Identification of a novel nonsense mutation in kyphoscoliosis peptidase gene in an Iranian patient with myofibrillar myopathy.

Myofibrillar myopathies (MFMs) are rare genetic and slowly progressive neuromuscular disorders. Several pathogenic mutations have been reported in MFM-related genes including DES, CRYAB, MYOT, LDB3 or ZASP, FLNC, BAG3, FHL1 and DNAJB6. Although MFMs is commonly inherited in an autosomal dominant manner, the inheritance pattern and novel mutated genes are not thoroughly elucidated in some cases. Here, we report discovery of a novel nonsense mutation in a 29-year-old Iranian male patient with motor disorders and deformity in his lower limbs. His parents are second cousins. Hereditary Motor Sensory Neuropathy as initial genetic diagnosis was ruled out. Whole exome sequencing using NGS on Illumina HiSeq4000 platform was performed to identify the disease and possible mutated gene(s). Our data analysis identified a homozygous nonsense unreported c.C415T (p.R139X) variant on kyphoscoliosis peptidase (KY) gene (NM_178554: exon4). Sanger sequencing of this mutation has been performed for his other related family members. Sequencing and segregation analysis was confirmed the NGS results and autosomal recessive inheritance pattern of the disease.

Introduction

Myofibrillar myopathies (MFMs) are among rare genetic neuromuscular disorders which are characterized by slowly progressive weakness in both proximal (near to the center of the body) and distal (the hands and feet) skeletal muscles and smooth muscular dysfunctions. According to the genetic background of affected individuals, MFM could be associated with diverse clinical manifestations. Although MFM may be diagnosed during childhood, the clinical features of the disease often appear in mid-adulthood (Ferrer & Olivé, 2008). In addition to the major skeletal muscle disorders, facial muscles weakness usually leads to different problems in speech and swallowing. Moreover, overt cardiomyopathy with arrhythmia, conduction defects and/or congestive heart failure has been reported in some cases. Sensory symptoms, muscle stiffness, aching and cramps are uncommon complications in MFM patients.

The diagnosis of MFM is usually based on clinical symptoms, electromyogram (EMG), nerve conduction studies and muscle biopsy. Several mutations have been reported across exons and exon-intron boundaries of MFMs related genes. They are mostly associated with filament proteins of muscle fibers, including DES (desmin), CRYAB (alpha-crystallin B chain),7, 8 MYOT (myotilin), LDB3 or ZASP(LIM domain-binding protein 3), FLNC (filamin-C), BAG3 (BAG family molecular chaperone regulator 3), FHL1 (four and a half LIM domains protein 1) and DNAJB6 (DnaJ homolog subfamily B member 6). MFMs usually inherited in an autosomal dominant manner. Although, FHL1 gene inherited in X-linked recessive pattern. However, the inheritance pattern in some families is not thoroughly elucidated which needs the characterization of pathogenic variants and segregation analysis in the family. Regarding the complications in screening the mutations of MFM-related genes by conventional methods, molecular genetics approaches using next generation sequencing (NGS) can be more precise and helpful in identification of affected patients. In this article, whole exome sequencing has been performed to identify the possible causing mutations for a male patient with foot deformities and muscular weakness.

Material and methods

Case presentation

A 29-year-old male patient with motor disorders and deformities in his lower limbs was referred to Hakim hospital (Neyshabour, Iran) from the welfare administration. He was unable to normal movement and has walking difficulties. His muscular weakness symptoms appeared at the age of about 3. His lower limbs were completely affected with weakness and muscle atrophy. His both legs have equinovarus foot deformity. He has mild scoliosis and joint contracture. His facial appearance and upper limbs muscles were normal. His IQ was normal.

His parents are second cousins. His affected sister has muscle weakness. His aunt (mother-side) was died with similar clinical symptoms. His lactate dehydrogenase (LDH = 575 IU/L) and creatine phosphokinase (CPK = 308 IU/L) showed abnormally high level in the serum. He did not perform electromyogram (EMG) and muscle biopsy. Written informed consent form was obtained from the all of the participant from this study.

Hereditary motor sensory neuropathy NGS panel

Molecular diagnosis of hereditary motor sensory neuropathy was performed using targeted next generation sequencing panel including 37 genes to identify mutations; AARS, DNM2, DYNC1H1, EGR2, FGD4, FIG4, GARS, GDAP1, GJB1, HSPB1, HSPB8, KARS, KIF1B, LITAF, LMNA, LRSAM1, MED25, MFN2, MPZ, MTMR2, NDRG1, NEFL, PMP22, PRPS1,PRX, RAB7A, SBF2, SBF1, SH3TC2, TRPV4, YARS, BSCL2, INF2, AIFM1, DHTKD1, PDK3, GNB4).This panel was selected per request and diagnosis of his neurologist. The panel was performed using a custom designed Nimblegen chip capturing the genes of interest followed by Next Generation Sequencing (NGS). A heterozygous c.3681C > T (p. Ala1227=) variant with uncertain clinical significance on SH3TC2 gene was detected. After segregation analysis and pedigree validation, clinical significance of this variant was excluded.

Whole exome sequencing (WES)

Whole exome paired-end sequencing analysis with 100X coverage was performed on extracted genomic DNA from peripheral blood of the proband to identify the disease causing mutations. Human whole exome enrichment was performed using Agilent Sure Select V6 Target Enrichment Kit, followed by highly specific and sensitive NGS method using Illumina HiSeq4000 platform. This method allows detection of point mutations and small indels (≤20 bp). Sequencing of all human exons and flanking 10 bp sequence plus UTR regions of >23000 genes was accomplished.

Sanger sequencing

The genomic DNA was extracted from peripheral blood using high pure PCR template preparation kit (Roche) according to the manufacturer's protocol. The specific forward (5′-GCCATGGTTCCCAAACTAAC-3′) and reverse (5′-CTGCCTAGTGCCTTCTGTCC-3′) primers were designed to validate the c.C415T (p. R139X) mutation on KY gene using Sanger sequencing. Each PCR cycle consisted of denaturation at 94 °C for 20 s, primer annealing at 58 °C and extension at 72 °C for 30 s for each one, followed by a final extension at 72 °C for 5 min to amplify 449 bp PCR products. The PCR reactions were performed using ready to use 2X master mix as manufacturer's protocol (Promega). The purified PCR products were sequenced by ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Results

WES results indicated a homozygous nonsense c.C415T (p. R139X) variant on kyphoscoliosis peptidase (KY) gene. This variant has not been reported for its pathogenicity. Its clinical significance is not thoroughly elucidated. Its frequency in normal population is very low (0.00008 in the NHLBI Exome Sequencing Project, and it is not found in GnomAD). It is predicted to be harmful because the nonsense variant which resulted in the conversion of Arg-139 amino acid to termination codon most likely leads to activate nonsense-mediated mRNA-decay pathway. Also, this abnormal nonsense variant may leads to produce a truncated polypeptide and loss of normal function of protein.

The KY gene is located on long arm of the chromosome 3 (3q22.2) and has 11 exons. This variant there is in exon 4. The KY gene is associated with autosomal recessive myopathy myofibrillar7 (MFM7) (Hedberg-Oldfors et al, 2016). Sanger sequencing of c.C415T variant has been performed for his parents (II.2 and II.3), his normal sister (III.1) and his affected sister (III.2) to validate its clinical significance. Also, carrier testing using Sanger sequencing has been performed for his wife (III.4) (Fig. 1). His aunt most likely had same mutation on KY gene. Although she may be had another type of neuromuscular disease since it is not possible to determine genetic status for her.

Figure 1

Pedigree of this family was shown. The arrow indicates the proband. His parents are second cousins.

Sanger sequencing results indicated heterozygous genotype for his parents and homozygous T/T mutant genotype for his affected sister. His clinically normal sister and his wife showed homozygous C/C normal genotype (Fig. 2).

Figure 2

Sanger sequencing results of detected variant for related family members of the patient; Patient's sister has homozygous normal genotype (C/C) for c.C415T (p.R139X) variant on KY gene (A). Patient's father has heterozygous genotype (C/T) for c.C415T variant (p.R139X) on KY gene (B).

Discussion

Kyphoscoliosis peptidase enzyme is involved in the function, maturation and stabilization of neuromuscular junctions. It is required for normal muscle growth. KY gene mutations are associated with the same spectrum of autosomal recessive MFM7 disorder. MFM7 (MIM: 617114) is a muscular disorder which is characterized by disintegration of the sarcomeric Z-disc and myofibrils. MFM7 is clinically characterized by early childhood onset of slowly progressive muscle weakness and mild atrophy primarily affecting the lower limbs which is associated with joint contractures. We have reported a novel homozygous nonsense c.C415T (p. R139X) variant on KY gene. Mutations and variants in KY gene have been also reported in the similar symptoms' congenital myopathy and neuromuscular disorders.16, 17 The patient of this study has equinovarus foot deformity in his two legs and muscular weakness in lower limbs and also mild scoliosis and joint contracture that all of this phenotypes were described as the symptoms of KY mutations in MFM7 and spastic paraplegia.15, 16

Molecular diagnosis to identify pathogenic mutations in affected patients with MFMs should be considered. Therefore, genetic counseling could be recommended for all individuals with MFMs and their related family members. The carrier couples with identified mutated genes should performed prenatal diagnosis to prevent of born new affected child.

Conclusions

A rare pathogenic mutation in KY gene was identified in a 29-year-old patient with MFM disorder which supports the link between KY gene mutations and neuromuscular disorders of patients with MFM. Our report can be helpful in finding an appropriate genetic counseling and molecular diagnosis for affected individuals with MFM.

Conflict of interests

The authors declare that they have no conflict of interests.