Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55–74, 75–89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG (adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75–89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.     

Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual

Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5′ untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.     

CAG/CTG and CGG/GCC Repeats in Human Brain Reference cDNAs: Outcome in Searching for New Dynamic Mutations

CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]nand [CGG]nwith more than seven to nine units were rare (1/2000), and perfect [CAG]n9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n> 9 and [CGG]n> 7 were characterized. The comparison of our data with other [CAG]nand [CGG]nresources suggests that the screening of reference cDNAs leads to unique sources of new [CAG]nand [CGG]nand will enhance the study of enlarged triplet repeats in human genetic diseases.     

PCR approach for detection of Fragile X syndrome and Huntington disease based on modified DNA: limits and utility

A group of mutations characterized by trinucleotide repeat expansion causes human diseases such as the Fragile X syndrome, Huntington disease (HD), and myotonic dystrophy. Methods based on PCR amplification of the CGG and CAG repeats region could facilitate the development of a rapid screening assay; unfortunately, amplification across CGG and CAG repeats can be inefficient and unreliable due to the G + C base composition. The utility of the PCR on modified DNA for amplification of the CGG and CAG repeats at the Fragile X syndrome and HD has been reported. In the present study, we analyzed the utility of PCR on modified DNA as a rapid screening method for diagnosis of patients with Fragile X syndrome and HD. A comparative analysis realized with 38 Fragile X and 29 HD patients showed that the molecular diagnosis by simple PCR on modified DNA has a sensitivity and specificity of 100% in Fragile X patients and 94.1% and 91.6% in HD patients. The results achieved from the statistical analysis allowed us to conclude that the amplification by simple PCR on modified DNA is a reliable and useful method for the molecular diagnosis of the Fragile X syndrome, but not for the HD.     

Incidence of fragile X in 5,000 consecutive newborn males

Fragile X syndrome (FXS) is the commonest cause of inherited mental retardation in males. Even though this affirmation is repeated in virtually all papers referring to FXS, the precise frequency of this syndrome in the general population is unknown. We present a general population screening analyzing an anonymous series of 5,000 consecutive newborn males from the neonatal screening program of the population of Catalonia in Spain. The aim of the study is to determine the incidence of FXS via a simple and economical methodology based on the nonamplification of the fragment containing the CGG repeats of the FRAXA locus in the samples carrying alleles over 52 repeats. From the initial 5,000 samples, 4,920 were in the normal range, 15 gave rise to bands with more than 52 repeats (11 corresponded to intermediate alleles and four premutated alleles). After further studies, two samples were considered to be carriers of full mutations. According to these results, the incidence of FXS affected newborn males is 1 in 2,466, and 1 in 1,233 males is a carrier of the premutation. We can deduce that 1 in 8,333 is an affected female with clinical manifestations and 1 in 411 will be a premutation carrier woman. Upon reviewing the literature, there seems to be variability in the frequencies found by the different groups. Therefore, given that our study is limited to the Catalan population in Spain, these results should be taken as valid for the Catalan region and should only be extrapolated to other populations with caution.     

Stable DNA Methylation Boundaries and Expanded Trinucleotide Repeats: Role of DNA Insertions

The human genome segment upstream of the FMR1 (fragile X mental retardation 1) gene (Xq27.3) contains several genetic signals, among them is a DNA methylation boundary that is located 65–70 CpGs upstream of the CGG repeat. In fragile X syndrome (FXS), the boundary is lost, and the promoter is inactivated by methylation spreading. Here we document boundary stability in spite of critical expansions of the CGG trinucleotide repeat in male or female premutation carriers and in high functioning males (HFMs). HFMs carry a full CGG repeat expansion but exhibit an unmethylated promoter and lack the FXS phenotype. The boundary is also stable in Turner (45, X) females. A CTCF-binding site is located slightly upstream of the methylation boundary and carries a unique G-to-A polymorphism (single nucleotide polymorphism), which occurs 3.6 times more frequently in genomes with CGG expansions. The increased frequency of this single nucleotide polymorphism might have functional significance. In CGG expansions, the CTCF region does not harbor additional mutations. In FXS individuals and often in cells transgenomic for EBV (Epstein Barr Virus) DNA or for the telomerase gene, the large number of normally methylated CpGs in the far-upstream region of the boundary is decreased about 4-fold. A methylation boundary is also present in the human genome segment upstream of the HTT (huntingtin) promoter (4p16.3) and is stable both in normal and Huntington disease chromosomes. Hence, the vicinity of an expanded repeat does not per se compromise methylation boundaries. Methylation boundaries exert an important function as promoter safeguards.     

A Dual-Mode Single-Molecule Fluorescence Assay for the Detection of Expanded CGG Repeats in Fragile X Syndrome

Fragile X syndrome is the leading cause of inherited mental impairment and is associated with expansions of CGG repeats within the FMR1 gene. To detect expanded CGG repeats, we developed a dual-mode single-molecule fluorescence assay that allows acquisition of two parallel, independent measures of repeat number based on (1) the number of Cy3-labeled probes bound to the repeat region and (2) the physical length of the electric field-linearized repeat region, obtained from the relative position of a single Cy5 dye near the end of the repeat region. Using target strands derived from cell-line DNA with defined numbers of CGG repeats, we show that this assay can rapidly and simultaneously measure the repeats of a collection of individual sample strands within a single field of view. With a low occurrence of false positives, the assay differentiated normal CGG repeat lengths (CGG _N , N = 23) and expanded CGG repeat lengths (CGG _N , N = 118), representing a premutation disease state. Further, mixtures of these DNAs gave results that correlated with their relative populations. This strategy may be useful for identifying heterozygosity or for screening collections of individuals, and it is readily adaptable for screening other repeat disorders.     

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

Premutation huntingtin allele adopts a non-B conformation and contains a hot spot for DNA damage

The expansion of a CAG trinucleotide repeat (TNR) sequence has been linked to several neurological disorders, for example, Huntington's disease (HD). In HD, healthy individuals have 5-35 CAG repeats. Those with 36-39 repeats have the premutation allele, which is known to be prone to expansion. In the disease state, greater than 40 repeats are present. Interestingly, the formation of non-B DNA conformations by the TNR sequence is proposed to contribute to the expansion. Here we provide the first structural and thermodynamic analysis of a premutation length TNR sequence. Using chemical probes of nucleobase accessibility, we found that similar to (CAG)(10), the premutation length sequence (CAG)(36) forms a stem-loop hairpin and contains a hot spot for DNA damage. Additionally, calorimetric analysis of a series of (CAG)(n) sequences, that includes repeat tracts in both the healthy and premutation ranges, reveal that thermodynamic stability increases linearly with the number of repeats. Based on these data, we propose that while non-B conformations can be formed by TNR tracts found in both the healthy and premutation allele, only sequences containing at least 36 repeats have sufficient thermodynamic stability to contribute to expansion.     

<Emphasis Type="Italic">HTT</Emphasis> Gene Premutation Allele Frequencies in the Russian Federation

Huntington disease (Huntington chorea, HD) is a severe neurodegenerative disease determined by polyglutamine. Polyglutamine expansion in exon 1 of the HTT gene causes Huntington disease. To date, less than 35 CAG triplets are suggested to be present in normal alleles. Alleles bearing from 27 to 35 CAG repeats are generally considered as intermediate or premutation. Alleles with the number of CAG repeats varying from 36 to 39 demonstrate reduced penetrance and result in late manifestation of the disease. To date, no studies based on representative samples of Russian residents estimating the frequencies of premutation and alleles with reduced penetrance have been published. Meanwhile, this is extremely important for genetic counseling of patients bearing such alleles and for the calculation of risks for their offspring. In the present study, the analysis of samples of Russian patients with incoming diagnoses of Huntington chorea (N = 1092), Wilson–Konovalov disease (N = 333), Hallervorden–Spatz disease (N = 33) and a control group consisting of 230 unexamined individuals from the Russian Federation under 45 years of age was carried out. A spectrum of CAG repeat length in the HTT gene in patients with HD was obtained. Patients with HD were detected in the samples of patients with incoming diagnoses of Wilson–Konovalov disease and Hallervorden–Spatz disease. This observation indicates the complexity of differential diagnosis of these diseases. An assumption was made that an allele with 36 CAG repeats should be classified as premutation. We confirmed the previously observed trend for the increased number of CAG repeats in the case of paternal transmission of the mutant allele. The frequency of premutation allele of 2.6% in Russian residents was established.     

The risk of fragile X premutation expansion is lower in carriers detected by general prenatal screening than in carriers from known fragile X families

The Fragile X syndrome is the most common cause of inherited mental retardation. For a female premutation carrier, the risk of having a child with a full mutation is positively correlated with the size of the premutation. The current study was performed to evaluate the risk of premutation expansion in the offspring of average-risk carriers detected by general prenatal screening. Over a 4-year period, 9,660 women underwent DNA screening for FMR1 mutation/premutation at the Tel Aviv Sourasky Medical Center. A premutation was defined as a CGG repeat number >50 in the 5' untranslated region (UTR) of exon 1 in the FMR1 gene. The study included only individuals with no family history of X-linked mental retardation or known FMR1 mutations. A premutation was found in 85 women (1 in 114), 68 of whom consented to have prenatal diagnoses in 74 pregnancies. The abnormal allele was transmitted to the offspring in 44 pregnancies. Of these, no change in allele size was noted in 35 pregnancies (79.6%), and expansion within premutation range was evident in 4 pregnancies (9%). In 5 pregnancies (11.4%), expansion to the full mutation was noted. This occurred only in carriers having more than 90 repeats. We conclude that the likelihood of Fragile X premutation expansion to full mutation is significantly lower in individuals ascertained by general prenatal carrier testing than in those from known Fragile X families.     

广西一脊髓小脑共济失调3型家系SCA3/MJD基因突变和多态性的分析

常染色体显性脊髓小脑型共济失调(Autosomal dominant spinocerebellar ataxias, ADCAs)是一种神经系统退行性疾病, 具有高度的遗传异质性, 其中脊髓小脑型共济失调3型(Spinocerebellar ataxias type 3, SCA3)是一种常见的类型。文章通过PCR扩增广西一个脊髓小脑共济失调家系SCA3/MJD基因片段, 用毛细管电泳和测序方法检测了SCA3/MJD基因的CAG重复序列大小、传递特点以及SCA3/MJD基因的变异。结果显示：家系的所有4名患者和3名无症状携带者(Asymptomatic carrier)的SCA3/MJD基因第10外显子中存在异常扩增的CAG重复序列, 重复次数为64~71次; CAG重复次数在具有cgg等位基因的正常个体间传递时保持不变, 提示cgg等位基因不是正常个体两代间CAG重复序列稳定性的影响因素。SCA3/MJD基因中另有两个单碱基点突变, 一个是内含子区的杂合性突变(IVS9-113 T>C), 另一个是外显子区域的错义突变(220 G>A, 220 Glu>Gly)。这两个点突变为首次报道, 但尚不能明确这两个新的点突变对SCA3表型的影响。     

Fragile-X Carrier Screening and the Prevalence of Premutation and Full-Mutation Carriers in Israel

Fragile-X syndrome is caused by an unstable CGG trinucleotide repeat in the FMR1 gene at Xq27. Intermediate alleles (51-200 repeats) can undergo expansion to the full mutation on transmission from mother to offspring. To evaluate the effectiveness of a fragile-X carrier-screening program, we tested 14,334 Israeli women of child-bearing age for fragile-X carrier status between 1992 and 2000. These women were either preconceptional or pregnant and had no family history of mental retardation. All those found to be carriers of premutation or full-mutation alleles were offered genetic counseling and also prenatal diagnosis, if applicable. We identified 207 carriers of an allele with >50 repeats, representing a prevalence of 1:69. There were 127 carriers with >54 repeats, representing a prevalence of 1:113. Three asymptomatic women carried the fully mutated allele. Among the premutation and full-mutation carriers, 177 prenatal diagnoses were performed. Expansion occurred in 30 fetuses, 5 of which had an expansion to the full mutation. On the basis of these results, the expected number of avoided patients born to women identified as carriers, the cost of the test in this study (U.S. $100), and the cost of lifetime care for a mentally retarded person (>$350,000), screening was calculated to be cost-effective. Because of the high prevalence of fragile-X premutation or full-mutation alleles, even in the general population, and because of the cost-effectiveness of the program, we recommend that screening to identify female carriers should be carried out on a wide scale.     

Screening for FXTAS in 95 Spanish patients negative for Huntington disease

Fragile X syndrome is the most common form of hereditary mental retardation. The molecular basis of this syndrome is mainly a CGG expansion in the 5' untranslated region of the FMR1 gene. Expansions with more than 200 CGG repeats abolish gene expression causing the classical fragile X phenotype. Premutation carriers (55-200 CGG) have normal cognitive function with increased risk of developing premature ovarian failure and fragile X-associated tremor-ataxia syndrome (FXTAS). Some clinical features associated with FXTAS, such as tremor, gait ataxia, cognitive decline, and generalized brain atrophy, are also seen in other movement disorders. Ninety-five patients referred for HD, who tested negative for the expansion in the IT15 gene, were screened for FMR1 CGG-repeat expansion. One FMR1 premutation male carrier was detected, giving an FXTAS frequency of 1.6%. Our results highlight that FXTAS is still not well diagnosed; therefore, we recommend FMR1 premutation screenings in all patients with late-onset tremor, ataxia, and cognitive dysfunction.