Disruption of CTNND2, encoding delta-catenin, causes a penetrant attention deficit disorder and myopia

Abstract

Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable neurodevelopmental disorder with poorly understood pathophysiology and genetic mechanisms. A balanced chromosomal translocation interrupts CTNND2 in several members of a family with profound attentional deficit and myopia, and disruption of the gene was found in a separate unrelated individual with ADHD and myopia. CTNND2 encodes a brain-specific member of the adherens junction complex essential for postsynaptic and dendritic development, a site of potential pathophysiology in attentional disorders. Therefore, we propose that the severe and highly penetrant nature of the ADHD phenotype in affected individuals identifies CTNND2 as a potential gateway to ADHD pathophysiology similar to the DISC1 translocation in psychosis or AUTS2 in autism.

Figure 1

A family with highly penetrant attention deficit and childhood myopia (A) Pedigree and clinical manifestations of the individuals from family 1 with ADHD and myopia. Black, attention deficit; blue, autistic traits; red, childhood onset myopia. Karyotypes of tested individuals are indicated. For others, DNA was not available. Asterisk, translocation; filled triangle, translocation plus additional genetic findings, text; filled circle, normal karyotype. (B) Karyotypes of individual F1 II.2 and F1 III.3 demonstrating a translocation between chromosomes 5 and 6.

Figure 2

Analysis of copy number variation in the chromosome 5;6 translocation (A) Illumina Chip data demonstrating balanced sequence of chromosome 5 in the region that contains CTNND2 (chr5:109,000,000–119,000,000) in three F1 translocation carriers (II.2 in brown, III.2 in gray, and III.3 in blue). (B) 1.2 Mb duplication (chr6:126121880–127921880) in the more severely-affected III.2 (gray), which was not present in other translocation carriers (II.2 in brown and III.3 in blue). This region encompasses ECHDC1, RNF146, RSPO3, and CENPW genes.

Figure 3

Chromosome 5;6 translocation (A) Graphic representation of the results obtained from the quantitative analysis of derivative chromosomes by short-read and long-read sequencing, which demonstrated chromosome breakpoints on Chr5 and Chr6 and flanking genes (left). Final positions after rearrangements are shown on the right. (B) Breakpoint sequences were confirmed by Sanger sequencing of PCR products of the regions flanking the breakpoints in blood cells from F1 II.2. Connecting arrows from Der 5 to Der 6 indicate hg38 coordinates at breakpoint and final reattachment locations on both chromosomes, with novel sequences inserted at junctions. There is a 3 bp loss in CTNND2 intron 2 between derivative chromosome 5 and derivative chromosome 6. A loss of approximately 30 kb on chromosome 6 is in a non-genic region.

Figure 4

Genetic analysis of family 2 and summary of genetic findings (A–E) The presence of a CTNND2 exon 2 deletion was established by low-coverage whole genome sequencing, exome sequencing, and qPCR as described in Subjects and methods. (A) Pedigree and phenotypes of family 2. (B) F2 I.2: relative read count for all exome-sequenced CTNND2 exons. Alt. refers to alternate exons not found in the longest transcript. (C) F2 I.2: qPCR of exon 2 of CTNND2 and estimated gene dosage ratios. For F2 I.2, the autosomal AMT and X-linked DMD control targets were within the normal expected range, while two different targets within CTNND2 exon 2 were indicative of a heterozygous deletion (CTNND2-E2 and CTNND2-E2B). Pink, biallelic exon 6 of AMT gene; brown and blue, two probes for CTNND2 exon 2; green, CTNND2 intron 2; blue, DMD gene exon 13. Error bars are ± 1 SD from 3 replicates. (D) Analysis of depth of coverage ratios for F2 I.2 CTNND2 exons. Top panel: frequency distribution histogram of log-transformed coverage ratio values of all exons from CTNND2. Bars represent frequency of observations within bins of depth of coverage values on the horizontal axis. The red vertical line marks the position of the depth of coverage ratio value of CTNND2 exon 2 within that specific bin. Bottom panel: frequency distribution of probability values (p values) from all CTNND2 exons from the resampling analysis. The horizontal axis represents bins of p values of the CTNND2 exons getting a depth of coverage ratio less than or equal to the depth of coverage ratio of a particular exon from CTNND2. The green vertical line marks the p value of CTNND2 exon 2 within its specific bin. Exon 2 had a coverage ratio value of 0.54, consistent with a hemizygous deletion (p = 0.0415) and 3 standard deviations (SDs) below the expected value for this exon, while all other exons are within ± 1 SD. (E) Summary schematic of qPCR and next-generation sequencing (NGS) approaches showing loss of CTNND2 exon 2. Results from three methods are shown: quantitative PCR (qPCR), low-pass whole genome sequencing (LG), and exon-by-exon copy number variant calling (CNVexon). Concordance across all methods for a heterozygous deletion is demonstrated. The critical region confirmed by all three methods includes exon 2 of CTNND2. (F) Summary of CTNND2 genetic findings in individuals with ADHD, supporting a haploinsufficiency mechanism. Red, translocation breakpoint in family 1; blue line, deletion in family 2; dashed brown line, translocation breakpoint from Hofmeister et al.