Tightly linked polymorphic markers for fragile X syndrome and prenatal cytogenetic diagnostic experience.

Linkage analysis using the polymorphic loci DXS369, DXS296, DXS297 and DXS306 was carried out on a cohort of 17 families segregating for fragile X syndrome. The observed recombination fractions at: DXS369 (Zmax = 3.02; theta = 0.06), DXS297 (Zmax = 2.92; theta = 0.0), DXS296 (Zmax = 3.82; theta = 0.0), DXA306 (Zmax = 4.55; theta = 0.05) confirm that these loci are tightly linked to FRAXA. Our experience in the cytogenetic analysis of 58 at risk pregnancies by chorionic villus or fetal blood sample examination documents a false negative rate in obligate carrier male pregnancies for CVS of 11% and for FBS of 3%.     

Klinefelter syndrome and two fragile X chromosomes

Two fragile X chromosomes were found in 20% of the cells in a moderately mentally retarded patient with Klinefelter syndrome.     

Effect of folic acid treatment in the fragile X syndrome

The effect of folic acid intake on the frequency of fragile X positive cells and some behavioural characteristics were evaluated in 5 boys and 4 adult males with the fragile X syndrome. The expression of fragile X was nullified in 6 and decreased in 3 of the 9 patients. Behavioural and motor ability were considered to have improved in 4 of the 5 boys but not in the 4 adults with fragile X syndrome.     

The strength of association between fragile(X) chromosome presence and mental retardation

In order to obtain a quantitative estimate of the degree of association between presence of fragile X chromosome (fra(X)) and mental retardation (MR), existing data from nonretarded males were analyzed. Clearly, fra(X) occurs less frequently among nonretarded compared to MR males. However, incidence estimates for fra(X) based upon existing data hold open the possibility that there may be significant numbers of nonretarded males with fra(X). Additional analyses of data from families with a pattern of fra(X) linked MR showed: (a) the probability that nonretarded male offspring will have fra(X) is very small, and (b) the probability that MR male offspring will have fra(X) is very large. Thus, accurate prognostic decisions can be based upon prenatal diagnosis of fra(X) presence, especially in families with a pattern of fra(X) linked MR.     

Prenatal diagnosis of 30 fetuses at risk for fragile X syndrome

The results of 30 prenatal diagnoses for fragile X syndrome are reported. Amniotic fluid cells were examined in 1 case, fetal blood in 4, and chorionic villi samples in the others. Of the 5 fetuses analyzed by cytogenetic methods, 1 had showed 4% of fraXq27.3 expression sites and the pregnancy was terminated. For 1 diagnosis, linkage analysis was used: the female fetus turned out to be normal. In 24 fetuses, the direct analysis of the mutation by StB12.3 probe was performed: 6 female and 3 male fetuses were found to carry a full mutation and 1 female fetus was found to carry a premutation. In 3 cases, the diagnoses were verified on fetal blood samples. Several tissues of 2 aborted male fetuses were analyzed for the fragile X mutation. The results are reported and discussed. © 1996 Wiley-Liss, Inc.     

The fragile X syndrome: A study of 83 families

The present report summarizes the experience on the mar(X) syndrome in a total of 157 male patients (44 prepubertal and 113 postpubertal) ascertained through 83 index patients from 83 families under investigation.
1. In one third of the families pedigree data were consistent with X-linked recessive inheritance. In the further two thirds of the families the presenting symptom was familial mental retardation with a mentally retarded mother, or mental subnormality with hyperkinetic behaviour in the male patient.
2. No more than 60% of the adult males presented the typical clinical triad (mental retardation -long face - megalotestes). The most characteristic finding in the mar(X) boy is the psychological profile with severe hyperkinetism, hypersensitivity, handbiting and autistic features in some of them.
3. In 4 of the 27 large mar(X) pedigrees strong evidence was present of a possible transmission of the mar(X) through normal males.
4. The high incidence of mental subnormality in the female offspring of heterozygote carriers, and the relationship between mental status, phenotype, age and expression of the mar(X) in different culture conditions is discussed.     

Fragile X syndrome in mildly mentally retarded children in a Northern Swedish county. A prevalence study

In an extensive etiological study of an unselected series of mildly mentally retarded children (MMR) (IQ 50–70) born 1959–1970 in a northern Swedish county, 5 of 110 boys (4.5%) and none of 61 girls had a fragile site on the distal end of the X-chromosome (Fra Xq 28). Consequently fragile X was seen in 2.9% of the total series of 171 children. In a combined series of severe and mild mental retardation, the incidence of the fragile X syndrome was calculated to be 1:3000 in the county of Vasterbotten. Next to trisomy 21 the fragile X syndrome was the most common single identified cause of MMR in boys. A cytogenetic investigation using special cultural conditions and banding techniques should be performed in cases of mental retardation of unclear etiology and in possible female carriers.     

Screening of mentally retarded males for macro-orchidism and the fragile X chromosome

Four hundred forty-four male residents of a state mental retardation institution were screened for macro-orchidism. Twenty-six white males (8.3%) and two black males (1.5%) had marked macro-orchidism (>34 ml). Seven of 17 whites tested for the fragile X were positive; the one black tested was negative. Thus, a minimum of 7/26 or 27% (whites) are fragile X positive indicating potential population variability, also evident from previous reports. Concurrent testing of institutionalized brother pairs indicated over half of the fragile X-positive males had a strong family history consistent with X-linked mental retardation.     

Multiple displacement amplification improves PGD for fragile X syndrome

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.     

Dilemmas in counselling females with the fragile X syndrome

The dilemmas in counselling a mildly retarded female with the fragile X syndrome and her retarded partner are presented. The fragile X syndrome is an X linked mental retardation disorder that affects males and, often less severely, females. Affected females have an increased risk of having affected offspring. The counselling of this couple was complicated by their impaired comprehension which subsequently impaired their thinking on the different options. The woman became pregnant and underwent CVS, which showed an affected male fetus. The pregnancy was terminated. Whether nondirective counselling for this couple was the appropriate method is discussed and the importance of a system oriented approach, through involving relatives, is stressed.     

The fragile X chromosome: Current methods

At the International Congress of Human Genetics (Jerusalem, September, 1981) a Workshop was held on the fragile X chromosome; it was entitled “The Fragile X Chromosome: Current Methods.” It was to focus on laboratory methods required to detect or to enhance the detection of the fragile X. The fragile site on the X chromosome is one of 13 proven fragile sites known in humans. (A diagram of these sites is provided.) The fragile site on the X is sensitive to the concentration of folic acid in the medium in which cells are cultured. Cytologically, gaps and breaks are increased at the X fragile site, which is located at band Xq27–28. To enhance expression in lymphocytes, prolongation of culture time to 96 hours, elevation of the pH, diminution of the colcemid effect, and air drying of slides are helpful. The need for methionine in low-folate media can be overridden by the addition of fluorodeoxyuridine (FUdR). Detection of the fragile X in males requires meticulous attention to methods of lymphocyte culture and metaphase preparation and then the examination of a sufficient number of mitoses, eg, 50–100 metaphases per individual. Detection of the fragile X in female carriers is often more difficult. Uniform detection of all obligate female carriers has not been achieved. Difficulty may correlate with increasing age or intelligence of females. Key methodologic advances with the fragile X include the addition of methotrexate, trifluorothymidine or, especially of FUdR to the culture medium. FUdR, for example, is helpful in demonstrating the fragile X in lymphoblastoid cell lines and fibroblasts. Both of these cell types now represent an opportunity to study the biochemistry of the fragile X. The success of the FUdR technique with skin fibroblasts heralds the feasibility of demonstrating the fragile X chromosome in cultured amniocytes. Since the Workshop, it has been reported that with FUdR the fragile X could in fact be detected in 46,XY amniotic fluid cells.     

Prenatal diagnosis of fragile X syndrome and the risk of expansion of a premutation

The aim of the present study was to evaluate prospectively the dynamics of the FMR1 gene. The risk of full mutation among pregnant women and the carriers, and the risk of expansion of a premutation allele to a full mutation were estimated. We identified 89 pregnant women with an expanded FMR1 gene seeking prenatal diagnosis. Amniocentesis or chorion villus sampling (CVS) was offered and a DNA test of the FMR1 gene was carried out in such pregnancies. The overall risk of full mutation among women (N = 21) with a repeat size between 60 and 80 was 4.8% (one fetus with mosaicism), and the risk of expansion of the premutation allele to a full mutation was 14% in those offspring to whom the premutation allele was transmitted. The risk of full mutation among the carriers (N = 13) with a repeat size between 81 and 100 was 61.5% (8/13), and the risk of expansion of a premutation allele to a full mutation was 89%. Only one case fell into the category of 101-200 repeats, and expansion to a full mutation was recorded. Fetuses of full mutation mothers inherited the larger allele in 64% (14/22) of the cases. The range of 40-59 repeats was safe: there were no fetal full mutations. The risk of full mutation was also low among the subjects with a repeat size between 60 and 80, whereas the risk increased significantly after 80 repeats. Maternal premutation size was positively correlated with the risk of having a full mutation offspring.     

Fragile X syndrome with extra microchromosome

A mentally retarded male with Martin-Bell syndrome, who has an extra microchromosome and is fra X negative in cytogenetic study is reported. Because of its small size, the origin of the microchromosome could not be determined. Two other affected males in this family (a cousin and a nephew of the proband) were fragile X positive, 24% and 26%, respectively. Cytogenetic studies and DNA analysis with the probe St B 12.3 were performed on several members of the family. The proband and the two other affected males showed a similar full mutation on the molecular study. This study emphasizes the importance of molecular analysis in the diagnosis of fragile X syndrome, particularly when cytogenetic studies demonstrate fra X negative in individuals in families likely to have X-linked mental retardation.     

Fragile site X chromosomes and X-linked mental retardation in severely retarded boys in a northern Swedish county. A prevalence study

In an unselected series of 96 severely mentally retarded boys (IQ < 50) born 1959–70 in a northern Swedish county, six had a fragile site on the distal end of the X chromosome (FraXq 28). The prevalence of the fragile X syndrome in severely retarded boys was 6 %. Next to trisomy 21, this fragile X syndrome appears to be the most common single cause of severe mental retardation in boys.     

Detection of the fragile X chromosome and other fragile sites

To assist in cell sample size selection and detect the fragile X chromosome, statistical tables have been presented. A comparable approach had been suggested earlier for the diagnosis of chromosome mosaicism. The cytologic detection of the fragile X or any fragile site is simply a special case in the detection of mosaicism. Minimum numbers of metaphases are recommended for fragile X analysis to have 95% confidence that the fragile X is not manifest in a given proportion of cells and similar recommendations apply to all other fragile sites.