Pierre Walrafen

Molecular combing reveals allelic combinations in facioscapulohumeral dystrophy.

The genetic variation underlying facioscapulohumeral muscular dystrophy (FSHD), 1 of the most common hereditary neuromuscular disorders, is complex, and associated with the contraction of a repeat array (D4Z4) at the subtelomeric end of chromosome 4q. Nonpathogenic variants of 4q and the presence of a homologous array on chromosome 10q make FSHD diagnosis extremely challenging, at least in individuals with nonstandard D4Z4 arrays. We aimed to improve FSHD molecular analysis by proposing an alternative technique to the Southern blot.

A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2

The BRCA1 and BRCA2 genes are involved in breast and ovarian cancer susceptibility. About 2 to 4% of breast cancer patients with positive family history, negative for point mutations, can be expected to carry large rearrangements in one of these two genes. We developed a novel diagnostic genetic test for the physical mapping of large rearrangements, based on molecular combing (MC), a FISH‐based technique for direct visualization of single DNA molecules at high resolution. We designed specific Genomic Morse Codes (GMCs), covering the exons, the noncoding regions, and large genomic portions flanking both genes. We validated our approach by testing 10 index cases with positive family history of breast cancer and 50 negative controls. Large rearrangements, corresponding to deletions and duplications with sizes ranging from 3 to 40 kb, were detected and characterized on both genes, including four novel mutations. The nature of all the identified mutations was confirmed by high‐resolution array comparative genomic hybridization (aCGH) and breakpoints characterized by sequencing. The developed GMCs allowed to localize several tandem repeat duplications on both genes. We propose the developed genetic test as a valuable tool to screen large rearrangements in BRCA1 and BRCA2 to be combined in clinical settings with an assay capable of detecting small mutations. Hum Mutat 33:998–1009, 2012.

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.

O.6 Beyond counting copies: direct visualization of copy number variations in a specific genomic context to explore facio-scapulo-humeral dystrophy (FSHD)

‘‘calpainopathy”) is caused by defects in p94/calpain 3, which is the skeletal muscle-specific member of Ca-dependent cysteine protease ‘‘calpain”. Mammals have 15 genes for calpains (CAPN1 CAPN3, CAPN5 CAPN16), which are involved in a variety of cellular functions, and p94 is encoded by CAPN3. Particularly, mouse gene knock-out of Capn2, which encodes catalytic subunit of ubiquitous m-calpain, causes embryonic lethality, and that ofCapn3 results in calpainopathy inmice, indicating an essential role of calpain inmammalian life and health. However, the mechanism of p94’s function in the pathogenesis of calpainopathy remains unclear. Here, we demonstrate that the stretch-dependent p94 distribution plays a crucial role in the pathogenesis of calpainopathy, using p94:C129S ‘‘knock-in” (p94CS-KI) mice, in which the endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129Smutant protein. The p94CS-KImice developed a progressivemuscular dystrophy, which was exacerbated by ageing or exercise, although their phenotype (myofibril integrity, serum CK levels, etc.) is less severe than that of p94 knock-out (p94KO) mice. The exercise-induced muscle degeneration in p94CS-KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94CS-KI mice showed impaired adaptation to physical stress, accompaniedbyanexercise-dependent alteration in themuscle ankyrin-repeat protein-2 (MARP2) level. These findings indicate that the stretch-induced dynamic redistribution of p94 is affected by its protease activity and is essential to protect skeletal muscle cells from degeneration, particularly under physical stress.