Abstract
CAPN3 encodes a calcium-activated skeletal muscle-specific protease. Pathogenic variants in CAPN3 are associated with autosomal recessive and dominant limb-girdle muscular dystrophy. We report on three children and one adult from four unrelated Iraqi Jewish families, who harbor the same homozygous variant in CAPN3, p.Gln123Lys. Patients shared recognizable features of toe-walking and elevated creatine phosphokinase since childhood. The variant affects a conserved protein domain common to the calpain super family and likely represents a founder mutation in individuals of Iraqi Jewish ancestry. Our findings have potential implications on screening in relevant populations, allowing for more prompt diagnoses and future therapies.
Introduction
CAPN3 encodes calpain-3, a calcium-dependent protease of the calpain family, preferentially expressed in skeletal muscle. It binds and cleaves titin,1,2 interacts with dysferlin and AHNAK to regulate the dysferlin complex 3 and plays a key role in orchestrating sarcomere assembly and remodeling,4,5 early development, 6 myonuclear apoptosis 7 and more.
CAPN3 is implicated in autosomal dominant limb-girdle muscular dystrophy 4 (LGMDD4, MIM #618129) and autosomal recessive limb-girdle muscular dystrophy 1 (LGMDR1, MIM #253600, formerly known as LGMD2A 8 ) - the first form of recessive LGMD to be identified. 9 LGMDR1 is characterized by symmetric, progressive proximal limb girdle weakness with variable age of onset (2–50 years), toe walking, and shortening of the Achilles tendons, typically with no cardiac involvement. Severity and onset vary even within the same family, making genotype-phenotype correlations difficult.10–14
The worldwide prevalence of LGMDR1 ranges from approximately 1 to 9 cases per 100,000 15 and it is one of the most common LGMDs worldwide.16–18 In Israel, LGMDR1 is not known to be particularly prevalent in any ethnic group and is not included in the national carrier screening funded program.
We report on four probands from four unrelated families of Iraqi Jewish descent with autosomal recessive calpainopathy, with a distinct clinical presentation that includes childhood-onset toe walking and elevated CK.
Materials and methods
Patients
Probands1-3 were seen at the Neuromuscular and Genetic clinics at Schneider Children's Medical Center of Israel. Clinical information was gathered from clinic visit notes. Imaging studies were evaluated by a radiologist.
The fourth family was identified through ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). In this family, genetic testing was carried out due to a family history of LGMD in the late paternal uncle (Proband4) and additional family members. Information was provided by the family and gathered from available medical records.
The study was approved by the institutional review board (IRB) RMC-0191-21. Families provided consent for publication.
Genetic evaluation
In Probands 1, 3 and 4 genotyping was ascertained by a commercial multi-gene panel (Fulgent and Invitae). The variants reported were re-evaluated by the individuals’ clinical geneticists using online variant interpretation tools as well as public and local databases. In Proband 2, single variant testing was carried out by Sanger sequencing at the Raphael Recanati Genetics Laboratory, Rabin Medical Center, Israel.
Results
Clinical presentations
Clinical presentations of the patients are summarized in Table 1 and Figure 1.
A. Pedigrees of the four families harboring the CAPN3 c.367C > A; p.Gln123Lys variant. B-E. Proband 1's MRI scans. B + C- fatty infiltration of the gastrocnemius muscle bilaterally. The fat fraction (FF) was 12% in the right gastrocnemius muscle and 13% on the left side. D + E - fatty infiltration of the sartorius, biceps femoris and adductor magnus muscles, 15–29%. F-G - Images of Proband 3. F - Torso and upper limbs showing significant muscle wasting. G –hypertrophy of right gastrocnemius muscle. H. Schematic representation of the CAPN3 protein and the CAPN3 c.367C > A; p.Gln123Lys variant. Top: The p.Gln123Lys variant affects a conserved residue in PC1, one of the subdomains of the cysteine protease domain of CAPN3. Bottom: Multiple sequence alignment showing conservation of the glutamine residue (Q) affected by the variant across different species as well as sequence similarity to calpain-1 (CAPN1) and calpain-2 (CAPN2).
Clinical characteristics of the four probands harboring the p.Gln123Lys variant.
Bx: biopsy; ECG: electrocardiogram; EMG: electromyography; kg: kilograms; LBBB: left bundle branch block; LGMD: limb-girdle muscular dystrophy; mo: months; NA: not applicable; ND: no data; NSR: normal sinus rhythm; PVCs: premature ventricular contractions; U/L: units per liter; yr: years.
Probands 1-3 presented in the first-second decade of life with toe walking, calf hypertrophy and elevated CK levels of thousands. Proband 3 had significant atrophy of the pectoralis muscles as well as wasting in all four limbs. Proband 1 and 2 had a family history of undiagnosed muscle disorder.
Proband 4 underwent a comprehensive genetic evaluation in his fifties. He reported proximal muscle weakness since age 16 years. He was wheelchair bound since age 24 years. In his late fifties he suffered from respiratory difficulties. CK level was 210 U/L. Electromyography (EMG) indicated myopathy, more pronounced in proximal muscles. On muscle biopsy he had scattered muscle fibers in connective tissue and fat, with multiple central nuclei. Staining for alpha and gamma sarcoglycan, beta dystroglycan, merosin and dysferlin was negative.
Genetic workup
For Proband 1, a 43-gene neuromuscular gene panel (Fulgent Diagnostics, Temple City, CA) identified a rare, homozygous variant classified at the time as a variant of uncertain significance (VUS): NM_000070.3 c.367C > A (p.Gln123Lys) in CAPN3. Two healthy siblings were found to be heterozygous carriers. The variant was highly suspected of being causative due to its clinical correlation with the disease presentation.
Proband 2 was referred to genetics shortly after Proband 1. Given the significant overlap of the clinical presentations in the two cases and the shared Iraqi ancestry, single variant testing was sent for the p.Gln123Lys variant in CAPN3 and she was found to be homozygous.
For Proband 3, workup included multiplex ligation-dependent probe amplification (MLPA) for the DMD gene which was negative. A comprehensive neuromuscular gene panel containing 201 genes (Invitae Corporation, San Francisco, CA) detected multiple variants of uncertain significance, including the same homozygous variant in CAPN3 (p.Gln123Lys) as in Proband 1 and Proband 2.
In Proband 4, MLPA analysis of DMD and testing for spinal muscular atrophy (SMA) was negative. No conclusive diagnosis was reached before he died in his 60 s. Years later, a gene panel (Invitae Corporation, San Francisco, CA) was done using the patient's DNA at the request of the family in order to diagnose the familial muscular dystrophy for future pregnancy planning. This test identified the p.Gln123Lys homozygous variant in CAPN3 in the patient.
Analysis of the p.Gln123Lys variant
The p.Gln123Lys variant results in replacement of a neutral polar amino acid (glutamine) with a basic polar amino acid (lysine). This rare variant affects a highly conserved residue in the protease core domain 1 (PC1), which, together with PC2, comprises the cysteine protease domain of CAPN3 (Figure 1(H)). No functional studies assessing the effect of the variant have been published, but a variant affecting the same residue (Q123H or p.Gln123His) detected in random mutagenesis experiments was considered responsible for inactivating calpain-3. 1 The variant is reported in one out of 1,613,232 alleles (minor allele frequency 0.0000006199) in gnomAD v4.1.0 (https://gnomad.broadinstitute.org). It was seen in trans with a pathogenic variant in a cohort of patients with LGMD published by Nallamili et al.; 19 no additional phenotypic information was available. The variant segregates with disease in four affected individuals and more than five unaffected siblings. Multiple computational tools predict a deleterious effect on the protein, with a REVEL score of 0.96 predicting a deleterious effect on the protein. Taken together, we classify this variant as pathogenic based on the current American College of Medical Genetics and Genomics (ACMG) criteria 20 (PM1_moderate, PM2_supporting, PM3, PP1_moderate, PP3_strong).
The ClinVar database contains an entry for this variant from Eurofins (Accession: SCV000340121.4, corresponding to the case from Nallamili et al. 19 ) classifying it as a VUS. A second ClinVar entry was submitted by Invitae, initially classifying the variant as a VUS. However, following the diagnosis of Proband 3 and in light of supporting family segregation studies it was later reclassified as pathogenic (Accession: SCV000645501.5).
Population frequency of the p.Gln123Lys variant
Given the shared Iraqi ancestry of the patients and homozygosity for the variant with parental consanguinity reported only in Family 4, we hypothesized that the p.Gln123Lys variant is a founder mutation in the Iraqi population. Since the Iraqi Jewish population is not well-represented in international public databases, we queried our local database which holds genomic data of probands and family members of various ethnic backgrounds. We did not observe the p.Gln123Lys variant in our database in 1376 individuals (of which 32 are of full and 185 are of partial Iraqi Jewish ancestry). While this did not allow us to estimate the frequency of this variant in the Iraqi population, the fact that it was seen in four unrelated Iraqi families despite being so rare suggests that it is more likely to be an ethnic variant rather than a hotspot.
Discussion
All four individuals in our cohort presented with proximal lower limb weakness in childhood or teen years. Probands 1–3 had a distinct presentation with elevated CK and toe-walking. In fact, the similarity between the clinical presentations was so great that Proband 2 was diagnosed by single variant testing. The similar presentations suggest a strong genotype-phenotype correlation for this variant. Genotype-phenotype correlations have previously been suggested for CAPN3, with a milder phenotype appearing in patients carrying no or one null allele compared to individuals with two null alleles. 21 In this case, however, a homozygous missense variant presents with symptoms as early as age 2 years.
Our patients’ signs and symptoms fit with previous reports of CAPN3-related LGMD. The MRI typically shows involvement of the posterior compartment of the thigh muscles.22,23 Proband 1's MRI showed similar features. Proband 3 had significant atrophy of the pectoralis muscles as well as wasting in all four limbs. Wasting of the trunk muscles and generalized muscle atrophy was described in earlier reports.24–26 Studies have also reported fatty replacement of abdominal wall muscles in autosomal dominant calpainopathy. 27 The ubiquitin-proteasome system has been suggested as the main pathway leading to muscle atrophy, and studies are under way to investigate proteasome inhibition as a therapeutic strategy in calpainopathy. 28
The cysteine protease domain which is affected by the p.Gln123Lys variant is shared amongst the calpains. Interestingly, sequence alignment shows that the glutamine residue is also conserved in CAPN1 and CAPN2 (Figure 1(H)), and variants affecting the corresponding glutamine residues in calpain-1 and calpain-2 are also rare in population databases. While multiple prediction tools supported the pathogenicity of this variant, muscle biopsy from affected individuals was unavailable and therefore we were unable to predict the level of CAPN3 in skeletal muscle.
The finding of the same homozygous variant in CAPN3 in four unrelated families of Iraqi Jewish descent suggests a founder effect in this population. In countries like Israel, where ethnicity-based carrier screening is widely implemented, detection of founder mutations can have direct implications on prenatal screening, and could also allow for more precise, faster and more cost-effective diagnostic testing. Data sharing through local and international databases plays an important role in identifying such variants.
Our work adds to the knowledge of calpainopathy, suggests genotype-phenotype correlations and identifies p.Gln123Lys as a putative founder mutation in the Iraqi Jewish population. As novel gene therapies for LGMD are under development, 29 it is important to diagnose patients in the early or presymptomatic stages of the disease and thus present an opportunity to participate and access investigational therapies in a rare disease that is currently incurable.
Footnotes
Acknowledgements
The authors wish to thank the patients and families who participated in this study.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data availability
The data supporting the findings of this study are available on request from the corresponding author.