Long-Read Whole-Genome Sequencing Uncovers a Deletion Upstream to HOXD13 Causing Synpolydactyly

Synpolydactyly (SPD) is a heterogeneous distal-limb malformation syndrome, characterized by webbing and duplication of adjacent digits. SPD1, the most common type, is attributed to disease-causing variants in HOXD13, a transcription factor in the HOXD cluster that is essential for limb development. Here, we present a challenging exome-negative case of familial SPD. The case was resolved using long-read genome sequencing, which revealed a microdeletion in a conserved noncoding regulatory region upstream of the HOXD cluster. Oxford Nanopore WGS-LRS was pursued on two affected sisters, followed by CMA-Cytoscan HT validation. WGS-LRS for two affected siblings revealed a shared microdeletion of ~5.6 kb upstream of EVX2 and HOXD13 [chr2:176073523-176,079,120 (hg38)]. The deletion is predicted to affect the enhancer of several HOXD genes (EH38E2053988) and overlaps with a short stretch of high-sequence homology between human and mouse, called R1. CMA-Cytoscan HT analysis and PCR validation approved segregation of the deletion in the siblings and revealed a paternal inheritance. Our findings expand the genotype-phenotype correlation of the regulatory region upstream of HOXD13 in SPD and highlight the utility of long-read WGS for detecting noncoding variants that are undetectable by exome sequencing.