Novel Mutation (c.8725T>C) in Two Siblings With Late-Onset LAMA2-Related Muscular Dystrophy

Dear Editor,

LAMA2-related muscular dystrophy can be classified into two clinical phenotypes: severe early-onset congenital muscular dystrophy and mild late-onset muscular dystrophy [1]. Several mild form muscular dystrophy cases have been diagnosed since Tan et al [2] reported the first case of late-onset LAMA2-related muscular dystrophy, thus expanding the spectrum of this disease [3,4,5,6]. Late-onset LAMA2-related muscular dystrophy presents clinically as limb girdle muscular dystrophy patterns and is associated with the partial expression of laminin α2 [6]. Here, we describe a novel missense mutation (c.8725T>C/p.Cys2909Arg) in the LAMA2 in a patient diagnosed as having late-onset LAMA2-related muscular dystrophy. This study was approved by the institutional research review board of the Catholic University of Korea, Incheon St. Mary's Hospital. Written informed consent was obtained from the patient's parents after they had been briefed about the study.

The proband was a 4-yr-old girl who visited our clinic for evaluation because of a delay in motor milestone acquisition. Although there was no familial history of hereditary disorders, the patient's younger sister had similar proximal lower extremity weakness symptoms. The patient exhibited gross motor developmental delay, but no difficulties with fine motor functions, and her cognitive and speech skills corresponded with her age. Her serum creatine kinase concentration was 1,105 IU/L. Nerve conduction and needle electromyography studies did not reveal any definitive myopathy or peripheral neuropathy findings. Following the first visit, the patient was referred to the genetic clinic.

A gene panel test using next-generation sequencing for autosomal recessive limb girdle muscular dystrophy (30 genes: ANO5, CAPN3, CAV3, DAG1, DES, DNAJB6, DYSF, FHL1, FKRP, FKTN, GAA, GMPPB, HNRPDL, ITGA7, LIMS2, LMNA, MYOT, PLEC, POMGNT1, POMT1, POMT2, SGCA, SGCB, SGCD, SGCG, TCAP, TNPO3, TRAPPC11, TRIM32, and TTN), a multiplex ligation-dependent probe amplification test for Duchenne muscular dystrophy, and a dried blood spot test (lysosomal enzyme acid α-glucosidase activity assay) for Pompe disease were performed; all test results were normal. The patient was subjected to further genetic testing using next-generation sequencing analyzing 4,813 genes (approximately 62,000 exons) associated with several genetic diseases in humans [7]. Genomic DNA was extracted from the peripheral blood of the patient as well as her parents and sister. The genomic DNA was enriched by using the TruSight One Sequencing Panel (Illumina, Inc., San Diego, CA, USA) [7]. This panel provides comprehensive coverage of approximately 102 known protein-encoding genes involved in congenital myopathy and muscular dystrophy.

We found compound heterozygous variations in the patient's LAMA2: a heterozygous nonsense variation in exon 49 (c.6955C>T/p.Arg2319^*), previously reported as a pathogenic variation [8,9], and a heterozygous missense variation in exon 62 (c.8725T>C/p.Cys2909Arg), which is a novel variation. The patient's parents were identified as heterozygous carriers for each variation, and the patient's sister had the same variations as the patient. Each variation was confirmed by conventional Sanger sequencing. One of the compound heterozygous variations present in the LAMA2 of both siblings was a novel missense variation (c.8725T >C/p.Cys2909Arg). This variation has not been reported in control databases such as the 1,000 Genomes Project, Exome Variant Server, Exome Aggregation Consortium, and the dbSNP Database, and has been predicted to be deleterious by several in silico analysis tools. This nucleotide variation is relatively conserved (GERP 5.22, phyloP7 0.991). Genetic analysis of the parents confirmed transconfiguration of the missense variant with the known pathogenic variant (c.6955C>T/p.Arg2319^*).

Brain magnetic resonance images (MRI) of the patient and her sister revealed diffuse symmetric high signal unmyelinated changes (Fig. 1). A biopsy of the patient's left vastus lateralis muscle showed muscular dystrophic patterns. Immunohistochemical staining was performed by using antibodies against the following proteins: the C-terminus of dystrophin, the rod domain of dystrophin, the N-terminus of dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, dysferlin, α-dystroglycan, caveolin-3, and laminin α2 (whole laminin α2). The laminin α2 staining in the patient was reduced compared with an age-matched normal control subject, whereas the staining of the other antibodies was non-specific (Fig. 2).

Fig. 1. Axial T2-weighted magnetic resonance images (A, the proband; B, the proband's sister) showing diffuse high signal intensity in the bilateral periventricular and subcortical white matter.

Fig. 2. Histologic examination of a muscle biopsy sample. (A) Hematoxylin and eosin (H-E) staining showing mild muscle fiber size variations, a few necrotic and regenerative muscle fibers, and endomysial edema (H-E stain, ×100). Immunohistochemical staining with the anti-laminin α2 antibody detected by 3,3′-diaminobenzidine (×200) showing decreased staining in the proband (B) compared with an age-matched normal control subject (C).

The proband and her sister showed similar clinical features, and the MRI findings of the two siblings and immunohistochemical findings of the proband were compatible with LAMA2-related muscular dystrophy. The evidence presented above and the cosegregation of the variants in the affected sister, suggest that this missense variant is the pathogenic element, based on the American College of Medical Genetics and Genomics guidelines regarding the interpretation of sequence variations [10].

In conclusion, we present a patient diagnosed as having late-onset LAMA2-related muscular dystrophy as a result of mutations in LAMA2 (including a novel mutation: c.8725T>C/p.Cys2909Arg) identified by next-generation sequencing. We suggest that this novel missense mutation (c.8725T>C/p.Cys2909Arg) is the pathogenic mechanism underlying the genotype-phenotype correlation and annotation process.