Molecular Combing for Fascioscapulohumeral Dystrophy Type 1: Benefits of Direct Visualization of DNA Fibers. C. M. Strom, J. C. Wang, X. J. Yang, B. H. Nguyen, V. Sulcova, P. Chan, Y. Liu, A. Anguiano, F. Z. Boyar Department of Cytogenetics, Nichols Institute, Quest Diagnostics, 33608 Ortega Highway, San Juan Capistrano, CA 92690.

   Fascioscapulohumeral dystrophy (FSHD) is the third most common muscular dystrophy. Type-1 FSHD is due to a contraction of the D4Z4 microsatellite repeat motif at chromosome 4q subtelomeric region. Chromosome 10q also contains copies of this repeat motif, of which contractions are not associated with FSHD. Two common haplotypes exist at 4q locus: 4qA and 4qB. Differentiation is important for diagnosis, as contractions of the 4qA but not the 4qB allele are associated with FSHD. Prior to the advent of DNA combing, molecular diagnosis relied on a series of pulsed-field Southern blots. These analyses can lead to false-positive results, because they cannot differentiate the A allele from the B allele on chromosome 4q nor can they differentiate chromosome 4q from chromosome 10q. Molecular combing is a technique in which DNA is uniformly stretched and then hybridized with gene-specific probes of various colors to create a molecular bar code. We developed and validated a molecular combing test to identify and measure contractions of the 4qA allele. Here we report the first 44 suspected FSHD cases tested with the molecular combing assay in our laboratory. In all 44, we were able to unambiguously identify all 4q and 10q alleles and determine the size of the D4Z4 repeat. Of the 44 samples, 13 (30%) were clearly affected, with 4qA repeat sizes between 3 and 8; 28 (63%) were clearly normal, with 4qA repeat sizes between 12 and 68; and 3 (7%) had a 4qA allele of 10-11 repeats, which is a borderline result. Two patients had a 4qB contraction and 15 had either 10qA or 10qB contraction, which could have led to a false-positive result if Southern blot had been used. We conclude that a molecular combing assay for FSHD is capable of determining the 4qA repeat size in clinical samples and can prevent false-positive results by differentiating 4qA from 4qB, 10qA, 10qB alleles. Additionally, more accurate measurement of repeat number by DNA combing methods may help correlate the size of the contracted 4qA allele with the timing of FSHD1 onset.

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