Genetic analysis and multimodal imaging identify novel mtDNA 12148T>C leading to multisystem dysfunction with tissue-specific heteroplasmy.

Patients with mitochondrial disorders present with clinically diverse symptoms, largely driven by heterogeneous mutations in mitochondrial-encoded and nuclear-encoded mitochondrial genes. These mutations ultimately lead to complex biochemical disorders with a myriad of clinical manifestations, often accumulating during childhood on into adulthood, contributing to life-altering and sometimes fatal events. It is therefore important to diagnose and characterize the associated disorders for each mitochondrial mutation as early as possible since medical management might be able to improve the quality and longevity of life in mitochondrial disease patients. Here we identify a novel mitochondrial variant in a mitochondrial transfer RNA for histidine (mt-tRNA-his) [m.12148T>C], that is associated with the development of ocular, aural, neurological, renal, and muscular dysfunctions. We provide a detailed account of a family harboring this mutation, as well as the molecular underpinnings contributing to cellular and mitochondrial dysfunction. In conclusion, this investigation provides clinical, biochemical, and morphological evidence of the pathogenicity of m.12148T>C. We highlight the importance of multiple tissue testing and in vitro disease modeling in diagnosing mitochondrial disease.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

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< 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.

Mutations in MINAR2 encoding membrane integral NOTCH2-associated receptor 2 cause deafness in humans and mice.

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2, encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein (Minar2tm1b/tm1b) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.

Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

SARS-CoV-2 Susceptibility and ACE2 Gene Variations Within Diverse Ethnic Backgrounds.

There is considerable variability in the susceptibility and progression for COVID-19 and it appears to be strongly correlated with age, gender, ethnicity and pre-existing health conditions. However, to our knowledge, cohort studies of COVID-19 in clinically vulnerable groups are lacking. Host genetics has also emerged as a major risk factor for COVID-19, and variation in the ACE2 receptor, which facilitates entry of the SARS-CoV-2 virus into the cell, has become a major focus of attention. Thus, we interrogated an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (United Kingdom) to assess the association between variants in the ACE2 locus and COVID-19 risk. We analysed whole-genome sequencing (WGS) data of 1,837 cases who were tested positive for SARS-CoV-2, and 37,207 controls who were not tested, from the UK's 100,000 Genomes Project (100KGP) for the presence of ACE2 coding variants and extract expression quantitative trait loci (eQTLs). We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = 0.015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = 0.029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing the risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p. H378R, p. Y515N) alter receptor affinity for the viral Spike (S) protein. Variant p. N720D, more prevalent in the European population (p < 0.001), potentially increases viral entry by affecting the ACE2-TMPRSS2 complex. The spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups.

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.

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

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.

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.

A missense mutation in TRAPPC6A leads to build-up of the protein, in patients with a neurodevelopmental syndrome and dysmorphic features.

Childhood onset clinical syndromes involving intellectual disability and dysmorphic features, such as polydactyly, suggest common developmental pathways link seemingly unrelated phenotypes. We identified a consanguineous family of Saudi origin with varying complex features including intellectual disability, speech delay, facial dysmorphism and polydactyly. Combining, microarray based comparative genomic hybridisation (CGH) to identify regions of homozygosity, with exome sequencing, led to the identification of homozygous mutations in five candidate genes (RSPH6A, ANKK1, AMOTL1, ALKBH8, TRAPPC6A), all of which appear to be pathogenic as predicted by Proven, SIFT and PolyPhen2 and segregate perfectly with the disease phenotype. We therefore looked for differences in expression levels of each protein in HEK293 cells, expressing either the wild-type or mutant full-length cDNA construct. Unexpectedly, wild-type TRAPPC6A appeared to be unstable, but addition of the proteasome inhibitor MG132 stabilised its expression. Mutations have previously been reported in several members of the TRAPP complex of proteins, including TRAPPC2, TRAPPC9 and TRAPPC11, resulting in disorders involving skeletal abnormalities, intellectual disability, speech impairment and developmental delay. TRAPPC6A joins a growing list of proteins belonging to the TRAPP complex, implicated in clinical syndromes with neurodevelopmental abnormalities.

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 A1 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.