Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD.

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Clinical array comparative genomic hybridization: a new paradigm.

BACKGROUND: Although the clinical utility of array comparative genomic hybridization (aCGH) is undisputed, the implementation of this technology is a unique experience for each laboratory. OBJECTIVE: Endeavors to construct a bacterial artificial chromosome (BAC)-based CGH microarray targeting microdeletion and duplication syndromes related to mental retardation and developmental delay are described. METHOD: Covering each chromosome at the 650-band level, the array comprises 1360 BAC clones with emphasis on the subtelomeric and pericentromeric regions and enrichment of genomic hot spots containing genes associated with specific constitutional disorders. During development of the array, fluorescence in situ hybridization (FISH) and end-sequencing analysis eliminated 24% of BACs that were mismapped or cross-hybridized, underscoring the need rigorously to assess arrayed elements. Performance of the BACs was tested further with chromosome-specific add-in experiments. CONCLUSION: Of the first 500 clinical cases, 54 (11%) showed chromosome abnormalities, which were confirmed by FISH with BACs from the aberrant loci or by conventional cytogenetics. Array CGH is a powerful tool that is now being implemented in the realm of diagnostic testing.