Investigating postzygotic de novo mutations and somatic mosaicism in monozygotic twins discordant for complex disorders

Monozygotic (MZ) twins were long thought to be genetically identical, however recent studies have demonstrated genetic differences between them. To test the hypothesis that early post-twinning mutational events leads to phenotypic discordance, thirteen MZ twins discordant for a range of complex disorders were investigated at the genomic and proteomic level. Whole-exome sequencing data was analysed using a union of VarScan2 and MuTect2 variant calling algorithms. Copy-number variation (CNV) analysis from Illumina HumanCore array data was also carried out using PennCNV and cnvPartition to identify structural variants that would not be detected by exome sequencing. All single nucleotide variants (SNVs), indels and CNVs were evaluated for functional consequence, evolutionary conservation, population frequency and overlap with known disease-susceptibility genes. Twenty-two putative discordant SNVs and indels, but no discordant structural variants, were identified. Parent-offspring trio analysis was implemented to assess potential association of germline de novo mutations with susceptibility to disease. A rare, highly conserved de novo mutation in RASD2 was detected in twins discordant for attention deficit hyperactivity disorder (ADHD). RASD2 is enriched in the striatum and involved in the modulation of dopaminergic transmission. In the twins discordant for Tourette’s syndrome, an inherited stop loss mutation was detected in AADAC, a known candidate gene for the disorder. Further, a de novo CNV duplication was identified in a twin pair discordant for ADHD overlapping CD38, a gene implicated in social amnesia and autism. When analysing the burden of shared CNVs among the twins, a rare hemizygous deletion in region 15q13.2 was detected in twins with schizophrenia, overlapping ARHGAP11B, a human-specific gene involved in basal progenitor amplification and neocortex expansion. To investigate potential downstream consequences of (epi)genetic mechanisms and underlying biochemical pathways, proteomic profiling of serum samples obtained from an MZ twin pair discordant for ischaemic stroke was analysed through a label-free pipeline. Biological processes overrepresented in the affected twin predominantly corresponded to stroke-related processes, including wound healing, blood coagulation and haemostasis. Further, a comparison of blood chemistries showed a >10- and >18-fold elevation of γ-glutamyltransferase (GGT) and erythrocyte sedimentation rate (ESR) levels respectively in the affected twin

A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377–5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5–12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux

Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy

BACKGROUND: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. METHODS: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. RESULTS: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. CONCLUSIONS: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically

De novo single-nucleotide and copy number variation in discordant monozygotic twins reveals disease-related genes

Recent studies have demonstrated genetic differences between monozygotic (MZ) twins. To test the hypothesis that early post-twinning mutational events associate with phenotypic discordance, we investigated a cohort of 13 twin pairs (n = 26) discordant for various clinical phenotypes using whole-exome sequencing and screened for copy number variation (CNV). We identified a de novo variant in PLCB1, a gene involved in the hydrolysis of lipid phosphorus in milk from dairy cows, associated with lactase non-persistence, and a variant in the mitochondrial complex I gene MT-ND5 associated with amyotrophic lateral sclerosis (ALS). We also found somatic variants in multiple genes (TMEM225B, KBTBD3, TUBGCP4, TFIP11) in another MZ twin pair discordant for ALS. Based on the assumption that discordance between twins could be explained by a common variant with variable penetrance or expressivity, we screened the twin samples for known pathogenic variants that are shared and identified a rare deletion overlapping ARHGAP11B, in the twin pair manifesting with either schizotypal personality disorder or schizophrenia. Parent-offspring trio analysis was implemented for two twin pairs to assess potential association of variants of parental origin with susceptibility to disease. We identified a de novo variant in RASD2 shared by 8-year-old male twins with a suspected diagnosis of autism spectrum disorder (ASD) manifesting as different traits. A de novo CNV duplication was also identified in these twins overlapping CD38, a gene previously implicated in ASD. In twins discordant for Tourette's syndrome, a paternally inherited stop loss variant was detected in AADAC, a known candidate gene for the disorder

Distinct proteomic profiles in monozygotic twins discordant for ischaemic stroke

Stroke is a common disorder with significant morbidity and mortality, and complex aetiology involving both environmental and genetic risk factors. Although some of the major risk factors for stoke, such as smoking and hypertension, are well-documented, the underlying genetic and detailed molecular mechanisms remain elusive. Exploring the relevant biochemical pathways may contribute to the clinical diagnosis of stroke and shed light on its aetiology. A comparative proteomic analysis of blood serum of a pair of monozygotic (MZ) twins discordant for ischaemic stroke (IS) was performed using a label-free quantitative proteomics approach. To overcome the limit of reproducibility in the serum preparation, two separate runs were performed, each consisting of three technical replicates per sample. Biological processes associated with proteins differentially expressed between the twins were explored with gene ontology (GO) classification using the functional analysis tool g:Profiler. ANOVA test performed in Progenesis LC-MS identified 179 (run 1) and 209 (run 2) proteins as differentially expressed between the affected and unaffected twin (p <0.05). Furthermore, the level of serum fibulin 1, an extracellular matrix protein associated with arterial stiffness, was on average 13.37-fold higher in the affected twin. Each dataset was then analysed independently, and the proteins were classified according to GO terms. The categories overrepresented in the affected twin predominantly corresponded to stroke-relevant processes, including wound healing, blood coagulation and haemostasis, with a high proportion of the proteins overexpressed in the affected twin associated with these terms. By contrast, in the unaffected twin diagnosed with atopic dermatitis, there were increased levels of keratin proteins and GO terms associated with skin development. The identification of cellular pathways enriched in IS as well as the upregulation of fibulin 1 sheds new light on the underlying disease-causing mechanisms at the molecular level. Our findings of distinct proteomic signatures associated with IS and atopic dermatitis suggest proteomic profiling could be used as a general approach for improved diagnostic, prognostic and therapeutic strategies

De novo and inherited variants in coding and regulatory regions in genetic cardiomyopathies

BACKGROUND: Cardiomyopathies are a leading cause of progressive heart failure and sudden cardiac death; however, their genetic aetiology remains poorly understood. We hypothesised that variants in noncoding regulatory regions and oligogenic inheritance mechanisms may help close the diagnostic gap. METHODS: We first analysed whole-genome sequencing data of 143 parent-offspring trios from Genomics England 100,000 Genomes Project. We used gene panel testing and a phenotype-based, variant prioritisation framework called Exomiser to identify candidate genes in trios. To assess the contribution of noncoding DNVs to cardiomyopathies, we intersected DNVs with open chromatin sequences from single-cell ATAC-seq data of cardiomyocytes. We also performed a case-control analysis in an exome-negative cohort, including 843 probands and 19,467 controls, to assess the association between noncoding variants in known cardiomyopathy genes and disease. RESULTS: In the trio analysis, a definite or probable genetic diagnosis was identified in 21 probands according to the American College of Medical Genetics guidelines. We identified novel DNVs in diagnostic-grade genes (RYR2, TNNT2, PTPN11, MYH7, LZR1, NKX2-5), and five cases harbouring a combination of prioritised variants, suggesting that oligogenic inheritance and genetic modifiers contribute to cardiomyopathies. Phenotype-based ranking of candidate genes identified in noncoding DNV analysis revealed JPH2 as the top candidate. Moreover, a case-control analysis revealed an enrichment of rare noncoding variants in regulatory elements of cardiomyopathy genes (p = .035, OR = 1.43, 95% Cl = 1.095-1.767) versus controls. Of the 25 variants associated with disease (p&lt; 0.5), 23 are novel and nine are predicted to disrupt transcription factor binding motifs. CONCLUSION: Our results highlight complex genetic mechanisms in cardiomyopathies and reveal novel genes for future investigations

Truncating mutation in intracellular phospholipase A₁ gene (DDHD2) in hereditary spastic paraplegia with intellectual disability (SPG54)

BACKGROUND: Hereditary spastic paraplegias (HSP), a group of genetically heterogeneous neurological disorders with more than 56 documented loci (SPG1-56), are described either as uncomplicated (or pure), or complicated where in addition to spasticity and weakness of lower extremeties, additional neurological symptoms are present, including dementia, loss of vision, epilepsy, mental retardation and ichthyosis. We identified a large consanguineous family of Indian descent with four affected members with childhood onset HSP (SPG54), presenting with upper and lower limb spasticity, mental retardation and agenesis of the corpus callosum. RESULTS: A common region of homozygosity on chromosome 8 spanning seven megabases (Mb) was identified in the affected individuals using the Illumina human cytoSNP-12 DNA Analysis BeadChip Kit. Exome sequencing identified a homozygous stop gain mutation (pR287X) in the phospholipase A(1) gene DDHD2, in the affected individuals, resulting in a premature stop codon and a severely truncated protein lacking the SAM and DDHD domains crucial for phosphoinositide binding and phospholipase activity. CONCLUSION: This mutation adds to the knowledge of HSP, suggests a possible founder effect for the pR287X mutation, and adds to the list of genes involved in lipid metabolism with a role in HSP and other neurodegenerative disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-015-1227-4) contains supplementary material, which is available to authorized users