Dystroglycanopathies arise when α-dystroglycan, a membrane protein that anchors muscle fibers to their surrounding basement membrane, is not properly decorated with sugar chains. At least 19 causative genes have been identified to date, reflecting the complexity of the glycosylation pathway that builds and attaches these critical sugar modifications. One of these genes is CRPPA, formerly known as ISPD, which encodes CDP-L-ribitol pyrophosphorylase A. This enzyme synthesizes CDP-ribitol, a substrate that downstream glycosyltransferases require to attach ribitol phosphate groups to α-dystroglycan. When that enzymatic step fails, dystroglycan loses its ability to bind ligands and interact with the basement membrane, destabilizing the muscle membrane and paving the way for progressive degeneration.

Variants in CRPPA produce a spectrum of disease severity. At the severe end sit congenital muscular dystrophies, often accompanied by brain and eye abnormalities such as cerebellar hypoplasia, ocular defects, and developmental delay. At the milder end lies limb-girdle muscular dystrophy, a clinically diverse group of disorders with an estimated prevalence of roughly 2 per 100,000 people, characterized by progressive proximal weakness and dystrophic features on muscle biopsy, typically emerging in late childhood, adolescence, or adulthood. Most reported individuals with CRPPA-related disease, however, manifest symptoms in infancy or early childhood, frequently with additional systemic or neurodevelopmental involvement. Although biallelic single-nucleotide variants in CRPPA are well documented, only a handful of reports describe compound heterozygous genotypes that combine a single-nucleotide variant with a copy number variant, and the phenotypic consequences of such combinations remain incompletely understood.

The diagnostic journey began with whole exome sequencing of the proband at the Kariminejad-Najmabadi Pathology and Genetics Center. Genomic DNA extracted from peripheral blood was enriched using the Twist Exome V2.0 kit and sequenced on an Illumina NovaSeq 6000 platform, with reads aligned to the GRCh38 reference genome. Variant annotation and classification followed American College of Medical Genetics and Genomics guidelines. Two heterozygous variants emerged in CRPPA: a missense single-nucleotide variant, c.267G>C, which substitutes cysteine for tryptophan at residue 89 (p.Trp89Cys), and a gross deletion spanning approximately 641 kilobases of chromosome 7p21.2 that removes exons 9 and 10 of the gene, along with neighboring genes including MEOX2 and several antisense transcripts.

Neither variant had been seen before in population databases. The missense variant was absent from the Genome Aggregation Database v4.1 and from Iranome, an Iranian genomic population database, and multiple in silico prediction tools, including PolyPhen-2, MutationTaster, CADD, FATHMM, REVEL, and PrimateAI, consistently flagged it as deleterious. CADD assigned it a score of 29.7, and REVEL returned a supporting score of 0.75. Nevertheless, because direct functional evidence was lacking, the variant was classified as a variant of uncertain significance under ACMG recommendations, supported by criteria PM2, PP3, and PP1. The deletion, absent from the Database of Genomic Variants and never previously reported, was classified as likely pathogenic based on its size, gene content, and segregation data.

Structural modeling added mechanistic texture to these predictions. AlphaFold2 localized residue Trp89 to a highly conserved region within the N-terminal catalytic domain of the enzyme, immediately adjacent to a β-strand, with very high prediction confidence, indicated by a per-residue confidence score above 90. The HOPE server predicted that replacing the bulky tryptophan side chain with the smaller cysteine would leave an empty space within the protein core, potentially reducing local structural stability and disrupting important domain contacts. Because the N-terminal catalytic domain houses the enzyme’s active-site machinery, missense variants in this region may compromise the integrity of the site that synthesizes CDP-ribitol. The authors caution, however, that functional studies will be required to confirm the biological significance of these computational findings.

Sanger sequencing and array comparative genomic hybridization traced the inheritance pattern through the family. The affected sister carried both variants, consistent with a compound heterozygous state. The missense variant was absent from the mother, while array-CGH confirmed that the mother carried the 641-kilobase deletion, meaning each parent contributed one defective allele. The proband’s clinical picture fit the limb-girdle pattern: progressive proximal lower limb weakness beginning at 41, elevated serum creatine kinase at 1193 units per liter against a normal range of 24 to 195, and electromyography showing features of a chronic non-irritable myopathy. Neurological examination revealed no calf hypertrophy, scapular winging, or joint hypermobility, and no ocular or central nervous system involvement was observed, with normal cognition.

His sister’s course, despite the identical genotype, diverged sharply. She developed symptoms at approximately 10 years of age, with progressive proximal weakness involving both upper and lower limbs. Her serum creatine kinase was only mildly elevated at 460 units per liter, notably lower than her brother’s, and her muscle biopsy revealed dystrophic changes consistent with a myopathic process. Her electromyography findings were comparable to her brother’s, showing chronic non-irritable myopathy, and she too had no evidence of brain or eye involvement and normal cognitive function. Where the brother’s weakness was restricted to the proximal lower limbs, hers spanned both limb girdles. Both siblings received supportive metabolic therapy with coenzyme Q10, riboflavin, and L-carnitine, and at follow-up the proband demonstrated only mild proximal lower limb weakness.

To place the family in context, the researchers reviewed published studies from 2012 through 2026 reporting CRPPA variants with sufficient clinical information. Among 59 patients, the majority, 37 patients or 62.7 percent, were compound heterozygous, while 20 patients, or 33.9 percent, carried homozygous variants, and in two patients only a single identifiable CRPPA allele was reported. Pathogenic variants were distributed throughout the coding region, but exon 9 emerged as a recurrent site of variation, underscoring its functional importance. The Human Gene Mutation Database currently lists 54 disease-associated CRPPA variants, with missense variants predominating and gross deletions forming a smaller proportion. Reported deletions most frequently involve exon 9 and less commonly extend to exon 10, and most published cases carrying such deletions presented with severe congenital dystroglycanopathy accompanied by cerebellar hypoplasia, ocular abnormalities, and developmental delay.

Milder phenotypes, however, have also been described, particularly when exon-level deletions occur in compound heterozygosity with missense variants. One recent report described a patient carrying a deletion involving exons 6 through 9 who presented with a limb-girdle phenotype accompanied by cerebellar and brainstem hypoplasia and cardiac involvement. Although genotype-phenotype correlations remain incompletely defined, previous studies suggest that specific compound heterozygous genotypes, particularly those involving frameshift or truncating variants, may lead to early-onset severe disease, whereas other combinations can produce later-onset, milder limb-girdle presentations. This variability is likely related to differing degrees of residual protein function. In the present family, the combination of the novel p.Trp89Cys variant and the exon 9-10 deletion is consistent with, although it does not prove, a hypomorphic disease mechanism in which partial enzyme activity survives.

The age at onset of 41 years in the male proband is markedly later than that reported in most CRPPA-related limb-girdle cases, broadening the known age-of-onset spectrum of the disease. Because most reported cases are childhood-onset and severe, the authors suggest that milder adult-onset presentations may be underrecognized, meaning some adults with unexplained proximal weakness could be carrying undiagnosed CRPPA-related disease. The finding carries a practical message for clinicians: this diagnosis should be considered even in adult-onset proximal muscle weakness that lacks the brain or eye abnormalities typically associated with more severe dystroglycanopathy phenotypes. It also underscores the importance of comprehensive genetic evaluation that assesses both single-nucleotide and copy number variants, since exome sequencing alone would have missed the gross deletion that completed the compound heterozygous genotype.

Beyond its diagnostic implications, the family demonstrates marked intrafamilial phenotypic variability within a single CRPPA-affected household, with childhood and late-onset presentations arising from the same compound heterozygous genotype. What determines whether one sibling loses substantial muscle function in childhood while another remains symptom-free into middle age remains an open question that current genetic testing cannot fully answer. For now, the case expands the mutational and clinical spectrum of CRPPA-related disease and adds a compelling data point to the ongoing effort to untangle the genotype-phenotype relationships of the dystroglycanopathies, a task made harder by the sheer diversity of the glycosylation pathway that keeps muscle membranes anchored and intact.

Subject of Research: Genetics of CRPPA-related limb-girdle muscular dystrophy and intrafamilial phenotypic variability

Article Title: Variable Phenotypic Manifestations of Novel Compound Heterozygous CRPPA Variants in Two Iranian Siblings With Limb‐Girdle Muscular Dystrophy

Article References: Alagha, P., Ahangari, F., Kariminejad, A., Ghasemi, A., Gholami, S., Sadatian, N., Kariminejad, R., Kahrizi, K., Okhovat, A. A., & Najmabadi, H. (2026). Variable Phenotypic Manifestations of Novel Compound Heterozygous CRPPA Variants in Two Iranian Siblings With Limb‐Girdle Muscular Dystrophy. Molecular Genetics & Genomic Medicine, 14(10), Article e70312. https://doi.org/10.1002/mgg3.70312

Image Credits: AI Generated

DOI: 10.1002/mgg3.70312

Keywords: CRPPA, limb-girdle muscular dystrophy, dystroglycanopathy, alpha-dystroglycan, compound heterozygous, copy number variant, missense variant, whole exome sequencing, phenotypic variability, genotype-phenotype correlation, Iranian siblings, muscular dystrophy