Functional Properties of Rare Missense Variants of Human <i>CDH13</i> Found in Adult Attention Deficit/Hyperactivity Disorder (ADHD) Patients

<div><p>The <i>CDH13</i> gene codes for T-cadherin, a GPI-anchored protein with cell adhesion properties that is highly expressed in the brain and cardiovascular system. Previous studies have suggested that <i>CDH13</i> may be a promising candidate gene for Attention Deficit/Hyperactivity Disorder (ADHD). The aims of this study were to identify, functionally characterize, and estimate the frequency of coding <i>CDH13</i> variants in adult ADHD patients and controls. We performed sequencing of the <i>CDH13</i> gene in 169 Norwegian adult ADHD patients and 63 controls and genotyping of the identified variants in 641 patients and 668 controls. Native and green fluorescent protein tagged wild type and variant CDH13 proteins were expressed and studied in CHO and HEK293 cells, respectively. Sequencing identified seven rare missense <i>CDH13</i> variants, one of which was novel. By genotyping, we found a cumulative frequency of these rare variants of 2.9% in controls and 3.2% in ADHD patients, implying that much larger samples are needed to obtain adequate power to study the genetic association between ADHD and rare CDH13 variants. Protein expression and localization studies in CHO cells and HEK293 cells showed that the wild type and mutant proteins were processed according to the canonical processing of GPI-anchored proteins. Although some of the mutations were predicted to severely affect protein secondary structure and stability, no significant differences were observed between the expression levels and distribution of the wild type and mutant proteins in either HEK293 or CHO cells. This is the first study where the frequency of coding <i>CDH13</i> variants in patients and controls is reported and also where the functional properties of these variants are examined. Further investigations are needed to conclude whether <i>CDH13</i> is involved in the pathogenesis of ADHD or other conditions.</p></div

<i>In silico</i> analysis of the effect of CDH13 variants.

<p>The analysis was based on the protein sequence. SIFT scores below 0.05 were considered damaging. I-mutant-3.0 predicted the effects of the variants on protein stability by calculating the unfolding Gibbs free energy value of the mutant proteins minus that of the wild type protein (ΔΔG = ΔG mutant – ΔG wild type), given in kcal/mol. A negative change indicates decreased stability. The reliability index (RI) for a large decrease (ΔΔG<−0.5) ranged from 0–10. For the G113R* a reliability index for a neutral stability change was given. Ternary classification (SVM3) −0.5< = ΔΔG< = 0.5 corresponds to neutral stability, ΔΔG<−0.5 to large decrease of stability and ΔΔG >0.5 to a large increase of stability.</p

Schematic description of the CDH13 proteins expressed in CHO and HEK293 cells.

<p>CDH13 was expressed with or without a C-terminal tGFP tag in HEK293 and CHO cells, respectively. The location of the identified variants is also shown (A). According to the general model of processing of GPI anchored proteins in the ER, a C-terminal transmembrane domain is cleaved off and is then replaced by a GPI anchor. The protein with the attached GPI anchor is then directed to the external side of the plasma membrane (B). Wild type and variant CDH13 proteins were expressed on the cell membrane in HEK293 and CHO cells. In HEK293 cells, the C-terminal GFP tag of the GFP-CDH13 fusion proteins was cleaved off as a result of GPI anchoring at the c- terminal of CDH13 and the fully processed protein was subsequently transferred to the cell membrane.</p

Frequency of <i>CDH13</i> variant alleles identified in Norwegian adult patient and control groups.

<p>Seven <i>CDH13</i> variants were identified in the sequencing study, three of which were only detected in patients. All variants were genotyped in a larger sample. Two-tailed P-values for genotype frequencies were calculated by Fisher’s exact test in a 2×2 contingency table.</p

CDH13 stained CHO cells expressing wild type and variant CDH13 on the plasma membrane.

<p>Cells were permeabilised before staining. A) Mock transfected cells, B) wild type CDH13, C) A376T, D) G113R, E) I585V, F) L643R, G) N39S, H) R174W, I) V112I. Wild type and variant CDH13 proteins were expressed on the cell membrane. Mock transfected cells did not express CDH13.</p

Genetic and molecular findings.

<p>A: coverage analysis of whole-genome sequencing data in patient 3 shows a clear regional drop in sequencing depth (red line) corresponding to the 45,049 bp deletion in the 5’-UTR of the <i>SLC19A3</i> gene. The two arrows mark the position of the break-points. Coverage in the same region of a control is shown in the bottom lane for comparison. The blue diagram depicts the SLC19A3 gene in a 3’-5’ alignment. Vertical rectangulars show the proportional position of the exons. B: Western blot analysis in frontal cortex homogenate of patient 1 using two primary antibodies (left and right panel) shows an increase of SLC19A3 protein immunoreactivity in the patient (P) compared to two age and gender matched controls (C1, C2).</p

Neuroimaging findings.

<p>A: axial T2 weighted MRI of patient 2 taken during an episode with seizures, increasing encephalopathy and exacerbation of the dystonia. Images show bilateral high T2-signal changes and swelling in the putamen and caudate head. In addition there are multiple cortical lesions preferentially occurring in the depths of sulci, which is typical of BBGD. B: axial T2 and coronal T2-FLAIR weighted MRI of patient 1 taken during an episodic exacerbation show a similar pattern with bilateral striatal and multiple cortical lesions. The signal abnormalities regressed completely on later scans (not shown). C (upper lane): FDG-PET scan of the brain of patient 1 taken ~5 years later shows multiple foci of decreased glucose metabolism that correlate to the localization of the transient cortical signal changes on MRI. The striatum shows minimal uptake consistent with severe neuronal loss. A normal scan is shown in the lower lane for comparison.</p

Partial pedigrees of the nine MODY families investigated.

<p>Squares represent male family members, circles female, and diamonds sex unknown. Numbers inside diamonds show the number of siblings. Black and grey symbols represent persons with diabetes and impaired glucose tolerance, respectively. An arrow denotes the proband in each family and stars indicate those subjects for whom DNA was available. Data under the symbols represent from top to bottom: age at diabetes diagnosis, mutation carrier status (N = Normal allele, M = Mutation), BMI and treatment (INS =  Insulin, OHA = Oral Hypoglycemic Agents).</p

Rare coding variants identified in the 111 target candidate genes using whole exome sequencing in nine patients with suspected MODY.

a<p>SIFT: − tolerated, + not tolerated/PolyPhen-2: − benign, + possibly damaging, ++ probably damaging/Align-GVGD: the Grantham variation (GV), and the Grantham deviation (GD) are combined to provide graded classifiers from most likely to interfere with function (class C65) to least likely (class C0).</p>b<p>Allele frequencies from the interim analysis of phase I of the 1000 Genomes Project, 2010.08.04 sequence index, which included 629 samples (SNPs released in November 2010, indels released in February 2011).</p><p>Abbreviation: Chr, chromosome number; 1000 G, the 1000 Genomes Project; n.a, not analysed due to insufficient number of alignments to make prediction.</p

Overview of all substitutions and indels detected in the nine probands before and after variant reduction.

<p>Overview of all substitutions and indels detected in the nine probands before and after variant reduction.</p