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546


Pangenome graphs improve the analysis of structural variants in rare genetic diseases

Groza, Cristian; Schwendinger-Schreck, Carl; Cheung, Warren A; Farrow, Emily G; Thiffault, Isabelle; Lake, Juniper; Rizzo, William B; Evrony, Gilad; Curran, Tom; Bourque, Guillaume; Pastinen, Tomi
Rare DNA alterations that cause heritable diseases are only partially resolvable by clinical next-generation sequencing due to the difficulty of detecting structural variation (SV) in all genomic contexts. Long-read, high fidelity genome sequencing (HiFi-GS) detects SVs with increased sensitivity and enables assembling personal and graph genomes. We leverage standard reference genomes, public assemblies (n = 94) and a large collection of HiFi-GS data from a rare disease program (Genomic Answers for Kids, GA4K, n = 574 assemblies) to build a graph genome representing a unified SV callset in GA4K, identify common variation and prioritize SVs that are more likely to cause genetic disease (MAF < 0.01). Using graphs, we obtain a higher level of reproducibility than the standard reference approach. We observe over 200,000 SV alleles unique to GA4K, including nearly 1000 rare variants that impact coding sequence. With improved specificity for rare SVs, we isolate 30 candidate SVs in phenotypically prioritized genes, including known disease SVs. We isolate a novel diagnostic SV in KMT2E, demonstrating use of personal assemblies coupled with pangenome graphs for rare disease genomics. The community may interrogate our pangenome with additional assemblies to discover new SVs within the allele frequency spectrum relevant to genetic diseases.
PMCID:10803329
PMID: 38253606
ISSN: 2041-1723
CID: 5624712

Tissue-specific and tissue-agnostic effects of genome sequence variation modulating blood pressure

Lee, Dongwon; Han, Seong Kyu; Yaacov, Or; Berk-Rauch, Hanna; Mathiyalagan, Prabhu; Ganesh, Santhi K; Chakravarti, Aravinda
Genome-wide association studies (GWASs) have identified numerous variants associated with polygenic traits and diseases. However, with few exceptions, a mechanistic understanding of which variants affect which genes in which tissues to modulate trait variation is lacking. Here, we present genomic analyses to explain trait heritability of blood pressure (BP) through the genetics of transcriptional regulation using GWASs, multiomics data from different tissues, and machine learning approaches. Approximately 500,000 predicted regulatory variants across four tissues explain 33.4% of variant heritability: 2.5%, 5.3%, 7.7%, and 11.8% for kidney-, adrenal-, heart-, and artery-specific variants, respectively. Variation in the enhancers involved shows greater tissue specificity than in the genes they regulate, suggesting that gene regulatory networks perturbed by enhancer variants in a tissue relevant to a phenotype are the major source of interindividual variation in BP. Thus, our study provides an approach to scan human tissue and cell types for their physiological contribution to any trait.
PMCID:10726310
PMID: 37910504
ISSN: 2211-1247
CID: 5590312

Cardiac muscle-restricted partial loss of Nos1ap expression has limited but significant impact on electrocardiographic features

Smith, Alexa; Auer, Dallas; Johnson, Morgan; Sanchez, Ernesto; Ross, Holly; Ward, Christopher; Chakravarti, Aravinda; Kapoor, Ashish
Genome-wide association studies have identified sequence polymorphisms in a functional enhancer of the NOS1AP gene as the most common genetic regulator of QT interval and human cardiac NOS1AP gene expression in the general population. Functional studies based on in vitro overexpression in murine cardiomyocytes and ex vivo knockdown in zebrafish embryonic hearts, by us and others, have also demonstrated that NOS1AP expression levels can alter cellular electrophysiology. Here, to explore the role of NOS1AP in cardiac electrophysiology at an organismal level, we generated and characterized constitutive and heart muscle-restricted Nos1ap knockout mice to assess whether NOS1AP disruption alters the QT interval in vivo. Constitutive loss of Nos1ap led to genetic background-dependent variable lethality at or right before birth. Heart muscle-restricted Nos1ap knockout, generated using cardiac-specific alpha-myosin heavy chain promoter-driven tamoxifen-inducible Cre, resulted in tissue-level Nos1ap expression reduced by half. This partial loss of expression had no detectable effect on the QT interval or other electrocardiographic and echocardiographic parameters, except for a small but significant reduction in the QRS interval. Given that challenges associated with defining the end of the T wave on murine electrocardiogram can limit identification of subtle effects on the QT interval and that common noncoding NOS1AP variants are also associated with the QRS interval, our findings support the role of NOS1AP in regulation of the cardiac electrical cycle.
PMCID:10627271
PMID: 37708408
ISSN: 2160-1836
CID: 5593352

RET enhancer haplotype-dependent remodeling of the human fetal gut development program

Chatterjee, Sumantra; Fries, Lauren E; Yaacov, Or; Hu, Nan; Berk-Rauch, Hanna E; Chakravarti, Aravinda
Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.
PMCID:10664930
PMID: 37948459
ISSN: 1553-7404
CID: 5607952

Ret deficiency decreases neural crest progenitor proliferation and restricts fate potential during enteric nervous system development

Vincent, Elizabeth; Chatterjee, Sumantra; Cannon, Gabrielle H; Auer, Dallas; Ross, Holly; Chakravarti, Aravinda; Goff, Loyal A
The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.
PMCID:10451519
PMID: 37585461
ISSN: 1091-6490
CID: 5595682

Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort

Cheung, Warren A; Johnson, Adam F; Rowell, William J; Farrow, Emily; Hall, Richard; Cohen, Ana S A; Means, John C; Zion, Tricia N; Portik, Daniel M; Saunders, Christopher T; Koseva, Boryana; Bi, Chengpeng; Truong, Tina K; Schwendinger-Schreck, Carl; Yoo, Byunggil; Johnston, Jeffrey J; Gibson, Margaret; Evrony, Gilad; Rizzo, William B; Thiffault, Isabelle; Younger, Scott T; Curran, Tom; Wenger, Aaron M; Grundberg, Elin; Pastinen, Tomi
Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.
PMCID:10226990
PMID: 37248219
ISSN: 2041-1723
CID: 5541202

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity

Bae, Jin H; Liu, Ruolin; Roberts, Eugenia; Nguyen, Erica; Tabrizi, Shervin; Rhoades, Justin; Blewett, Timothy; Xiong, Kan; Gydush, Gregory; Shea, Douglas; An, Zhenyi; Patel, Sahil; Cheng, Ju; Sridhar, Sainetra; Liu, Mei Hong; Lassen, Emilie; Skytte, Anne-Bine; Grońska-Pęski, Marta; Shoag, Jonathan E; Evrony, Gilad D; Parsons, Heather A; Mayer, Erica L; Makrigiorgos, G Mike; Golub, Todd R; Adalsteinsson, Viktor A
Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.
PMID: 37106072
ISSN: 1546-1718
CID: 5465432

Biochemical characterization of two novel mutations in the human high-affinity choline transporter 1 identified in a patient with congenital myasthenic syndrome

Rizvi, Midhat; Truong, Tina K; Zhou, Janet; Batta, Manav; Moran, Ellen S; Pappas, John; Chu, Mary Lynn; Caluseriu, Oana; Evrony, Gilad D; Leslie, Elaine M; Cordat, Emmanuelle
Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to re-uptake choline. Bi-allelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, that we identified in an 11 year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life.
PMID: 36611016
ISSN: 1460-2083
CID: 5433572

Interleukin-1 receptor antagonist gene ( IL1RN ) variants modulate the cytokine release syndrome and mortality of SARS-CoV-2

Attur, Mukundan; Petrilli, Christopher; Adhikari, Samrachana; Iturrate, Eduardo; Li, Xiyue; Tuminello, Stephanie; Hu, Nan; Chakravarti, Aravinda; Beck, David; Abramson, Steven B
OBJECTIVE/UNASSIGNED:, the gene encoding the anti-inflammatory IL-1 receptor antagonist (IL-1Ra), on the cytokine release syndrome and mortality. METHODS/UNASSIGNED:gene were assessed for association with laboratory markers of the cytokine release syndrome (CRS) and mortality. RESULTS/UNASSIGNED:rs419598 CC SNV exhibited lower inflammatory biomarker levels, and was associated with reduced mortality compared to the CT/TT genotype in men (OR 0.49 (0.23 - 1.00); 0.052), with the most pronounced effect observed between the ages of 55-74 [5.5% vs. 18.4%, p<0.001]. CONCLUSION/UNASSIGNED:modulates the COVID-19 cytokine release syndrome via endogenous " anti-inflammatory" mechanisms. SIGNIFICANCE STATEMENT/UNASSIGNED:merits further evaluation in severe SARS-CoV-2 infection.
PMCID:9882468
PMID: 36711766
CID: 5602052

Quality assessment and refinement of chromatin accessibility data using a sequence-based predictive model

Han, Seong Kyu; Muto, Yoshiharu; Wilson, Parker C; Humphreys, Benjamin D; Sampson, Matthew G; Chakravarti, Aravinda; Lee, Dongwon
Chromatin accessibility assays are central to the genome-wide identification of gene regulatory elements associated with transcriptional regulation. However, the data have highly variable quality arising from several biological and technical factors. To surmount this problem, we developed a sequence-based machine learning method to evaluate and refine chromatin accessibility data. Our framework, gapped k-mer SVM quality check (gkmQC), provides the quality metrics for a sample based on the prediction accuracy of the trained models. We tested 886 DNase-seq samples from the ENCODE/Roadmap projects to demonstrate that gkmQC can effectively identify "high-quality" (HQ) samples with low conventional quality scores owing to marginal read depths. Peaks identified in HQ samples are more accurately aligned at functional regulatory elements, show greater enrichment of regulatory elements harboring functional variants, and explain greater heritability of phenotypes from their relevant tissues. Moreover, gkmQC can optimize the peak-calling threshold to identify additional peaks, especially for rare cell types in single-cell chromatin accessibility data.
PMID: 36508674
ISSN: 1091-6490
CID: 5381942