Exome sequencing and CRISPR/Cas genome editing identify mutations of <i>ZAK</i> as a cause of limb defects in humans and mice

Spielmann, Malte; Kakar, Naseebullah; Tayebi, Naeimeh; Leettola, Catherine; Nürnberg, Gudrun; Sowada, Nadine; Lupiáñez, Darío G.; Harabula, Izabela; Flöttmann, Ricarda; Horn, Denise; Chan, Wing Lee; Wittler, Lars; Yılmaz, Rüstem; Altmüller, Janine; Thiele, Holger; Bokhoven, Hans van; Schwartz, Charles E.; Nürnberg, Peter; Bowie, James U.; Ahmad, Jamil; Kubisch, Christian; Mundlos, Stefan; Borck, Guntram

doi:10.1101/gr.199430.115

Cited by 54 publications

(51 citation statements)

References 45 publications

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“…The differences between human and mouse phenotypes are most likely a result of species differences, as well as enhancer redundancy in mice, which has been described in several recent studies. 3,43 Second, the CNV detection rate of 10% in patients with congenital limb malformation reported here might be slightly overestimated since our cohort is partially biased by the initial clinical selection. In this study, array CGH was used as a first screening test for all samples, but some samples were sent to us by collaborating laboratories only after candidate gene testing was performed and yielded no result.…”

Section: Discussionmentioning

confidence: 85%

Noncoding copy-number variations are associated with congenital limb malformation

Flöttmann

Kragesteen

Geuer

et al. 2018

Genetics in Medicine

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PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

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Section: Discussionmentioning

confidence: 85%

Noncoding copy-number variations are associated with congenital limb malformation

Flöttmann

Kragesteen

Geuer

et al. 2018

Genetics in Medicine

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“…ZAK is a direct target of Tp63, and the deletion of its SAM domain has been shown to be associated with downregulation of Tp63 in the developing limb bud (Figure 2). 42 The absence of split hands but the presence split feet in patients with ZAK mutations supports the control of ZAK by TP63 genes because the hindlimbs of the Tp63 KO mice are more severely affected than the forelimbs. 17,18 In addition, having split feet without split hands suggest the association of ZAK, PITX1, and TBX4 genes in the formation of feet.…”

Section: Zak and Shfmmentioning

confidence: 86%

Genetic regulatory pathways of split‐hand/foot malformation

Kantaputra

Carlson

2018

Clinical Genetics

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Split-hand/foot malformation (SHFM) is caused by mutations in TP63, DLX5, DLX6, FGF8, FGFR1, WNT10B, and BHLHA9. The clinical features of SHFM caused by mutations of these genes are not distinguishable. This implies that in normal situations these SHFM-associated genes share an underlying regulatory pathway that is involved in the development of the central parts of the hands and feet. The mutations in SHFM-related genes lead to dysregulation of Fgf8 in the central portion of the apical ectodermal ridge (AER) and subsequently lead to misexpression of a number of downstream target genes, failure of stratification of the AER, and thus SHFM. Syndactyly of the remaining digits is most likely the effects of dysregulation of Fgf-Bmp-Msx signaling on apoptotic cell death. Loss of digit identity in SHFM is hypothesized to be the effects of misexpression of HOX genes, abnormal SHH gradient, or the loss of balance between GLI3A and GLI3R. Disruption of canonical and non-canonical Wnt signaling is involved in the pathogenesis of SHFM. Whatever the causative genes of SHFM are, the mutations seem to lead to dysregulation of Fgf8 in AER cells of the central parts of the hands and feet and disruption of Wnt-Bmp-Fgf signaling pathways in AER.

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“…The gene of interest in this study, ZAK, has been shown, in two consanguineous Pakistani pedigrees and in confirmatory animal models, to be relevant to SHFM with associated hearing loss. 7 The 38 patients selected for study in this cohort were screened for phenotypic match to patients with the reported pathogenic variant p.Phe368Cys in ZAK. All 38 patients in this cohort manifested the SHFM phenotype and had variable degrees of sensorineural hearing loss.…”

Section: Discussionmentioning

confidence: 99%

“…The authors generated a ZAK -/knockout mouse model which caused all mice to die during embryonic development. 7 Deletion of the SAM domain (which contains p.Phe368Cys) by CRISPR/Cas caused a hindlimb defect that was similar to the defect observed in mice with mutant Trp63, a known SHFM gene. 7 These studies demonstrate the relevance of the SAM domain within ZAK to SHFM.…”

Section: Introductionmentioning

confidence: 85%

Rare missense variant p.Ala505Ser in the ZAK protein observed in a patient with split-hand/foot malformation from a non-consanguineous pedigree

et al. 2020

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Objective: Split-hand/foot malformation (SHFM) is a rare, often debilitating, congenital limb malformation. A single nucleotide polymorphism within the leucine zipper containing kinase AZK (ZAK) gene was recently associated with SHFM in two consanguineous Pakistani pedigrees. We hypothesized that additional unrelated patients with the phenotype may carry a pathogenic mutation in ZAK. Methods: DNA samples were collected from 38 patients with SHFM and associated hearing loss for Sanger DNA sequencing and in silico analysis. Results: Two missense mutations within ZAK were detected in 11 patients, but only one missense variant, p.Ala505Ser, occurred with a presumed rare allele frequency. In silico modeling of the ZAK protein with the p.Ala505Ser substitution indicated a negative binding free energy change (mean

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Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

Cited by 54 publications

References 45 publications

Noncoding copy-number variations are associated with congenital limb malformation

Noncoding copy-number variations are associated with congenital limb malformation

Genetic regulatory pathways of split‐hand/foot malformation

Rare missense variant p.Ala505Ser in the ZAK protein observed in a patient with split-hand/foot malformation from a non-consanguineous pedigree

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