Small rare recurrent deletions and reciprocal duplications in 2q21.1, including brain-specific ARHGEF4 and GPR148

Abstract

We have identified a rare small (~450 kb unique sequence) recurrent deletion in a previously linked attention-deficit hyperactivity disorder (ADHD) locus at 2q21.1 in five unrelated families with developmental delay (DD)/intellectual disability (ID), ADHD, epilepsy and other neurobehavioral abnormalities from 17 035 samples referred for clinical chromosomal microarray analysis. Additionally, a DECIPHER (http://decipher.sanger.ac.uk) patient 2311 was found to have the same deletion and presented with aggressive behavior. The deletion was not found in either six control groups consisting of 13 999 healthy individuals or in the DGV database. We have also identified reciprocal duplications in five unrelated families with autism, developmental delay (DD), seizures and ADHD. This genomic region is flanked by large, complex low-copy repeats (LCRs) with directly oriented subunits of ~109 kb in size that have 97.7% DNA sequence identity. We sequenced the deletion breakpoints within the directly oriented paralogous subunits of the flanking LCR clusters, demonstrating non-allelic homologous recombination as a mechanism of formation. The rearranged segment harbors five genes: GPR148, FAM123C, ARHGEF4, FAM168B and PLEKHB2. Expression of ARHGEF4 (Rho guanine nucleotide exchange factor 4) is restricted to the brain and may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function. GPR148 encodes a G-protein-coupled receptor protein expressed in the brain and testes. We suggest that small rare recurrent deletion of 2q21.1 is pathogenic for DD/ID, ADHD, epilepsy and other neurobehavioral abnormalities and, because of its small size, low frequency and more severe phenotype might have been missed in other previous genome-wide screening studies using single-nucleotide polymorphism analyses.

Figure 1.

Schematic representation of the genomic architecture in 2q21.1. Genomic coordinates correspond to the hg18 build of the human genome. Red bars represent deletions and blue bars represent reciprocal duplications. The NAHR sites identified in patient 2 mapped to directly oriented ∼109 kb subunits, shown as green horizontal arrows.

Figure 2.

Chromosomal microarray, FISH, MLPA and PCR results. (A) Genome-wide and (B) 2q21.1 aCGH plots showing the identified deletion in patient 2. Probes are arranged on the x-axis according to physical mapping positions, with the most proximal 2q21.1 probes to the left and the most distal 2q21.1 probes to the right. Values along the y-axis represent log2 ratios of patient: control intensities. (C) Partial metaphase chromosomes after FISH with a 2q21.1-specific BAC clone RP11-675I14 (red) and a control chromosome 2 alpha satellite centromere probe (green), showing the absence of red signal on the deleted chromosome 2 in patient 2. (D) Agarose electrophoresis gel showing patient 2-specific junction fragment (∼ 10 kb) and absence of patient-specific junction fragment in control DNA. MLPA analysis plots confirming the 2q21.1 deletion in patients (E) 1 and (F) 2, and the reciprocal duplication in patients (G) 8 and (H) 10.

Figure 3.

DNA sequence homology between the proximal LCR2q21.1 (chr2:130,394,886–131,193,978) and distal LCR2q21.1q21.2 (chr2:131,646,166–132,838,104) clusters for paralogous subunits larger than 1 kb in size (hg18). (Top) UCSC segdup track representing the proximal LCR2q21.1 cluster. (Middle) Results of Miropeats program analysis between the proximal LCR2q21.1 and distal LCR2q21.1q21.2 clusters. (Bottom) UCSC seg dup track representing the distal LCR2q21.1q21.2 cluster.

Figure 4.

Genomic structure of the ∼109 kb LCR2q21.1 subunits of 97.7% DNA sequence identity, in which NAHR sites were mapped in patient 2. (A) The NAHR site regions in patient 2 were narrowed to a 181 bp interval by sequencing the long-range PCR products obtained with the forward primer F specific to the proximal subunit and the reverse primer R, specific to the distal subunit (arrows depicting primers are not shown to scale). (B) Schematic representation of the GC content of the ∼109 kb LCR2q21.1 subunits with NAHR sites mapping to over 60% GC-rich fragment.