A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy

Figures

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  Fig 1. Clinical data. A. Clinical data of the proband (III:3). A1, A2: Hand extensors weakness. A3: Palmar atrophy, mainly in thenar muscles. A4: Peroneus lateralis, markedly affected, as all legs muscles except for tibialis anterior and posterior muscles. A5: Leg atrophy. B. MRI of the proband. B1: Thigh. Hamstrings and adductors severely involved; quadriceps partially spared, except right vastus lateralis and intermedialis. B2: Legs. Peroneal lateralis severely affected, as all muscles except tibialis anterior and tibialis posterior, B3: Pelvic muscles spared in comparison with femoro-distal muscles. C. Morphological studies. C1-C4 Radialis muscle biopsy of proband (III:3). C1. Haematoxylin & Eosin staining: presence of some nuclear internalization and fiber size variation. C2 NADH: lobulated fibers are indicated by an arrow; uneven staining of the intermyofibrillar network in some fibers is indicated by a star. C3 desmin: mild diffuse desmin surcharge in several fibers C4 myotilin: normal staining. C5-C8 Peroneal muscle biopsy from the proband’s son (IV:2). C5 NADH: atrophic lobulated fibers are indicated by an arrow. C6 desmin: mild diffuse desmin surcharge in several fibers. C7…

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  Fig 2. Genetic analysis of OBSCN c.13330 C>T and FLNC c.5161delG mutations. A. Family pedigree. Black filled symbols represent affected family members. The genotype of individuals is shown as follows: +/+ wild type, OBSCN +/- heterozygous for the c.13330 C>T mutation, FLNC +/- heterozygous for the c.5161delG mutation. Arrow indicates the proband. B. Electropherogram of the OBSCN gene sequence (upper panel) and of the FLNC gene sequence (lower panel). C. Phylogenetic alignment of the OBSCN orthologs. Sequences represent the Ig59 domains of obscurin in distinct mammalian species. The arginine in position 4444 in the human OBSCN sequence is highlighted in yellow and is conserved in mammalian species. D. Interaction between titin domains ZIg9/ZIg10 and obscurin domains Ig58/Ig59. In vitro transcribed and translated myc-tagged titin domains ZIg9/ZIg10 were used in pull-down experiments with GST-fusion proteins containing either wild type Ig58/Ig59 domains (GST-obscurin-Ig58/Ig59WT) or R4444W mutated Ig58/Ig59 domains (GST-obscurin-Ig58/Ig59MUT). Proteins were separated by SDS-PAGE, transferred to membranes and detected by mouse anti-myc antibodies. E. Quantification of GST pull-down…

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  Fig 3. Structure and analysis of human obscurin Ig59. A. Cartoon of the Ig59 crystal structure, showing the typical Ig-like fold. B. Comparison between the lowest RMSD Ig59 NMR structure and the X-ray structure. C. CD plot of WT obscurin Ig58-59 (black squares) and p.Arg4444Trp (open circles). D. MD simulated average models of WT (blue) and p.Arg4444Trp (red). Ca position. The side chains for Arg4444/Trp4444 are shown. E. RMSD vs residue number comparison of the mutant model to the wild-type model. The Arg4444Trp site is colored in red.

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  Fig 4. Both domains of obscurin Ig58/Ig59 are involved in binding titin ZIg9/ZIg10. HSQC chemical shift changes of Ig58/Ig59 upon the addition of 0.5x human titin ZIg9/ZIg10, mapped onto a model of obscurin Ig58/Ig59 (A) or by residue number (B). Gray denotes residues with no data; green denotes residues with no significant chemical shift changes; yellow denotes residues chemical shift changes greater than 2x standard deviation; red denotes residues experiencing a large drop in peak intensity of greater than 2.5x the average intensity. The two obscurin mutations linked to muscle disease, Arg4344 and Arg4444, are labeled and colored blue.