George E. Houck

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.

A Comparison of Adult and Childhood Progerias: Werner Syndrome and Hutchinson-Gilford Progeria Syndrome

The Werner syndrome, also known as progeria of the adult, and the childhood Hutchinson-Gilford Progeria Syndrome (hereafter Progeria), both serve as genetic disease models of human aging (Brown, 1979). A comparison of their similarities and differences may be useful in order to gain insight into the nature of the genetic mutations underlying these conditions. Their modes of inheritance indicate the involvement of a single gene. This implies that some specific genes may lead to a phenotype of greatly accelerated senescence and that such genes may have direct effects on the rate of aging. Determining the basic mechanisms involved in producing their phenotypes may point the way to an understanding of important pathogenetic aspects underlying the aging process.

Reverse mutations in the fragile X syndrome.

Three females were identified who have apparent reversal of fragile X premutations. Based on haplotype analysis of nearby markers, they were found to have inherited a fragile X chromosome from their premutation carrier mothers, and yet had normal size FMR1 repeat alleles. The changes in repeat sizes from mother to daughter was 95 to 35 in the first, 145 to 43 in the second, and 82 to 33 in the third. In the first family, mutations of the nearby microsatellites FRAXAC2 and DXS548 were also observed. In the other two, only mutations involving the FMR1 repeats were found. We suggest differing mutational mechanisms such as gene conversion versus DNA replication slippage may underlie such reversions. We estimate that such revertants may occur among 1% or less of premutation carrier offspring. Our results indicate that women identified to be carriers by linkage should be retested by direct DNA analysis.

FMR1 CGG-Repeat Instability in Single Sperm and Lymphocytes of Fragile-X Premutation Males

To determine the meiotic instability of the CGG-triplet repeat in the fragile-X gene, FMR1, we examined the size of the repeat in single sperm from four premutation males. The males had CGG-repeat sizes of 68, 75, 78, and 100, as determined in peripheral blood samples. All samples showed a broad range of variations, with expansions more common than contractions. Examination of single lymphocytes indicated that somatic cells were relatively more stable than sperm. Surprisingly, the repeats in sperm from the 75- and 78-repeat males had very different size ranges and distribution patterns despite the similarity of the repeat size and AGG interruption in their somatic cells. These results suggest that cis or trans factors may have a role in male germline repeat instability.

Fine mapping of an Alzheimer Disease-associated gene encoding beta-amyloid protein

We have sublocalized an Alzheimer Disease-associated gene, which encodes for cerebrovascular beta-amyloid protein, to the region from the centromere through the proximal half of band 21q21 using both somatic cell and in situ mapping techniques. In addition we found repeatedly significant but weaker hybridization of the beta-amyloid protein probe to the short arm of chromosome 20. 794 cells were analyzed from whole blood, lymphoblastoid and skin cultures. The latter two types of cultures had parts of the 21st chromosome translocated to other chromosomes facilitating sublocalization.

Fragile X full mutation alleles composed of few alleles: implications for CGG repeat expansion.

Southern analysis of the FMR1 repeat region has suggested that individuals with the full mutation usually carry a heterogeneous array of FMR1 alleles in somatic tissue that can range from 200 to more than 1,000 repeats. Our studies indicate that this heterogeneity is an artifact generated by ethidium bromide commonly used in Southern analysis. When analyzed in the absence of ethidium bromide, nearly all full mutation individuals carried only one to four major alleles and did not exhibit the heterogeneity often referred to as a “smear” in the literature. Full mutations in chorionic villi, however, exhibited much greater heterogeneity. Nine transmissions from mothers with full mutation alleles to offspring indicated that the full mutations continued to expand in transmission to the next generation. In contrast, analysis of leukocyte DNA from three full mutation males revealed no change in somatic full mutation alleles over many years. Our studies support the hypothesis that the FMR1 CGG repeat instability is limited to very early embryogenesis in the soma. These studies also have clinical importance because the omission of ethidium bromide will facilitate the diagnosis of females with full mutation alleles.

Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.

Accelerated prenatal diagnosis of fragile X syndrome by polymerase chain reaction restriction fragment detection.

Prenatal diagnosis of fragile X syndrome requires detection of the full FMR1 mutation in chorionic villus or amniotic fluid cell samples. Although analysis of genomic DNA restriction fragment pattern is a highly reliable technique for identification of the full FMR1 mutation, standard Southern blot determination of this pattern requires significantly more genomic DNA than is initially available from a prenatal sample. To overcome this limitation we developed a method that determines the diagnostic pattern of genomic restriction fragments from a fraction of a prenatal specimen. The prenatal DNA sample is first digested with EcoRI and EagI, and after agarose gel electrophoresis, the 2- to 10-kb region of the gel is serially sectioned and amplified by polymerase chain reaction. Analysis of prenatal samples from an unaffected male and from a full mutation male showed that this approach generated a diagnostic pattern comparable with a Southern blot of 100-fold more material. This innovation enables laboratories to prenatally diagnose the full FMR1 mutation sooner than standard techniques.

Rapid Fragile X Carrier Screening and Prenatal Diagnosis Using a Nonradioactive PCR Test

OBJECTIVE To develop a rapid, nonradioactive test using the polymerase chain reaction (PCR) capable of detecting full fragile X mutations, premutations, and resolving normal alleles and to apply this to prenatal diagnosis and carrier screening of pregnant women at risk for fragile X carrier status. DESIGN Prenatal and blood sample PCR analysis with confirmation by direct Southern blotting and cytogenetic techniques. SETTING Samples sent to a DNA diagnostic research laboratory at a tertiary referral center. PARTICIPANTS Pregnant women with a family history of undiagnosed mental retardation or known fragile X syndrome and controls. RESULTS A rapid, nonradioactive PCR screening protocol for the fragile X mental retardation-1 gene for both normal and mutant alleles was developed. Analysis of 570 control X chromosomes showed a modal number of 30 CGG repeats (range, 12 to 52 repeats) and a calculated heterozygosity of approximately 80%. No excess of homozygosity was found, indicating the test was accurate for normal allele resolution. In addition, 150 unrelated pregnant women were screened. Within known fragile X families, five of 20 pregnant women were diagnosed as carriers. Two new fragile X families were diagnosed among relatives of 130 females with family histories of undiagnosed mental retardation, although no carriers were identified. Prenatal PCR testing of 28 carriers accurately detected nine fetuses with full mutations. CONCLUSIONS This rapid, nonradioactive PCR protocol allows accurate resolution of normal alleles as well as simultaneous detection of carrier alleles and full mutations. With this approach, efficient screening of pregnant women at risk for fragile X carrier status, subsequent genetic counseling of identified carriers, and reliable prenatal diagnosis can be offered.