Analysis of linkage relationships between genetic markers around the fragile X locus with special reference to the daughters of normal transmitting males

Summary Genetic linkage data from loci around the fragile X locus at Xq27.3 are analysed in the light of the hypothesis of Pembrey et al. (1985) concerning the generation of the fragile X mutation. Recombination between the four loci 52A, F9, fragile X, and ST14 is significantly decreased in meioses giving rise to the affected grandsons of normal transmitting males, when compared to families where there are no apparent normal transmitting males. There are at least two possible explanations for this phenomenon. Either the established fragile site at Xq27.3 promotes increased recombination in the distal part of the X chromosome as a secondary event, unrelated to the mechanism of formation of the fragile site itself, or an event involving recombination at or around Xq27.3 is the mechanism of formation of the full fragile X mutation, and the decreased recombination seen amongst flanking marker loci in meioses giving rise to the affected grandsons of normal transmitting males is the result of interference.     

Unaffected carrier males in families with fragile X syndrome.

Males who transmit the fragile X chromosome but are themselves clinically normal have occasionally been observed. We have studied three families segregating the fragile X. In one family, there are three unaffected carrier males, and in each of the other two families, there is one unaffected carrier male. Three of these carrier males were studied cytogenetically, and none exhibited the fra(X)(q27) marker. The occurrence of carrier males and of other unusual genetic features in fragile X families suggest that this condition is not inherited as a standard recessive trait linked to the X chromosome.     

A cytogenetic study of a population of retarded females with special reference to the fragile(X) syndrome

Summary A cytogenetic survey of a population of 278 mentally retarded females on community placement is described. Thirty-five females had an aneuploid chromosome constitution and a single female was found to have the fra(X) syndrome. The frequency of the fra(X) syndrome among female retardates is discussed together with the apparent absence of de novo mutants among this class of fra(X) probands.     

Frequency of the fragile X syndrome in Japanese mentally retarded males

Summary Among 243 institutionalized mentally retarded males in Japan, 13 patients (5.3%) with the fre(X)(q27) from nine families were detected. These 13 patients accounted for 8.6% of 152 male inmates with unknown causes of mental retardation in the population. One out of nine pedigrees had an apparently unaffected male transmitter of this disorder. Our data agree with the frequencies of the fra(X) syndrome in various retarded populations, most of which were Caucasians, suggesting that the prevalence of the fra(X) syndrome in Japanese is not significantly different from those in Causasians.     

Multilocus analysis of the fragile X syndrome

Summary A multilocus analysis of the fragile X (fra(X)) syndrome was conducted with 147 families. Two proximal loci, DXS51 and F9, and two distal loci, DXS52 and DXS15, were studied. Overall, the best multipoint distances were found to be DXS51-F9, 6.9%, F9-fra(X), 22.4%; fra(X)-DXS52, 12.7%; DXS52-DXS15, 2.2%. These distances can be used for multipoint mapping of new probes, carrier testing and counseling of fra(X) families. Consistent with several previous studies, the families as a whole showed genetic heterogeneity for linkage between F9 and fra(X).     

Further evidence for genetic heterogeneity in the fragile X syndrome

Summary The X-linked fragile X[fra(X)] syndrome, associated with a fragile site at Xq27.3, is the most common Mendeban inherited form of mental deficiency. Approximately 1 in 1060 males and 1 in 677 females carry the fra (X) chromosome. However, diagnosis of carrier status can be difficult since about 20% of males and 44% of females are nonpenetrant for mental impairment and/or expression of fra (X). We analyzed DNA from 327 individuals in 23 families segregating fra (X) for linkage to three flanking polymorphic probes: 52A, F9, and ST14. This allowed probable nonpenetrant, transmitting males and carrier females to be identified. A combined linkage analysis was conducted using these families and published probe information on F9 in 27 other families, 52A in six families, and ST14 in five families. The two-point recombination fraction for 52A-F9 was 0.13 (90% confidence interval, 0.10–0.16), for F9-fra(X) was 0.21 (0.17–0.24), and for fra(X)-ST14 was 0.12 (0.07–0.17). Tight linkage between F9 and fra(X) was observed in some families; in others loose linkage was seen suggesting genetic linkage heterogeneity. Risk analysis of carrier status using flanking DNA probes showed that probable nonpenetrant transmitting males were included in families showing both tight and loose linkage. Thus, in contrast to our previous conclusions, it appears that the presence or absence of nonpenetrant, transmitting males in a family is not an indicator of heterogeneity. To determine if heterogeneity was present, we employed the admixture test. Evidence for linkage heterogeneity between F9 and fra(X) was found, significant at P<0.0005. Nonsignificant heterogeneity was seen for 52A-F9 linkage. No heterogeneity was found for fra(X)-ST14. The frequency of fra(X) expression was significantly lower in families with tight F9-fra(X) linkage than in families with loose linkage. Cognition appeared to relate to linkage type: affected males in tight linkage families had higher IQs than those in loose linkage families. These findings of genetic heterogeneity can account in part for the high prevalence and apparent high new mutation rate of fra(X). They will affect genetic counseling using RFLPs. An understanding of the basis for genetic heterogeneity in fra(X) will help to clarify the nature of the unusual pattern of inheritance seen in this syndrome.     

A cytogenetic study of mentally retarded school children in taiwan with special reference to the fragile X chromosome

Summary A cytogenetic study was made on 341 mentally retarded children in the Provincial Nantou Rehabilitation Center for the Mentally Retarded and the St. Raphael Opportunity Center in Tainan. Of the 89 mentally retarded children with chromosomal abnormalities, 63 had Down syndrome, 13 had the fragile X [fra(X)] syndrome, and the remaining had other aneuploid constitutions. Family studies were possible for 2 of the 13 fra(X) probands. The results of this study illustrate the contribution of chromosomal abnormalities to the pathogenesis of mental retardation in children.     

Recurrent and unexpected segregation of the FMR1 CGG repeat in a family with fragile X syndrome

Fragile X syndrome, the most common cause of hereditary mental retardation, results from amplification of a CGG trinucleotide repeat in the FMR1 gene. The transmission of the CGG repeat from premutated individuals to their premutated descendants is usually unstable, showing an increase in the size of the repeat. We report here a family which exhibits recurrent and unexpected transmission of the maternal premutation to three daughters. The first daughter exhibited mosaicism with two premutated alleles, one contracted and the other expanded. The second daughter showed a reversion from the maternal premutation to the normal range, and the third carried an expanded premutated allele associated with an expanded paternal allele within the normal range. These variations in the size of the CGG repeat may result from many different mechanisms such as DNA polymerase slippage on the leading or lagging strand during replication, large contractions of repeats on the parental strand during replication, or recombination through unequal crossover between sister chromatids. Our results suggest that the variation of the CGG premutated alleles in this family may be the result of intrinsic instability associated with a trans-acting factor such as a mismatch repair gene product. Received: 21 August 1995 / Revised: 21 September 1995     

Molecular analysis of a ring chromosome X in a family with fragile X syndrome

The phenotypically normal sister of a patient affected by fragile X syndrome was referred for genetic counselling and was found to carry a mosaic karyotype 46,X,r(X)/45,X. Because the probability of the simultaneous chance occurrence of fragile X syndrome and a ring chromosome X in the same family is very low, we postulated that the breakpoint of the ring chromosome X originated in the cytogenetic break in Xq27.3 responsible for fragile X syndrome. In order to determine the relative positions of the breakpoint on the ring chromosome X and the (CGG)n unstable sequence responsible for the fragile X mutation, we used molecular markers to analyse the telomeric regions of chromosome X in this family. The results showed that the ring chromosome X was the maternal fragile X chromosome and that the telomeric deletion on the long arm encompassed the (CGG)n sequence. This suggests that the cytogenetic break in Xq27.3 is distinct from the unstable (CGG)n sequence, or that the break followed by the end-to-end fusion creating the ring chromosome was not completely conservative. Analysis of DNA markers on the short arm of chromosome X evidenced a deletion of a large part of the pseudoautosomal region, allowing us to position the genes involved in stature and in some syndromes associated with telomeric deletions of Xp on the proximal side of the pseudoautosomal region.     

Diagnosis of fragile X syndrome by direct mutation analysis

A total of 27 fragile X pedigrees consisting of over 100 nuclear families were analyzed by Southern blotting methods and probes StB12.3 and StB12.3xx to detect the expansion of the (CGG) _n repeat within the FMR-1 gene and the abnormal methylation pattern of the adjacent DNA region responsible for the fragile X syndrome. Clinical expression was found to be associated with the presence of a full mutation ( > 500 bp, associated with abnormal methylation) in all the males and 50% of the females studied, whereas individuals carrying a premutation ( = 100–700 bp) were normal. A preferential size increase in the enlarged (CGG) _n repeat was detected in successive generations, the instability being stronger when transmitted from a female than from a male. No expansion of the premutation to the full mutation occurred in the paternal transmissions, and the size increase was significantly smaller than in the maternal transmissions. This could partly explain the stability of the premutation through several generations in families with transmitting males. In the maternal transmissions, the risk of expansion of a premutation to a full mutation appeared to depend on its size. The critical maternal premutation size leading invariably to the full mutation was between = 175–200 bp. This is important for genetic counseling and also explains the commonly observed clustering of affected individuals in fragile X families.     

A fragile X female with Down syndrome

Summary Clinical and cytogenetic aspects of a female infant with trisomy 21 and the fragile X [fra (X)] chromosome are reported. Most of the facial characteristics of the patient are those observed in Down syndrome, but some features such as long face with prominent forehead and lower jaw, and large ears are related to the fra (X) syndrome. The origin of an additional chromosome 21 may be ascribed to maternal first meiotic nondisjunction in our case. It has been suspected that female carriers of the fra (X) chromosome may be predisposed to meiotic nondisjunctional events. However, there is probably no relationship between the two chromosomal abnormalities in our case because of the maternal age at the delivery.     

A clinical and molecular genetic analysis of the fragile X syndrome

Results of phenotypical, patho-psychological and molecular-genetic analysis of the 53 probands with clinical features of the fragile X syndrome and 10 female carriers are presented. The clinical heterogeneity, diagnostic criteria, methods of genetic risk estimation, perspectives of prevention of this disease are discussed.     

A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)₂₉ allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males. Received: 31 May 1995 / Revised: 4 October 1995     

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.     

Spermatogenesis in two patients with the fragile X syndrome

Summary Light and electron microscopic studies on testicular biopsies were carried out in two men, 40 and 44 year old, with the fra(X) form of mental retardation and macroorchidism. Distinct interstitial edema, an increased amount of lysosomal inclusions in Sertoli cells, and disturbance of spermatid differentiation were found in both probands. Additionally, some extent of tubular atrophy was demonstrated in one patient. The impairment of spermatogenesis is discussed with respect to pressure effects on the germinal epithelium due to the edema.     

Spermatogenesis in two patients with the fragile X syndrome

Summary Chromosomes at first meiosis from two males with the fra(X) form of mental retardation were studied using pachytene surface spreads and air-dried preparations. The pachytene sex bivalents showed no discontinuation of the synaptonemal complex in the terminal part of Xq corresponding to band Xq27–28 of the mitotic chromosomes. In both cases the frequency of a secondary association of Xq and Yq appeared to be increased compared with controls. The pairing behavior of autosomal bivalents in pachytene and the frequency and distribution of chiasmata in diakinesis were normal. The impairment of spermatogenesis found in these males may not be caused by a meiotic disorder, but could be related to peritubular or intratubular pressure effects on germ cells.     

Of mice and the fragile X syndrome

Fragile X syndrome is the most common cause of inherited mental retardation, and recently a number of mouse models have been generated to study the condition. Knockout of the gene associated with fragile X, Fmr1, results in mild, but consistent abnormalities, analogous to the clinical and pathological symptoms observed in human patients. Thus, many aspects of the syndrome can now be studied in mice, taking full advantage of the benefits of this model organism, including the short generation time and unlimited supply of tissue. The experimental data suggest that knockout of Fmr1 mildly disturbs a variety of processes in different brain regions.