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

Abstract

Objective: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes.

Methods: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest.

Results: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations.

Conclusions: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.

Figure 1. Skeletal muscle histopathology in patients with centronuclear myopathy carrying TTN mutations

(A–E) Hematoxylin & eosin (H&E) staining of transverse muscle sections from patients 314-1, 966-1, 979-1, 1044-1, and 1093-1 show multiple cells with internal and central nuclei. (F) Percentages of fibers with central nuclei (i.e., nuclei in the geometric center of the fiber), internal nuclei (i.e., nuclei anywhere in the cytoplasm but the geometric center), and peripheral nuclei (i.e., nuclei underneath the sarcolemma, at the cell periphery) were calculated based on a count of at least 200 fibers. (G–K) Histochemical staining of muscle from patient 1044-1. (G) H&E staining on longitudinal section shows several fibers with multiple internal nuclei arranged in a row (arrows). (H) Adenosine triphosphatase (ATPase) staining at pH 4.3 demonstrates predominance and hypotrophy of darkly stained type I fibers. (I) Cytochrome c oxidase–succinate dehydrogenase (CCO-SDH) and (J) nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-TR) stains show several fibers with central core-like areas devoid of oxidative reaction (arrows). (K) No inclusions or depositions were detected in Gomori trichrome staining. (L–Q) Electron micrograph of muscle from patient 1044-1 (L–O) and from patient 314-1 (P, Q). (L) Fibers with central nuclei (asterisk) demonstrate varying degrees of myofibrillar disorganization, compared with the normal sarcomeres that lie in parallel in a fiber with peripheral nuclei (bottom left corner). (M) Two internal nuclei (asterisk) arranged in a row in a fiber with severe sarcomeric disorganization, and regions devoid of mitochondria (arrow). (N) Z-disk streaming (arrows). (O–Q) Focus on regions with disintegrated sarcomeres showing disrupted I- and A-band regions (arrowheads). Black scale bars = 40 μm; white scale bars = 10 μm. Pt = patient.

Figure 2. TTN mutations associated with centronuclear myopathy cause a loss of titin C-terminal region in patient muscles

(A) Locations of the antibody epitopes used in immunofluorescence analysis are depicted on the skeletal muscle titin isoform N2A (NM_133378). The Z-disk (red), I-band (yellow), A-band (dark blue), and M-band (light blue) regions of a titin molecule spanning halfway of a sarcomere are represented. (B) Locations of the TTN mutations in each patient are represented on the N2A isoform. Arrows depict the type of mutations: frameshift (green), nonsense (red), splicing (black), in-frame insertion/deletion (blue). (C) Immunofluorescence analysis of patient and age-matched healthy control muscles. Frozen muscle sections were stained with titin N-terminal, A–I junction, C-terminal, or calpain 3 antibody (red); and with anti-α-actinin antibody (green). Staining with the control anti-α-actinin antibodies are presented in the inset lower right panels where the titin C-terminal or calpain 3 antibodies showed no signal. (D) Overview of the hybrid minigene splicing assay. Wild-type exon 168 and 192 (numbering based on N2A isoform) were cloned with their flanking introns into the pZW4 splicing reporter construct between HpaI and KpnI restriction sites. The mutations at the donor or acceptor splice sites were introduced by site-directed mutagenesis. Wild-type or mutant hybrid minigene containing plasmids were transfected into HEK293 cells, followed by RNA extraction and reverse transcriptase–PCR using primers flanking the hybrid constructs. (E) Results of the hybrid minigene splicing assay. The minigenes containing wild-type exon 168 or exon 192 were spliced correctly. The c.32854G>C mutation at the donor splice site of exon 168 caused exon skipping (left panel). The c.37112-1G>A mutation at the acceptor splice site of exon 192 resulted in the majority of transcripts to remain unspliced, as well as exon skipping or intron retention in a subset of transcripts (right panel). Scale bar = 40 μm. For = forward; mut = mutation; Pt = patient; Rev = reverse; WT = wild-type.

Figure 3. Reduced size and amount of titin in patients with centronuclear myopathy carrying TTN mutations

Gel electrophoresis of patient muscle proteins with control human soleus (HS) and cardiac (HC) muscle proteins. Both controls were run on the same gel to observe the various known titin isoforms present in the 2 muscle types that have been sequenced and therefore can serve as high-molecular-weight markers. Their molecular masses (in MDa) are shown in parentheses. The predominant titin isoforms expressed in skeletal (N2A, 3.8 MDa) and cardiac (N2BA, ∼3.5 MDa and N2B, 3.0 MDa) muscles are marked. The titin degradation product T2 (2.1 MDa) and nebulin (0.77 MDa) are shown as well. The known molecular masses were used to estimate the molecular masses of titin species present in the patients. Patient 1044-1: ∼3.8, ∼2.7, and ∼2.1 MDa; patient 1093-1: ∼3.5 and 2.3 MDa. Myosin heavy chain (MHC) was used as a loading control. Pt = patient.