DLX4is associated with orofacial clefting and abnormal jaw development

Abstract

Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son—c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.

MOZ directs the distal-less homeobox gene expression program during craniofacial development

“…The DLX transcription factors are crucial for craniofacial development (Acampora et al, 1999;Depew et al, 1999Depew et al, , 2005Jeong et al, 2012Jeong et al, , 2008Qiu et al, 1997Qiu et al, , 1995. One-third of individuals with copy number variation at the DLX5/DLX6 locus present with mild to severe palatal abnormalities, such as high palate or cleft palate and cleft lip (Elliott and Evans, 2006), and a DLX4 mutation has been described in a child with bilateral cleft lip and palate (Wu et al, 2015). Single homozygous mutant mice for Dlx1, Dlx2 and Dlx5 display cleft palate with varying penetrance (Acampora et al, 1999;Depew et al, 1999Depew et al, , 2005Han et al, 2009;Qiu et al, 1997Qiu et al, , 1995.…”

A novel PTCH1 mutation underlies nonsyndromic cleft lip and/or palate in a Han Chinese family

“…Moreover, GWAS identifies only the association between the risk locus and the disease, rather than the actual causal relationship, making it difficult to interpret the underlying mechanisms of phenotype occurrence. In recent years, next‐generation sequencing, including whole‐genome and WES, has been used to determine causal variation and interpret the aetiologic mechanisms of the NSCL/P hereditary pedigree (Tian et al., ; Wu et al., ). These techniques complement GWASs and together cover the spectrum of NSCL/P mutations.…”

“…Therefore, it is difficult to determine the association between NSCL/P and those rare variants with lower allele frequencies (Vieira, 2008 (Tian et al, 2017;Wu et al, 2015). These techniques complement GWASs and together cover the spectrum of NSCL/P mutations.…”

Zebrafish models of orofacial clefts

“…Moreover, a breakpoint within a regulatory element distal to DLX5 and DLX6 was identified in a multi‐generational family presenting with deafness and craniofacial defects, including micrognathia and cleft palate (Brown et al, ) (OMIM #616788). A novel truncating variant (p.Gln183Argfs*57) of DLX4 was also identified in a mother–son pair that presented with enlarged palpebral fissure, lagophthalmos and bilateral CL/P (Wu et al, ). Together, these findings support a role for DLX family members in the etiology of CL/P.…”