Abstract

Limb weakness is an uncommon symptom in children, with multiple factors contributing to related diseases, particularly genetic disorders. A nine-year-old boy presented with slowly progressive muscle weakness of the limb-girdle muscles. We evaluated the clinical symptoms, laboratory tests, imaging examinations, and pathological examinations of this proband. We combined whole-exome and Sanger sequencing to identify the novel compound heterozygous pathogenic mutations NM 001849.3: c.1970-10_1978 del CGGCTTGCAGGGACGCGTG and c.2462-3C>A inCOL6A2 in this proband inherited from the mother and father, respectively. Mutational confirmation at the mRNA level demonstrated that the proband carried a homozygous abnormal sequence with 23bp deletions (c.2462-2484 del GGACGCGTGTGGGCGTGGTGCAG) at the beginning of exon 26. In contrast, both parents and sibling have normal sequences with no clinical symptoms. The results of this study further expand the mutational spectrum and will be helpful for further molecular diagnosis.
Keywords: COL6A2; Ullrich congenital muscular dystrophy (UCMD); compound heterozygous mutations; whole exome sequencing; sanger sequencing;
Congenital muscular dystrophies (CMDs) are a group of rare genetic diseases that primarily affect the muscle and are characterized by progressive degeneration and weakness(Kirschner, 2013). Clinical symptoms typically manifest at birth or within the first few months of life(Carakushansky et al., 2012). Ullrich congenital muscular dystrophy (UCMD) is a rare type of autosomal dominant or recessive CMDs, mainly caused by mutations in the related genes leading to loss of collagen VI with an earlier onset time and progressive clinical symptoms(Kirschner, 2013; Park et al., 2014). Collagen type VI is consistently linked to the development of congenital muscular dystrophy(Bushby et al., 2014; Antoniel et al., 2020; Simsek-Kiper et al., 2020), composed of three distinct alpha chains encoded by COL6A (Lampe & Bushby, 2005). Most patients with UCMD carry mutations in COL6A that result in the abnormal production of collagen type VI(Bushby et al., 2014). However, the relationship between genotype and phenotype remains unclear.
The nine-year-old boy (II-2, Figure 1 A ) was the second child of healthy non-consanguineous parents. The patient presented myasthenia and was initially diagnosed with CMD. His symptoms of gait instability had not improved and became increasingly severe as the proband got older; intellectual development was normal compared with peers. Physical examination shows a clear mind, mental reactivity, normal nutritional condition, bilateral lower extremity weakness, the inability to stand, and the left positive Babinski sign. The proband presented equinus when he stood with auxiliary assistance. The muscle strength of the upper limbs was grade IV, and that of the lower limbs was III. Deep tendon reflexes were decreased in the knees. Auxiliary examinations yield the following results: Laboratory examinations revealed no obvious abnormalities in the blood routine or coagulation parameters. Biochemical assays were as follows: creatine kinase isoenzyme MB 12.03 (Reference value: 0–5 ng/ml); creatinine 19 (Reference value: 62–115 µmol/L); creatine kinase 370 (Reference value: 38–174 U/L). On the biceps brachii, vastus medialis, and anterior muscle, needle electromyography revealed that the duration of voluntary motor unit potential on volition was reduced by 32%, 27%, and 38%, respectively. There were no discernible abnormalities in the upper and lower limb sensory nerve conduction. Similarly, no abnormalities were detected during brain MRI or diffusion-weighted imaging (DWI). The left biceps brachii muscle biopsy was performed Figure 1 B . The pathological outcome was the atrophy of both types of fibers. Part of the muscle fiber was replaced by connective and adipose tissues, and the size of the remaining muscle fiber was noticeably different. A small amount of regenerative and moth-eaten muscle fiber also appeared. Immunohistochemistry results indicated that dystrophin-Rod, dystrophin-N, and β-sarcoglycan were reduced in muscle fiber membranes compared to the normal control. A proportion of muscle fiber membranes were absent of dystrophin-C reporter staining. Collectively, the proband is highly probable to have muscular dystrophy, and genetic testing is extremely helpful for disease diagnosis.
Our whole-exome sequencing (WES) results indicate that the proband carries two novel compound heterozygous pathogenic mutations inCOL6A2 , one of which was a frameshift mutation (NM 001849.3: c.1970-17_1971 del ACGCGTGCGGCTTGCAGGG), and the other was a splice site mutation (NM 001849.3: c.2462-3C>A) (Figure 1 C ). Subsequently, the mutations of COL6A2 in the proband, his parents, and his sibling were validated by Sanger sequencing. Notably, the frameshift mutation (c.1970-10_1978 del CGGCTTGCAGGGACGCGTG) was confirmed at the DNA level in the proband and his mother by Sanger sequencing and was different from the WES results Moreover, another site was observed in the father and proband. The mode of inheritance complies with the rules of autosomal dominant inheritance. The mRNA was extracted from the peripheral blood to further verify the functional effect of the genetic variants. RNA sequence analyses showed wild-type sequences in the parents and sibling. However, the proband has a homozygous but unusual sequence, a 23bp (c.2462–2484 del GGACGCGTGTGGGCGTGGTGCAG) deletion at the beginning of exon 26 compared to wild-type (Figure 1 D ). We speculated that mutations affect the phenotype only when two mutations are present together.
Zhang et al. reported two UCMD patients with different mutations inCOL6A2 ; one carried a homozygous c.1870G>A (p.E624K) variant and the other a homozygous c.2626C>A (p.R876S). Both patients presented with myasthenia, joint contractures, and joint laxity(Zhang et al., 2010). Lucarini et al. reported a proband who carried a homozygous A > G mutation at −10 of intron 12 inCOL6A2 that goes along with generalized muscle weakness, arthrogryposis, and mild spine rigidity(Lucarini et al., 2005). In our study, we described a UCMD proband with slowly progressive muscle weakness of the limb-girdle muscles and identified novel compound heterozygous pathogenic mutations of COL6A2 in a Chinese family. Subsequently, we directly extracted RNA from peripheral blood to verify the effect of the two different novel types of COL6A2 mutations at the mRNA level. Our results demonstrated that the proband carried a homozygous aberrant mRNA sequence, suggesting that two different novel mutations may together contribute to the partial deletion at the beginning sequences of the same exon 26. This might be attributed to the defective pre-mRNA splicing and phenotype caused only when both mutations are present, indicating that RNA sequencing is also essential to diagnose the disease. However, the action mechanism of those mutations is not entirely clear. Therefore, an ideal method combining DNA and RNA sequencing has been suggested as an optimal tool for prenatal diagnosis. In summary, our research demonstrated the novel pathogenicity of mutations and expanded the variant spectrum of COL6A2 , further improving the accurate diagnosis of UCMD.