RAPID FRAGILE-X CARRIER SCREENING AND PRENATAL-DIAGNOSIS USING A NONRADIOACTIVE PCR TEST

Objective.-To develop a rapid, nonradioactive test using the polymeras e chain reaction (PCR) capable of detecting full fragile X mutations, premutations, and resolving normal alleles and to apply this to prenat al diagnosis and carrier screening of pregnant women at risk for fragi le X carrier status. Design.-Prenatal and blood sample PCR analysis wi th confirmation by direct Southern blotting and cytogenetic techniques . Setting.-Samples sent to a DNA diagnostic research laboratory at a t ertiary referral center. Participants.-Pregnant women with a family hi story of undiagnosed mental retardation or known fragile X syndrome an d controls. Results.-A rapid, nonradioactive PCR screening protocol fo r the fragile X mental retardation-1 gene for both normal and mutant a lleles was developed. Analysis of 570 control X chromosomes showed a m odal number of 30 CGG repeats (range, 12 to 52 repeats) and a calculat ed 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 carrie rs. Two new fragile X families were diagnosed among relatives of 130 f emales with family histories of undiagnosed mental retardation, althou gh no carriers were identified. Prenatal PCR testing of 28 carriers ac curately detected nine fetuses with full mutations. Conclusions.-This rapid, nonradioactive PCR protocol allows accurate resolution of norma l alleles as well as simultaneous detection of carrier alleles and ful l mutations. With this approach, efficient screening of pregnant women at risk for fragile X carrier status, subsequent genetic counseling o f identified carriers, and reliable prenatal diagnosis can be offered.