Background
Limb-girdle muscular dystrophies (LGMDs) are clinically and genetically heterogeneous muscle disorders inherited as an autosomal recessive or dominant pattern. Clinically, patients are characterized by symmetrical weakness of pelvic, scapular and trunk muscles [
1,
2]. LGMDs also show clinical overlapping with other muscle disorders like Emery-Dreifuss Muscular Dystrophy (EDMD; MIM: 310300), recessive congenital myopathy [MIM: 612540], myofibrillar myopathy (MFM; MIM: 601419) and late onset dominant distal myopathy [MIM: 604454] [
3,
4]. More than 30 recessively and dominantly inherited forms have been identified for LGMDs [
3]. The prevalence of LGMDs is about 4–7/100,000 and may have childhood, teenage or adulthood onset [
3,
4]. The prevalence of autosomal recessive muscle disorders like LGMD and congenital muscular dystrophies are rare in Pakistani populations. LGMD shows severe clinical manifestations such as proximal muscle weakness, loss of ambulation between third and sixth decade, severe disability within 20 years of onset, and muscle biopsy might reveal dystrophic changes [
3,
4]. Patient with LGMD had a similar disease course as Duchene muscular dystrophy (DMD), had calf hypertrophy and were non-ambulatory after age 15. Pathogenic mutations in
TTN has also been associated with other severe disorders such as cardiomyopathy, dilated, 1G (MIM:604145), cardiomyopathy, familial hypertrophic 9 (MIM:613765), muscular dystrophy, limb-girdle, autosomal recessive 10 (MIM:608807), myopathy, proximal, with early respiratory muscle involvement (MIM:603689), salih myopathy (MIM:611705), tibial muscular dystrophy, tardive (MIM:600334) [
5‐
10] .
In this study, we documented a clinical and molecular investigation of a consanguineous Pakistani family segregating LGMD in an autosomal recessive form and identified a novel homozygous missense mutation in the TTN gene located on chromosome 2q31.2. To the best of our knowledge the molecular studies on mutation in the TTN gene is reported for the first time from Pakistan.
Discussion
LGMD is an inherited genetic disorder characterized by limb and girdle weakness and transmitted in either an autosomal recessive or an autosomal dominant pattern [
1,
2]. Several genes are associated with the LGMD phenotype and the next generation sequencing (NGS) technology can be the best choice for definitive diagnosis of LGMD [
15,
16]. The affected individuals reported here, exhibit several phenotypes such as difficulty in rising from the floor, delay in motor milestones, and muscle weakness, mild microcephaly, intellectual disability, generalized muscle hypertrophy and developmental delay (Table
1). Such features were also reported previously [
15,
16]. Cardiomyopathy also was observed in our patients [
17]. Recently, Younus et al (2019) reported a nonsense mutation in the SGCD gene among Pakistani population having LGMD features that shows variability with features in comparison the cases reported here [
18]. Through WES, we detected a homozygous missense mutation (c.98807G > A; p.Arg32936His) in the exon 353 of the
TTN gene known to be associated with LGMD phenotypes.
The titin protein is organized into four structurally and functionally distinct regions that correlate with the muscle sarcomere [
19‐
21]. These regions, located at the amino terminus to the carboxy terminus of the protein, include the Z-disk, I-band, A-band, and the M-line [
21‐
23] (Fig.
1). Carriage of the mutation c.98807G > A which is very close to the M domain of the
TTN gene, results in amino acid change of the Arg32936 residue into the His32936 and alter the secondary structure of the TTN protein causing protein instability. Using homology modelling; three-dimensional models of wild-type and the mutated
TTN protein (p.Arg32936His) revealed a Z scores between 0.5–1.0, indicating no significant deviation from the scores determined for proteins of similar size. The entire
TTN gene consists of 364 exons, located on chromosome 2q31, and transcribes an mRNA over 100 kb long that could hypothetically produce around 38,138 residues and 4200 kDa proteins [
24].
TTN has multiple key roles in all striated muscle cells, well suited for its role as an architectural protein and provide specific attachment to a plethora of essential proteins [
23]. A total of 346
TTN disease-causing mutations (259 missense/nonsense, 23 splicing, 13 small insertions, 47 small deletions, 1 small indels and 2 gross deletions) have been reported in the human gene mutation database (HGMD) with at least 10 different conditions, including isolated cardiomyopathies, purely skeletal muscle phenotypes and infantile diseases affecting both types of striated muscles (Table
3) [
17,
18]. A majority of patients with
TTN mutations have normal intelligence quotient (IQ), but our patients showed poor language development, mild microcephaly and developmental delay (Table
1).
Table 3HGMD reported mutations in TTN gene associated with LGMD disorders
TTN | c.187G > A | p.A63T | Missense | Muscular dystrophy, limb-girdle |
TTN | c.3100G > A | p.V1034 M | Missense | Muscular dystrophy |
TTN | c.7961G > A | p.R2654K | Missense | Muscular dystrophy |
TTN | c.22771A > T | p.K7591* | Nonsense | Muscular dystrophy |
TTN | c.28730C > T | p.P9577L | Missense | Muscular dystrophy |
TTN | c.46363C > T | p.R15455* | Nonsense | Muscle weakness |
TTN | c.49243G > A | p.A16415T | Missense | Muscular dystrophy |
TTN | c.63658G > A | p.A21220T | Missense | Muscle weakness |
TTN | c.76850G > A | p.R25617Q | Missense | Muscle weakness |
TTN | c.78320C > T | p.P26107L | Missense | Muscle weakness |
TTN | c.87483G > C | p.W29161C | Missense | Muscle weakness |
TTN | c.97332C > A | p.Y32444* | Nonsense | Muscular dystrophy |
TTN | c.98456C > G | p.S32819* | Nonsense | Muscle weakness |
TTN | c.99274C > T | p.Q33092* | Nonsense | Muscular dystrophy |
TTN | c.100133A > C | p.H33378P | Missense | Tibial muscular dystrophy |
TTN | c.100136 T > A | p.I33379N | Missense | Tibial muscular dystrophy |
TTN | c.100163 T > C | p.L33388P | Missense | Tibial muscular dystrophy |
TTN | c.100186C > T | p.Q33396* | Nonsense | Tibial muscular dystrophy |
TTN | c.57871 + 2 T > G | – | Splice site | Muscular dystrophy |
TTN | c.99673 + 1G > C | – | Splice site | Muscle weakness |
TTN | c.6379_6380delTA | p.(Tyr2127Leufs*8) | Deletion | Muscular dystrophy |
TTN | c.43733-4_43740del12 | – | Deletion | Muscular dystrophy |
TTN | c.59385delT | p.(Lys19796Argfs*24) | Deletion | Tibial muscular dystrophy |
TTN | c.90401delC | p.(Pro30134Leufs*15) | Deletion | Tibial muscular dystrophy |
TTN | c.93409delT | p.(Ser31137Leufs*4) | Deletion | Muscular dystrophy, limb girdle 2 J |
TTN | c.98807G > A | p.Arg32936His | Missense | Muscular dystrophy, limb-girdle |
TTN | c.99943delT | p.(Ser33315Glnfs*10) | Deletion | Tibial muscular dystrophy |
TTN | c.100185delA | p.(Lys33395Asnfs*9) | Deletion | Tibial muscular dystrophy |
TTN | c.32190dupT | – | Duplication | Muscular dystrophy, limb girdle 2 J |
TTN | c.92854_92857dupACTG | – | Duplication | Tibial muscular dystrophy |
TTN | c.100076_100086delins11 | – | Indels | Tibial muscular dystrophy |
TTN | c.1662 + 15_3101–3 | – | Gross deletion | Muscular dystrophy |
TTN | ex. 34–41 | – | Gross deletion | Muscle weakness |
Homozygous knockdown mice
(ttn−/−) had a degeneration of both distal and proximal skeletal muscles by 2–3 weeks of age [
25].
ttn−/−mice developed a rigid gait, kyphosis due to axial skeletal muscle association and normally does not survive long. Histological studies indicated that degeneration was specific to skeletal muscles with no other symptoms such as cardiomyopathy or impairment of the central or peripheral nervous system [
25]. Both fore and hind limbs skeletal muscles had severe and progressive dystrophic phenotypes indicating that
TTN plays a pivotal role in skeletal system development [
25].
Conclusion
This is the first report of TTN pathogenesis causing LGMD type 10 from Pakistani population. Failure for detection of c.98807G > A (p. Arg32936His) in 200 ethnically matched control individuals chromosomes outside of the family or in the public databases, designate that this homozygous missense mutation (is probably pathogenic and deleterious. However, further studies regarding LGMDs among large number of Pakistani population might lead to a deeper understanding, genetic mechanisms and future therapeutic interventions.
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