Syndactyly (SPD) is a hereditary distal limb anomaly marked by webbing or duplication of digits, which can occur in isolation or as part of a genetic syndrome. The most common form, SPD type 1 (SPD1), is linked to pathogenic variants in the HOXD13 gene, part of the HOXD gene cluster crucial for limb development. This cluster’s expression is regulated by topologically associated domains (TADs) that sequentially activate gene groups essential for early and distal limb formation. Disruptions in HOXD genes or their regulatory elements – through missense mutations, deletions, or repeat expansions – can lead to limb malformations like SPD and polydactyly. While traditional sequencing techniques often miss noncoding variants, advanced methods such as long-read genome sequencing (GS) have improved diagnostic capabilities. This report highlights a familial SPD case undetected by exome sequencing (ES) but resolved through long-read sequencing (LRS), which identified a microdeletion in a conserved noncoding region upstream of the HOXD cluster.

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The  Exome Sequencing (ES) was performed on the proband and two affected siblings using the Agilent V4 kit and NovaSeq6000 platform, with data analyzed through the Geneyx Analysis platform. Reads were aligned, and single nucleotide variants as well as copy number variants were called using Illumina DRAGEN, then annotated and filtered in Geneyx Analysis software. To further investigate, long-read GS was conducted on the proband and one sibling using Oxford Nanopore’s PromethION48. Variant calling employed Clair3 for small variants and Sniffles2 for structural variants, with phasing by Whatshap and thorough annotation performed on Geneyx Analysis platform. Geneyx’s integrated enhancer analysis – leveraging ENCODE data – enabled direct correlation of phenotypic findings with a disrupted enhancer region. Additional testing included chromosomal microarray (CMA) using Cytoscan HT and PCR validation.

Figure 1. taken from the publication.

In an unrelated family of Ashkenazi Jewish descent, four out of six siblings presented with a consistent form of nonsyndromic distal limb anomaly resembling a form of SPD, primarily affecting the toes with structural abnormalities and milder, variable hand involvement. While Exome Sequencing (ES) and Chromosomal Microarray Analysis (CMA) were negative, long-read Genetic Sequencing in two affected siblings revealed a shared 5.6 kb deletion upstream of HOXD13, partially affecting the 3′UTR of EVX2. This deletion, absent from public CNV databases, encompasses a highly conserved enhancer (EH38E2053988) with strong regulatory scores and predicted interaction with HOXD genes. Using Geneyx’s enhancer visualization and scoring framework, the deletion was confidently linked to disruption of the limb development regulatory architecture. TAD analysis confirmed likely regulatory disruption. The variant segregated with all affected siblings and was inherited from a mosaic, unaffected father, as confirmed by CMA, PCR, and preimplantation genetic testing. Based on ACMG criteria, this deletion was classified as Likely Pathogenic.

In summary, this paper highlights the diagnostic advantages and limitations of next-generation sequencing, emphasizing that Exome Sequencing (ES) often fails to detect noncoding variants, structural rearrangements, or repeat expansions. While short-read GS provides some improvement, Long Read Sequencing (LRS) offers key advantages, particularly for conditions like SPD, by detecting challenging variants such as polyalanine tract expansions in HOXD13. In the reported Ashkenazi Jewish kindred, LRS identified a likely pathogenic 5.6 kbp microdeletion upstream of HOXD13 and partially involving the 3′UTR of EVX2. The pathogenic mechanism may involve disrupted enhancer activity, altered EVX2 expression, or a combined effect on both genes.  

This case illustrates how Long Read Sequencing, when paired with Geneyx’s phenotype-to-enhancer analysis tools, can resolve complex diagnoses involving regulatory noncoding regions and supports its broader implementation in clinical genetics.

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