Mental retardation linked to fragility of chromosome X: current knowledge

The association of the fragile X chromosome with X-linked mental retardation is now well established. The main clinical features are mental retardation, typical facial dysmorphism and macroorchidism. Cytogenetically there is a fragile site in band Xq27-28 which can be demonstrated using specific techniques. The genetic studies are compatible with a X-linked dominant inheritance with an incomplete penetrance. A preliminary estimation of an overall frequency of 1: 2000 males for the fra(X)(q) condition is suggested.     

Fragile X chromosome and X-linked mental retardation.

A family is described in which three normal females transmitted to seven males X-linked mental retardation associated with macro-orchidism and a fragile site on the long arm of the X chromosome -- fra(X)(q27). The affected males also had minor clinical features in common: a large forehead, long face, large ears, a long upper lip and large extremities.     

Fragile site Xq27 and mental retardation. Clinical and cytogenetic manifestation in heterozygotes and hemizygotes of five kindreds

Summary Clinical and cytogenetic data of five kindreds with X-linked mental retardation and a methotrexate-inducible fragile site at the distal long arm of the X chromosome fra(X)(q27) are reported; comprising a total of 26 individuals studied cytogenetically, 10 hemizygotes, five obligate heterozygotes, seven facultative heterozygotes, and four normal males, i.e., fathers and brothers of affected hemizygotes. The heterozygotes in two of these sibships show partial phenotypic and/or mental manifestation. Two of them, who are obligate heterozygotes, expressed fra(X)(q27) in 23% and 16% of their metaphases at the ages of 27 and 53 years. In the obligate and facultative heterozygotes, who are mentally normal, the marker X chromosome could not be detected in lymphocyte cultures. We conclude from these findings that the occurrence of fra(X)(q27) might correlate with the phenotypic expression in heterozygotes rather than with the age of the individual.This investigation was supported in part by the Deutsche Forschungsgemeinschaft     

Further delineation of X-linked mental retardation

Summary Chromosomal, clinical, and psychological data are presented on members of six families with X-linked mental retardation. Affected males in three of these families express the fra(X)(q28) marker, while the retarded males in the other three do not. Similar variable physical and psychological charateristics, such as lop ears, large testes, and perseverative speech, are present in affected males in all six families. Preliminary analysis of the psychological data also shows that males with and without marker expression cannot be differentiated with certainty. On this basis we suggest that there is a type of X-linked mental retardation with many phenotypic features of marker-X mental retardation but without expression of the X chromosome fragile site.     

Chromosomal characteristics of X-linked recessive mental retardation. I. The X chromosome

The X-chromosome was studied in blood lymphocytes of 68 males with aspecific mental retardation (MR), their 57 relatives and 15 intellectually normal males. The incidence of a fragile X-chromosome (fra(X)) was found to be 4.7% in an unselected group of 42 patients, 50% among 10 probands in which pedigree data were suggestive of X-linked MR diagnosis, and 75% in the group of 15 patients selected for phenotype characteristic of the fragile X syndrome. The fra(X) was present in 1-43% of metaphases in different individuals, no such marker being observed in cells of 15 normal individuals. No significant difference was found when the incidence of the fra(X) was compared in cells cultured in the medium 199 with low folic acid content and the Eagle's medium supplemented with 5-fluorodeoxyuridine (10.62 +/- 2.94 SEM and 13.53 +/- 2.85 SEM, respectively). The possibility of false-positive diagnosis of the fragile X syndrome was quantitatively appreciated. A half of the patients showing a fra(C) in conventionally stained chromosomes were found to have fragile 6 autosome as the only marker in these cells, and in patients with the evident fragile (X) syndrome the fra(6) constituted about one-third of the fra(X) frequency. Both culture media employed were similar in the fra(6) induction.     

Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications

Summary The distribution and frequency of aphidicolin-induced common fragile sites were studied in chromosomes of cultured skin fibroblasts and PHA-stimulated lymphocytes from five normal individuals; 0.2 M aphidicolin was added for the last 26 h of culture. Skin fibroblasts from five fra(X)-positive patients were also studied in the same manner. Fragile sites most frequently found in fibroblasts from normal individuals were 3q26.2, 7q11.23, 16q23, 1p31, 10q11.2, 12q23 and 7q31, whereas those in lymphocytes from the same individuals were 3p14, 16q23, Xp22, 7q32 and 14q24. The distribution of fragile sites in fibroblasts from fra(X)-positive patients was essentially identical with that in normal individuals. The average number of gaps and breaks in 100 metaphases was 36.8 in fibroblasts from normal individuals, 113.8 in those from fra(X)-positive patients, and 279 in lymphocytes from normal individuals. Their rates of chromosome-type breaks and gaps were 7.9%, 29.7% and 54.5%, respectively. Thus, the distribution and frequency of aphidicolin-induced fragile sites were different between skin fibroblasts and lymphocytes, possibly reflecting differences in their DNA replication sequence or gene activity.     

X-linked mental retardation. I. Martin-Bell syndrome (report of 18 families)

Eighteen families with X-linked mental retardation (MR) with or without macroorchidism, fragile-X positive at Xq27 (Martin-Bell syndrome) have been studied clinically and cytogenetically. All 58 affected males presented variable degrees of MR, fra(X) (q27) of their peripheral lymphocytes, macroorchidism in all adult patients with the exceptions of one with microorchidism as 47,XXY sex chromosome complement and the other with borderline testes, and characteristic facial appearance. The expression of the marker X in the heterozygotes seems to be more related to the mental development rather than the age of the individual. In two families the transmission of the syndrome through unaffected males seems probable.     

Human chromosome hot points

Summary Peripheral lymphocytes from 16 healthy adults, 9 pregnant women, and 3 fragile X syndrome patients were cultured in Eagle's minimum essential medium without folic acid (MEM-FA). The addition of 2mM, 4mM, or 8mM uridine 24h or 72h prior to harvest resulted in increases of chromosome gaps or breaks, especially at hot points 3p14, 16q23-24, and at fragile site Xq27. Pregnant women showed higher frequencies of 3p14 breaks and total chromosome breaks than men and non-pregnant women. The other chromosome regions, such as 6q26, 7q23, 7q35, 6p25, Xp22, 14q23 and 11p13, also frequently showed gaps or breaks. The results indicated that the unbalance of nucleotide pools was one of the causes of chromosome breakages. The higher frequencies of chromosome gaps and breaks under the condition of thymidylate stress may be due to the misincorporation of uracil instead of thymine into DNA.     

Variability in the expression of the fragile site of the (fra)X chromosome in 2 consecutive cell cycles

The number and morphology of X chromosomes were analysed in tetraploid cells induced with colcemid in cultured blood lymphocytes obtained from a patient with fra(X) syndrome of mental retardation. In contrast to diploid cells containing fra(X) chromosome in 22.7% of cells, the marker X was found in 51.6% of tetraploids, each cell containing only one fragile X out of the two expected ones. The data obtained indicate an extreme lability of the expression of fragile site (X) (q 27) in consecutive lymphocyte generation.     

Apparent homozygosity for the fragile site at Xq28 in a normal female

Summary A 29-year-old obligate carrier for X-linked mental retardation associated with the marker X, fra(X)(q28), showed the fragile site on both X chromosomes in two cells from independent cultures grown with methotrexate. Possible explanations include true homozygosity, artifact, and transposition of the fragile site.     

Frequency of fragile X chromosomes,fra(X), in lymphocytes in relation to blood storage time and culture techniques

Summary The fra(X) frequencies in metaphases of lymphocytes from seven male patients with X-linked mental retardation and macroorchidism were scored after use of different culture techniques and different times of blood storage. No statistically significant differences were found between the fra(X) rates of lymphocytes grown either in folic acid deficient medium (TC 199) or in medium containing folic acid as well as a folic acid antagonist (methotrexate: MTX, aminopterine: AP). With respect to the effect of the time interval between blood sampling and culture set-up, a statistically significant decrease in the fra(X) frequency was observed in all culture types after 4 or 7 days of blood storage.     

Evidence that a dodecamer duplication in the gene HOPA in Xq13 is not associated with mental retardation

A recent study suggested that a dodecamer duplication in exon 42 of the HOPA gene in Xq13 may be a significant factor in the etiology of X-linked mental retardation. In an effort to investigate this possibility, we determined the incidence of the dodecamer duplication in cohorts of non-fragile X males with mental retardation from three countries, cohorts of fragile X males from two countries, 43 probands from families with X-linked mental retardation and control cohorts from three countries. The duplication was found in 3.6-4.0% of male patients from two non-fragile X groups (Italy and South Carolina), in 1.2% from another non-fragile X group (South Africa), but in no male patients from families with X-linked mental retardation (South Carolina). The dodecamer duplication was also found in several white males with fragile X syndrome from France (5%) and South Africa (22.2%). Additionally, the duplication was found in 1.5% of South Carolinian newborn males, 2.5% South Carolinian male college students, 5% Italian male controls and 4.5% of the white South African controls. None of the black South African non-fragile X individuals with mental retardation, the fragile X or the control samples tested carried the duplication, suggesting that the duplication is rare in the black South African population. The incidence of the duplication was not significantly different between any of the groups in the study. Therefore, results of our studies in four different populations do not corroborate the findings of the previous study, and indicate that the HOPA dodecamer duplication does not convey an increased susceptibility to mental retardation.     

Fragile X syndrome: search for phenotypic manifestations at loci for hypoxanthine phosphoribosyltransferase and glucose-6-phosphate dehydrogenase.

The subjects of this study were individuals with the form of X-linked mental retardation that is associated with the presence of a cytologically variant X chromosome having a secondary constriction or "fragile site" at Xq 27-28 (Fra X). Studies were carried out to test the hypothesis that deletions or modifications at neighboring loci occur as a consequence of events at the fragile site. Skin fibroblasts and peripheral blood lymphocytes from affected males were analyzed with respect to the expression of two X-lined enzymes: glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyltransferase (HPRT); loci for these enzymes are known to be located in the region of the fragile site. Although the number of cells resistant to thioguanine (HPRT-deficient) obtained from some cultures from one Fra X male and blood cells of another was greater than expected, the frequency of these cells was not increased in cultures from other Fra X males. Furthermore, our results indicate that the G6PD activity and electrophoretic mobility in Fra X males is similar to that in normal cells, thus providing no evidence for the loss of the long-arm telomere in the fragile X syndrome.     

Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

The combined effects of FUdR addition and methionine depletion on the X-chromosome fragile site.

The fragile site on the X chromosome [fra(X)] associated with mental retardation is not normally seen in lymphocytes cultured in media containing folic acid or lacking methionine. The requirement for methionine has been taken to mean that amino acid metabolism or one-carbon transfer via S-adenosyl-methionine is most important in fra(X) expression. The inhibitory effect of folic acid can be overcome, as we have shown, by the addition of 5'-fluorodeoxyuridine (FUdR) to the culture medium, suggesting that depletion of dTMP available for DNA synthesis is most important in expression. We tested the combined effects of FUdR addition and methionine depletion on the fra(X). We found expression of the fra(X), indicating that methionine per se is not necessary for expression. Our work supports the suggestion that expression of the fra(X) is due to specific limitation of the dTMP pool available for DNA synthesis.     

Folic acid and chromosome breakage III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

The fragile site (16)(q22)

Summary In the lymphocytes of heterozygous carriers of the rare autosomal fragile site (16)(q22) an exceptionally high frequency of sister chromatid exchanges was demonstrated at the induced fragile site by means of simultaneous berenil and BrdU treatment of the cultures. The rate of sister chromatid exchanges at q22 is also increased in the fragile chromosome 16 by treating the cells with BrdU alone. The possible reasons for the preferential occurrence of induced and spontaneous sister chromatid exchanges at fra (16)(q22) are discussed.     

Induction of sister chromatid exchanges at common fragile sites.

Experiments were performed to gain further insight into chromosome structure and behavior at common fragile sites by testing the hypothesis that gaps at these sites predispose to intrachromosomal recombination as measured by sister chromatid exchanges (SCEs). Human lymphocytes were concurrently treated with aphidicolin, for determination of fragile site expression, and with 5-bromodeoxy-uridine, for SCE analysis. Aphidicolin induced chromosome gaps nonrandomly, with the great majority of gaps occurring at common fragile sites. On average, 66% of gaps were accompanied by an SCE at the site of the lesion. Analysis of two specific common fragile sites at 3p14 and 16q23 showed the same pattern; that is, on average 70% of gaps at these sites were accompanied by an SCE. These results show that common fragile sites are hot spots not only for chromosomal lesions such as gaps but also for SCE formation.     

Enhanced sensitivity of lymphoblastoid cells from individuals carrying the mutation for the fragile X syndrome to the clastogenic effects of FUdR

Individuals known to carry the mutation for the fragile X syndrome can sometimes be identified cytogenetically by the presence of a fragile site on the X chromosome at q27.3. The frequency of cells bearing this fragile site is known to be enhanced by culturing the cells in folic acid deficient medium and/or by introducing folic acid metabolism inhibitors such as FUdR. In this study FUdR induction of chromosomal aberrations other than the fragile X was investigated. Lymphoblastoid cells from an obligate carrier, a mentally retarded male and a control were cultured in folic acid deficient medium in the presence of FUdR and harvested at various times after culture initiation. The frequency of chromosome and chromatid breaks was found to be higher in cells from the individuals carrying the mutation for the fragile X syndrome. The frequency of micronuclei, an indirect index of chromosome breakage, was also more elevated in cells from these individuals than in cells from the control. These findings are of potential importance to carrier detection of this common genetic disorder.     

Frequency of the fragile X syndrome in institutionalized mentally retarded females in Japan

Summary The fragile X [fra(X)] syndrome was screened on 190 Japanese institutionalized females with moderate to severe mental retardation. Two inmates with severe mental retardation (IQ 20) had the fra(X) chromosome in 26% and 15% of the cells examined, indicating that the prevalence of the fra(X) syndrome was about 1% in all female inmates and was about 3.27% in severely mentally retarded females with known causes. However, no female with fra(X) syndrome was found in 35 moderately retarded females. Both had brothers with the fra(X) syndrome and the prevalence was 10% in females with a family history of mental retardation. In addition, the replication study of the fra(X) chromosome in the patients supported the proposal that an excess of the early replicated fra(X) chromosome is related to the mental capacity in heterozygous females. Therefore, the fra(X) syndrome should not be ignored even in severely mentally retarded females with a family history, though the heterozygotes are commonly normal to subnormal in their mental development. in addition, the replication study of the fra(X) chromosome may help to estimate mental development in the carrier children.