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

TLDR

The study aimed to develop a rapid, nonradioactive PCR assay capable of detecting full fragile X mutations, premutations, and normal alleles for use in carrier screening and prenatal diagnosis of at-risk pregnancies. The authors implemented a PCR-based screening protocol, confirmed by Southern blotting and cytogenetics, and applied it to 150 unrelated pregnant women and 570 control X chromosomes, with samples processed at a tertiary diagnostic laboratory. The assay accurately resolved normal alleles (modal 30 CGG repeats, ~80% heterozygosity, no excess homozygosity), identified carriers in 5 of 20 known families and 2 new families, and detected nine full‑mutation fetuses among 28 carriers, demonstrating reliable screening, counseling, and prenatal diagnosis.

Abstract

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.Prenatal and blood sample PCR analysis with confirmation by direct Southern blotting and cytogenetic techniques.Samples sent to a DNA diagnostic research laboratory at a tertiary referral center.Pregnant women with a family history of undiagnosed mental retardation or known fragile X syndrome and controls.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.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.