecular study of 82 patients affected with HD from 46 pedigrees within the Grampian region, Scotland. Our results show a correlation between age of onset and size of the CAG expansion. This study has produced no overlap in mutation size between affected and unaffected alleles. The sex of the parent transmitting the mutated allele and the size of the normal allele have no significant effect on the clinical features of the disease. In the three juvenile cases the affected parent was the father but the number of cases is too small to produce statistical significance. An increase in the CAG repeat size is shown in the transmission of the gene in five cases, accompanied by an earlier age of onset in four; in three of these cases, the affected parent was the father. Eleven sib pairs were studied and there is a negative correlation between the difference in age at onset and the difference in repeat size. Thus there is some evidence of a relationship, but this is not statistically significant because of the small numbers involved. The presence of the same or different normal allele had no effect on age of onset in this small group. We suggest that additional factors, as yet unrecognised, influence the age of onset and clinical presentation of HD.     

安徽入亨廷顿病CAG三核苷酸重复的分子分析

为在分子水平了解安徽人亨廷顿病(HD)的发病机制,为该病的基因诊断和遗传咨询提供科学依据.应用巢式PCR,变性聚丙烯酰胺凝胶电泳以及DNA测序等方法,对6例正常安徽人以及6例HD家系的研究结果表明中国人正常IT15基因(CAG)n重复序列的拷贝数为13-26,而所有被分析的HD患者都携带一个CAG序列高度重复的IT15基因,其(CAG)n的拷贝数为40-94;且CAG重复序列的拷贝数与发病年龄呈现一定的相关性.     

A probable case of mutation in Huntington''s disease.

A patient is described in whom Huntington's disease was diagnosed at the age of 34 years. No evidence of the disorder was found in either parent. Their parentage of the alleged mutant could not be excluded from a study of the inheritance of 25 genetic markers.     

Huntington''s disease: implications of associated cellular radiosensitivity

Ionizing radiation sensitivity was studied in a series of Huntington's Disease (HD) patients and controls by measurement of radiation-induced chromosome aberrations in lymphocytes and by clonogenic survival of lymphoblastoid cell lines. As a group, HD patients were found to be significantly more radiosensitive than controls (p less than 0.001), but there was an overlap between values for the two groups such that an absolute distinction is not possible. These data are consistent with an association between HD and radiosensitivity but not with identity between HD and a radiosensitive phenotype, so that cellular radiosensitivity cannot be used for individual diagnosis. Analysis of three families including 5 HD patients and 11 first-degree relative confirmed this conclusion and demonstrated that even within a given family presymptomatic diagnosis cannot be based on measurement of radiosensitivity. However, the common association of cellular radiosensitivity with HD probands and their families provides a potential lead to the identification of HD gene(s) and so to an eventual understanding of the aetiopathogenesis of this disease at the molecular level.     

Moderate instability of the trinucleotide repeat in spino bulbar muscular atrophy.

Increased length of a protein-coding CAG repeat within the androgen receptor gene appears to be the only type of mutation responsible for spino-bulbal muscular atrophy (SBMA or Kennedy disease). We have analysed a large 4-generation SBMA family and found that the mutant allele was unstable upon transmission from parent to child, with a documented variation from 46 to 53 repeats and a tendency to increase in size (7 increases and a single decrease in 17 events), which appeared stronger upon transmission from a male than from a female. Our results suggest also limited somatic instability of the abnormal allele, with observable variation of up to 2-3 repeats. This indicates that the behavior of the CAG repeat is similar to that observed for small premutations in the fragile X syndrome, or small abnormal alleles in myotonic dystrophy, two diseases which are caused by expansion of an unstable trinucleotide repeat.     

Instability of the FMR2 trinucleotide repeat region associated with expanded FMR1 alleles

The fragile sites FRAXA and FRAXE, located ∼600 kb apart on Xq27.3 and Xq28, respectively, are due to a CGG trinucleotide repeat expansion. Although the expansion mechanism for these and other trinucleotide repeat disorders remains unknown, the similarities between the FRAXA and FRAXE regions suggest a possible association between the 2 sites. DNA from 953 individuals was analyzed to determine the distribution of FRAXE repeat sizes in this population and to ascertain potential association between FRAXA and FRAXE repeat sizes. Thirty-four FMR2 alleles ranging from 3–42 repeats were identified. No FRAXE expansions were found in this population, supporting previous findings that FRAXE expansions are rare. However, in the fragile X syndrome affected group, a FMR2 delection, 2 cases of FRAXE repeat instability and a FRAXE mosaic male were identified. Also, a previously identified, rare FMR2 polymorphism was observed. Statistical analyis showed no correlation between normal FRAXA and FRAXE repeat sizes studied, although there was a significant size difference in larger FMR2 alleles that segregated with expanded FMR1 alleles. These findings support the idea of an association between repeat expansion in the FMR1 gene and instability or deletions in the FMR2 gene. Am. J. Med. Genet. 73:447–455, 1997. © 1997 Wiley-Liss, Inc.     

Candidate DNA replication initiation regions at human trinucleotide repeat disease loci

The positions of DNA replication initiation regions (IRs) at three human trinucleotide repeat (TNR) disease loci were examined in order to characterize the role played by IRs in explaining the known locus-specific variation in TNR instability levels. Using three different normal cell lines, candidate IRs were identified at the HD, SCA-7 and SBMA loci. At each locus the IR is less than 3.6 kb from the CAG/CTG repeat tract. Preliminary studies with a cell line homozygous for an HD disease mutation indicated no change in the position of the candidate IR in spite of the mutation. Comparison with experimental results from model systems suggests that a complex relationship may exist between instability and the proximity and/or orientation of the repeats with respect to an IR.     

Population genetics of trinucleotide repeat polymorphisms

Trinucleotide repeats at five disease loci (DM, DRPLA, HD, SBMAand SCA1) were surveyed in phenotypically normal individualsfrom three continental populations. This is the first analysisto examine the population dynamics of these five disease-relatedtrinucieotide repeats in the same individuals from worldwidepopulations. Roughly half of all alleles observed at each locusare shared between all continental groups. For three loci, diseaseprevalence in each population corresponds with the number ofalleles in the upper tail of the allele-size distribution. Theallele-size distributions of African, Asian and Caucasian groupsshow a high degree of variation, and gene diversity estimatesfor trinucieotide repeat loci exceed estimates derived fromdinucleotide or tetranucleotide repeats. Analyses that comparedinfinite alleles and stepwise mutation models suggest that normalvariation at trinucieotide loci is not generated by stepwisemutation alone. Trees constructed for subpopulations using trinucieotiderepeat loci show accurate continental clustering. Interpopu-lationgenetic distance estimates show remarkable similarity to distanceestimates produced from tetranucleotide repeats or nuclear restrictionsite polymorphisms. This finding is especially noteworthy inlight of the fact that trinucieotide repeat polymorphisms atthese loci can cause disease, while restriction site and tetranucleotidepolymorphisms appear to be selectively neutral. In contrast,genetic distance estimates from trinucieotide loci are poorlycorrelated with genetic distance estimates from mitochondrialsequence data.