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) [5,6,7,8,9,10] .

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.

Methods

Family recruitment and DNA isolation

The present family has two affected individuals lives in the Bannu district of Khyber Pakhtunkhwa province, Pakistan. Pedigree was drawn (Fig. 1a) and the affected individuals were thoroughly examined by a local medical doctor. Clinical information including age, gender, family history and consanguinity was recorded. Blood samples were drawn from the two affected (IV: 3, IV: 5) and normal individuals of the family. Genomic DNA was extracted using the QIAquick DNA extraction kit (QIAamp, Qiagen, Valencia, CA, USA) and quantified using Nanodrop-2000 spectrophotometer (Thermo Scientific, Schaumburg, IL, USA).

Fig. 1
figure 1

a A consanguineous pedigree showing two affected members (IV:3 and IV:5) in the fourth generation having limb girdle muscular dystrophy. Affected individuals in the pedigree are shown with shaded symbols and unaffected with open symbols. Double lines indicate consanguineous union. b Sequence chromatogram of the TTN gene is showing segregation of c.98807G > A; p. Arg32936His in all family members c Ribbon representation of three-dimensional structure of human titin with close-up view of mutant (right) and wild type (left) at position 32,936 showing the local conformation induced by the substitution of arginine by histidine. d Ramachandran plots of wild and mutant types. e Schematic view of the functional domain of the TTN gene and localization of known mutation (Arg32936His). The novel missense variant p. Arg32936His reported here is indicated in red localized in the M domain. f The panel also shows the evolutionary conservation of Arg32936 across different species

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Library preparation and whole-exome sequencing

A 100 ng of genomic DNA were needed to amplify the targeted amplicon. Exome libraries of the DNA product were created using the Ion AmpliSeq™ Exome Panel [11,12,13]. Emulsion polymerase chain reaction (emPCR) was performed using a OneTouch 2 instrument with an Ion PI Template OT2 200 Kit V3.The Ion OneTouch ES enrichment system (Life Technologies, Carlsbad, USA) was used for ISP enrichment step. The manufacturer’s instructions of Life Technologies company were followed to prepare and load the Ion Proton I chip [11,12,13].

Data processing

Sequencing data were aligned to the Homo sapiens hg 19 (GRCh37/hg19). Torrent Variant Caller software (version 4.4.3) was used to analyze the genotyping data and call the multi-allelic variations and indels. Post detection of variant was performed using wANNOVAR (http://wannovar.usc.edu/). An Integrative Genome Viewer (IGV, http://www.broadinstitute.org/igv/) was used to visualize sequencing data. Variant frequencies were obtained from various databases such as the 1000 Genomes Project, dbSNP142, Exome Aggregation Consortium (ExAC) and gnomAD (Additional file 1: Table S1).

Bioinformatics analysis

Different prediction programs including Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), PROVEAN (http://provean.jcvi.org) and Mutation Taster (http://www.mutationtaster.org/) predicted this mutations to be probably damaging. Finally, for the interpretation of variants, American College of Medical Genetics and Genomics (ACMG) 2015 guidelines were used [14].

Mutation confirmation

To validate the detected variant, specific fragments were PCR-amplified using site-specific primers using primer3 software (http://primer3.ut.ee) and analyzed by Sanger sequencing (Fig. 1b). The identified variant was analyzed in 200 ethnically matched control individuals (Fig. 1).

Protein modeling

The primary sequence of TTN was retrieved in FASTA format through UniProtKB/SwissProt database (https://www.uniprot.org/uniprot/O95672). Retrieved sequence was used to predict the three-dimensional (3D) protein structure using I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). The three-dimensional model of mutated TTN protein (p.Arg32936His) was generated by MODELLER 9.17 (https://salilab.org/modeller/9.17/release.html). The recognition of errors in experimental and theoretical models of protein structures is a major problem in structural bioinformatics. Different evaluation tools were used for the assessment of protein structure. The model was further processed by RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) ERRAT (https://servicesn.mbi.ucla.edu/ERRAT/) and Protein Structure Analysis (ProSA; https://prosa.services.came.sbg.ac.at/prosa.php). RAMPAGE generates Ramachandran plot for the assessment of models along with distribution of residues in favoured, allowed and outlier regions. ERRAT generated a plot indicating the confidence and overall quality of model. ProSA calculated an overall quality score of the predicted structure (Fig. 1c & d).

Results

Clinical description of patients

Clinical examination was performed for both affected individuals (IV: 3; IV: 5). They were born to first-cousin parents with a normal pregnancy and delivery. They were 20–25 years old, and had severe LGMD. Notable clinical findings include difficulty in rising from the floor, delay in motor milestones, and muscle weakness. They had mild microcephaly, intellectual disability (ID), generalized muscle hypertrophy and developmental delay (Table 1). Follow up clinical examination of the patients revealed cardiomyopathy, proximal and distal weakness, inability to stand, loss of ambulation, and both were confined to a wheelchair. They also had a triangular face, low set of ears, and clinodactyly in lower limb digits. In addition, both were suffering pelvic and shoulder girdle muscular dystrophy, muscular pain and also facial muscles weakness when doing a usual muscle exercise. Features such as skin, teeth, nails, eyes, reproductive and cardiac deformities were not observed in both of them. Their parents showed no abnormalities and were healthy.

Table 1 Clinical features of the affected individuals
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Whole exome sequencing

In the present study, clinical diagnosis was confirmed by genetic analysis. Of these, both patients (IV: 3; IV: 5) and their parents (III-1;III-2) were subjected to whole exome sequencing (WES) as described earlier using Ion Torrent platform [11,12,13]. WES results indicated a novel homozygous missense variant (c.98807G > A; p.Arg32936His) in TTN (MIM: 188840) gene responsible for LGMD phenotype (Table 2). Sanger sequencing perfectly confirmed segregation of the disease phenotype. The variation G-to-A transversion results into the substitution of arginine (R) to histadine residues (Arg32936His). This mutation is conserved across different species and can affect greatly the amino acid (aa) sequence located in the M domain of TTN gene that might change the protein features and also affect the splice site (Fig. 1 e& f). Different online bioinformatics tools were used to analyze the pathogenicity of the variant (Table 2).

Table 2 Homozygous variant on chromosome 2 from exome data of TTN family
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Using homology modelling, 3D models of wild type and mutated TTN protein (p.Arg32936His) were predicted and evaluated by online structure analysis tools as described above. Ramachandran plot generated by RAMPAGE indicated that approximately 93% of residues in the model lie in allowed regions of torsion angles. ERRAT and ProSA showing overall quality of model and quality score of the predicted structure (Fig. 1 c & d).

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 [19,20,21]. 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 [21,22,23] (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 3 HGMD reported mutations in TTN gene associated with LGMD disorders
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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.