Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: implications for molecular diagnosis of the fragile X syndrome.

The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.

Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data.

The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.

Impact of carrier status determination for Duchenne/Becker muscular dystrophy by computer-assisted laser densitometry.

Carrier status determination for Duchenne and Becker muscular dystrophies (D/BMD), disorders caused by mutations in the dystrophin gene at Xp21, is complicated by a number of factors. These include a high mutation rate and a 5-10% recombination frequency across the dystrophin gene. For these reasons, linkage analysis frequently gives an inconclusive result, and a direct mutation detection method for females at risk is desirable. Because 65% of the mutations that cause D/BMD are deletions of one or more exons of the dystrophin gene, diagnosis in most affected males is relatively easy using multiplex polymerase chain reaction (PCR) analysis. However, deletion analysis in females is more difficult because of the interference of the normal X chromosome in the deletion assay. We have developed a quantitative PCR-based analysis designated computer-assisted laser densitometry (CALD), which uses the automated fluorescent fragment analysis application of the Applied Biosystems (Foster City, California) automated sequencer. This method has proved to be 100% accurate in retrospective blind studies analysing a total of 351 samples. Subsequent analysis of more than 800 women from more than 400 D/BMD families has shown that a highly accurate carrier risk can be given in more than 90% of cases.

Tissue-specific methylation differences and cognitive function in fragile X premutation females.

Tissue-specific variation in (CGG)n repeat size and methylation status of the FMR1 gene was investigated in 17 female premutation carriers. Minor variation in premutation repeat size among leukocyte, lymphoblast, and fibroblast tissues was noted in some subjects. One subject exhibited a premutation size allele of (CGG)64 in leukocyte and fibroblast tissues by polymerase chain reaction analysis but a normal-size allele of (CGG)46 in lymphoblast cells, suggesting low-level mosaicism in blood and clonality of the lymphoblast cell line. Six subjects exhibited differences in methylation pattern between leukocytes and lymphoblasts but not between leukocytes and fibroblasts, whereas 2 subjects showed large differences in methylation pattern between leukocytes and fibroblasts. Cognitive function was studied in 14 subjects using the Wechsler Adult Intelligence Scale-Revised. Mean Verbal and Performance IQs were well within the average range as was the mean Full Scale IQ; nevertheless, a trend toward lower Performance IQ compared with Verbal IQ was observed. No significant correlation was apparent between Full Scale IQ and (CGG)n repeat size; however, a significant positive correlation was observed between Full Scale IQ and the proportion of the active X carrying the normal FMR1 allele in fibroblasts but not in leukocytes or lymphoblasts.

FRAXE expansion is not a common etiological factor among developmentally delayed males.

Expansion of a (CGG)n trinucleotide repeat unit at FRAXE, a newly defined fragile site distal to FRAXA, at Xq28, is reported to be associated with mild mental retardation. Three hundred developmentally delayed male patients referred for fragile X testing but negative for the FMR-1 gene trinucleotide expansion were screened for the FRAXE expansion. This group of patients had a wide range of intellectual or behavioral problems and included 19 patients who had low-level fragile site expression detected cytogenetically at Xq27-q28. None of the patients tested positive for the FRAXE expansion. These results suggest that FRAXE is not a common etiological factor among this group of patients. The data support the hypothesis that FRAXE is either very rare or a benign fragile site that is not associated with any clinical phenotype, similar to the FRAXF and FRA16A sites.

Heterogeneity in Roberts syndrome.

Roberts syndrome (RS) is a rare, autosomal recessive condition characterized primarily by growth retardation, developmental delay, and limb anomalies. Some RS patients (RS+), but not others (RS-), have an abnormality of their constitutive heterochromatin (the "RS effect"). RS+ patients also show a cellular hypersensitivity to DNA damaging agents such as mitomycin C (MMC). Lymphoblastoid cell lines from 2 unrelated RS+ patients were fused and hybrid cells examined for correction of the RS effect and MMC hypersensitivity. Neither cellular defect was corrected in the 2 hybrid cell lines examined, suggesting that these 2 patients represent a single complementation group. Fusions were also performed between one RS+ cell line and 2 different RS- cell lines. In both fusions, the hybrids demonstrated correction of both the heterochromatin abnormality and MMC hypersensitivity. These observations suggest that RS+ and RS- patients belong to different complementation groups and do not arise from the same single gene mutation.

Somatic cell hybridization of Roberts syndrome and normal lymphoblasts resulting in correction of both the cytogenetic and mutagen hypersensitivity cellular phenotypes.

Roberts syndrome (RS) is a rare recessive condition of limb deformities, growth retardation, and developmental delay. Cultured cells from approximately half of RS patients exhibit a "puffing" of the constitutive heterochromatin and a hypersensitivity to mitomycin C (MMC). Patients exhibiting these cellular phenomena are designated RS+. Somatic cell hybridization with normal cells has been shown to correct the heterochromatin abnormality in RS+ cells. To determine if the MMC hypersensitivity could also be corrected by hybridization to normal cells, we fused two different RS+ lymphoblastoid cell lines (LCLs) to a ouabain-resistant, HAT-sensitive, normal LCL. Cytogenetic analyses of hybrid cell lines (HCLs) revealed complete correction of the heterochromatin abnormality. MMC cell killing assays revealed correction of the mutagen hypersensitivity as well. Five of the six HCLs tested exhibited D10 values (the dose at which 10% of the cells survive) that were not significantly lower than that of the normal parent but that were 6- to 18-fold greater than those of the RS+ parents. Correction of both of these cellular phenotypes in RS+ cells by fusion with normal cells supports the hypothesis that both of these phenomena are caused by a common defect in the Roberts syndrome gene (RBS).