A System for Phenotype Harmonization in the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine (TOPMed) Program.

Genotype-phenotype association studies often combine phenotype data from multiple studies to increase statistical power. Harmonization of the data usually requires substantial effort due to heterogeneity in phenotype definitions, study design, data collection procedures, and data-set organization. Here we describe a centralized system for phenotype harmonization that includes input from phenotype domain and study experts, quality control, documentation, reproducible results, and data-sharing mechanisms. This system was developed for the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine (TOPMed) program, which is generating genomic and other -omics data for more than 80 studies with extensive phenotype data. To date, 63 phenotypes have been harmonized across thousands of participants (recruited in 1948-2012) from up to 17 studies per phenotype. Here we discuss challenges in this undertaking and how they were addressed. The harmonized phenotype data and associated documentation have been submitted to National Institutes of Health data repositories for controlled access by the scientific community. We also provide materials to facilitate future harmonization efforts by the community, which include 1) the software code used to generate the 63 harmonized phenotypes, enabling others to reproduce, modify, or extend these harmonizations to additional studies, and 2) the results of labeling thousands of phenotype variables with controlled vocabulary terms.

Mechanical Birth Injuries: A Comprehensive Five-Year Analysis From a Tertiary Care Hospital.

BACKGROUND: Birth injury or birth trauma refers to physical damage or trauma that occurs to a newborn during the birthing process. To ensure continuous care and improve neonatal outcomes, it is crucial to know the incidence, types, relation to the mode of delivery, and their management. METHODOLOGY: This is a retrospective cohort study conducted at Aga Khan University Hospital, Pakistan from January 2018 to December 2022. Neonates aged from birth to 28 days of life identified to sustain any form of mechanical birth injuries were included. Data analysis was done using SPSS version 19 (IBM Corp., Armonk, NY). RESULTS: In the last five years, 51 mechanical birth injuries were found among 27,854 deliveries, which accounts for one in 546 births with an overall prevalence of 0.001%. Out of the total mechanical birth injuries, 12 (23.5%) were noticed in spontaneous vaginal delivery, six (11.8%) had instrumental delivery, and 33 (64.7%) patients had cesarean sections. More birth injuries were noticed in emergency cesarean section as compared to vaginal deliveries. There were 40 babies (78%) with soft tissue injuries, seven (14%) had musculoskeletal injuries/fractures, two (4%) babies had intracranial bleeding, and two (4%) had fractures along with intracranial bleeding. There was no mortality reported among these neonates. CONCLUSION: The overall rate of birth injuries was significantly lower as compared to other low and middle-income countries. Most of the birth injuries were soft tissue injuries in patients with cesarean sections. The rate of birth injury did not show any association with the time of delivery. More frequent obstetric emergency drills would improve complications associated with shoulder dystocia.

Rare variant contribution to the heritability of coronary artery disease.

Whole genome sequences (WGS) enable discovery of rare variants which may contribute to missing heritability of coronary artery disease (CAD). To measure their contribution, we apply the GREML-LDMS-I approach to WGS of 4949 cases and 17,494 controls of European ancestry from the NHLBI TOPMed program. We estimate CAD heritability at 34.3% assuming a prevalence of 8.2%. Ultra-rare (minor allele frequency ≤ 0.1%) variants with low linkage disequilibrium (LD) score contribute ~50% of the heritability. We also investigate CAD heritability enrichment using a diverse set of functional annotations: i) constraint; ii) predicted protein-altering impact; iii) cis-regulatory elements from a cell-specific chromatin atlas of the human coronary; and iv) annotation principal components representing a wide range of functional processes. We observe marked enrichment of CAD heritability for most functional annotations. These results reveal the predominant role of ultra-rare variants in low LD on the heritability of CAD. Moreover, they highlight several functional processes including cell type-specific regulatory mechanisms as key drivers of CAD genetic risk.

Dual diagnosis of UQCRFS1-related mitochondrial complex III deficiency and recessive GJA8-related cataracts.

Biallelic pathogenic variants in UQCRFS1 underlie a rare form of isolated mitochondrial complex III deficiency associated with lactic acidosis and a distinctive scalp alopecia previously described in two unrelated probands. Here, we describe a participant in the Undiagnosed Diseases Network (UDN) with a dual diagnosis of two autosomal recessive disorders revealed by genome sequencing: UQCRFS1-related mitochondrial complex III deficiency and GJA8-related cataracts. Both pathogenic variants have been reported before: UQCRFS1 (NM_006003.3:c.215-1 G>C, p.Val72_Thr81del10) in a case with mitochondrial complex III deficiency and GJA8 (NM 005267.5:c.736 G>T, p.Glu246*) as a somatic change in aged cornea leading to decreased junctional coupling. A multi-modal approach combining enzyme assays and cellular proteomics analysis provided clear evidence of complex III respiratory chain dysfunction and low abundance of the Rieske iron-sulfur protein, validating the pathogenic effect of the UQCRFS1 variant. This report extends the genotypic and phenotypic spectrum for these two rare disorders and highlights the utility of deep phenotyping and genomics data to achieve diagnosis and insights into rare disease.

The Genetic Determinants and Genomic Consequences of Non-Leukemogenic Somatic Point Mutations.

Clonal hematopoiesis (CH) is defined by the expansion of a lineage of genetically identical cells in blood. Genetic lesions that confer a fitness advantage, such as point mutations or mosaic chromosomal alterations (mCAs) in genes associated with hematologic malignancy, are frequent mediators of CH. However, recent analyses of both single cell-derived colonies of hematopoietic cells and population sequencing cohorts have revealed CH frequently occurs in the absence of known driver genetic lesions. To characterize CH without known driver genetic lesions, we used 51,399 deeply sequenced whole genomes from the NHLBI TOPMed sequencing initiative to perform simultaneous germline and somatic mutation analyses among individuals without leukemogenic point mutations (LPM), which we term CH-LPMneg. We quantified CH by estimating the total mutation burden. Because estimating somatic mutation burden without a paired-tissue sample is challenging, we developed a novel statistical method, the Genomic and Epigenomic informed Mutation (GEM) rate, that uses external genomic and epigenomic data sources to distinguish artifactual signals from true somatic mutations. We performed a genome-wide association study of GEM to discover the germline determinants of CH-LPMneg. After fine-mapping and variant-to-gene analyses, we identified seven genes associated with CH-LPMneg (TCL1A, TERT, SMC4, NRIP1, PRDM16, MSRA, SCARB1), and one locus associated with a sex-associated mutation pathway (SRGAP2C). We performed a secondary analysis excluding individuals with mCAs, finding that the genetic architecture was largely unaffected by their inclusion. Functional analyses of SMC4 and NRIP1 implicated altered HSC self-renewal and proliferation as the primary mediator of mutation burden in blood. We then performed comprehensive multi-tissue transcriptomic analyses, finding that the expression levels of 404 genes are associated with GEM. Finally, we performed phenotypic association meta-analyses across four cohorts, finding that GEM is associated with increased white blood cell count and increased risk for incident peripheral artery disease, but is not significantly associated with incident stroke or coronary disease events. Overall, we develop GEM for quantifying mutation burden from WGS without a paired-tissue sample and use GEM to discover the genetic, genomic, and phenotypic correlates of CH-LPMneg.

Perceptions of forensic scientists on statistical models, sequence data, and ethical implications for DNA evidence evaluations: A qualitative assessment.

With the introduction of next generation sequencing (NGS) technology in the forensic field, it will be of interest to assess if forensic scientists feel equipped to interpret and present DNA evidence for sequence data. Here, we describe perceptions of sixteen U.S.-based forensic scientists on statistical models, sequence data, and ethical implications for DNA evidence evaluations. To get an in-depth understanding of the current situation, we used a qualitative research approach with a cross-sectional study design. Semi-structured interviews (N = 16) were conducted with U.S. forensic scientists working with DNA evidence. Open-ended interview questions were used to explore participants' views and needs surrounding the use of statistical models and sequence data for forensic purposes. We conducted a conventional content analysis using ATLAS. ti software and employed a second coder to ensure reliability of our results. Eleven themes emerged: 1) a statistical model that maximizes the value of the evidence is preferred; 2) a high-level understanding of the statistical model used is generally sufficient; 3) transparency is key in minimizing the risk of creating black boxes; 4) training and education should be an ongoing effort; 5) the effectiveness of presenting results in court can be improved; 6) NGS has the potential to become revolutionary; 7) some hesitations surrounding the use of sequence data remain; 8) there is a need for a concrete plan to alleviate barriers to the implementation of sequencing techniques; 9) ethics plays a major part in the role of a forensic scientist; 10) ethical barriers for sequence data depend on the application; 11) DNA evidence has its limitations. The results of this study give insight into the perceptions of forensic scientists regarding the use of statistical models and sequence data, providing valuable information in the move towards implementing sequencing methods for DNA evidence evaluations.

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

Full-length Isoform Sequencing for Resolving the Molecular Basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient-derived samples has been shown to improve the diagnostic yield for solving cases of suspected Mendelian conditions, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length transcript sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts the branch point critical for intron 6 splicing. Full-length long-read isoform complementary DNA (cDNA) sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates 5 distinct altered splicing transcripts. All 5 altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

Carriers of rare damaging CCR2 genetic variants are at lower risk of atherosclerotic disease.

Background: The CCL2/CCR2 axis governs monocyte trafficking and recruitment to atherosclerotic lesions. Human genetic analyses and population-based studies support an association between circulating CCL2 levels and atherosclerosis. Still, it remains unknown whether pharmacological targeting of CCR2, the main CCL2 receptor, would provide protection against human atherosclerotic disease. Methods: In whole-exome sequencing data from 454,775 UK Biobank participants (40-69 years), we identified predicted loss-of-function (LoF) or damaging missense (REVEL score >0.5) variants within the CCR2 gene. We prioritized variants associated with lower monocyte count (p<0.05) and tested associations with vascular risk factors and risk of atherosclerotic disease over a mean follow-up of 14 years. The results were replicated in a pooled cohort of three independent datasets (TOPMed, deCODE and Penn Medicine BioBank; total n=441,445) and the effect of the most frequent damaging variant was experimentally validated. Results: A total of 45 predicted LoF or damaging missense variants were identified in the CCR2 gene, 4 of which were also significantly associated with lower monocyte count, but not with other white blood cell counts. Heterozygous carriers of these variants were at a lower risk of a combined atherosclerosis outcome, showed a lower burden of atherosclerosis across four vascular beds, and were at a lower lifetime risk of coronary artery disease and myocardial infarction. There was no evidence of association with vascular risk factors including LDL-cholesterol, blood pressure, glycemic status, or C-reactive protein. Using a cAMP assay, we found that cells transfected with the most frequent CCR2 damaging variant (3:46358273:T:A, M249K, 547 carriers, frequency: 0.14%) show a decrease in signaling in response to CCL2. The associations of the M249K variant with myocardial infarction were consistent across cohorts (ORUKB: 0.62 95%CI: 0.39-0.96; ORexternal: 0.64 95%CI: 0.34-1.19; ORpooled: 0.64 95%CI: 0.450.90). In a phenome-wide association study, we found no evidence for higher risk of common infections or mortality among carriers of damaging CCR2 variants. Conclusions: Heterozygous carriers of damaging CCR2 variants have a lower burden of atherosclerosis and lower lifetime risk of myocardial infarction. In conjunction with previous evidence from experimental and epidemiological studies, our findings highlight the translational potential of CCR2-targeting as an atheroprotective approach.

Evaluating the use of blood pressure polygenic risk scores across race/ethnic background groups.

We assess performance and limitations of polygenic risk scores (PRSs) for multiple blood pressure (BP) phenotypes in diverse population groups. We compare "clumping-and-thresholding" (PRSice2) and LD-based (LDPred2) methods to construct PRSs from each of multiple GWAS, as well as multi-PRS approaches that sum PRSs with and without weights, including PRS-CSx. We use datasets from the MGB Biobank, TOPMed study, UK biobank, and from All of Us to train, assess, and validate PRSs in groups defined by self-reported race/ethnic background (Asian, Black, Hispanic/Latino, and White). For both SBP and DBP, the PRS-CSx based PRS, constructed as a weighted sum of PRSs developed from multiple independent GWAS, perform best across all race/ethnic backgrounds. Stratified analysis in All of Us shows that PRSs are better predictive of BP in females compared to males, individuals without obesity, and middle-aged (40-60 years) compared to older and younger individuals.

Whole-genome sequencing uncovers two loci for coronary artery calcification and identifies ARSE as a regulator of vascular calcification.

Coronary artery calcification (CAC) is a measure of atherosclerosis and a well-established predictor of coronary artery disease (CAD) events. Here we describe a genome-wide association study (GWAS) of CAC in 22,400 participants from multiple ancestral groups. We confirmed associations with four known loci and identified two additional loci associated with CAC (ARSE and MMP16), with evidence of significant associations in replication analyses for both novel loci. Functional assays of ARSE and MMP16 in human vascular smooth muscle cells (VSMCs) demonstrate that ARSE is a promoter of VSMC calcification and VSMC phenotype switching from a contractile to a calcifying or osteogenic phenotype. Furthermore, we show that the association of variants near ARSE with reduced CAC is likely explained by reduced ARSE expression with the G allele of enhancer variant rs5982944. Our study highlights ARSE as an important contributor to atherosclerotic vascular calcification, and a potential drug target for vascular calcific disease.

Recommendations on the use and reporting of race, ethnicity, and ancestry in genetic research: Experiences from the NHLBI TOPMed program.

How race, ethnicity, and ancestry are used in genomic research has wide-ranging implications for how research is translated into clinical care and incorporated into public understanding. Correlation between race and genetic ancestry contributes to unresolved complexity for the scientific community, as illustrated by heterogeneous definitions and applications of these variables. Here, we offer commentary and recommendations on the use of race, ethnicity, and ancestry across the arc of genetic research, including data harmonization, analysis, and reporting. While informed by our experiences as researchers affiliated with the NHLBI Trans-Omics for Precision Medicine (TOPMed) program, these recommendations are applicable to basic and translational genomic research in diverse populations with genome-wide data. Moving forward, considerable collaborative effort will be required to ensure that race, ethnicity, and ancestry are described and used appropriately to generate scientific knowledge that yields broad and equitable benefit.

Mendelian randomization supports bidirectional causality between telomere length and clonal hematopoiesis of indeterminate potential.

Human genetic studies support an inverse causal relationship between leukocyte telomere length (LTL) and coronary artery disease (CAD), but directionally mixed effects for LTL and diverse malignancies. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by expansion of hematopoietic cells bearing leukemogenic mutations, predisposes both hematologic malignancy and CAD. TERT (which encodes telomerase reverse transcriptase) is the most significantly associated germline locus for CHIP in genome-wide association studies. Here, we investigated the relationship between CHIP, LTL, and CAD in the Trans-Omics for Precision Medicine (TOPMed) program (n = 63,302) and UK Biobank (n = 47,080). Bidirectional Mendelian randomization studies were consistent with longer genetically imputed LTL increasing propensity to develop CHIP, but CHIP then, in turn, hastens to shorten measured LTL (mLTL). We also demonstrated evidence of modest mediation between CHIP and CAD by mLTL. Our data promote an understanding of potential causal relationships across CHIP and LTL toward prevention of CAD.

Chromosome Xq23 is associated with lower atherogenic lipid concentrations and favorable cardiometabolic indices.

Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10-72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10-4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10-5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids.

Dynamic incorporation of multiple in silico functional annotations empowers rare variant association analysis of large whole-genome sequencing studies at scale.

Large-scale whole-genome sequencing studies have enabled the analysis of rare variants (RVs) associated with complex phenotypes. Commonly used RV association tests have limited scope to leverage variant functions. We propose STAAR (variant-set test for association using annotation information), a scalable and powerful RV association test method that effectively incorporates both variant categories and multiple complementary annotations using a dynamic weighting scheme. For the latter, we introduce 'annotation principal components', multidimensional summaries of in silico variant annotations. STAAR accounts for population structure and relatedness and is scalable for analyzing very large cohort and biobank whole-genome sequencing studies of continuous and dichotomous traits. We applied STAAR to identify RVs associated with four lipid traits in 12,316 discovery and 17,822 replication samples from the Trans-Omics for Precision Medicine Program. We discovered and replicated new RV associations, including disruptive missense RVs of NPC1L1 and an intergenic region near APOC1P1 associated with low-density lipoprotein cholesterol.