Mutation in CUL4B, which encodes a member of cullin-RING ubiquitin ligase complex, causes X-linked mental retardation

Abstract

We reevaluated a previously reported family with an X-linked mental retardation syndrome and attempted to identify the underlying genetic defect. Screening of candidate genes in a 10-Mb region on Xq25 implicated CUL4B as the causative gene. CUL4B encodes a scaffold protein that organizes a cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. A base substitution, c.1564C-->T, converted a codon for arginine into a premature termination codon, p.R388X, and rendered the truncated peptide completely devoid of the C-terminal catalytic domain. The nonsense mutation also results in nonsense-mediated mRNA decay in patients. In peripheral leukocytes of obligate carriers, a strong selection against cells expressing the mutant allele results in an extremely skewed X-chromosome inactivation pattern. Our findings point to the functional significance of CUL4B in cognition and in other aspects of human development.

Figure 1.

Updated pedigree of a family with XLMR. V-1 was aged 1 year at the time of examination and was found to carry the mutation. IV-4 was proband (arrow). Blackened symbols represent affected individuals, and symbols with dots represent carriers.

Figure 2.

Mutation in CUL4B that causes XLMR. A, Minimal candidate region of 10.26 Mb between XSTR3 and XSTR4. The candidate genes screened are shown at the bottom. The location of CUL4B is highlighted. B, Partial sequence chromatograms of exon 9 of CUL4B in a patient and a healthy male from the family. The normal and altered nucleotides at 1564 are labeled with an asterisk (*). C, Genomic structure of CUL4B and schematic representation of CUL4B protein. The position of the introduced stop codon is indicated by a red arrow. The position of serine-rich and cullin domains are indicated. D, Confirmation of the c.1564C→T mutation by PCR-RFLP assay. All patients and carriers exhibited the 172-bp fragment representing the 1564T allele. Part of exon 9 of CUL4B was amplified from genomic DNA and was digested with BclI, to distinguish between the 1564C allele (absence of BclI site) and the 1564T allele (presence of BclI site). Sizes of the resulting restriction fragments are indicated. The primers used in this experiment were 5′-CCAATCATCATGCTTCTCAACT-3′ (forward) and 5′-CGGTTAGTTTCTTCCAATGATC-3′ (reverse).

Figure 3.

X-chromosome inactivation that was extremely skewed in leukocytes of the carriers with the CUL4B mutation. A, Methylation status of an HpaII site near the CAG repeat of AR was analyzed. U = undigested; D = digested with HpaII. Methylated DNA on the inactive X chromosome is resistant to HpaII digestion and thus can be PCR amplified with primers flanking the restriction site. DNA on the active X chromosome, as in patient III-16, is cleavable by HpaII and gives no PCR products. X-chromosome inactivation pattern was measured by densitometry as the ratio between the PCR products from two X chromosomes and was scored as “random” (ratios 50:50–80:20) or “skewed” (ratios >80:20–95:5). When only one band was visible, the pattern was scored as “extremely skewed” (ratio >95:5). Random X inactivation was observed in unaffected females III-6 (ratio 51:49), III-12 (ratio 52:48), and III-15 (59:41). X inactivation was extremely skewed toward one chromosome in CUL4B carriers III-2, III-4, III-8, and III-10, in which only one allele was visible. B, Chromatograms of the partial sequences from genomic DNA (gDNA) and leukocyte cDNA in the carriers. Although heterozygous status was observed in genomic DNA from the carriers (left panel), only the wild-type allele was detected in their leukocyte cDNA (right panel). The normal and altered nucleotides at 1564 are labeled with an asterisk (*).