Manifestation of the fragile site Xq27 in fibroblasts

Summary Fibroblasts from a heterozygous carrier for the Martin-Bell syndrome, who manifests the fragile site Xq27, were cloned to separate the population carrying the primary defect on the active X chromosome from the population with this defect on the inactive X. Clones with this defect on the active X manifest the fra(X)(q27) whereas clones from the other population are fra(X)-negative (Steinbach et al. 1983b). In this project, the replication status of the X chromosome manifesting the fra(X)(q27) was studied in seven clones with this defect on the active X.The results obtained on the clones were very similar to the results obtained from uncloned fibroblasts and lymphocytes. In the clones the fragile site was found manifested on the early replicating X in 73 cells and on the late replicating X in four cells.Since the defect is located on the active X chromosome of these cells the manifestation of the fragile site on the late replicating X suggests that the defect and the fragile site cannot be identical. It is concluded that there is no obligate synteny of this defect and the manifested fragile site.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Fragile (X) X-linked mental retardation. II. Frequency and replication pattern of fragile (X)(q28) in heterozygotes

The frequency of cytologic expression and the replication pattern of the fragile (X) [fra(X)] were investigated in 28 fra(X) heterozygotes, of which 25 agreed to psychological assessment. One-third of the heterozygotes in this study are mentally retarded. The intellectually impaired carriers had a higher frequency of fra(X) and a higher proportion of early-replicating fra(X) than the normally intelligent carriers. The early-replicating fra(X) accounted for 39% of the variability in IQ and the late-replicating fra(X) for 12%. Age had a minimal inverse effect on fra(X) expression and replication pattern. Thus, it appears that mental retardation in females heterozygous for the fra(X) may largely be a function of the proportion of cells with an early-replicating, active X chromosome possessing the fragile site.     

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.     

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.     

Chromosome characteristics of X-linked mental retardation. II. Autosomes

The frequencies of chromosome and chromatid breaks and gaps were studied in blood lymphocytes of three groups of individuals: 21 males with X-linked mental retardation characterized by fragile X chromosome; 52 males with non-differentiated X-linked mental retardation having no fra(X) chromosome in their cells; 15 intellectually normal males. The lymphocytes were cultured both in medium 199 and in Eagle's medium supplemented with fluoro-deoxyuridine. The significantly higher frequencies of various autosomal lesions were observed in the individuals with the fragile X chromosome syndrome and in those with mental retardations without fra(X) chromosome, in comparison with normal males. The significant difference in some autosome lesions was also found between both groups of the patients. The distribution of chromosome lesions in autosomes of different groups was significantly higher in chromosomes A and lower in groups B, E, F and G, than expected in accordance with their relative length in the haploid set. In all the groups of individuals studied, the predominant localization of chromosome and chromatid breaks and gaps was observed in fragile sites 1p31, 3p14, 6q26 and 16q23.     

Variations in the presence of a fragile site on X--fra(X)--according to cases and methods used

In the nearly last two years, using ten different media combinations in our research for the fragile X, we have observed cytogenetic variations, the knowledge of which may be useful for correct diagnosis. This experience, based on more than 6000 metaphases in 41 cases, includes 5 typical probands, 4 obligate and 4 possible heterozygotes, 19 deficient psychopaths, and 9 controls. Three main techniques were retained as shown in the text. In this study a fragile site was documented on 239 chromosomes of the C or X group, among which 180 could be specifically identified by trypsin Giemsa banding. Of these 180 fragile sites, X involvement was shown in 101 cells, and 55 other lesions were found to affect a chromosome 6. In our experience, none of 1180 cells from 9 control individuals were positive. It thus may be that under rigorous culture conditions the occurrence of one single fragile X at q27 or q28 is suggestive of the presence of the underlying mutation. In 19 cases studied because of mental and psychotic problems, nonetheless considered as clinically negative, only 2 out of 2510 cells had a fragile X, whereas frequency among cytogenetically verified X in our positive cases varied from 4 to 20% cells. We have come to distinguish five variations in the cytogenetic aspect of this site on the X, shown from A to E in a figure, two of which (D and E) may not have been described before. In rare cases this fragility usually seen as a chromatid or isochromatid gap may be present as a break with a resulting "double minute" found elsewhere in the metaphase field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication status of the fragile X chromosome,fra(X)(q27), in three heterozygous females

Summary Investigation of lymphocyte cultures from three females heterozygous for fra(X)(q27) shows widely differing proportions of early and late replicating X chromosomes having the fragile site, and suggests that the replication status of the fragile X may be related to the mental capacity of the patient. The study has utilised a sequential staining technique to reduce ascertainment bias, and evidence is presented to suggest that the expression of the fragile site is independent of the differential incorporation of BUdR into the early and late replicating X chromosomes.     

Manifestation of the fragile site Xq27 in fibroblasts

A protocol is reported which allows the efficient induction of bromodeoxyuridine (BrdU)-induced R-type replication patterns in fibroblast cultures prepared to demonstrate the fragile site fra(X)(q27). The technique includes partial synchronization of the culture by fluorodeoxyuridine (FdU) blocking at the G1/S transition. This block does not impair the induction of the fragile site in medium 199 containing methotrexate. The marked increase of the mitotic index in the synchronized culture may be an advantage in the study of folic acid sensitive fragile sites in fibroblasts. Adding BrdU after block removal leads to an efficient labeling of replicating chromosomes without severely impairing the manifestation of fra(X)(q27).     

The fragile site (16) (q22)

Summary The rare fragile site at 16q22 was experimentally induced in lymphocyte cultures with various AT-specific, non-intercalating DNA-ligands. The optimum conditions for the induction of fra (16)(q22) were determined. The best expression of fra (16)(q22) was found with the aromatic diamidine berenil which is recommended for further studies on this fragile site. The results indicate that fra (16)(q22) is a region with AT-rich, late replicating DNA. The simultaneous treatment of lymphocytes with berenil and aphidicolin (inhibitor of DNA polymerase ) induces both the rare fra (16) (q22) and the common fra (16) (q23) within the same chromosome. A population study on 350 unselected individuals showed that fra (16)(q22) is the most common of all rare autosomal fragile sites in man. The frequency of individuals heterozygous for fra (16)(q22) is 5.1% no homozygosity for fra (16) (q22) was detected. Statistical analysis indicates that the population is in Hardy-Weinberg equilibrium with respect to the fragile and non-fragile chromosomes 16.     

New deletion syndrome: 1q43.

A male infant showed dysmorphology of the head and face, neck, extremities, and genitalia, as well as growth and mental retardation. His G-banded karyotype was 46,XY,--1+der(1),t(1;16)(q43;q24)mat. Combined with five previously reported cases involving similar terminal deletions beginning at 1q42 or 43, we show that the homology of phenotypic characteristics permits identification of a new deletion syndrome, the first involving chromosome 1.     

X-inactivation pattern in carriers of X-linked retinitis pigmentosa: A valuable means of prognostic evaluation?

In a large family with X-linked retinitis pigmentosa 2 (XLRP2), we reexamined 7 obligate carrier females and 6 daughters of obligate carriers, whose linkage relationships suggested that they carried the XLRP2 gene. The phenotype varied from totally normal eyes through mild retinal changes to complete loss of vision. The X-inactivation analysis was carried out with the highly informative probe M27 on DNA from blood lymphocytes. This probe detects a locus DXS 255 that is differentially methylated on the active and inactive X chromosomes. In 5 blind heterozygotes (aged 43 to 68 years), we found that the X chromosome carrying the RP2 gene was methylated and active in nearly all their cells. The opposite X inactivation pattern was found in a carrier female (aged 45 years) who gave normal findings on eye examination. Carriers with less skewed X inactivation had a less severe clinical outcome. However, we found little or no correlation between their phenotypes and the methylation status of their X chromosomes. Our results suggest that it may be possible to develop a predictive test that could identify cases with severe outcome and perhaps cases with normal outcome.     

X-X translocation in a patient with gonadal dysgenesis and the problem of phenotype-karyotype correlations

Summary A sex-chromatin-positive woman without stunted growth, but with primary amenorrhea, and some stigmas of pure gonadal dysgenesis had the chromosome constitution 45,X/46,Xt(X;X)(q27;q27). The abnormal chromosome formed a large Barr body and was late-labeling. The chromosome consisted of two X chromosomes attached by their long arms (end-to-end), both apparently having the partial distal deletion. Both centromeric regions showed C-staining but only one constriction. The chromosome is interpreted as an isodicentric with only one centromere functioning. Some problems of phenotype-karyotype correlations are discussed.