Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy

SApproximately 95% of individuals with spinal muscular atrophy (SMA) lack b oth copies of the SMNt gene at 5q13, The presence of a nearly identical cen tromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polyme rase chain reaction approach to direct carrier testing, Adapting a radioact ivity-based method described previously, multiplex polymerase chain reactio n was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 p resumed carriers (50 parents of affected individuals and 10 relatives impli cated by linkage analysis) and 40 normal control individuals were tested. N ormalized results (to the mean of five or more control samples harboring tw o copies of the SMNt gene) were consistently within the ranges of 0.4 to 0. 6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test result s demonstrated de novo deletions associated with crossovers, unaffected ind ividuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copi es of the SMNt gene. This report demonstrates that fluorescence-based carri er testing for SMA is accurate, reproducible, and useful for genetic risk a ssessment, and that carrier testing may need to be combined with linkage an alysis in certain circumstances. (C) 1999 Wiley-Liss, Inc.