Expression of the fragile site Xq27 in fibroblasts. I. Detection of Fra(X)(q27) in fibroblast clones from males with X-linked mental retardation

Summary Twelve fibroblast clones from two males with X-linked mental retardation expressed the fragile site Xq27 in 3%–38% of metaphases analyzed. The number of in vitro doublings during the cloning procedure had no evident influence on the induction of fragile X expression. The variability of fragile X expression seems to depend on cell properties acquired during culture rather than on properties originally inherent in the cells.     

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.     

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.     

Screening and diagnosis for the fragile X syndrome among the mentally retarded: an epidemiological and psychological survey. Collaborative Fragile X Study Group.

The fragile X syndrome is an X-linked mental retardation disorder caused by an expanded CGG repeat in the first exon of the fragile X mental retardation (FMR1) gene. Its frequency, X-linked inheritance, and consequences for relatives all prompt for diagnosis of this disorder on a large scale in all affected individuals. A screening for the fragile X syndrome has been conducted in a representative sample of 3,352 individuals in schools and institutes for the mentally retarded in the southwestern Netherlands, by use of a brief physical examination and the DNA test. The attitudes and reactions of (non)consenting parents/guardians were studied by (pre- and posttest) questionnaires. A total of 2,189 individuals (65%) were eligible for testing, since they had no valid diagnosis, cerebral palsy, or a previous test for the FMR1 gene mutation. Seventy percent (1,531/2,189) of the parents/guardians consented to testing. Besides 32 previously diagnosed fragile X patients, 11 new patients (9 males and 2 females) were diagnosed. Scoring of physical features was effective in preselection, especially for males (sensitivity .91 and specificity .92). Major motives to participate in the screening were the wish to obtain a diagnosis (82%), the hereditary implications (80%), and the support of research into mental retardation (81%). Thirty-four percent of the parents/guardians will seek additional diagnostic workup after exclusion of the fragile X syndrome. The prevalence of the fragile X syndrome was estimated at 1/ 6,045 for males (95% confidence interval 1/9,981-1/ 3,851). On the basis of the actual number of diagnosed cases in the Netherlands, it is estimated that >50% of the fragile X cases are undiagnosed at present.     

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.     

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。}     

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.     

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.     

The clinical phenotype in institutionalised adult males with X-linked mental retardation (XLMR)

In an institutionalised population of 471 mentally retarded adult residents (436 males and 35 females), 22 males (i.e. 5 % of the male population) had XLMR, accounting for 36.1 % of the residents diagnosed with a monogenic disorder (n = 61). Fragile X syndrome (FRAXA) was diagnosed in 16 residents, X-linked mental retardation with marfanoid habitus (Lujan-Fryns syndrome) in 2, and non-specific X-linked mental retardation (MRX) in 4 males. The 4 MRX-patients included 3 male sibs of a family, carrying a mutation in the IL-1 receptor accessory protein-like gene, and one male patient member of the MRX-44 family (linkage with LOD-score of 2.90). In the group of 215 males with idiopathic mental retardation (MR), family histories and pedigree data were compatible with XLMR in 35 males (35/215 = 16.3 %) from 32 families. Of these 35 males, 5.7 % were microcephalic with dysmorphic features and 5.7 % macrocephalic; micro-orchidism and macro-orchidism were each found in 11.4 %. One macrocephalic male had also macro-orchidism and dysmorphic features. In this study, the diagnosis of XLMR could thus be proposed in 57 males i.e. 13.1 % of the total male population. The clinical phenotype, behavioural problems and follow-up data in these different subgroups of XLMR are presented.     

Segregation of FRAXE in a large family: clinical, psychometric, cytogenetic, and molecular data.

During an ongoing study on X-linked mental retardation, we ascertained a large family in which mild mental retardation was cosegregating with a fragile site at Xq27-28. Clinical, psychometric, cytogenetic, and molecular studies were performed. Apart from mild mental retardation, affected males and females did not show a specific clinical phenotype. Psychometric assessment of four representative affected individuals revealed low academic achievements, with verbal and performance IQs of 61-75 and 70-82, respectively. Cytogenetically the fragile site was always present in affected males and was not always present in affected females. With FISH the fragile site was located within the FRAXE region. The expanded GCC repeat of FRAXE was seen in affected males and females either as a discrete band or as a broad smear. No expansion was seen in unaffected males, whereas three unaffected females did have an enlarged GCC repeat. Maternal transmission of FRAXE may lead to expansion or contraction of the GCC repeat length, whereas in all cases of paternal transmission contraction was seen. In striking contrast to the situation in fragile X syndrome, affected males may have affected daughters. In addition, there appears to be no premutation of the FRAXE GCC repeat, since in the family studied here all males lacking the normal allele were found to be affected.     

A common fragile site at Xq27: theoretical and practical implications.

The fragile site at Xq27 (FRAXA) is associated with a common form of X-linked mental retardation (Martin-Bell syndrome). It is induced in culture by conditions of thymidylate stress and is generally considered a rare fragile site found only in association with an X-linked form of mental retardation. Using a somatic cell hybrid system, we previously demonstrated that fragile-X expression can be induced by thymidylate stress in normal X chromosomes at low levels (4%-5%). In the present report, significantly higher levels of fragile-X expression (6%-28%) have been induced in lymphocytes or lymphoblasts of all seven control males using high doses of aphidicolin (1.5 microM). Similar high levels of expression (10%-12%) were observed in both of two normal male chimpanzees (Pan troglodytes). These data demonstrate that Xq27 contains a common fragile site (FRAXD) that is ancestral to the divergence of man and the chimpanzee. Presence of a common and a rare fragile site in the same metaphase chromosome band does not prove that they are identical and may, in fact, represent two unrelated fragile sites. However, the possibility exists that the common fragile site at Xq27 may be the substrate for unequal recombination events that produces the rare fragile site associated with Martin-Bell syndrome. In addition, presence of a common fragile site at Xq27 may explain the occasional observation of low-frequency fragile-X expression in normal control individuals. Caution is therefore warranted in the interpretation of low-level fragile-X expression in diagnostic and prenatal diagnostic settings.     

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.     

Fragile (X) expression: Relationship to the cell cycle

Summary The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.This work was supported by Grant HD 07879 from the National Institutes of Health     

A study of FRAXE in mentally retarded individuals referred for fragile X syndrome (FRAXA) testing in the United Kingdom.

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.     

X-linked mental retardation: many genes for a complex disorder

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR is very heterogeneous, and about two-thirds of patients have clinically indistinguishable non-syndromic (NS-XLMR) forms, which has greatly hampered their molecular elucidation. A few years ago, international consortia overcame this impasse by collecting DNA and cell lines from large cohorts of XLMR families, thereby paving the way for the systematic study of the molecular causes of XLMR. Mutations in known genes might already account for 50% of the families with NS-XLMR, and various genes have been pinpointed that seem to be of particular diagnostic importance. Eventually, even therapy of XLMR might become possible, as suggested by the unexpected plasticity of the neuronal wiring in the brain, and the recent successful drug treatment of a fly model for fragile X syndrome.     

A systematic cytogenetic study of a population of 1170 mentally retarded and/or behaviourly disturbed patients including fragile X-screening. The Hondsberg experience

A cytogenetic study was performed in a population of 1170 mentally retarded and/or behaviourly disturbed patients of the Hondsberg Institute in the south of the Netherlands. The cytogenetic data are presented and discussed. In all patients chromosomal evaluation was performed with Giemsa-banding and Quinacrine fluorescence, and additional banding techniques were performed whenever they were necessary to clarify the chromosomal abnormality. A fragile X screening with M199 cultures was performed in 311 males. In 22.1% of the patients a chromosomal basis was found for their developmental retardation: 14.3% Down syndrome patients, 6.1% other chromosomal abnormalities (mainly partial autosomal trisomies and monosomies and sex-chromosome abnormalities). In 24 males, through 21 index patients, a positive fragile X screening was found, i.e. 6.7% of the screened population and 1.8% of the total population. These results indicate that the diagnostic contribution of the fragile X screening is numerically of equal importance as are advanced chromosome banding techniques, and its contribution to the diagnosis of fragile X syndrome in one index male patient in general leads to the detection of several female relatives at risk to be carrier of this X-linked recessively inherited condition. The causal relationship between the occurrence of mental retardation and chromosomal aberration in genera i.e. autosomal trisomies, partial autosomal trisomies and monosomies, and Xq27-28 fragility is well established and is, to some extent, easy to understand. Whether carriers of other chromosomal rearrangements, mainly of balanced reciprocal and Robertsonian translocations, small extra chromosomes, paracentric inversions and chromosomal variants, have increased risk for mental handicap and/or congenital malformations in their progeny, remains unclear at the present time. Some of these residual problems and questions are discussed in the perspective of their importance for genetic counseling. Detailed data will be presented about the mental development and psychological profile of patients with these different types of chromosomal abnormalities and rearrangements.     

A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation

X-linked nonspecific mental retardation (MRX) has a frequency of 0.15% in the male population and is caused by defects in several different genes on the human X chromosome. Genotype-phenotype correlations in male patients with a partial nullisomy of the X chromosome have suggested that at least one locus involved in MRX is on Xp22.3. Previous deletion mapping has shown that this gene resides between markers DXS1060 and DXS1139, a region encompassing approximately 1.5 Mb of DNA. Analyzing the DNA of 15 males with Xp deletions, we were able to narrow this MRX critical interval to approximately 15 kb of DNA. Only one gene, VCX-A (variably charged, X chromosome mRNA on CRI-S232A), was shown to reside in this interval. Because of a variable number of tandem 30-bp repeats in the VCX-A gene, the size of the predicted protein is 186-226 amino acids. VCX-A belongs to a gene family containing at least four nearly identical paralogues on Xp22.3 (VCX-A, -B, -B1, and -C) and two on Yq11.2 (VCY-D, VCY-E), suggesting that the X and Y copies were created by duplication events. We have found that VCX-A is retained in all patients with normal intelligence and is deleted in all patients with mental retardation. There is no correlation between the presence or absence of VCX-B1, -B, and VCX-C and mental status in our patients. These results suggest that VCX-A is sufficient to maintain normal mental development.     

Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome

The most common genetic cause of mental retardation after Down's syndrome, the fragile X syndrome, is associated with the occurrence of a fragile site at Xq27.3. This X-linked disease is intriguing because transmission can occur through phenotypically normal males. Theories to explain this unusual phenomenon include genomic rearrangements and methylation changes associated with a local block of reactivation of the X chromosome. Using microdissected markers close to the fragile site, we have been able to test these hypotheses. We present evidence for the association of methylation with the expression of the disease. However, there is no simple relationship between the degree of methylation and either the level of expression of the fragile site or the severity of the clinical phenotype.     

Molecular diagnosis of the fragile X and FRAXE syndromes in patients with mental retardation of unknown cause in Mexico

The fragile X syndrome (Fra-X) is the most common cause of inherited mental retardation with X-linked semi-dominant inheritance. The prevalence of Fra-X in the Mexican population is unknown. The aim of this population screening study was to determine if Fra-X or FRAXE mutations are the cause of a number of cases of mental retardation in a sample of Mexican children with mental retardation of unknown cause (MRUC) and to stress the importance of performing molecular analysis of the FMR-1 gene in all patients with MRUC. We report here the direct analysis of CGG and GCC repeats within the FMR-1 and FMR-2 genes, respectively, in 62 unrelated patients with MRUC. Two male index cases had the CGG expansion, although they did not express the Xq27.3 fragile site cytogenetically. Fra-X diagnosis was highly suspected on a clinical basis in one of the patients, but not in the other. Both mothers were found to be premutation carriers. The molecular studies of FMR-1 showed that the proportion of MRUC patients with Fra-X is 3.2%. This frequency was not significantly different to that reported in most populations. As reported in other series, no patients with FRAXE were found in our sample. Our findings confirm that the molecular analysis of the FMR-1 gene is necessary in MRUC patients to achieve unequivocal diagnosis of fragile X syndrome, carrier premutation detection and for accurate genetic counseling.