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.