Congenital Titinopathy: Comprehensive characterization and pathogenic insights

Figures

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  Figure 1. Schematic representation of titin isoforms and location of patient mutations. (A) Size (relative to number of amino acids) of the 4 main titin regions encoded by the inferred complete metatranscript (Refseq transcript NM_001267550.1), the single characterized skeletal muscle isoform N2A (Refseq transcript NM_133378.4), the principal cardiac long isoform N2BA (NM_001256850.1) and the principle cardiac short isoform N2B (NM_003319.4). The Z‐disc region of titin (green) interacts with α‐actinin, telethonin, and other Z‐disc–related proteins. The I‐band region (blue) contains multiple tandem immunoglobulin‐like domains and the “PEVK” domain (yellow), which is rich in proline (P), glutamic acid (E), valine (V), and lysine (K). The PEVK domain unravels when stretched, giving titin its elastic properties. The A‐band region (red) contains multiple myosin and C‐protein binding sites, and alternating fibronectin type III and immunoglobulin repeats that form a shape that complements myosin. The M‐band region (purple) is encoded by the last 6 exons [M‐band exon (Mex) exons 1–6 (exons 359–364)] and contains a kinase domain, immunoglobulin domains, and binding sequences for calpain 3,…

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  Figure 2. Examples of common and clinically significant features. Lower limb magnetic resonance imaging (MRI) is also shown. (A) Female Family 25 proband. The pregnancy for this infant was complicated by reduced fetal movements and prenatal ultrasound detection of limb contractures. The image shows typical “frog leg” positioning due to marked congenital hypotonia and weakness. Note also the nasogastric feeding tube and reduced palmar creases (inset). This infant also had mild pulmonary stenosis and needed intubation and ventilation for a short period following delivery. (B, C) The older of the 2 affected brothers (Sibling 1) from Family 1 at age 34 years. Both brothers had congenital scoliosis, which progressed during childhood. Spinal surgery was not possible in the pictured brother due to concurrent development of severe respiratory insufficiency. His respiratory deficit was diagnosed at age 13 years following an out of hospital respiratory arrest, and he has since relied on nocturnal ventilation via a tracheostomy tube. This patient has slowly progressive mild to moderate limb weakness but remains ambulant despite significant axial involvement. He also has left ventricular cardiac…

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  Figure 3. Examples of histopathological and ultrastructural features. All brightfield scale bars = 50 µm unless otherwise stated. (A) Centralized nuclei in the pattern of centronuclear myopathy in a hematoxylin and eosin (H&E)‐stained quadriceps section from the Family 3 female proband at age 5 years. Scale bar = 20 µm. (B) Both internalized and centralized nuclei in an H&E‐stained quadriceps section from the male proband from Family 5, at age 14 years. One fiber (arrow) has multiple internalized nuclei, a feature that was much more prominent in biopsies from older patients. This image also shows a very mild increase in endomysial connective tissue. (C) Electron micrograph (EM) image showing internalized nuclei and scattered, small, poorly defined areas of sarcomeric disruption consistent with minicores (Sibling 1/Family 1, quadriceps, age = 14 years, scale bar = 20 µm). (D) Lucent areas compatible with multiminicores in a succinate dehydrogenase–stained section from the same biopsy as shown in C. The multiminicores in D were confirmed ultrastructurally, as shown in F, which presents 2 minicores in a longitudinally orientated fiber (scale bar = 5 µm). (E) Multiminicores as discrete…

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  Figure 4. Western blot analysis of patient muscle samples. Antititin antibodies were used that were specific for the titin N‐terminus (Z1Z2; top panel) or C‐terminus (M8M9; bottom panel). The 4 left lanes are controls, and the 7 right lanes are biopsies from patients (from Families 4, 5, 13, 15, and 16), or segregation‐inconclusive cases (Families 28 and 29). All patients have mutations in both of their TTN alleles that produce proteins that are predicted to vary in size. The segregation‐inconclusive cases also have 2 TTN mutations that are predicted to produce proteins of different sizes if their mutations are (as suspected) in trans. The bottom of the figure shows the expected protein mass, assuming that the wild‐type full‐length titin is 3.8MDa and that the mutant protein is reduced by the size of the missing exons(s). The largest predicted proteins are in patient biopsies from Families 28, 29, 4, 13, and 16 at nearly full size (in‐frame deletion of a single small exon near the middle of titin), consistent with their observed expression of a large titin that reacts with both Z1Z2 and M8M9. The largest predicted proteins in the remaining 2 patients (from Families 5 and 15) are slightly…