Copy-number gains of HUWE1 due to replication- and recombination-based rearrangements

Abstract

We previously reported on nonrecurrent overlapping duplications at Xp11.22 in individuals with nonsyndromic intellectual disability (ID) harboring HSD17B10, HUWE1, and the microRNAs miR-98 and let-7f-2 in the smallest region of overlap. Here, we describe six additional individuals with nonsyndromic ID and overlapping microduplications that segregate in the families. High-resolution mapping of the 12 copy-number gains reduced the minimal duplicated region to the HUWE1 locus only. Consequently, increased mRNA levels were detected for HUWE1, but not HSD17B10. Marker and SNP analysis, together with identification of two de novo events, suggested a paternally derived intrachromosomal duplication event. In four independent families, we report on a polymorphic 70 kb recurrent copy-number gain, which harbors part of HUWE1 (exon 28 to 3' untranslated region), including miR-98 and let-7f-2. Our findings thus demonstrate that HUWE1 is the only remaining dosage-sensitive gene associated with the ID phenotype. Junction and in silico analysis of breakpoint regions demonstrated simple microhomology-mediated rearrangements suggestive of replication-based duplication events. Intriguingly, in a single family, the duplication was generated through nonallelic homologous recombination (NAHR) with the use of HUWE1-flanking imperfect low-copy repeats, which drive this infrequent NAHR event. The recurrent partial HUWE1 copy-number gain was also generated through NAHR, but here, the homologous sequences used were identified as TcMAR-Tigger DNA elements, a template that has not yet been reported for NAHR. In summary, we showed that an increased dosage of HUWE1 causes nonsyndromic ID and demonstrated that the Xp11.22 region is prone to recombination- and replication-based rearrangements.

Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Pedigrees of Families with Xp11.22 Copy-Number Gains (A) Pedigrees of six unreported families with a nonrecurrent microduplication at Xp11.22, including HUWE1. All tested individuals are marked with Nrl if the duplication was not present or Dup if the duplication was found in this individual. X-inactivation ratios in females are provided between brackets. Ni, not informative; dn, de novo event. (B) Pedigrees of the four families with the partial HUWE1 copy-number gain. Segregation of the aberrations was analyzed by qPCR in family members from whom DNA was available. Family members with weak cognitive levels are indicated in gray.

Figure 2

Overview of the 12 Nonrecurrent and 4 Recurrent Microduplications at Xp11.22 The locations and sizes of each nonrecurrent duplication, as defined by high-resolution oligo array and iterative qPCR mapping, are illustrated by horizontal blue bars. The sizes of each copy-number gain are indicated, as well as the sequence used as microhomology substrates for rearrangement, if defined. Where LCR was involved, the minimal size of the LCR is provided between brackets. The positions of the SNPs and markers that we analyzed are indicated at the top. The genes present within this region are shown at the bottom, in red. The shortest region of overlap is indicated in gray and harbors HUWE1 and the intronically located microRNAs miR-98 and let-7f-2. The recurrent polymorphic duplication identified in the four unrelated families is indicated by the yellow box shown above the genes. Positions are according to UCSC hg18. Nd, not determined due to insufficient DNA, repeat-rich, or nonreference breakpoint regions.

Figure 3

A Recurrent Partial HUWE1 Copy-Number Gain Is Identified in Four Unrelated Families Mapping of the recurrent copy-number gain was performed by iterative rounds of qPCR, for which each result is indicated as “−” for a normal copy number of 1.0 or “+” when the locus had a copy-number gain. The duplication starts at 53.57 Mb, which is upstream of HUWE1, and ends at 53.63 Mb within intron 28 of this gene. Analysis of both breakpoint regions by RepeatMasker identified the presence of 2.9 and 2.5 kb TcMAR-Tigger DNA elements (indicated as vertical-striped boxes) at the distal (TcMAR-Tigger-Dist) and proximal (TcMAR-Tigger-Prox) side, respectively. The locations of HSD17B10, part of HUWE1, and both microRNAs are at the bottom. Positions (in kb) are according to UCSC hg18.

Figure 4

HUWE1 but Not HSD17B10 mRNA Expression Is Increased in EX469 The microduplication in EX469 is the only one that does not include HSD17B10, thereby limiting the minimal duplicated region to HUWE1 only. The qPCR data are the mean of four independent experiments. Four controls were used in each experiment. SDs are provided.