Intrafamilial variability of ZRS‐associated syndrome: characterization of a mosaic ZRS mutation by pyrosequencing

Abstract

During limb development, the spatio-temporal expression of sonic hedgehog (SHH) is driven by the Zone of polarizing activity Regulatory Sequence (ZRS), located 1 megabase upstream from SHH. Gain-of-function mutations of this enhancer, which cause ectopic expression of SHH, are known to be responsible for congenital limb malformations with variable expressivity, ranging from preaxial polydactyly or triphalangeal thumbs to polysyndactyly, which may also be associated with mesomelic deficiency. In this report, we describe a patient affected with mirror-image polydactyly of the four extremities and bilateral tibial deficiency. The proband's father had isolated preaxial polydactyly type II (PPD2). Using Sanger sequencing, a ZRS point mutation (NC_000007.14, g.156584153A>G, UCSC, Build hg.19) was only identified in the patient. However, pyrosequencing analysis enabled the detection of a 10% somatic mosaic in the blood and saliva from the father. To our knowledge, this is the first description of a ZRS mosaic mutation. This report highlights the complexity of genotype-phenotype correlation in ZRS-associated syndromes and the importance of detecting somatic mosaicism for accurate genetic counselling.

Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

“…All ZRS variants tested in this study, including previously published human variants (Al-Qattan et al, 2012;Albuisson et al, 2011;Baas et al, 2017;Cai et al, 2019;Cho et al, 2013;Farooq et al, 2010;Furniss et al, 2008;Girisha et al, 2014;Gurnett et al, 2007;Heutink et al, 1994;Lettice et al, 2003;Lodder, 2009;Norbnop et al, 2014;Semerci et al, 2009;VanderMeer et al, 2012;Vanlerberghe et al, 2015;Wieczorek et al, 2010;Wu et al, 2016;Zguricas et al, 1999;Zhang et al, 2019), variants from animals (Dorshorst et al, 2010;Dunn et al, 2011;Knudsen and Kochhar, 1981;Lettice et al, 2008;Masuya et al, 2007;Zhao et al, 2009) and novel variants reported in this study. Table S1 contains the following columns: variant coordinate in GRCh38 genome assembly (1 st column), relative position within the ZRS enhancer (2 nd column), reference and variant alleles (3 rd column), variant name (4 th column), organism of origin (5 th column), VISTA ID of the tested construct containing the human ZRS enhancer with the variant (6 th column), variant classification based on the enSERT result (7 th column), reference (8-9 th columns), clinical phenotypes and human genetics data for variants from human patients (10-24 th columns), PhyloP scores (25 th column), predicted TF binding sites…”

Zone of Polarizing Activity Regulatory Sequence Mutations/Duplications with Preaxial Polydactyly and Longitudinal Preaxial Ray Deficiency in the Phenotype: A Review of Human Cases, Animal Models, and Insights Regarding the Pathogenesis

“…The ZRS 417 A>G mutation presented with a very unique phenotype of mirror-image polydactyly of the four extremities and bilateral tibial deficiency [ 20 ]. Interestingly, the same mutation in a mosaic form resulted in isolated triphalangeal thumb or thumb duplication with a triphalangeal component (PPD type II) [ 20 ].…”

“…Previous authors have emphasized that ZRS point mutations resulting in Werner syndrome (which has PPD-LPAD association in the phenotype) have “strong” ectopic anterior SHH expression [ 16 ]. Further evidence regarding this strong anterior expression comes from appearance of mirror-image duplication of the hands and feet in some patients with ZRS point mutations [ 20 ]. Experimentally, transplantation of few SHH expressing cells to the anterior limb bud results in PPD.…”

Variable expression of subclinical phenotypes instead of reduced penetrance in families with mild triphalangeal thumb phenotypes

“…However, it remains unclear why the disruption of SHH causes TPT in one family member and a subclinical phenotype in another. One example of how intrafamilial variability can be explained is based on a reported family, where different degrees of somatic mosaicism were associated with various phenotypes in affected family members 22. As the regulatory function of ZRS on SHH is extremely delicate and affected by timing, location and level of activity, it is plausible that the slightest alteration of one of these factors can cause this interindividual phenotypical variation.…”