Background

Tibial muscular dystrophy (TMD) is a late-onset distal myopathy with an autosomal dominant inheritance pattern. It was first described in a Finnish patient affecting at least one in 10,000 people [1, 2]. In patients with the mildest form of TMD, symptoms may go unnoticed, are usually confined to the lower leg in particular, the tibialis anterior muscle, and appear between the ages of 35 to 45 years. The strength of the muscles just below the ankle can be affected by muscle weakness in the ankle [2, 3].

With the progressive onset, weakness and atrophy of the long toe extensors make it difficult to lift the foot while walking, causing a condition called foot drop. This can cause clumsiness and difficulty in walking. But despite the difficulties, most patients retain the ability to walk [4, 5]. In rare cases, this condition can weaken the arm muscles, but cardiomyopathy and involvement of the facial muscles have not been diagnosed in patients with TMD [2, 6].

TMD is caused by mutations in the TTN gene that carries the instructions for making a protein called titin [7]. The TTN gene is on chromosome 2 (2q31); the entire coding region consists of 363 exons encoding 38,138 amino acid residues (4200 kD) [8]. The terminal ends of titin are embedded in two specific sarcomeric regions; the N-terminus within the Z-disc, and the C-terminus within the M-line. Mutation in extreme C terminus of TTN, situated at the end of M-band of the TTN, results in TMD. One of the best characterized functions of titin is that of a scaffold protein aiding myofibrillar assembly during myogenesis, and it is responsible for muscle stretching [9]. However, it is also the backbone to keep the contractile elements of the sarcomere in place [10, 11]. An important role in the myofibrillar signalling pathway has also been demonstrated, and Titin appears to integrate or coordinate multiple signalling pathways involved in gene activation and/or protein folding, quality control and degradation [12,13,14].

Mutations in the TTN gene lead to the production of a defective titin protein, the structure and function of which are altered. This defective protein titin impairs the function of sarcomeres and normal muscle contraction. The severity of the symptoms of TMD is determined by the type of TTN mutation and varies from patient to patient. The effects of mutations in the TTN gene in the muscles of the lower leg are yet to be ascertained [15].

Many additional TTN-related muscular phenotypes are emerging as a consequence of next-generation sequencing (NGS) screening in patients with myopathy [16]. Whole exome sequencing (WES) is rapidly being implemented in genetic diagnostic practice for patients with neuromuscular diseases [17,18,19]. TTN gene is huge in size, makes it difficult to sequence the entirely on a routine basis in diagnostic laboratories. Therefore, before the introduction of NGS technology, only a limited number of TTN mutations were identified. WES has enabled the rapid identification of new TTN variants [20]. However, NGS screenings reveal many rare titin variants but their clinical interpretation is a challenge [16, 21, 22].

The present study detects the novel TTN gene variant in patient with neuromuscular disorders and provides a path for further functional studies to establish clinical diagnosis.

Case presentation

A 20-year-old male with no bilateral facial weakness/ ophthalmoplegia was presented with 2-year long history of walking difficulties. While walking he has to drag the feet and upon examination was notable for severe lower limb weakness and occasional tongue fasciculation. Further examination also showed moderate weakness of abductor digiti minimi (ADM) and mild scoliosis. In clinical investigation, magnetic resonance imaging (MRI) was conducted for Cervico Dorsal Spine and showed no abnormality, cervical spine was normal in curvature and alignment, and intervertebral discs were also normal in height. While in blood test, marker for the detection of skeletal muscle disease, CK-NAC [N-acetyl-cystein-(NAC)-activated creatinkinase (CK)] was elevated to 672 U/L and uric acid was found to be in normal range along with non-reactive HBsAg (Hepatitis B surface antigen). Nerve conduction study (NCS) was normal. Needle electromyography (EMG) showed denervation active patterns in First Dorsal Interosseous (FDI) muscles. To further investigate the diagnosis, Whole Exome Sequencing was performed which revealed a novel heterozygous missense variant (c.41529G > C;p.Arg13843Ser) in TTN gene. DNA was extracted using Qiagen DNA mini kit, as per the manufacturer’s instructions. The quality of extracted genomic DNA (gDNA) was measured with a NanoDrop2000 spectrophotometer and then with a Qubit 3.0 fluorometer for more accurate DNA quantification using the Qubit dsDNA High Sensitivity (HS) Assay Kit. Approximately 100 ng of genomic DNA was taken for exome libraries construction employing the Ion Ampliseq Exome RDY panel (Thermo Fisher Scientific) according to the manufacturer's protocol.

Sequencing was done using Hi-Q chemistry on Ion Proton platform (Thermo Fisher Scientific). Sequences were aligned against the reference genome (GRCh37/hg19) in Torrent Suite v.5.12.0 and Torrent Suite Variant Caller v.5.2.1 software (Thermo Fisher Scientific), with default parameters followed by annotation of VCF file using Ion Reporter v5.18 (Thermo Fisher). A total of 38,767 variants composed of 54% synonymous, 43% missense and 2% frameshift/indels were found in the WES data. For WES data filtering procedures, first phase consisted of benign and synonymous variant filtering, and the second phase was based on variant impact (nonsynonymous, truncating), allele frequency (< 0.1%) and pathogenicity prediction tools for missense variants (score > 3). Since, there were still many candidate variants and genes, we performed a second prioritization step based on manual regulation of biological function and filtered out variants of genes not relevant to the patient's phenotype. WES results indicated a novel heterozygous missense variant (c.41529G > C;p.Arg13843Ser) in TTN gene responsible for Tibial muscular dystrophy. Identified variant was located in GC region and variant coverage and alignment at position chr2:179,484,592 was viewed using the Integrative Genomics Viewer (IGV, http://www.broadinstitute.org/igv/) (Fig. 1B). Confirmation of the identified variant was done by Sanger sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing kit and loaded on an ABI 3500Dx automated Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific). Primer sequences for the pathogenic variant in the TTN gene (NM_001256850.1) were designed using Primer 3.0 online as follow: Forward 5′-ATGCTTGCGGGGTAGAAGAC-3′ and Reverse 5′- CCTGGTCACGGGGCTTAAT-3′. Sanger sequencing analysis confirmed that the proband carried the variant in heterozygous state (Fig. 1C). The pathogenicity of detected variant was evaluated using different online bioinformatics tools; SIFT (https://sift.bii.a-star.edu.sg/), Mutation Taster (http://www.mutationtaster.org), PROVEAN (http://provean.jcvi.org/index.php), and FATHMM-XF (http://fathmm.biocompute.org.uk/fathmm-xf/) which predicted this variant to be damaging (Table 1). Finally, for the interpretation of variant, American College of Medical Genetics and Genomics (ACMG) 2015 guidelines were followed [23].

Fig. 1
figure 1

A Pedigree of the probands' family. B IGV plot showing the mutation region in WES data in the proband. Track comprises two parts: a histogram of the read depth and the reads as aligned to the reference sequence. Reads are colored according to the aligned strand (red = forward strand; blue = reverse strand). C Sanger sequence chromatogram showing a novel heterozygous missense variant in exon 200 of the TTN gene (c.41529G > C;p.Arg13843Ser) associated with tibial muscular dystrophy

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Table 1  WES analysis of identified the TTN gene variant in the proband
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Discussion

TTN performs several critical functions in all skeletal muscle cells that are well adapted to its role as an architectural protein and provide specific binding to a variety of essential proteins [24]. TTN had previously been linked to cardiovascular syndromes until Hackman and his colleagues identified it as a cause of tibial muscular dystrophy (TMD) (OMIM #600334) in a Finnish population in the year 2002 [25]. In addition to Finnish population, disease-causing TTN variants in TMD patients were also reported in other European families, enlisted in Table 2. TMD is a dominant, distal myopathy which typically presents in adulthood. Weakness usually affects the anterior tibial muscle and does not cause cardiomyopathy. Hackman and co-workers were the first to report TTN as a cause of skeletal muscle disease and demonstrated that a variant of Mex6 (the last exon of TTN) causes a functional defect in the titin M line and results in a predominantly inherited TMD phenotype.

Table 2 Reported variants in TTN gene causing TMD in European families
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Several research groups have used next generation sequencing to assess the role of TTN variants in skeletal disease. Savarese and his group attempted to distinguish positive TTN variants from pathogenic variants by iteratively sequencing patients with skeletal myopathy (n = 504) with uncharacterized disease [28]. The classification of TTN variants identified by WES is problematic because of the large number of variants of this gene, both in size and predominance of heterozygous variants, which are reduced to 2% of the normal population [29,30,31]. Using WES, we detected heterozygous missense variant (c.41529G > C;p.Arg13843Ser) which was confirmed by Sanger DNA sequencing in exon 200 of TTN gene, associated with TMD phenotypes. In order to assess the degree of pathogenicity, functionality and stability of the protein, the identified variant was subjected to various in silico functional prediction algorithms based on criteria such as the location of the variant on the genome, sequence homology, degree of conservation and physicochemical properties and structure. SIFT, MutationTaster, PROVEAN and FATHMM-XF protein function prediction softwares were used, and all agreed on the pathogenicity of the variant. The results from these tools classified the variant as deleterious and disease-causing, and boosted the variant pathogenicity level on the protein structure.

However, definitive evidence of pathogenicity for missense variants can only be established by functional study, segregation studies in very large families and/or by identifying unrelated patients or families with the same mutation. The interpretation of the missense variant of TTN could also benefit from establishing a clinical and research consortium able to incorporate a group of patients into a larger cohort [32].

Diagnostic laboratories today are making widespread use of next-generation whole sequencing, which is increasing the number of TTN variants (particularly those of uncertain significance) enrolled in clinical testing. This then presents a challenge for clinicians who need to assess the importance of TTN variants as part of their diagnostic assessment. Furthermore, the high rates of TTN variants in the general population limits the understanding of pathogenicity [33]. It is estimated that at least three rare, non-synonymous TTN variants are identified in any one individual, which is certainly related to the sheer size of the gene [16]. Often these rare/novel variants, as well as missense variants are classified as variants of uncertain significance (VUS). Uncertainties associated with VUS in TTN, especially those associated with skeletal myopathy, inevitably complicate the diagnostic work-up, including genetic counselling and clinical management.

Previous studies have confirmed the association of TTN variants with several life-threatening neuromuscular and/or cardiovascular disease, though both interpretation and clinical utility of TTN variants are often challenging in this setting [16, 31, 34, 35]. Although in-silico predictions of the effects of any missense variant are frequently unreliable, functional validation of missense changes in TTN presents unique challenges as its large size prevents cloning and expression of full-length protein in in-vitro systems [16].

However, despite recent reports reducing overall population estimates of TTN mutations, historical awareness of the prevalence of TTN mutations remains unexcavated which further exacerbate the problem of diagnostic interpretation.

Conclusions

WES has dramatically expanded the spectrum of skeletal muscle disorders associated with TTN causative mutation. Our study sensitizes the neurologists and geneticists on the potential role of TTN gene and titinopathies thereby, aiding in better understanding and more consistent interpretation of titin mutations.