title: Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD.
abstract: 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.
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Vasale
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Fatih
authors.initials: F
authors.lastname: Boyar
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Jocson
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Vladimira
authors.initials: V
authors.lastname: Sulcova
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Chan
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Khalida
authors.initials: K
authors.lastname: Liaquat
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Hoffman
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Meservey
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Isabell
authors.initials: I
authors.lastname: Chang
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tsao
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Kerri
authors.initials: K
authors.lastname: Hensley
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Renius
authors.initials: R
authors.lastname: Owen
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Corey
authors.initials: C
authors.lastname: Braastad
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Weimin
authors.initials: W
authors.lastname: Sun
authors.affiliation: Genomic Vision, 92220 Bagneux, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Walrafen
authors.affiliation: Genomic Vision, 92220 Bagneux, France.
authors.forename: Jun
authors.initials: J
authors.lastname: Komatsu
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Jia-Chi
authors.initials: JC
authors.lastname: Wang
authors.affiliation: Genomic Vision, 92220 Bagneux, France.
authors.forename: Aaron
authors.initials: A
authors.lastname: Bensimon
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Arturo
authors.initials: A
authors.lastname: Anguiano
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Jaremko
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Zhenyuan
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA.
authors.forename: Sat
authors.initials: S
authors.lastname: Batish
authors.affiliation: Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Strom
authors.affiliation: Quest Diagnostics, Athena Diagnostics, Marlborough, MA, USA. Electronic address: joseph.j.higgins@questdiagnostics.com.
authors.forename: Joseph
authors.initials: J
authors.lastname: Higgins
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D002894:Chromosomes, Human, Pair 4; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020391:Muscular Dystrophy, Facioscapulohumeral / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: Blotting; Chromosome mapping; Electrophoresis; Facioscapulohumeral; Fluorescence; Gel; Gene mapping; In situ hybridization; Muscular dystrophy; Pulsed-field; Southern