Identification of a DLG3 stop mutation in the MRX20 family

Abstract

Here, we identified the causal mutation in the MRX20 family, one of the larger X-linked pedigrees that have been described in which no gene had been identified up till now. In 1995, the putative disease gene had been mapped to the pericentromeric region on the X chromosome, but no follow-up studies were performed. Here, whole exome sequencing (WES) on two affected and one unaffected family member revealed the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM_021120.4) that segregated with the affected individuals in the family. DLG3 mutations have been consequently associated with intellectual disability and are a plausible explanation for the clinical abnormalities observed in this family. In addition, we identified two other variants co-segregating with the phenotype: a stop gain mutation in SSX1 (c.358G>T/p.(Glu120Ter)) (NM_001278691.2) and a nonsynonymous SNV in USP27X (c.56 A>G/p.(Gln19Arg)) (NM_001145073.3). RNA sequencing revealed 14 differentially expressed genes (p value < 0.1) in 7 affected males compared to 4 unaffected males of the family, including four genes known to be associated with neurological disorders. Thus, in this paper we identified the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM_021120.4) as likely responsible for the phenotype observed in the MRX20 family.

Fig. 1. Pedigree of the MRX20 family.

Pedigree is shown as in original publication [6]. Segregation of variants in DLG3 (c.195del/p.(Thr66ProfsTer55)) (NM_021120.4), SSX1 (c.358G>T/p.(Glu120Ter)) (NM_001278691.2) and USP27X (c.56A>G/p.(Gln19Arg)) (NM_001145073.3) are shown for all family members of which DNA was available as well as X-inactivation patterns of seven women of the family. The ratio of X chromosome inactivation for the AR and RP2 alleles were interpreted as follows: <80:20 = random (R); 80:20 to 90:10 = moderately skewed (MS); >90:10 = highly skewed (HS). NI Non-informative.

Fig. 2. DNA sequencing chromatogram of the variants found in DLG3, SSX1, and USP27X in the MRX20 family.

The reverse complement sequence is shown. An example of the wild-type allele, an alternative allele, which refers to a mutant allele for DLG3, but to a non-reference allele for SSX1 and USP27X, and a heterozygous combination of both alleles in a carrier female are represented. The chromosomal positions (GRCh38) of these variants are: NC_000023.11:g.70445396del, NC_000023.11:g.48263809G>T and NC_000023.11:g.49880363A>G, respectively.

Fig. 3. Differential expression and enriched pathway analysis.

A Differential expression analysis of the MRX20 family. Hierarchical clustering heatmap showing the differential expressed genes; all genes with an adjusted p value < 0.1 are represented. * Represents the differentially expressed genes with an adjusted p value < 0.05. B Enriched pathways based on the differential expression data. Bar chart shows enrichment ratio or NES of results with direction. Enriched pathways with a False Discovery Rate (FDR) ≤ 0.05 are shown in a darker shade. Analysis was performed using WebGestalt [14]. C Validations of differential expression of genes CDCA4, WWTR1, PEX26, LDHA, and NMT2 using RT-PCR normalized to ACTB, UBC, and YWHAZ in the MRX20 family. Statistical significance was obtained by a two-tailed Mann-Whitney U test. * represents a p value < 0.05 and ** represents a p value < 0.01.

Fig. 4. Representation of the synapse-associate protein 102 (SAP102) encoded by the Discs Large MAGUK Scaffold Protein 3 (DLG3) gene.

The protein contains three tandem PDZ domains, an scr homology (SH3) domain and a guanylate kinase (GK) domain. Known protein coding mutations are indicated by arrows (NM_021120.4/ NP_066943.2) (Supplementary Table 5 gives a representation of all published mutations in the DLG3 gene).