Identification of a novel LAMA2 c.2217G > A, p.(Trp739*) mutation in a Moroccan patient with congenital muscular dystrophy: a case report

Background Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a rare autosomal recessive genetic condition caused by deleterious mutations in the LAMA2 gene encoding the laminin-α2 chain. It is the most frequent subtype of congenital muscular dystrophies (CMDs) characterized by total laminin-α2 deficiency with muscle weakness at birth or in the first six months of life. To the best of our knowledge, this study reports the first molecular diagnosis and genetic defect of this heterogeneous form of CMD performed in a Moroccan medical genetic center using next-generation sequencing (NGS). It allows us to expand the mutational spectrum of the LAMA2 gene. Case presentation We report the case of a female Moroccan child with clinical and paraclinical features in favor of a CMD. She has global congenital hypotonia with generalized muscle weakness, psychomotor retardation, increased serum creatine kinase, and normal brain scan at the age of six months. Targeted NGS leads to the identification of a novel homozygous nonsense mutation c.2217G > A, p.(Trp739*) in the exon 16 of LAMA2. Sanger sequencing confirmed this mutation in the affected patient and showed that her parents are heterozygous carriers. Conclusions A modern genetic analysis by NGS improves the genetic diagnosis pathway for adequate genetic counseling of affected families more precisely. An accession number from the National Center for Biotechnology Information (NCBI) ClinVar database was retrieved for this novel LAMA2 mutation.


Background
Laminin α2-related muscular dystrophy (LAMA2 MD) is a rare autosomal recessive neuromuscular disorder caused by homozygous or compound heterozygous mutations in the LAMA2 gene (MIM*156225) on chromosome 6q22-q23, encoding the laminin-α2 chain [1]. A total loss of merosin function due to pathogenic LAMA2 mutations causes mainly a severe phenotype with early onset of merosin-deficient congenital muscular dystrophy type 1A (MDC1A). It is the most frequent form of congenital muscular dystrophy (CMD), responsible for 30% to 40% of all CMD cases in Europe [2]. MDC1A includes a broad range of phenotypes with different degrees of severity due to variability in the onset symptoms, clinical manifestations, and mutation effects on the laminin-α2 synthesis [3].
Herein, we report the case of a Moroccan female patient with a severe form of CMD caused by a novel homozygous nonsense mutation in the LAMA2 gene identified by next-generation sequencing (NGS).

Case presentation
The proband (VI.3) is a female of 2 years and 7 months at the time of her genetic assessment. She is consanguineous, the third liveborn of a couple of half-first cousins (V.1 and V.8), sharing the same grand-mother (III.2). The grand-mother (III.2) was firstly married to her maternal cousin (III.1), grand-father of (V.1) and then to her paternal cousin (III.3), grand-father of (V.8). The proband's siblings (VI.1 and VI.2) aged 10 and 6 respectively, were normal at the time of assessment. Grand-parents, parents, uncles, aunts and cousins of the proband (VI.3) were healthy with no particular disease history (Fig. 1a).
The proband's pregnancy was medically followed and presented without complication, but the baby was born prematurely at 33 weeks of gestation by vaginal delivery. Since the neonatal period, her parents have noticed a muscle weakness with hypotonia.
At 1 year of age, the neurologic evaluation showed a delay in motor development, tetraparesis with hypotonia, and an inability to sit. The tendon and bone reflexes were absent in lower limbs, and Babinski's sign was negative. Her serum creatine kinase (CK) level was increased to 537 IU/l (normal range < 170 IU/l [4]). An electroneuromyographic analysis showed normal nerve conduction at the time of examination and the electromyogram (EMG) showed a myogenic pattern in muscles of upper and lower limbs. The computerized tomography (CT) scan at 6 months was normal and did not reveal any cerebral changes. Her intellectual development was normal and she never had seizures. She had no ocular signs and did not have cardiac nor respiratory dysfunction. She was unable to raise her head until 18 months and she was unable to stand up or stay up without support until now.
Clinical and paraclinical symptoms enable us to conclude that the patient's phenotype may be compatible with a CMD. Thus, molecular genetic analysis was performed in the proband through a custom gene panel of 24 genes known to be related to inherited neuromuscular disorders in order to reach a precise molecular diagnosis ( Table 1). The informed consent was obtained from the proband's parents prior to molecular analysis. Samples of peripheral blood were collected from the affected child and her parents.
Confirmation of this mutation was performed by conventional Sanger sequencing in the patient and her parents, using the BigDye ™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Thermo Fisher Scientific) and loaded on an ABI 3500 automated Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific). Exon 16 and its flanking regions were amplified using the set of primers (ex16F: 5′GCT TTG ACA AGC ACA TTT GATG3′/ex16R: 5′GGT CCC CAG GGT AGT AGC TG3′). A direct nucleotide sequence analysis confirmed that the proband carried the mutation in a homozygous state (Fig. 1b). Both parents were heterozygous (Fig. 1c, d).

Discussion and conclusions
The CMDs are a group of inherited muscle diseases, genetically and clinically heterogeneous, mainly with an autosomal recessive type of inheritance. They are manifested in early life or infancy [5]. Some severe forms of CMD can be fatal in infancy, whereas others may have a milder course and can survive into adulthood [6]. To date, there are at least 32 genetic forms of CMD that have been associated with mutations in more than 30 genes coding for structural and functional proteins of skeletal muscle fibers [7,8]. MDC1A, is the most common subtype of CMD in Caucasian populations due to laminin-α2 defects, accounting for 30 to 40 percent of total cases [2,9].
We report here the clinical and molecular data of a patient with a severe phenotype of MDC1A. The patient manifested congenital hypotonia, muscle weakness, and had serum CK level which was three times the upper normal value. Similar values of CK levels were found in two unrelated Tunisian families with the same phenotype severity as our case, which had 3-5 times the normal range [10].
At the age of 6 months, our patient had a normal cerebral function without any specific change, observed by the CT scan. In contrast, Oliveira et al. reported a case of an MDC1A patient with white matter changes (WMC) observed in the CT scan [11].
In previous studies, patients with LAMA2 mutations, showed normal brain magnetic resonance imaging (MRI) when has been detected during the neonatal period or in the first 6 months of life [11][12][13]. However, white matter abnormalities have been shown in the later period of life except for the case of an Italian patient reported by Saredi et al. [14], who carried a compound heterozygous nonsense mutation with an atypical phenotype and nearly normal brain MRI performed at 16 years of age. Thus, in our case, we could not completely exclude the absence of WMC, as well as the first detection by CT, which was done at an early age, it was considered as a period in which the changes are not always visible even with MRI observation in some patients.
Because of clinical heterogeneity and overlap between the different forms of CMD, NGS technology may be more efficient to increase the molecular diagnostic success rate, bypassing immunohistochemistry, which is not always available like in the Moroccan context [15,16].
To the best of our knowledge, we report here the first molecular study performed on a Moroccan female proband underlining a novel nonsense mutation in the LAMA2 gene using an Ion Torrent PGM platform. Our mutation c.2217G > A may cause a complete deficit in laminin-α2 function due to a premature termination codon (PTC) at 739 amino acid residue. The current mutation has never been reported in public human databases.
To date, more than 400 different pathogenic mutations in the LAMA2 gene have been reported in HGMD professional release 2019.4 (accessed Nov 2020), (http:// www. hgmd. cf. ac. uk/ ac/ gene. php? gene= LAMA2), with no noticeable mutational hotspot. The most frequently reported genotype in the literature for patients with MDC1A were mutations that create a PTC associated with a total absence of laminin-α2 and detected in a homozygous state [11,12,17].
We have illustrated in (Fig. 2), all pathogenic LAMA2 nonsense mutations reported in the public version of HGMD in patients with severe merosin-deficient CMD or milder myopathy according to zygosity state. We observed that the effect of mutations on phenotype development and its severity differs between patients regardless of their mode of inheritance, which makes the understanding of the physiopathological mechanisms so far unknown. Interestingly, in the same exon 16, a nonsense mutation c.2230C > T (p.Arg744*) was previously reported at a homozygous state in two adult siblings with a mild MD phenotype [18]. Whereas the same mutation at compound heterozygous state together with another nonsense mutation c.4048C > T (p.Arg1350*) in exon 27 is responsible for causing a severe CMD [17].
In conclusion, NGS has revolutionized molecular genetic research and has become an essential genetic tool for molecular diagnosis of heterogeneous disorders as CMDs. It allows us to improve our understanding of the origin of MDC1A-causing by the identification of a mutation in the LAMA2 gene. The novel mutation identified here provides an appropriate course of management to the patient to offer genetic counseling to the family, to expand the genetic spectrum of LAMA2-related CMDs, and to raise awareness to the pediatric neurologists on the morbidity of this severe form of CMD that is so far under-diagnosed in the Moroccan population, and to show them the added value of NGS technology in reducing diagnostic wandering.
Abbreviations MDC1A: Merosin-deficient congenital muscular dystrophy type 1A; CMD: Congenital muscular dystrophy; CK: Creatine kinase; NGS: Next-generation sequencing; MD: Muscular dystrophy; EMG: Electromyogram; CT: Fig. 2 Schematic representation of the LAMA2 gene structure and localization of identified LAMA2 nonsense mutations that are listed in the public version of HGMD. Two classifications, homozygous and heterozygous state, of LAMA2 nonsense mutations scattered along the coding sequence containing 65 exons (only those containing nonsense mutations are described and marked in black) are shown in patients with severe or milder phenotype of CMD (in dark blue) and patients with mild myopathy (in light blue). Nonsense mutations listed in HGMD are shown in black. Mutations of the second allele are illustrated in the following manner: nonsense (in red), frameshift (in green), missense (in yellow), splice site (in purple), and in-frame deletion (in grey). [?] refers to an unknown second mutation from the other allele due to using classical tests that could not identify the second mutation in the proband. The asterisk (*) refers to the novel mutation identified in our Moroccan patient. cDNA reference sequence that has been used to identify these mutations was NM_000426.3