Reduced kinase function in two ultra-rare TNNI3K variants in families with congenital junctional ectopic tachycardia.

Genetic missense variants in TNNI3K, encoding troponin-I interacting kinase, have been associated with dilated cardiomyopathy (DCM) and observed in families with supraventricular tachycardias (SVT). Previously, a family harboring the TNNI3K-c.1615A > G (p.Thr539Ala) variant presented with congenital junctional ectopic tachycardia (CJET), an arrhythmia that arises from the atrioventricular (AV) node and His bundle. However, this was a relatively small four-generational family with limited genetic testing (N = 3). We here describe a multigenerational family with CJET harboring a novel ultra-rare TNNI3K variant: TNNI3K-c.1729C > T (p.Leu577Phe). Of all 18 variant carriers, 13 individuals presented with CJET, resulting in a genetic penetrance of 72%. In addition, CJET is reported in another small family harboring TNNI3K-c.2225C > T (p.Pro742Leu). Similar to the previously published CJET family, both TNNI3K variants demonstrate a substantial reduction of kinase activity. Our study contributes novel evidence supporting the involvement of TNNI3K genetic variants as significant contributors to CJET, shedding light on potential mechanisms underlying this cardiac arrhythmia.

A novel leaky splice variant in centromere protein J (CENPJ)-associated Seckel syndrome.

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823T>C) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.

PPA2-associated sudden cardiac death: extending the clinical and allelic spectrum in 20 new families.

PURPOSE: Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants. METHODS: Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized. RESULTS: Among the 34 individuals, only 6 remain alive. Twenty-three died before the age of 2 years while five died between 14 and 16 years. Within these 28 cases, 15 died of sudden cardiac arrest and 13 of acute heart failure. One case was diagnosed prenatally with cardiomyopathy. Four teenagers drank alcohol before sudden cardiac arrest. Progressive neurological signs were observed in 2/6 surviving individuals. For 11 variants, recombinant PPA2 enzyme activities were significantly decreased and sensitive to temperature, compared to wild-type PPA2 enzyme activity. CONCLUSION: We expand the clinical and mutational spectrum associated with PPA2 dysfunction. Heart failure and sudden cardiac arrest occur at various ages with inter- and intrafamilial phenotypic variability, and presentation can include progressive neurological disease. Alcohol intake can trigger cardiac arrest and should be strictly avoided.

An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis.

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results: We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion: Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target.

Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment.

There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.

Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin 2, cause nonsyndromic autosomal-recessive intellectual disability.

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

Mutations in the genes for thyroglobulin and thyroid peroxidase cause thyroid dyshormonogenesis and autosomal-recessive intellectual disability.

We have used single-nucleotide polymorphism microarray genotyping and homozygosity-by-descent (HBD) mapping followed by Sanger sequencing or whole-exome sequencing (WES) to identify causative mutations in three consanguineous families with intellectual disability (ID) related to thyroid dyshormonogenesis (TDH). One family was found to have a shared HBD region of 12.1 Mb on 8q24.21-q24.23 containing 36 coding genes, including the thyroglobulin gene, TG. Sanger sequencing of TG identified a homozygous nonsense mutation Arg2336*, which segregated with the phenotype in the family. A second family showed several HBD regions, including 6.0 Mb on 2p25.3-p25.2. WES identified a homozygous nonsense mutation, Glu596*, in the thyroid peroxidase gene, TPO. WES of a mother/father/proband trio from a third family revealed a homozygous missense mutation, Arg412His, in TPO. Mutations in TG and TPO are very rarely associated with ID, mainly because TDH is generally detectable and treatable. However, in populations where resources for screening and detection are limited, and especially where consanguineous marriages are common, mutations in genes involved in thyroid function may also be causes of ID, and as TPO and TG mutations are the most common genetic causes of TDH, these are also likely to be relatively common causes of ID.

15q11.2 Duplication Encompassing Only the UBE3A Gene Is Associated with Developmental Delay and Neuropsychiatric Phenotypes.

Duplications of chromosome region 15q11-q13 with the maternal imprint are associated with a wide spectrum of neuropsychiatric disorders, including autism spectrum disorders, developmental delay, learning difficulties, schizophrenia, and seizures. These observations suggest there is a dosage-sensitive imprinted gene or genes within this region that explains the increased risk for neuropsychiatric phenotypes. We present a female patient with developmental delay in whom we identified a maternally inherited 129-Kb duplication in chromosome region 15q11.2 encompassing only the UBE3A gene. Expression analysis in cultured fibroblasts confirmed overexpression of UBE3A in the proband, compared with age- and sex-matched controls. We further tested segregation of this duplication in four generations and found it segregated with neuropsychiatric phenotypes. Our study shows for the first time clinical features associated with overexpression of UBE3A in humans and underscores the significance of this gene in the phenotype of individuals with 15q11-q13 duplication.

Truncation of the E3 ubiquitin ligase component FBXO31 causes non-syndromic autosomal recessive intellectual disability in a Pakistani family.

In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.

Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder.

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.

Mutations in MECP2 exon 1 in classical Rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2.

The overwhelming majority of Rett syndrome cases are caused by mutations in the gene MECP2. MECP2 has two isoforms, termed MECP2_e1 and MECP2_e2, which differ in their N-terminal amino acid sequences. A growing body of evidence has indicated that MECP2_e1 may be the etiologically relevant isoform in Rett Syndrome based on its expression profile in the brain and because, strikingly, no mutations have been discovered that affect MECP2_e2 exclusively. In this study we sought to characterize four classical Rett patients with mutations that putatively affect only the MECP2_e1 isoform. Our hypothesis was that the classical Rett phenotype seen here is the result of disrupted MECP2_e1 expression, but with MECP2_e2 expression unaltered. We used quantitative reverse transcriptase PCR to assay mRNA expression for each isoform independently, and used cytospinning methods to assay total MECP2 in peripheral blood lymphocytes (PBL). In the two Rett patients with identical 11 bp deletions within the coding portion of exon 1, MECP2_e2 levels were unaffected, whilst a significant reduction of MECP2_e1 levels was detected. In two Rett patients harboring mutations in the exon 1 start codon, MECP2_e1 and MECP2_e2 mRNA amounts were unaffected. In summary, we have shown that patients with exon 1 mutations transcribe normal levels of MECP2_e2 mRNA, and most PBL are positive for MeCP2 protein, despite them theoretically being unable to produce the MECP2_e1 isoform, and yet still exhibit the classical RTT phenotype. Altogether, our work further supports our hypothesis that MECP2_e1 is the predominant isoform involved in the neuropathology of Rett syndrome.

De novo deletion in MECP2 in a monozygotic twin pair: a case report.

BACKGROUND: Rett syndrome (RTT) is a severe, progressive, neurodevelopmental disorder predominantly observed in females that leads to intellectual disability. Mutations and gross rearrangements in MECP2 account for a large proportion of cases with RTT. A limited number of twin pairs with RTT have also been reported in literature. CASE PRESENTATION: We investigated 13 year old, monozygotic twin females with RTT and some noticeable differences in development using a combinatorial approach of sequencing and Taqman assay. Monozygosity status of the twins was confirmed by informative microsatellite markers. CONCLUSIONS: The twins shared a de novo deletion in exon 3 in the MBD domain of MECP2. To the best of our knowledge, this is only the second report of genetic analysis of a monozygotic twin pair.

Distinct de novo deletions in a brother-sister pair with RTT: a case report.

Rett syndrome (RTT), a neurodevelopmental disorder caused by mutations in the X-linked gene encoding methyl-CpG-binding protein2 (MeCP2), is a leading cause of mental retardation in females. Majority of cases are sporadic (99%) but some familial cases have also been observed. We describe a familial study with a brother-sister pair with symptoms of RTT and exhibiting distinct deletions in the MECP2. The non-shared de novo deletion in the two sibs provides important insights into the disease etiology, especially for male sibs showing varied phenotypes as compared to the classical ones seen in the females.

Cadherin 2-Related Arrhythmogenic Cardiomyopathy: Prevalence and Clinical Features.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the right and left ventricle, often causing ventricular dysfunction and life-threatening arrhythmias. Variants in desmosomal genes account for up to 60% of cases. Our objective was to establish the prevalence and clinical features of ACM stemming from pathogenic variants in the nondesmosomal cadherin 2 (CDH2), a novel genetic substrate of ACM. METHODS: A cohort of 500 unrelated patients with a definite diagnosis of ACM and no disease-causing variants in the main ACM genes was assembled. Genetic screening of CDH2 was performed through next-generation or Sanger sequencing. Whenever possible, cascade screening was initiated in the families of CDH2-positive probands, and clinical evaluation was performed. RESULTS: Genetic screening of CDH2 led to the identification of 7 rare variants: 5, identified in 6 probands, were classified as pathogenic or likely pathogenic. The previously established p.D407N pathogenic variant was detected in 2 additional probands. Probands and family members with pathogenic/likely pathogenic variants in CDH2 were clinically evaluated, and along with previously published cases, altogether contributed to the identification of gene-specific features (13 cases from this cohort and 11 previously published, for a total of 9 probands and 15 family members). Ventricular arrhythmic events occurred in most CDH2-positive subjects (20/24, 83%), while the occurrence of heart failure was rare (2/24, 8.3%). Among probands, sustained ventricular tachycardia and sudden cardiac death occurred in 5/9 (56%). CONCLUSIONS: In this worldwide cohort of previously genotype-negative ACM patients, the prevalence of probands with CDH2 pathogenic/likely pathogenic variants was 1.2% (6/500). Our data show that this cohort of CDH2-ACM patients has a high incidence of ventricular arrhythmias, while evolution toward heart failure is rare.

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal ß-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and ß-catenin. A pharmacological activator of the WNT/ß-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and ß-catenin, and evidence for targeted activation of the WNT/ß-catenin pathway as a potential treatment for this disease.

A comprehensive analysis of mitochondrial genes variants and their association with antipsychotic-induced weight gain.

Antipsychotic Induced Weight Gain (AIWG) is a common and severe side effect of many antipsychotic medications. Mitochondria play a vital role for whole-body energy homeostasis and there is increasing evidence that antipsychotics modulate mitochondrial function. This study aimed to examine the role of variants in nuclear-encoded mitochondrial genes and the mitochondrial DNA (mtDNA) in conferring risk for AIWG. We selected 168 European-Caucasian individuals from the CATIE sample based upon meeting criteria of multiple weight measures while taking selected antipsychotics (risperidone, quetiapine or olanzapine). We tested the association of 670 nuclear-encoded mitochondrial genes with weight change (%) using MAGMA software. Thirty of these genes showed nominally significant P-values (<0.05). We were able to replicate the association of three genes, CLPB, PARL, and ACAD10, with weight change (%) in an independent prospectively assessed AIWG sample. We analyzed mtDNA variants in a subset of 74 of these individuals using next-generation sequencing. No common or rare mtDNA variants were found to be significantly associated with weight change (%) in our sample. Additionally, analysis of mitochondrial haplogroups showed no association with weight change (%). In conclusion, our findings suggest nuclear-encoded mitochondrial genes play a role in AIWG. Replication in larger sample is required to validate our initial report of mtDNA variants in AIWG.

Identification of a homozygous missense mutation in LRP2 and a hemizygous missense mutation in TSPYL2 in a family with mild intellectual disability.

Non-syndromic autosomal recessive intellectual disability (ID) is a genetically heterogeneous disorder with more than 50 mutated genes to date. ID is characterized by deficits in memory skills and language development with difficulty in learning, problem solving, and adaptive behaviors, and affects ∼ 1% of the population. For detection of disease-causing mutations in such a heterogeneous disorder, homozygosity mapping together with exome sequencing is a powerful approach, as almost all known genes can be assessed simultaneously in a high-throughput manner. In this study, a hemizygous c.786C>G:p.Ile262Met in the testis specific protein Y-encoded-like 2 (TSPYL2) gene and a homozygous c.11335G>A:p.Asp3779Asn in the low-density lipoprotein receptor-related protein 2 (LRP2) gene were detected after genome-wide genotyping and exome sequencing in a consanguineous Pakistani family with two boys with mild ID. Mutations in the LRP2 gene have previously been reported in patients with Donnai-Barrow and Stickler syndromes. LRP2 has also been associated with a 2q locus for autism (AUTS5). The TSPYL2 variant is not listed in any single-nucleotide polymorphism databases, and the LRP2 variant was absent in 400 ethnically matched healthy control chromosomes, and is not listed in single-nucleotide polymorphism databases as a common polymorphism. The LRP2 mutation identified here is located in one of the low-density lipoprotein-receptor class A domains, which is a cysteine-rich repeat that plays a central role in mammalian cholesterol metabolism, suggesting that alteration of cholesterol processing pathway can contribute to ID.

Rapporteur summaries of plenary, symposia, and oral sessions from the XXIIIrd World Congress of Psychiatric Genetics Meeting in Toronto, Canada, 16-20 October 2015.

The XXIIIrd World Congress of Psychiatric Genetics meeting, sponsored by the International Society of Psychiatric Genetics, was held in Toronto, ON, Canada, on 16-20 October 2015. Approximately 700 participants attended to discuss the latest state-of-the-art findings in this rapidly advancing and evolving field. The following report was written by trainee travel awardees. Each was assigned one session as a rapporteur. This manuscript represents the highlights and topics that were covered in the plenary sessions, symposia, and oral sessions during the conference, and contains major notable and new findings.