Recessive Truncating Titin Gene, TTN, Mutations Presenting As Centronuclear Myopathy

Supplemental Data

Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy

Ozge Ceyhan, PhD*; Pankaj B. Agrawal, MD, MMSc*; Carlos Hidalgo, PhD; Klaus Schmitz-Abe,PhD; Elizabeth T. DeChene, MS, CGC; Lindsay C. Swanson,MS, CGC; Rachel Soemedi, PhD; Nasim Vasli, PhD; Susan T. Iannaccone, MD; Perry B. Shieh, MD, PhD; Natasha Shur, MD; Jane M. Dennison, MD; Michael W. Lawlor, MD, PhD; Jocelyn Laporte, PhD; Kyriacos Markianos,PhD; William G. Fairbrother, PhD; Henk Granzier, PhD; Alan H. Beggs,PhD

From the Division of Genetics and Program in Genomics, The Manton Center for Orphan Disease Research (O.C., P.B.A., K.S., E.T.D., L.C.S., K.M., A.H.B.), Boston Children’s Hospital, Harvard Medical School, Boston, MA; Division of Newborn Medicine (P.B.A.), Boston Children’s Hospital, Harvard Medical School, Boston, MA; Department of Physiology and Sarver Molecular Cardiovascular Research Program (C.H., H.G.), University of Arizona, Tucson, AZ; Center for Computational Molecular Biology and Department of Molecular and Cellular Biology and Biochemistry (R.S., W.G.F.), Brown University, Providence, RI; Department of Translational Medicine (N.V., J.L.), IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France;Departments of Pediatrics and Neurology and Neurotherapeutics (S.T.I.), University of Texas Southwestern Medical Center, Dallas, TX; Department of Neurology (P.B.S.), University of California, Los Angeles, CA; Division of Human Genetics (N.S.), Department of Pediatrics, Rhode Island Hospital, Providence, RI;Department of Pediatrics (J.M.D.), Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine (M.W.L), Medical College of Wisconsin, Milwaukee, WI; Hasbro Children’s Hospital, Brown University, Providence, RI; 1Center for Biomedical Engineering (W.G.F.), Brown University, Providence, RI

*These authors contributed equally to the manuscript.

Correspondence: Dr. Alan H. Beggs:

Figure e-1. Identification and Sanger sequencing confirmation of TTN variants in CNM patients

(A) Summary of whole-exome sequencing results for patients with TTN mutations. Non-synonymous variants include frameshift/in-frame deletions/insertions, stopgain/loss mutations, and non-synonymous SNPs.

(B) The TTN variants identified in fivepatients by next generation sequencing were confirmed by Sanger sequencing. All five individuals were found to be compound heterozygotes for different disruptingTTN variants.

Figure e-2. Structural integrity of the triad-associated protein complex in patient muscles

Immunofluorescence analysis of patient and age-matched healthy control muscles using ryanodine receptor (RyR), aldolase A (AldoA), and α-actinin antibodies. Muscle biopsy sample from patient 314-1showed reduced staining for aldolase A, while the levels of both proteins in patients1044-1 and 1093-1 were comparable with the controls. Representative figures of staining on three different sections for each patient are presented. Scale bar = 40 m.

Table e-1. Analysis of theTTNmutations disrupting splice sites
Patient / Allele / NM_133378 cDNA / NM_133378 Exon / MaxEnt score wild-type / MaxEnt score mutant
314-1 / P / c.32854G>C / 168 / 3.9 / -10.3a
M / c.37112-1G>C / int 191 / 9.27 / 0.57
979-1 / P / c.11764+1G>A / int 49 / 8.54 / 0.36
1093-1 / P / c.18229G>A / 72 / 4.3 / 0.33
Mutations within the consensus splice sites were evaluated by the degree of which the mutated site deviated from splice site models (i.e. scored with position weight matrices using MaxEntScan),e1 while mutations outside of the canonical splice sites were evaluated by Spliceman,e2,e3 a program that uses positional distribution to identify splicing mutations.
aA negative MaxEnt score indicates the sequence is more likely to occur in random sequence than in the set of splice sites.

e-References

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