Transition from normal to premutated alleles in fragile X syndrome results from a multistep process.

In fragile X syndrome, the most common cause of inherited mental retardation, phenotypic expression has been linked to a region containing a repetitive sequence, (CGG)n, that appears to lengthen dramatically in fragile X patients and to show length variation in normal individuals. In order to investigate possible mechanisms responsible for further expansion of CGG in the normal population, we selected 31 normal unrelated X chromosomes carrying either the high-risk DX204-AC155 or DX196-AC151 haplotypes, as defined by the flanking microsatellites, DXS548 and FRAXAC2. Nearly 100% of CGGs with more than 35 repeats were found on DX204-AC155 haplotypes, with a mean length significantly higher and much more variable than in normal individuals carrying other haplotypes including the high-risk haplotype DX196-AC151. These findings suggest that the transition from the normal to the abnormal range occurs by a multistep process, a primary event, such as unequal crossing-over, leading to increased size and moderate instability of the repeat, and from which DNA polymerase slippage could lead to recurrent premutations. Our results also suggest that the upper limit of the normal range is roughly 35 repeats in the fragile X gene. The 36-54 repeats range would define an intermediate allele only observed, up to now, in DX204-AC155 fragile X chromosomes.

Prenatal losses of trisomy 21.

Various cytogenetic studies of early spontaneous abortions have observed trisomy 21 in about 3% of the abortuses. Analyses of prenatal diagnosis performed during the 17th week of gestation and observations on late spontaneous abortions and perinatal deaths have also shown the importance of lethal trisomy 21 in the second half of pregnancy. From these studies, it can be estimated that at least four-fifths of conceptuses with trisomy 21 fail to survive. One important question is why the same chromosome anomaly in a zygote can lead either to an early arrest of development or to a liveborn infant. Pathologic examinations of spontaneously aborted embryos with trisomy 21 have rarely shown malformations that can explain the death of the embryo, but macroscopic and microscopic examinations of their placentas show growth retardation and hypoplasia. Analysis of in vitro growth characteristics of cell lines established from tissues of trisomy 21 embryos have demonstrated differences in cell population doubling times; this may explain variations in the developmental potentials of conceptuses with trisomy 21.

Affected sibs with fragile X syndrome exhibit an age-dependent decrease in the size of the fragile X full mutation.

The sizes of the fragile X mutation in 33 sib pairs affected with fragile X syndrome were determined by Southern blot analysis. An age-dependent decrease in the size of the mutation was found, suggesting positive selection of blood cells carrying small mutations during life or maternal imprinting.

[Prenatal diagnosis of genetic diseases using chorionic villi]. / Diagnostic prénatal des maladies géniques sur villosités choriales.

Chorionic villi biopsy allows first trimester prenatal diagnosis of some genetic diseases. In this study the results of 163 diagnoses are presented, and among these, 59 diagnoses of autosomal recessive metabolic diseases with 8 observations of congenital adrenal hyperplasia due to 21 OH deficiency and 6 observations of Fanconi anemia.

Identification of C trisomies in human abortuses.

Fifteen cell lines initiated from embryonic tissues of abortuses with C trisomy were frozen and stored. They were thawed and karyotyped again with banding techniques. Trisomies 7, 8, 9, 10, and 12 were identified. Some characteristics of these abortuses are discussed in connection with the chromosome identifications.

In vitro cultivation of cells from aneuploid human embryos. Initiation of cell lines and longevity of the cultures.

During a cytogenetic study of human spontaneous abortions, attempts were made to initiate cell lines from tissues of embryos with chromosomal anomalies. The rate of success of these attempts and the life-span of the cell cultures is correlated with the development attained by these aneusomic embryos.

[Antenatal diagnosis of structural chromosome anomalies. 226 cases (author's transl)]. / Diagnostic prénatal des anomalies de la structure chromosomique. 226 observations.

A series of 226 antenatal diagnoses of chromosome structural re-arrangements in 181 couples has shown the usefulness of such diagnosis for couples including a genitor carrying a balanced anomaly. Analysis of the data has elicited major variations in the incidence of unbalanced anomalies in foetal cells and has provided information which can usefully be applied to genetic counselling.

[Identification by Q and G bands of chromosome anomalies in spontaneous abortion]. / Identifaction par les bandes Q et G des anomalies chromosomiques dans les avortements spontanés

Banding technique were applied to cell lines which had been established earlier from spontaneous human abortions and preserved frozen. Most of the autosomes were shown to be involved in lethal trisomies. As a result of the precise identification of affected chromosomes, the exact time of developmental arrest as well as certain phenotypes could be correlated with the various trisomies.

Early prenatal diagnosis of 21-hydroxylase deficiency using amniotic fluid 17-hydroxyprogesterone determination and DNA probes.

The results of early prenatal diagnoses of congenital adrenal hyperplasia are reported. The determination of 17-hydroxyprogesterone values in amniotic fluid taken transabdominally at 11 weeks of gestation enabled prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency. There is a clear-cut difference between normal and pathological values at that time of pregnancy. This method of diagnosis can be combined with genotyping of the fetus by HLA-DNA probes on chorionic villus sampling or can be used alone. Prenatal diagnosis with a 21-OH probe is possible when a preliminary study has demonstrated that the index case is homozygous for the deletion.

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.

Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes.

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X-linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.