Fig. 1

Fig. 1 Workflow of the CNV study and additional individuals/families with ZFHX4 variants.

A Workflow of the CNV study. CNVs were called from an existing dataset including exome sequencing data for 50 nsCL/P trios (Ishorst et al. 2022). CNV filtering was performed as follows. (I) The CNV frequency threshold was set to ≤10% among all 50 nsCL/P trios. (II) De novo CNV calling was performed using PLINK/Seq. (III) Only CNVs that were extracted using either CoNIFER and/or EXCAVATOR2 in addition to XHMM were retained in the analysis. CNVs that (IV) did not span RefSeq Genes, (V) overlapped by >50% with regions of segmental duplication or >80% with genes in difficult to analyze regions (Segmental Dups track, UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly), and (VI) had population frequencies ≥0.01 were excluded. B Pedigree of index trio with heterozygous de novo CNV in ZFHX4. C Exon and protein domain structure of human ZFHX4. Exons are colored in alternating white and black, and positions of the start codon (ATG) and stop codon (TAG) are marked. ZFHX4 is a transcription factor with four homeodomains (orange) and 23 zinc finger domains (blue). Protein domains are positioned in proportion to the corresponding exons. Red bar, heterozygous ZFHX4 de novo deletion in the index patient with nsCLP. P1-P3, position of primer pairs spanning the CNV for qPCR verification; black arrows, positions of additional ZFHX4 variants including loss-of-function variants from targeted-sequencing and a homozygous missense variant from a collaboration with Girisha & colleagues. D Pedigrees of four additional families (targeted-sequencing study and collaboration with Girisha & colleagues). E Tyr653 is one out of seven conserved residues (drawn bold) in the fold of a canonical zinc-finger motif. F Model of the zinc-finger structure in ZFHX4 proposes the formation of a hydrogen bond between Tyr653 and His662. Mutation of Tyr653 to histidine could change this conformational arrangement of side chains and thus impair zinc-binding. CNV copy number variation, nsCL/P nonsyndromic cleft lip with/without cleft palate, qPCR quantitative polymerase chain reaction, ES exome sequencing.