A novel ZRS variant causes preaxial polydactyly type I by increased sonic hedgehog expression in the developing limb bud

Abstract

Purpose: Preaxial polydactyly (PPD) is a common congenital hand malformation classified into four subtypes (PPD I-IV). Variants in the zone of polarizing activity regulatory sequence (ZRS) within intron 5 of the LMBR1 gene are linked to most PPD types. However, the genes responsible for PPD I and the underlying mechanisms are unknown. Methods: A rare large four-generation family with isolated PPD I was subjected to genome-wide genotyping and sequence analysis. In vitro and in vivo functional studies were performed in Caco-2 cells, 293T cells, and a knockin transgenic mouse model. Results: A novel g.101779T>A (reference sequence: NG_009240.2; position 446 of the ZRS) variant segregates with all PPD I-affected individuals. The knockin mouse with this ZRS variant exhibited PPD I phenotype accompanying ectopic and excess expression of Shh. We confirmed that HnRNP K can bind the ZRS and SHH promoters. The ZRS mutant enhanced the binding affinity for HnRNP K and upregulated SHH expression. Conclusion: Our results identify the first PPD I disease-causing variant. The variant leading to PPD I may be associated with enhancing SHH expression mediated by HnRNP K. This study adds to the ZRS-associated syndromes classification system for PPD and clarifies the underlying molecular mechanisms.

Two nonsense GLI3 variants are associated with polydactyly and syndactyly in two families by affecting the sonic hedgehog signaling pathway

“…Shh is expressed at the posterior margin of the limb buds in vertebrates, specifying an anteroposterior pattern in developing limbs by stimulating the proliferation of mesenchyme and regulating the anteroposterior length of the apical ectodermal ridge. As we previously reported, ectopic anterior expression of SHH in the hindlimbs led to preaxial polydactyly in ZRS g.101779T>A homozygous mice models (Xu et al, 2020). Yoshiyuki et al demonstrated that a severe lack of SHH signaling led to the polydactyly, syndactyly, and brachydactyly in homozygote hereditary multiple malformation mutant (hmm −/− ) mouse embryos.…”

GLIS Family Zinc Finger 1 was First Linked With Preaxial Polydactyly I in Humans by Stepwise Genetic Analysis

“…PPD I is a frequent developmental defect worldwide, while disease-causing variants have only been identified in a few patients ( Ullah et al, 2019 ; Umair et al, 2019 ; Xu et al, 2020 ). Genetic etiologies of most cases remain to be studied.…”

“…PPD occurrence is accompanied by an increase in cell proliferation and migration in the anterior-proximal region of limb buds ( Crick et al, 2003 ). Variants in GLI3 and ZRS cause PPD by inducing the anterior ectopic expression of SHH ( Deng et al, 2015 ; Xu et al, 2020 ). SHH promotes cell proliferation and migration in vivo and in vitro ( Hayes et al, 2001 ; Wang et al, 2019 ).…”

“…Currently, six loci have been identified in nonsyndromic PPD cases, including GLI1 , GLI3 , STKLD1 , the zone of polarizing activity (ZPA) regulatory region (ZRS), which is a limb-specific enhancer of SHH that is located within intron five of LMBR1 , the pre-ZRS region (a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS), and a deletion located 240 kb from the SHH promotor. Genetic etiologies in most PPD patients have never been evaluated ( Petit et al, 2016 ; Potuijt et al, 2018 ; Ullah et al, 2019 ; Umair et al, 2019 ; Xu et al, 2020 ; Baas et al, 2021 ).…”

TATTOO-seq delineates spatial and cell type-specific regulatory programs during limb patterning

“…2H), suggesting that this partition of the ZPA in distinct sub-regions may correspond to alternative ZRS-associated TFs. This hitherto hidden level of spatial and regulatory diversity in the ZPA therefore may further contribute to the diversity of limb morphologies resulting from ZRS genetic variants [44][45][46] as they may change Shh expression modulate its impact on target genes in either the proximal or distal part of the ZPA [41].…”