HUWE1 mutations in Juberg-Marsidi and Brooks syndromes: the results of an X-chromosome exome sequencing study

Abstract

Background: X linked intellectual disability (XLID) syndromes account for a substantial number of males with ID. Much progress has been made in identifying the genetic cause in many of the syndromes described 20-40 years ago. Next generation sequencing (NGS) has contributed to the rapid discovery of XLID genes and identifying novel mutations in known XLID genes for many of these syndromes.

Methods: 2 NGS approaches were employed to identify mutations in X linked genes in families with XLID disorders. 1 involved exome sequencing of genes on the X chromosome using the Agilent SureSelect Human X Chromosome Kit. The second approach was to conduct targeted NGS sequencing of 90 known XLID genes.

Results: We identified the same mutation, a c.12928 G>C transversion in the HUWE1 gene, which gives rise to a p.G4310R missense mutation in 2 XLID disorders: Juberg-Marsidi syndrome (JMS) and Brooks syndrome. Although the original families with these disorders were considered separate entities, they indeed overlap clinically. A third family was also found to have a novel HUWE1 mutation.

Conclusions: As we identified a HUWE1 mutation in an affected male from the original family reported by Juberg and Marsidi, it is evident the syndrome does not result from a mutation in ATRX as reported in the literature. Additionally, our data indicate that JMS and Brooks syndromes are allelic having the same HUWE1 mutation.

Figure 1

Pedigrees of families with HUWE1 mutations. (A) Pedigree of K9149. Photographs show microcephaly, cupped ears and thin upper lip in infant (II-6); and 27-year-old nephew (III-2) from original family reported by Juberg and Marsidi. (B) Pedigree of family K9223. Photographs show microcephaly, cupped ears, blepharophimosis, deeply set eyes, depressed nasal bridge and bulbous nasal tip in affected male (III-1) from original family reported by Brooks et al at age 4 months and 4 years. (C) Pedigree of family 3. The affected boys' mother (I-2) and one sister (II-2) have mild learning disabilities. Photographs show microcephaly, frontal hair upsweep, deep-set eyes, broad nasal tip, wide mouth, thin upper lip and prominent jaw in brothers, ages 10 (II-5) and 13 years (II-4). The older brother also has cupped ears, thick eyebrows and coarse face. +, presence of mutation; −, normal; , proband; , affected; , carrier.

Figure 2

E3 ubiquitin ligase HUWE1 protein level is decreased in the affected male, III-2, from family K9149. (A) Immunoblot analysis of the HUWE1 protein level a lymphoblastoid cell line from the affected male (figure 1A, III-2) and in a normal male in the family (figure 1A, IV-1). The star indicates unspecific bands. (B) Quantification of three independent experiments as the one presented in (A); error bars indicate mean±SD; **p<0.01.

Figure 3

Protein levels of well-known HUWE1 substrates are upregulated in the affected male III-2, from family K9149. Immunoblot analysis of Mcl-1 (A) and p53 (C) protein levels in a normal male in the family (figure 1A, IV-1) and a lymphoblastoid cell line from the affected male (family 1A, III-2). (B) and (D) Quantification of three independent experiments as the ones presented in (A) and (C), respectively; error bars indicate mean±SD; *p<0.05.

Figure 4

Functional assessment of the p.R4063Q HUWE1 mutation in family 3. HUWE1 protein levels are increased in p.R4063Q lymphoblastoid cell lines (LCLs) derived from the affected brothers and results in moderate reduction in the levels of the p53 protein. Unaffected father I-1 (lane 1), affected sons, II-3 and II-4 (lanes 2 and 3). Total LCL protein lysates were western blotted and probed for HUWE1, p53 and B-tubulin (loading control).

Figure 5

Domains and missense mutations found in HUWE1 with the relative boundaries of each domain indicated. The known missense alterations are shown below the DUF4414 and HECT domains. The p.G4310R mutation reported in this paper is in red. The WWE domain is named for its conserved residues and is predicted to mediate specific protein–protein interactions in ubiquitin and ADP-ribose conjugation systems. The C-terminal catalytic HECT belongs to a subclass of ubiquitin-protein E3 ligases. DUF913 belongs to a family found in various ubiquitin protein ligases and DUF4414 is in a family of domains frequently found in DNA-binding proteins of the URE-B1 type. DUF, domains of unknown function.