Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability

Abstract

Copy number variants and indels in 251 families with evidence of X-linked intellectual disability (XLID) were investigated by array comparative genomic hybridization on a high-density oligonucleotide X chromosome array platform. We identified pathogenic copy number variants in 10% of families, with mutations ranging from 2 kb to 11 Mb in size. The challenge of assessing causality was facilitated by prior knowledge of XLID-associated genes and the ability to test for cosegregation of variants with disease through extended pedigrees. Fine-scale analysis of rare variants in XLID families leads us to propose four additional genes, PTCHD1, WDR13, FAAH2, and GSPT2, as candidates for XLID causation and the identification of further deletions and duplications affecting X chromosome genes but without apparent disease consequences. Breakpoints of pathogenic variants were characterized to provide insight into the underlying mutational mechanisms and indicated a predominance of mitotic rather than meiotic events. By effectively bridging the gap between karyotype-level investigations and X chromosome exon resequencing, this study informs discussion of alternative mutational mechanisms, such as noncoding variants and non-X-linked disease, which might explain the shortfall of mutation yield in the well-characterized International Genetics of Learning Disability (IGOLD) cohort, where currently disease remains unexplained in two-thirds of families.

Figure 1

Single-Nucleotide Variants Can Effect Probe Hybridization (A) Population distribution of mean log2 ratio for six probes overlying the ZDHHC9 IVS1+5G > C point mutation found in a single family. (B) Relative location of probes and point mutation. (C) Population distribution of mean log2 ratio for six probes overlying common SNP rs1329546. (D) Relative location of probes and SNP.

Figure 2

A Noncoding Deletion Eliminates CUL4B Expression (A) UCSC genome browser snapshot showing 6 kb deletion location relative to CUL4B RefSeq transcripts, repetitive elements, and regions with regulatory potential and vertebrate conservation. (B) Cosegregation of deletion and disease in family 506 could be tested in only a subsection of the extended X-linked pedigree because of limited sample availability. Arrow indicates individual analyzed by aCGH. (C) qPCR analysis indicates absence of CUL4B mRNA, but not control CUL4A transcript, in patient LCLs relative to a wild-type control. Expression analysis was performed as described in Kerzendorfer et al. with Applied Biosystems TaqMan gene expression assays Hs00757716_m1 (CUL4A) and Hs00186086_m1 (CUL4B).

Figure 3

Cosegregation of CNVs and Disease for Putative Novel XLID Genes Arrows indicate the individual subjected to aCGH analysis. Genotypes of all tested individuals are reported with alleles described as WT (wild-type), del (deletion), or dup (duplication). (A) Family 540, PTCHD1 deletion. (B) Family 494, FAAH2 intragenic deletion. (C) Family 206, WDR13 intragenic deletion. (D) Family 463, GSPT2 duplication.

Figure 4

Retroinsertion of LUZP4 (A) Increased log2 ratios were observed for LUZP4 exonic probes (highlighted by red arrows) but not for surrounding noncoding regions. Scatter plot shows individual probes, solid line shows six probe sliding average. (B) The retroinsertion (INS) is found in all three affected males and was inherited from their mothers. Both unaffected males did not have the retroinsertion (WT). The arrow indicates the individual analyzed by aCGH. Colored bars summarize nonpseudoautosomal X chromosome microsatellite and SNP linkage data and reveal no common region of haploidentity between the three affected males.