A Novel Bi-Allelic Mutation in the Paired Box 7 Gene Causing Congenital Myopathy 19 in an Indian Family

Dear Editor,

Congenital myopathies are a heterogenous group of inherited muscle disorders with onset at (or shortly after) birth that occur due to genetic defects in structural skeletal muscle proteins.[1] The paired box (PAX) gene family consists of nine nuclear transcription factor genes, all sharing a highly conserved DNA-binding region known as the “paired box.” These genes play a vital role in cephalic neural crest cell formation, myogenesis, and central nervous system development.[2] The PAX-7 transcription factor plays a key role in muscle homeostasis by maintaining normal satellite cell specification, differentiation, and function.[3] Pathogenic mutations in PAX-7 have been shown to cause congenital myopathy with scoliosis, muscle atrophy, hypotonia, and failure to thrive.[4] We report the first Indian case of congenital myopathy 19 (CMYO19) and highlight novel genetic mutations not previously described in the literature.

A 21-year-old female was born of a second-degree consanguineous marriage via full-term normal vaginal delivery and had normal milestones (independent sitting achieved around 9 months, standing with support at 10–11 months, and walking without support at 14–15 months of age). The parents noticed drooping of both eyelids shortly after birth. At 10 years of age, she noticed that soap frequently entered her eyes while washing her face. For the past 2 years, she complained of bilateral thigh and leg pain after stair climbing and prolonged walking. The symptoms progressed gradually, and for the past 1 year, she developed pain in the upper limbs upon holding heavy objects and placing them on an overhead shelf. Currently, she cannot climb one flight of stairs nor walk 100 m continuously without resting in between. There was no history of slippage of footwear or objects from the hands, sensory loss, limb stiffness, cola-colored urine, “second-wind” phenomenon, double vision, reduced extraocular movements, swallowing or breathing difficulty, or neck weakness. Her elder sister also had bilateral eyelid drooping and bifacial weakness since birth but had normal ambulation [Figure 1].

Figure 1.

Pedigree chart (up to four generations) of the family with the PAX7 gene mutation

She was asthenic, with a Body Mass Index (BMI) of 16.02 kg/m². Bilateral ptosis, bifacial weakness, broad forehead, bilateral temporal hollowing, and mild retrognathia were noted [Figure 2a-2d]. There were no joint contractures or spinal deformities. Extraocular movements were normal. Medical Research Council (MRC) grading of power was 5/5 across the shoulder, elbow, and wrist joints. Grip strength was 80% (as per normal age- and sex-matched reference limits on hand-grip dynamometry) in both hands. MRC grading of power at the hip joint in flexion and extension was 4+/5 and 4/5, respectively. Power at the knee and ankle joints was MRC grade 5/5. Deep tendon reflexes (DTRs) were preserved, and bilateral plantar responses were flexor. The neurological examination of the elder sister revealed bilateral ptosis, normal extraocular movements, and bifacial weakness. Power in the upper and lower limbs was MRC grade 5/5, with normal DTRs. Her gait was normal, and she was fully ambulatory.

Figure 2.

Clinical photographs highlighting the symptoms of the patient. 2a. Clinical photograph (front view) of the patient showing bilateral ptosis; 2b. Broad nasal bridge, broad forehead, and bitemporal wasting (hollowing); 2c. Profile view demonstrating mild retrognathia (white arrow); and 2d. Demonstration of bifacial weakness, noticeable upon attempted smiling (front view)

The complete hemogram, liver and renal function tests, and thyroid function tests were within normal limits. Serum creatine kinase (CK) and lactate dehydrogenase levels were 80 IU/L (<171) and 196 IU/L (140-280), respectively. Fasting venous lactate level was 9.8 mg/dl (10–20 mg/dl). Antiacetylcholine receptor antibodies and antimuscle-specific kinase antibodies were negative. Serum tandem mass spectroscopy and urinary gas chromatography–mass spectroscopy were negative for inborn errors of metabolism. Nerve conduction studies, repetitive nerve stimulation testing, and electromyography did not reveal any significant abnormalities. Whole-body muscle Magnetic Resonance Imaging (MRI) was normal. Muscle biopsy showed normal muscle morphology with mild fiber size variability and normal immunohistochemistry. Routine blood investigations in the elder sister were also within normal limits, and her CK value was 118 IU/L. Nerve conduction and electromyography studies were normal in the elder sister as well. Whole-exome and mitochondrial genome sequencing identified a novel homozygous missense variant, c. 812G > A (p. Arg271His), in exon 6 of the PAX7 gene (PAX7: location: Chr1: 19027172: c.812G > A: p.Arg271His: exon: 6: read depth: 145 [NM_002584.3]), which was confirmed by Sanger sequencing. The same variant was present in the homozygous state in the elder sister. The variant was predicted to have a damaging effect by SIFT (deleterious, score 0), PolyPhen-2 (deleterious, 0.98), Likelihood Ratio Test (LRT), Mutation Taster (deleterious, score 1), and Functional Analysis through Hidden Markov Models (FATHMM) (deleterious, score −4.25) in silico prediction tools. The minor allele frequency was 0% and 0.0008% in the 1000 Genomes and gnomeAD databases. Segregation analysis revealed the same variant in the heterozygous state in both parents [Figure 3a-3c]. Based on the segregation analysis, the variant was considered a likely pathogenic variant as per the American College of Medical Genetics and Genomics guidelines (PM1, PM2, PP1, PP2, PP3).[5] The patient was counselled regarding the disease and advised physiotherapy, oral calcium, and vitamin D3 supplementation. She is currently under follow-up.

Figure 3.

Sanger sequencing in the index case, elder sister, and the parents. 3a. Sequence chromatogram showing the presence of the c. 812G>A (p. Arg271His) variant in exon 6 of the PAX7 gene in the homozygous state in the index case (patient). The same variant was present in the homozygous state in the elder sister as well. Sequence chromatograms showing that the same variant (c. 812G > A[p. Arg271His]) in exon 6 of the PAX7 gene was present in the heterozygous state in the father (3b) and the mother (3c), demonstrating that the parents were asymptomatic carriers

Primary satellite cell-opathies are neuromuscular conditions caused by pathogenic mutations exclusively affecting satellite cell function, with little or no effect on muscle fibers.[6] Satellite cells are normally quiescent in adult skeletal muscle but become mitotically active in times of stress (growth cues, injury) to produce myoblasts that are vital for continual muscle growth, repair, and regeneration. PAX7 is the master regulator of satellite cell function, and loss-of-function mutations have been shown to reduce satellite cell survival, the overall satellite cell pool, and skeletal muscle fiber size.[7]

Biallelic variants in the PAX7 gene have been shown to cause CMYO19, which is associated with infantile-onset bilateral ptosis, prominent bifacial weakness, facial dysmorphism, scoliosis, axial weakness, and early loss of ambulation.[4] The affected individuals in this family had bilateral ptosis and bifacial weakness shortly after birth. However, scoliosis, prominent axial weakness, and early loss of ambulation were not observed. This less severe phenotype could be due to the nature of the genetic mutation. Similar to our case, serum Creatinine Phosphokinase (CPK) levels and muscle biopsy are usually normal in CMYO19, as the pathology is in the satellite cells without muscle fiber degeneration.

The mutation observed caused an amino acid substitution of histidine for arginine at position 271 in exon 6 of the PAX7 gene. This mutation occurred in the conserved homeodomain region of the gene involved in DNA binding.[8] Replacement of a highly polar amino acid (arginine) with a less polar amino acid (histidine) may result in impaired protein functionality and protein–protein interaction by weakening electrostatic interactions and noncovalent complex formation, possibly impairing the DNA–protein (PAX7 homeodomain) interaction and DNA-binding ability of PAX7.[9] The previously reported variants with more severe phenotypic manifestations were homozygous stop codon variants or splice-site variants in the paired-box domain (upstream in the gene), possibly resulting in premature termination or nonsense-mediated mRNA decay.[4] Figure 4 illustrates the variant in this case along with the previously reported variants. The variant reported in this case is an extremely rare variant (minor allele frequency of 0.0008% in the gnomAD database) and is absent from the 1000 Genomes and ClinVar general population databases. This could be due to the high prevalence of consanguinity and endogamy in the community and region (South Asia) to which our patient belonged. This may have resulted in restricted genetic diversity in a small/local population subgroup (“founder effect”) and caused increased prevalence of extremely rare homozygous variants that would otherwise be absent in the general population.[10]

Figure 4.

A schematic representation of the PAX7 gene, demonstrating the location of previously reported variants in the literature (mostly located upstream in the gene) and the location of the new variant reported in this case (encircled in black)

To conclude, this is the first case report of CMYO19 from India. Our case highlights a less severe form of the disease. Furthermore, we report a novel disease-causing homozygous PAX7 variant that has not been previously described. There were limitations to this study, such as the single-case report design and the lack of demonstration of actual satellite cell deficiency in the muscle biopsy specimen. Further studies—particularly those including satellite cell staining to conclusively show deficiency—are needed to better understand the phenotypic-genotypic correlation in PAX7-related disorders in the Indian population.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given her consent for her images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and that due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.