Homozygous variants in ZSWIM6 cause severe syndromic short stature and developmental delay

Introduction We investigated two siblings from a consanguineous Kurdish family presenting with a specific facial phenotype (prominent forehead, supraorbital ridges, broad nose, depressed nasal bridge), severe short stature with biochemical features of growth hormone (GH) deficiency, microcephaly, and developmental delay. Whole-exome sequencing identified a homozygous variant (c.3119G>A, p.Arg1040His) in gene ZSWIM6, a putative transcription factor. Previously, only de novo heterozygous ZSWIM6 variants were known, causing either acromelic frontonasal dysostosis (AFND) or severe intellectual disability without craniofacial or skeletal anomalies. We aimed to characterize the functional impact of these three distinct variants to understand the resulting genotype-phenotype correlations. Methods The three ZSWIM6 variants were prepared in vitro by site-directed mutagenesis. Transcriptional activity was assessed in HEK293 cells using dual-luciferase reporter assays for HECW2 and ZIC2 promoters, previously hypothesized to possess functional links to ZSWIM6. In addition, in silico 3D structural modelling was performed using AlphaFold to predict the impact of each mutation on protein conformation and surface charge. Results The siblings shared partial overlap with prior ZSWIM6-associated phenotypes but also exhibited a distinct clinical presentation. Functional assays confirmed ZSWIM6 is a transcriptional regulator of the HECW2 and ZIC2 promoters. The three variants showed different effects on transcriptional regulation: the p.Arg1040His variant reduced ZIC2 activation (loss-of-function), p.Arg1163Trp enhanced ZIC2 activation (gain-of-function), and p.Arg913Ter specifically increased HECW2 activation (derepression). The 3D structural modelling predicted that p.Arg1040His and p.Arg1163Trp alter local surface charge without disrupting the overall fold, while p.Arg913Ter truncates ZSWIM6, potentially affecting the DNA binding interactions. Conclusion We report the first-ever homozygous ZSWIM6 variant, causing a novel phenotype of severe syndromic short stature with biochemical features consistent with GH deficiency and developmental delay. Our findings demonstrate that ZSWIM6 is a key transcriptional regulator and that allele-specific disturbances drive the marked clinical heterogeneity of ZSWIM6-related disorders.