Mutations in EOGT confirm the genetic heterogeneity of autosomal-recessive Adams-Oliver syndrome

Abstract

Adams-Oliver syndrome (AOS) is a rare, autosomal-dominant or -recessive disorder characterized primarily by aplasia cutis congenita and terminal transverse limb defects. Recently, we demonstrated that homozygous mutations in DOCK6 cause an autosomal-recessive form of AOS. In this study, we sought to determine the contribution of DOCK6 mutations to the etiology of AOS in several consanguineous families. In two of the five families studied, we identified two homozygous truncating mutations (a splice-site mutation and a frameshift duplication). DOCK6 sequencing revealed no mutation in the remaining three families, consistent with their autozygosity mapping and linkage-analysis results, which revealed a single candidate locus in 3p14.1 on three different haplotype backgrounds in the three families. Indeed, exome sequencing in one family revealed one missense mutation in EOGT (C3orf64), and subsequent targeted sequencing of this gene revealed a homozygous missense mutation and a homozygous frameshift deletion mutation in the other two families. EOGT encodes EGF-domain-specific O-linked N-acetylglucosamine (O-GlcNAc) transferase, which is involved in the O-GlcNAcylation (attachment of O-GlcNAc to serine and threonine residues) of a subset of extracellular EGF-domain-containing proteins. It has a documented role in epithelial-cell-matrix interactions in Drosophila, in which deficiency of its ortholog causes wing blistering. Our findings highlight a developmental role of O-GlcNAcylation in humans and expand the genetic heterogeneity of autosomal-recessive AOS.

Figure 1

Identification of Five Consanguineous AOS-Affected Families The index individual is indicated with an arrow in each pedigree, and asterisks denote individuals whose DNA was available for analysis. Please note that the degree of consanguinity between III:1 and III:2 in AOS_F4 is uncertain. (A–E) Representative clinical images of the index individuals from families AOS_F2, AOS_F3, and AOS_F5. Clinical photographs of AOS_F2 IV:1 show the hypoplastic terminal phalanges (A and B) and periventricular calcifications, thick cortex, and posterior pachygyria (C). Clinical photographs show the cutis aplasia in AOS_F3 IV:4 (D) and AOS_F5 V:1 (E).

Figure 2

Identification of an AOS-Associated Locus on Chromosome 3 (A) AutoSNPa output for chromosome 3 reveals an ROH (boxed in red) exclusively shared among AOS_F3 IV:4, AOS_F4 IV:2, and AOS_F5 V:1, IV:4, and IV:5. (B) Combined genome-wide linkage analysis of the three families revealed a single maximal peak with a LOD score of ∼3.7 on chromosome 3. (C) Upper panel: diagram of EOGT (red color denotes coding exons, yellow denotes UTRs, and triangles denote mutation sites). Lower panel: sequence chromatograms of the three mutations in EOGT (control tracing is shown for comparison) show the two missense variants in AOS_F3 IV:4 and AOS_F5 V:1 (mutation sites are denoted by asterisks, and the 1 bp deletion in AOS_F4 IV:2 is denoted by a red line). Please note that the NCBI contains only an alternatively spliced version (RefSeq NM_173654.1), according to which the nomenclature of the three mutations will be as follows: c.620G>C (p.Trp207Ser) (unchanged), c.832–791delA (instead of c.1074delA [p.Gly359Aspfs^∗28]), and c.878G>A (p.Arg293Gln) (instead of c.1130G>A [p.Arg377Gln]).

Figure 3

Whole-Mount In Situ Hybridization of Eogt during Mouse Embryonic Development (A) An E10.5 mouse embryo shows expression in the growing edge of the limb bud. (B) An E11.5 mouse embryo shows expression in the apical ectodermal ridge of the limbs. (C) An E12.5 embryo shows the digit-condensation expression of Eogt mRNA (triangles) in the limbs. (C1 and C2) Close-up views of the expression in the digits of the hindlimbs (C1) and forelimbs (C2) of the same E12.5 embryo as in (C). (D) Sense control is shown for comparison in an E12.5 embryo. Eogt probes correspond to the area spanning nucleotides 91–816 and 830–1,537 of the coding region (RefSeq NM_175313.4).