脆性X综合征的基因诊断与产前诊断

为了探讨简便、快速、准确、价廉的脆性X综合征的诊断方法,对6个智能低下家系进行了细胞遗传学检查,以及PCR直接扩增FMR1 5^'端(CGG)_{n<\sub>重复序列、RT-PCR扩增FMR1基因的cDNA序列的分子遗传学检查。A家系先证者脆性X染色体高表达（35/273）,分子遗传学检查证实为脆性X综合征全突变患者;B家系先证者及其母亲无脆性X染色体表达,分子遗传学检查证实为非脆性X综合征患者;C家系的男性胎儿脆性X染色体表达（5/93）,先证者及其母亲未发现脆性X染色体,分子遗传学检查证实男性胎儿为脆性X综合征全突变患者,其母亲为前突变携带者,哥哥为嵌合体患者;D家系先证者脆性X染色体高表达17%,其姐姐脆性X染色体5%,分子遗传学检查证实先证者为脆性X综合征全突变患者,其姐姐为嵌合体患者;E家系先证者及其母亲,F家系先证者发现可疑脆性X染色体,分子遗传学检查证实为非脆性X综合征家系。结论： PCR直接扩增FMR1基因(CGG)n<\sub>重复序列联合RT-PCR扩增FMR1基因cDNA 序列简便、快速、价廉。可用于脆性X综合征的筛查、诊断及产前诊断,有推广应用价值。}     

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG•CCG repeat tract in the 5′ UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of γ-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.     

Fragile-X syndrome: Unique genetics of the heritable unstable element

The fragile site at Xq27.3 is an unstable microsatellite repeat, p(CCG)n. In fragile-X syndrome pedigrees, this sequence exhibits variable amplification, the length of which correlates with fragile-site expression. There is a direct relationship between increased p(CCG)n copy number and propensity for instability: individuals having large amplifications exhibit somatic variation due to increased instability. The instability of the p(CCG)n repeat, when transmitted through affected pedigrees, explains the unusual segregation patterns of fragile-X phenotype, referred to as the Sherman paradox. All individuals of fragile-X genotype were found (where testing was possible) to have a parent with amplified p(CCG)n repeat, indicating that few, if any, cases of fragile-X syndrome are not familial.     

Molecular understanding of aluminum-induced topological changes in (CCG)12 triplet repeats: relevance to neurological disorders

Recent studies have shown that gene mutations are involved in the pathology of neurological disorders. CCG repeats cause genetic instability and are localized at the 5' end of the non-coding regions of the FMR1 gene in fragile X syndrome. Our studies for the first time showed that aluminum (Al) levels were elevated in the serum samples of fragile X syndrome and also provide evidence for the interaction of aluminum with (CCG)12-repeats. Circular dichroism spectroscopic studies of (CCG)12 indicated B-DNA conformation and in the presence of Al (10(-5) M) CCG repeats attained Z-DNA conformation. Further spectroscopic studies, which included melting profiles, ethidium bromide binding patterns and interaction of Z-DNA specific polyclonal antibodies confirmed the Z-conformation in (CCG)12-repeats in the presence of Al (10(-5) M). It is interesting to mention that Al-induced Z-conformation is stable even after the total removal of Al from CCG by desferoximine, a chelating drug. This is the first report to proof the role of Al in modulating the DNA (CCG repeats) topology and this information provides a clue about the possible involvement of Al at a molecular level in neurological/neurodegenerative disorders.     

Expansion of the CGG repeat in fragile X in the FMR1 gene depends on the sex of the offspring.

Analysis of 139 mother-to-offspring transmissions of fragile X CGG triplet repeats revealed that the repeat expansion is enhanced in mother-to-son transmissions compared with mother-to-daughter transmissions. Evidence has been based on analysis of mother-offspring differences in the size of repeat (in kb), as well as on comparisons between proportions of male and female offspring with premutations, and full mutations, inherited from mothers carrying a premutation. Mean difference in the repeat size from mother-son transmissions was 1.45 kb, compared with mother-daughter transmissions of 0.76 kb. The difference is due primarily to a greater proportion of male than female offspring with full mutation from the premutation mothers and also to a higher frequency of reduction in repeat size from mothers to daughters than from mothers to sons. Our findings suggest the possibility of an interaction of the normal X homologue in a female zygote with the FMR1 sequence on the fragile X during replication to account for the lower level of expansion in mother-to-daughter transmissions relative to mother-to-son transmissions.     

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.     

Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats

The fragile X syndrome results from expansions as well as deletions of the repeating CGG.CCG DNA sequence in the 5'-untranslated region of the FMR1 gene on the X chromosome. The relative frequency of disease cases promoted by these two types of mutations cannot be ascertained at present because the routine clinical assay monitors only expansions. At least 30 articles have been reviewed that document the involvement of deletions of part or all of the CGG.CCG repeats along with varying extents of DNA flanking regions as well as very small mutations including single base pair changes. Studies of deletion mutants of CGG.CCG tracts in Escherichia coli plasmids revealed a similar spectrum of mutagenic products. The triplet repeat tract in a non-B conformation is the mutagen, not the sequence per se in the right-handed B helix. Hence, molecular investigations in a simple model organism may generate useful initial information toward therapeutic strategies for this disease.     

Noninvasive test for fragile X syndrome, using hair root analysis.

Identification of the FMR1 gene and the repeat-amplification mechanism causing fragile X syndrome led to development of reliable DNA-based diagnostic methods, including Southern blot hybridization and PCR. Both methods are performed on DNA isolated from peripheral blood cells and measure the repeat size in FMR1. Using an immunocytochemical technique on blood smears, we recently developed a novel test for identification of patients with fragile X syndrome. This method, also called "antibody test," uses monoclonal antibodies against the FMR1 gene product (FMRP) and is based on absence of FMRP in patients' cells. Here we describe a new diagnostic test to identify male patients with fragile X syndrome, on the basis of lack of FMRP in their hair roots. Expression of FMRP in hair roots was studied by use of an FMRP-specific antibody test, and the percentage of FMRP-expressing hair roots in controls and in male fragile X patients was determined. Control individuals showed clear expression of FMRP in nearly every hair root, whereas male fragile X patients lacked expression of FMRP in almost all their hair roots. Mentally retarded female patients with a full mutation showed FMRP expression in only some of their hair roots (<55%), and no overlap with normal female controls was observed. The advantages of this test are (1) plucking of hair follicles does no appreciable harm to the mentally retarded patient, (2) hairs can be sent in a simple envelope to a diagnostic center, and (3) the result of the test is available within 5 h of plucking. In addition, this test enabled us to identify two fragile X patients who did not show the full mutation by analysis of DNA isolated from blood cells.     

Fragile X Premutations Are Not a Major Cause of Early Menopause

Fragile X syndrome is an X-linked mental retardation condition that usually is due to a trinucleotide-repeat expansion in the FMR1 gene. Whereas full-mutation alleles (> 230 repeats) lead to fragile X syndrome, premutation alleles (approximately 60-200 repeats) are apparently non-penetrant. However, previous studies have suggested that female premutation carriers may have an increased incidence of premature menopause. To test this possible association, we screened for premutation alleles among 216 women with early menopause (at age < 47 years), 33 of whom had premature menopause (at age < 40 years), as well as among 107 control women, all of whom were ascertained solely on the basis of age at menopause. No full-mutation alleles were found; and only one premutation allele was found, but, it was in a member of the control group. These results are consistent with what would be expected on the basis of chance only. Our sample size was sufficient to rule out a > or = 3-fold increased risk of early menopause and a > or = 9-fold increased risk of premature menopause due to an FMR1 premutation, under a model considering the risk of both sporadic and familial early menopause. Likewise, our results rule out a > or = 4-fold increased risk of familial early menopause and a > or = 26-fold increased risk of familial premature menopause, under a less probable model in which only familial early menopause is considered. These results indicate that the fragile X premutation is not a major risk factor for early menopause and suggest that the risk of premature menopause to fragile X-premutation carriers may not be as great as that reported elsewhere.     

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.     

Rare fragile sites

Rare folate-sensitive fragile sites are the archetypal trinucleotide repeats. Although the CAG repeat in the androgen receptor, associated with spinobulbar muscular atrophy, was the first to be published in 1991, it was the publication in the same year of the molecular basis of fragile X that focused much attention on trinucleotide repeat expansion as a mutational mechanism. A number of rare fragile sites have had their repeat elements characterised since that time. The so-called "folate-sensitive" fragile sites are likely to be all CCG repeat expansions similar to the fragile X. The folate insensitive fragile sites have more complex longer repeat elements. Only two rare fragile sites (FRAXA and FRAXE) are of unequivocal clinical significance in that they are associated with intellectual disability.     

A fragile gene

Fragile X syndrome is the most common cause of inherited mental retardation in humans. The fragile X gene (FMR1) has been cloned and the mutation causing the disease is known. The molecular basis of the disease is an expansion of a trinucleotide repeat sequence (CGG) present in the first exon within the 5′ untranslated region of the FMR1 gene. Affected individuals have repeat CGG sequences of above 200. As a result the gene is not producing protein. It has been shown that the FMR1 protein has RNA binding activity, but the function of this RNA binding activity is not known. The timing and mechanism of repeat amplification are not yet understood. An animal model for fragile X syndrome has been generated, which can be used to study the clinical and biochemical abnormalities caused by absence of FMR1 protein product.     

Fragile X gene instability: anchoring AGGs and linked microsatellites.

Interspersed AGGs within the FMR1 gene CGG repeat region may anchor the sequence and prevent slippage during replication. In order to detect the AGG position variations, we developed a method employing partial MnlI restriction analysis and analyzed X chromosomes from 187 males, including 133 normal controls (117 with 20-34 and 16 with 35-52 repeats), plus 54 fragile X premutations with 56-180 repeats. Among controls, the interspersed AGG positions were highly polymorphic, with a heterozygosity of 91%. Among the control samples, 1.5% had no AGG positions, 25% had one, 71% had two, and 3% had three. Among the fragile X premutation samples, 63% had no AGG, while 37% had only one AGG. Analysis of premutation samples within fragile X families showed that variation occurred only within the 3' end of the region. Thus, the instability was polar. Controls with > or = 15 pure CGG repeats were associated with the longest alleles of two nearby microsatellites, FRAXAC1 with 20-21 repeats and DXS548 with 202-206 bp and with increased microsatellite heterozygosity. The association of long pure CGG regions, as with fragile X chromosomes, with the longer and more heterozygous microsatellite alleles suggests they may be related mechanistically. Further, our results do not support a recent suggestion that the frequency of fragile X alleles may be increasing. Finally, analysis of a set of nonhuman primate samples showed that long pure CGG tracks are variable in size and are located within the 3' region, which suggests that polar instability within FMR1 is evolutionarily quite old.