Charlotte J. Duncan

Prenatal diagnosis and carrier screening for fragile X by PCR

During the past three years, we have conducted fragile X DNA studies for carrier screening and prenatal diagnosis using a previously described PCR protocol that accurately resolves normal FMR1 alleles and premutations and detects most full mutations [Brown et al., JAMA 270:1569-1575, 1996]. A total of 344 pregnant women with a family history of mental retardation of unknown cause were screened and 6 fragile X carriers were identified: two had full mutations, and four had premutations. The mentally retarded relatives of two other women were found to be fragile X positive although the women themselves were not carriers. In all, 6 carriers and 8 fragile X families were identified by this screening. We have also screened 40 pregnant women who were members of previously identified fragile X families, but whose carrier status was unknown. Ten were found to be carriers and were offered prenatal diagnosis. Prospective prenatal testing of 84 carrier women correctly detected 31 fetal samples (19 females, 12 males) with full mutations and 6 with premutations (2 females, 4 males). No false positives but one false negative occurred early on due to undetected maternal cell contamination. In addition, screening of 806 males with developmental delays of unknown cause gave positive results in 33 (4.1%). Potential problems and pitfalls of direct DNA testing are discussed. Because of the proven success of fragile X screening with direct molecular analysis, screening of all undiagnosed individuals with mental retardation and at risk pregnant women should now be considered. The identification of fragile X carriers and prenatal diagnosis of their pregnancies should significantly reduce the prevalence of this syndrome.

Further evidence for genetic heterogeneity in the fragile X syndrome

SummaryThe X-linked fragile X[fra(X)] syndrome, associated with a fragile site at Xq27.3, is the most common Mendeban inherited form of mental deficiency. Approximately 1 in 1060 males and 1 in 677 females carry the fra (X) chromosome. However, diagnosis of carrier status can be difficult since about 20% of males and 44% of females are nonpenetrant for mental impairment and/or expression of fra (X). We analyzed DNA from 327 individuals in 23 families segregating fra (X) for linkage to three flanking polymorphic probes: 52A, F9, and ST14. This allowed probable nonpenetrant, transmitting males and carrier females to be identified. A combined linkage analysis was conducted using these families and published probe information on F9 in 27 other families, 52A in six families, and ST14 in five families. The two-point recombination fraction for 52A-F9 was 0.13 (90% confidence interval, 0.10–0.16), for F9-fra(X) was 0.21 (0.17–0.24), and for fra(X)-ST14 was 0.12 (0.07–0.17). Tight linkage between F9 and fra(X) was observed in some families; in others loose linkage was seen suggesting genetic linkage heterogeneity. Risk analysis of carrier status using flanking DNA probes showed that probable nonpenetrant transmitting males were included in families showing both tight and loose linkage. Thus, in contrast to our previous conclusions, it appears that the presence or absence of nonpenetrant, transmitting males in a family is not an indicator of heterogeneity. To determine if heterogeneity was present, we employed the admixture test. Evidence for linkage heterogeneity between F9 and fra(X) was found, significant at P<0.0005. Nonsignificant heterogeneity was seen for 52A-F9 linkage. No heterogeneity was found for fra(X)-ST14. The frequency of fra(X) expression was significantly lower in families with tight F9-fra(X) linkage than in families with loose linkage. Cognition appeared to relate to linkage type: affected males in tight linkage families had higher IQs than those in loose linkage families. These findings of genetic heterogeneity can account in part for the high prevalence and apparent high new mutation rate of fra(X). They will affect genetic counseling using RFLPs. An understanding of the basis for genetic heterogeneity in fra(X) will help to clarify the nature of the unusual pattern of inheritance seen in this syndrome.

Normal adaptive function with learning disability in duplication 8p including band p22.

Duplication 8p usually results in a syndrome characterized by profound mental retardation, mild facial anomalies, and malformations of hand, heart, and brain. We report on a large kindred segregating a Y;8 translocation in whom several individuals have duplication 8p22-->8pter. These individuals have normal adaptive function despite their unbalanced karyotype. The family was studied with G-banding and fluorescent in situ hybridization (FISH) using probes to chromosomes 8 and Y. Comparison of this family with other reported cases defines a mild clinical outcome for trisomy 8p22-->8pter in contrast to the severe findings when the duplication involves a longer, more proximal segment.

Low frequencies of apparently fragile X chromosomes in normal control cultures: A possible explanation

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.

Atypical Down syndrome and partial trisomy 21

A case of “atypical” Down Syndrome (DS), where the proposita did not exhibit all of the clinical features of DS and had de novo partial trisomy 21, was studied. Results from phenotypic, chromosome banding and superoxide dismutase (SOD) gene dosage studies suggest a karyotype of 46,XX,‐12, + t(12pter to 12qter::21q21 to 21q22.?2). Additional studies of such atypical cases will provide more precise sublocalization for both gene and phenotypic mapping of the bands that are responsible for the DS phenotype.

Distal duplication 14q: report of three cases and further delineation of the syndrome

SummaryThree cases of distal duplication 14q are presented. The first two cases are cousins in a kindred segregating a balanced translocation t(14;18)(q31;q23). The third case resulted from a maternal translocation t(14;18)(q24;p11). By review of these cases and those previously reported, a distal duplication 14q syndrome is further delineated. Common features include postnatal growth retardation, mental retardation, hypotonia, microcephaly, slanted palpebral fissures, ocular hypertelorism, sparse eyelashes and eyebrows, nasal dysmorphism, tented lip, micrognathia, posteriorly rotated ears, and minor skeletal anomalies.

Chromosomal abnormalities in amniotic fluid cell cultures: a comparison of apparent pseudomosaicism in Chang and RPMI‐1640 media

The finding of chromosome mosaicism is one of the most difficult problems in fetal chromosome analysis. Whether the finding indicates true mosaicism or pseudomosaicism must be investigated. Studies detailing the incidence of true mosaicism and pseudomosaicism have been reported (Hsu & Perlis 1984, Bui et al. 1984, Worton & Stern 1984) but do not correlate pseudomosaicism with any particular type of culture media. Benn & Hsu (1985) compared cell growth and chromosome abnormalities in amniotic fluid cell cultures grown in Chang medium and RPMI‐1640 medium and found no statistically significant difference in the rate of abnormalities in the two media. We have previously shown that Chang medium exhibited more abnormalities which were not verified in second and third cultures (Masia et al. 1986). In the current study we examined 212 cases grown in both Chang and RPMI‐1640 media, and compared apparent single and multiple cell pseudomosaic abnormalities to medium type. The number of observed abnormalities was 22, occurring in 19 of the cases studied. Apparent pseudomosaic chromosome anomalies were observed in 18 Chang cultures and in 4 RPMI‐1640 cultures. Statistical analysis found significant correlation between medium type and the degree of observed pseudomosaic cells. We conclude that the rate at which pseudomosaic cells are observed is partly a function of medium type, and in our laboratory Chang medium caused apparent pseudomosaicism at a greater level than RPMI‐1640 medium.

Progress toward an Internal Control System for Fragile-X Induction by 5-Fluorodeoxyuridine in Whole-Blood Cultures

We have been attempting to develop a consistently reliable internal control to assure the effectiveness of the 5-fluorodeoxyuridine (FUdR) fragile-X [fra(X)] induction system. We carried out a systematic study of whole-blood specimens cultured from 56 individuals from two different laboratories. An analysis of nearly 9,000 cells demonstrated: (1) the importance of establishing baseline levels of fragile sites in each laboratory, and (2) that a combination of common fragile sites (different for each laboratory) could serve as a consistently reliable indicator of the effectiveness of the FUdR fra(X) induction system. It was suggested that a non-FUdR culture(s) should be incorporated into a laboratorys fra(X)-screening protocol, so that if there are any doubts about the effectiveness of the FUdR system a comparison to background or spontaneously occurring fragile sites can be made within the laboratory. Repeat cultures are recommended where no increase in common fragile-site frequency is observed in the FUdR induction system, and where fra(X) was strongly suspected but not found. In addition, the necessity of using more than one fra(X) induction system in whole-blood cultures was demonstrated, including the effectiveness of an FUdR/excess thymidine double-induction system. Finally, 2 cases of apparent mosaicism for Klinefelter syndrome in fra(X) individuals were observed.