Next-generation sequencing (NGS), particularly whole-exome sequencing (WES) and whole-genome sequencing (WGS), has revolutionized the diagnosis of rare Mendelian disorders by uncovering novel molecular mechanisms and broadening the recognized phenotypic spectra (Baldridge et al., 2017). These advances have also highlighted multi-locus disease-causing genomic variations (MGVs) and multiple genetic diagnoses (MGDs), in which pathogenic variants at two or more independent loci give rise to blended clinical features (Posey et al., 2017). Such findings complicate diagnosis, management, and genetic counseling, as each condition carries distinct inheritance patterns and recurrence risks. The growing recognition of MGVs and MGDs marks a paradigm shift in Mendelian genetics and underscores the need for clinical geneticists to integrate molecular and bioinformatics expertise with traditional evaluation for accurate phenotype–genotype interpretation (Hennekam and Biesecker, 2012).

Through the lens of WES and WGS, MGDs have been detected in 1.4%–7.2% of cases, challenging the traditional single-diagnosis paradigm and highlighting the need for more refined, multilayered clinical approaches (Narayanan et al., 2021; Smith et al., 2019). In this context, trio-based whole-exome sequencing (trio-WES) has been shown to yield higher diagnostic rates than proband-only or traditional methods, further enhancing accuracy while highlighting the need for additional studies to evaluate its clinical utility and economic impact (Lee et al., 2014).

In this study, we report a proband with Duchenne muscular dystrophy (DMD) and congenital bilateral cataracts from a consanguineous family affected by oculodentodigital dysplasia (ODDD). Trio-based exome sequencing and clinical correlation revealed disease-causing variants at multiple loci, with additional phenotypes. These findings provide important insights for therapeutic planning and recurrence-risk counseling for the family.

DMD (OMIM 310200) is an X-linked disorder marked by progressive muscle weakness and wasting. Globally, it affects an estimated 7.1 per 100,000 male individuals (2.8 per 100,000 in the general population), with a birth prevalence of 19.8 per 100,000 live male births (Crisafulli et al., 2020). The DMD gene is the largest in the human genome; due to its size (∼2.5 Mb, 79 exons), it is prone to various mutations (Muntoni et al., 2003; Aartsma-Rus et al., 2016). The reported variants consist of large deletions, duplications, insertions, and point mutations, with nearly one-third occurring de novo (Aartsma-Rus et al., 2006; Gibbs et al., 2019; Bello et al., 2016; Zambon et al., 2022; Torella et al., 2020). There were 80% big rearrangements (69% deletions and 11% duplications) and 20% small variants (25% small deletions, 9% insertions, 14% splice-site, and 52% point mutations—mostly nonsense) among the 7,149 DMD mutations found in the TREAT-NMD database (Bladen et al., 2015). Individuals harboring these genetic defects typically exhibit progressive degeneration of skeletal, respiratory, and cardiac muscles, ultimately resulting in premature death (Birnkrant et al., 2018).

ODDD (OMIM 164200) is an extremely rare inherited disorder that develops due to pathogenic variations in the GJA1 gene, which codes the gap junction protein connexin 43 (Paznekas et al., 2003). A wide range of neurological and physical disorders are the outcome of the syndrome’s high penetrance and diverse clinical manifestations (Judisch et al., 1979). The defining skeletal characteristic is bilateral syndactyly of the fourth and fifth digits, which is often associated with camptodactyly, clinodactyly, and toe syndactyly. Craniofacial features consist of a slender nose with underdeveloped alae nasi, microcephaly, anteverted nares, and a large columella, while the hair and nails may exhibit thinness, sparsity, and brittleness (Reisner et al., 1969). Common signs of dental abnormalities include microdontia, enamel hypoplasia, caries, and early tooth loss (Hindu and Umer, 2023). The ocular phenotype may encompass microphthalmia, microcornea, iris abnormalities, cataracts, glaucoma, and optic atrophy (Kumar et al., 2020). Moreover, neurological problems may manifest, such as spastic paraparesis, ataxia, dysarthria, and neurogenic bladder, with certain individuals displaying mild intellectual disability. ODDD manifests as a multisystem illness characterized by significant phenotypic diversity, even among the affected individuals within the same family.

Congenital cataract is a rare yet significant cause of childhood blindness, occurring in 0.6–9.3 cases per 10,000 live births (Shee et al., 2016). It is the consequence of developmental defects or genetic mutations that result in a reduction in lens transparency. Congenital (within 1 year) and juvenile (within 10 years) forms are present; they are frequently isolated but may occasionally be syndromic with ocular or systemic anomalies (Chan et al., 2012). Diagnoses and treatments have been enhanced by advancements in pediatric surgery, imaging, and DNA sequencing technologies (Gillespie et al., 2016). Although amblyopia treatment and prompt intervention can help manage glaucoma and impaired vision, early surgery is essential for improved visual outcomes (Gasper et al., 2016).

This is the inaugural documentation instance of a proband in a consanguineous family possessing MGVs related to DMD, ODDD, congenital bilateral cataract, and their associated phenotypes.

The FYCO1 (FYVE and Coiled-Coil Domain Containing 1) gene, located on chromosome 3p21.31, encodes an autophagy adapter protein that interacts with LC3 (microtubule-associated protein 1A/1B-light chain 3) and Rab7, a small GTPase. Through these interactions, FYCO1 mediates the microtubule-dependent transport of autophagosomes toward the cell periphery, thereby facilitating the degradation of damaged α-crystallin proteins—major structural components that are essential for ocular lens transparency (Chen et al., 2011; Olsvik et al., 2015).

The identified variant, FYCO1:c.2482C>T (p.Gln828*), is located in exon 8 of the transcript NM_024513.4. This single-nucleotide substitution (C>T) at coding position 2,482 changes the codon for glutamine (CAG) to a premature stop codon (TAG), resulting in a nonsense (stop-gain) mutation. The alteration is predicted to generate a truncated protein lacking the downstream coiled-coil and FYVE domains that are crucial for vesicular trafficking or to trigger nonsense-mediated mRNA decay (NMD), leading to the complete loss of protein function and potentially causing autosomal recessive congenital cataract (OMIM #610019), which is characterized by early-onset bilateral lens opacity typically without systemic involvement. Accordingly, the FYCO1 variant is interpreted as likely pathogenic, consistent with the established disease mechanism. The FYCO1:c.2482C>T variant was absent from major population databases (gnomAD, ExAC, and IndiGenomes) and clinical databases (ClinVar and LOVD). Additionally, no entries were found in PubMed, Google Scholar, or the Franklin (Genoox) database; the variant was also absent from other population datasets (1000 Genomes, ESP6500, ExAC, and UK10K), indicating that it is a novel variant.

While cataracts have occasionally been reported in patients with ODDD (Kumar et al., 2020), the congenital bilateral cataract observed in the proband of the present study is most likely attributable to the novel FYCO1 variant. The likely pathogenic GJA1 variant, which is responsible for ODDD, is also present in the father and brother, neither of whom exhibits a cataract phenotype, supporting the independent contribution of the FYCO1 variant to the ocular manifestation.

The DMD: c.5124_5127del (p.Lys1708Asnfs*12; rs398123979) variant results in a frameshift and premature termination, leading to dystrophin loss of function. It fulfills PVS1 (predicted loss of function) and is classified as pathogenic according to the ACMG guidelines. This frameshift disrupts the open reading frame, resulting in a truncated dystrophin protein lacking essential functional domains required for normal muscle function, as observed in the proband. This variant represents a null allele in DMD, a gene with a well-established loss-of-function disease mechanism, supported by 1,841 pathogenic truncating variants reported in ClinVar across 77 exons (including 27 in exon 36) and strong loss of function (LoF) constraint (gnomAD observed/expected = 0.154). The variant affects coding exon 36/79 and is located upstream of the terminal exon, not within the last 50 bp of the penultimate exon, and is, therefore, predicted to undergo nonsense-mediated mRNA decay.

The DMD frameshift indel c.5124_5127del is recorded as pathogenic in clinical databases (ClinVar) with two independent submissions linking it to DMD, but no peer-reviewed case reports providing detailed genotype–phenotype correlation for this exact variant are currently available in accessible literature. Parental and sibling testing by WES with orthogonal Sanger sequencing did not detect the DMD variant, which is consistent with a de novo event. However, ultra-low level maternal mosaicism cannot be excluded as the currently used methodologies are not optimized for the reliable detection of low-variant allele fraction events and would require ultra-deep targeted sequencing for definitive exclusion.

Variations in GJA1 (connexin 43) act by disrupting gap junction communication through several mechanisms, including protein mislocalization, failure to form connexons, or altered channel conductance and gating. Many ODDD-associated missense variants cluster in a region homologous to the calmodulin-binding motif, affecting calcium-dependent regulation. Functional studies of related connexins indicate that these mutations often act in a dominant-negative manner, where mutant subunits interfere with wild-type connexin function during embryonic development (Paznekas et al., 2003; Sargiannidou et al., 2021). The GJA1:c.396_398del (p.Ile132_Lys133delinsMet; rs1773903559) variant, identified in ClinVar but not specifically linked to the ODDD phenotype, represents an in-frame deletion altering conserved residues in connexin 43. Based on the combined ACMG criteria codes, it is classified as likely pathogenic.

None of the three causal variants were detected in major population databases such as gnomAD or ExAC, highlighting their rarity. Similarly, Franklin (Genoox) searches showed no entries across other population datasets (1000 Genomes, ESP6500, ExAC, and UK10K), confirming their absence in global cohorts.

PLINK-based indicative ROHs and FROH analyses from exome data indicate extended autozygosity (up to 15 Mb–16 Mb), which is consistent with parental consanguinity equivalent to second cousins or closer. The novel homozygous FYCO1 variant was localized within one such ROH, indicating identity-by-descent, while the DMD variant arose de novo. The coexistence of recessive, dominant, and de novo variants highlights the diagnostic complexity of multi-locus pathogenicity in consanguineous families.

The presence of the mentioned three pathogenic or likely pathogenic variants within a single individual is extremely rare and a first report. Each gene is independently associated with a distinct Mendelian disorder: FYCO1 with autosomal recessive congenital cataract, DMD with de novo appearance, and GJA1 with autosomal dominant oculodentodigital dysplasia. This exceptional co-occurrence underscores the importance of comprehensive genomic analysis for accurate diagnosis and phenotypic correlation. Such multi-locus variant combinations are scarcely reported and emphasize the contribution of MGVs to phenotypic diversity and variable expressivity in Mendelian disorders.

The relevance of this type of case lies primarily in the diagnostic, interpretative, and counseling challenges it presents rather than its frequency. In patients with complex or multisystem phenotypes, the pursuit of a single unifying molecular diagnosis may lead to diagnostic anchoring, particularly when some clinical features are discordant or exceed the known spectrum of a single disorder. In this case, systematic phenotypic stratification and independent evaluation of major clinical manifestations enabled more accurate attribution of the findings to distinct genetic etiologies.

From a medical genetics perspective, the coexistence of multiple molecular diagnoses necessitates independent interpretation of each variant with respect to the inheritance pattern, prognosis, and recurrence risk, thereby complicating genetic counseling and family risk assessment. Furthermore, this study highlights the importance of distinguishing true phenotypic expansion of a genetic disorder from the coexistence of multiple molecular defects as apparently broadened phenotypes may be more parsimoniously explained by separate genetic conditions.

In some complex cases, additional diagnoses may escape standard NGS pipelines due to technical or interpretative limitations, thus underscoring the value of careful phenotyping, periodic data reanalysis, and close clinician–geneticist collaboration in the evaluation of complex cases.

The present study exemplifies the clinical and genetic complexity arising from multi-locus variation in a consanguineous pedigree and highlights the importance of trio/family-based exome sequencing for accurate diagnosis, genetic counseling, and phenotype interpretation in rare genetic disorders.