Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination.

BACKGROUND: Between November 2021 and February 2022, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. METHODS: We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. FINDINGS: We identified 18 co-infections, one of which displayed evidence of a low Delta-Omicron recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In the three cases, the 5' end of the viral genome was from the Delta genome and the 3' end from Omicron, including the majority of the spike protein gene, though the breakpoints were different. CONCLUSIONS: Delta-Omicron recombinant viruses were rare, and there is currently no evidence that Delta-Omicron recombinant viruses are more transmissible between hosts compared with the circulating Omicron lineages. FUNDING: This research was supported by the NIH RADx initiative and by the Centers for Disease Control Contract 75D30121C12730 (Helix).

Incomplete Penetrance of Population-Based Genetic Screening Results in Electronic Health Record.

The clinical value of population-based genetic screening projects depends on the actions taken on the findings. The Healthy Nevada Project (HNP) is an all-comer genetic screening and research project based in northern Nevada. HNP participants with CDC Tier 1 findings of hereditary breast and ovarian cancer syndrome (HBOC), Lynch syndrome (LS), or familial hypercholesterolemia (FH) are notified and provided with genetic counseling. However, the HNP subsequently takes a "hands-off" approach: it is the responsibility of notified participants to share their findings with their healthcare providers, and providers are expected to implement the recommended action plans. Thus, the HNP presents an opportunity to evaluate the efficiency of participant and provider responses to notification of important genetic findings, using electronic health records (EHRs) at Renown Health (a large regional hospital in northern Nevada). Out of 520 HNP participants with findings, we identified 250 participants who were notified of their findings and who had an EHR. 107 of these participants responded to a survey, with 76 (71%) indicating that they had shared their findings with their healthcare providers. However, a sufficiently specific genetic diagnosis appeared in the EHRs and problem lists of only 22 and 10%, respectively, of participants without prior knowledge. Furthermore, review of participant EHRs provided evidence of possible relevant changes in clinical care for only a handful of participants. Up to 19% of participants would have benefited from earlier screening due to prior presentation of their condition. These results suggest that continuous support for both participants and their providers is necessary to maximize the benefit of population-based genetic screening. We recommend that genetic screening projects require participants' consent to directly document their genetic findings in their EHRs. Additionally, we recommend that they provide healthcare providers with ongoing training regarding documentation of findings and with clinical decision support regarding subsequent care.

SARS-CoV-2 variant Delta rapidly displaced variant Alpha in the United States and led to higher viral loads.

We report on the sequencing of 74,348 SARS-CoV-2 positive samples collected across the United States and show that the Delta variant, first detected in the United States in March 2021, made up the majority of SARS-CoV-2 infections by July 1, 2021 and accounted for >99.9% of the infections by September 2021. Not only did Delta displace variant Alpha, which was the dominant variant at the time, it also displaced the Gamma, Iota, and Mu variants. Through an analysis of quantification cycle (Cq) values, we demonstrate that Delta infections tend to have a 1.7× higher viral load compared to Alpha infections (a decrease of 0.8 Cq) on average. Our results are consistent with the hypothesis that the increased transmissibility of the Delta variant could be due to the ability of the Delta variant to establish a higher viral load earlier in the infection as compared to the Alpha variant.

Positive predictive value highlights four novel candidates for actionable genetic screening from analysis of 220,000 clinicogenomic records.

PURPOSE: To identify conditions that are candidates for population genetic screening based on population prevalence, penetrance of rare variants, and actionability. METHODS: We analyzed exome and medical record data from >220,000 participants across two large population health cohorts with different demographics. We performed a gene-based collapsing analysis of rare variants to identify genes significantly associated with disease status. RESULTS: We identify 74 statistically significant gene-disease associations across 27 genes. Seven of these conditions have a positive predictive value (PPV) of at least 30% in both cohorts. Three are already used in population screening programs (BRCA1, BRCA2, LDLR), and we also identify four new candidates for population screening: GCK with diabetes mellitus, HBB with ß-thalassemia minor and intermedia, PKD1 with cystic kidney disease, and MIP with cataracts. Importantly, the associations are actionable in that early genetic screening of each of these conditions is expected to improve outcomes. CONCLUSION: We identify seven genetic conditions where rare variation appears appropriate to assess in population screening, four of which are not yet used in screening programs. The addition of GCK, HBB, PKD1, and MIP rare variants into genetic screening programs would reach an additional 0.21% of participants with actionable disease risk, depending on the population.

Using symptom-based case predictions to identify host genetic factors that contribute to COVID-19 susceptibility.

Epidemiological and genetic studies on COVID-19 are currently hindered by inconsistent and limited testing policies to confirm SARS-CoV-2 infection. Recently, it was shown that it is possible to predict COVID-19 cases using cross-sectional self-reported disease-related symptoms. Here, we demonstrate that this COVID-19 prediction model has reasonable and consistent performance across multiple independent cohorts and that our attempt to improve upon this model did not result in improved predictions. Using the existing COVID-19 prediction model, we then conducted a GWAS on the predicted phenotype using a total of 1,865 predicted cases and 29,174 controls. While we did not find any common, large-effect variants that reached genome-wide significance, we do observe suggestive genetic associations at two SNPs (rs11844522, p = 1.9x10-7; rs5798227, p = 2.2x10-7). Explorative analyses furthermore suggest that genetic variants associated with other viral infectious diseases do not overlap with COVID-19 susceptibility and that severity of COVID-19 may have a different genetic architecture compared to COVID-19 susceptibility. This study represents a first effort that uses a symptom-based predicted phenotype as a proxy for COVID-19 in our pursuit of understanding the genetic susceptibility of the disease. We conclude that the inclusion of symptom-based predicted cases could be a useful strategy in a scenario of limited testing, either during the current COVID-19 pandemic or any future viral outbreak.

Comprehensive Allele Genotyping in Critical Pharmacogenes Reduces Residual Clinical Risk in Diverse Populations.

Genomic-guided pharmaceutical prescribing is increasingly recognized as an important clinical application of genetics. Accurate genotyping of pharmacogenomic (PGx) genes can be difficult, owing to their complex genetic architecture involving combinations of single-nucleotide polymorphisms and structural variation. Here, we introduce the Helix PGx database, an open-source star allele, genotype, and resulting metabolic phenotype frequency database for CYP2C9, CYP2C19, CYP2D6, and CYP4F2, based on short-read sequencing of >86,000 unrelated individuals enrolled in the Helix DNA Discovery Project. The database is annotated using a pipeline that is clinically validated against a broad range of alleles and designed to call CYP2D6 structural variants with high (98%) accuracy. We find that CYP2D6 has greater allelic diversity than the other genes, manifest in both a long tail of low-frequency star alleles, as well as a disproportionate fraction (36%) of all novel predicted loss-of-function variants identified. Across genes, we observe that many rare alleles (<0.1% frequency) in the overall cohort have 10 times higher frequency in one or more subgroups with non-European genetic ancestry. Extending these PGx genotypes to predicted metabolic phenotypes, we demonstrate that >90% of the cohort harbors a high-risk variant in one of the four pharmacogenes. Based on the recorded prescriptions for >30,000 individuals in the Healthy Nevada Project, combined with predicted PGx metabolic phenotypes, we anticipate that standard-of-care screening of these 4 pharmacogenes could impact nearly half of the general population.

Genome-Wide Identification of Rare and Common Variants Driving Triglyceride Levels in a Nevada Population.

Clinical conditions correlated with elevated triglyceride levels are well-known: coronary heart disease, hypertension, and diabetes. Underlying genetic and phenotypic mechanisms are not fully understood, partially due to lack of coordinated genotypic-phenotypic data. Here we use a subset of the Healthy Nevada Project, a population of 9,183 sequenced participants with longitudinal electronic health records to examine consequences of altered triglyceride levels. Specifically, Healthy Nevada Project participants sequenced by the Helix Exome+ platform were cross-referenced to their electronic medical records to identify: (1) rare and common single-variant genome-wide associations; (2) gene-based associations using a Sequence Kernel Association Test; (3) phenome-wide associations with triglyceride levels; and (4) pleiotropic variants linked to triglyceride levels. The study identified 549 significant single-variant associations (p < 8.75 × 10-9), many in chromosome 11's triglyceride hotspot: ZPR1, BUD13, APOC3, APOA5. A well-known protective loss-of-function variant in APOC3 (R19X) was associated with a 51% decrease in triglyceride levels in the cohort. Sixteen gene-based triglyceride associations were identified; six of these genes surprisingly did not include a single variant with significant associations. Results at the variant and gene level were validated with the UK Biobank. The combination of a single-variant genome-wide association, a gene-based association method, and phenome wide-association studies identified rare and common variants, genes, and phenotypes associated with elevated triglyceride levels, some of which may have been overlooked with standard approaches.

Pathogenic variants in actionable MODY genes are associated with type 2 diabetes.

Genome-wide association studies have identified 240 independent loci associated with type 2 diabetes (T2D) risk, but this knowledge has not advanced precision medicine. In contrast, the genetic diagnosis of monogenic forms of diabetes (including maturity-onset diabetes of the young (MODY)) are textbook cases of genomic medicine. Recent studies trying to bridge the gap between monogenic diabetes and T2D have been inconclusive. Here, we show a significant burden of pathogenic variants in genes linked with monogenic diabetes among people with common T2D, particularly in actionable MODY genes, thus implying that there should be a substantial change in care for carriers with T2D. We show that, among 74,629 individuals, this burden is probably driven by the pathogenic variants found in GCK, and to a lesser extent in HNF4A, KCNJ11, HNF1B and ABCC8. The carriers with T2D are leaner, which evidences a functional metabolic effect of these mutations. Pathogenic variants in actionable MODY genes are more frequent than was previously expected in common T2D. These results open avenues for future interventions assessing the clinical interest of these pathogenic mutations in precision medicine.

Genome-wide rare variant analysis for thousands of phenotypes in over 70,000 exomes from two cohorts.

Understanding the impact of rare variants is essential to understanding human health. We analyze rare (MAF < 0.1%) variants against 4264 phenotypes in 49,960 exome-sequenced individuals from the UK Biobank and 1934 phenotypes (1821 overlapping with UK Biobank) in 21,866 members of the Healthy Nevada Project (HNP) cohort who underwent Exome + sequencing at Helix. After using our rare-variant-tailored methodology to reduce test statistic inflation, we identify 64 statistically significant gene-based associations in our meta-analysis of the two cohorts and 37 for phenotypes available in only one cohort. Singletons make significant contributions to our results, and the vast majority of the associations could not have been identified with a genotyping chip. Our results are available for interactive browsing in a webapp (https://ukb.research.helix.com). This comprehensive analysis illustrates the biological value of large, deeply phenotyped cohorts of unselected populations coupled with NGS data.

Evaluation for Genetic Disorders in the Absence of a Clinical Indication for Testing: Elective Genomic Testing.

The increasing quality and diminishing cost of next-generation sequencing has transformed our ability to interrogate large quantities of genetic information. This has led to a dramatic increase in the number of elective genomic tests performed. In this article, elective test denotes a test that a patient chooses to undertake without a clinical indication. The variety of elective genomic testing options is considerable. Because these offerings provide differing levels of sensitivity and specificity, it can be difficult to choose among them. A simple rubric to compare offerings is not readily available. We propose a framework designated completeness that evaluates both analytical and interpretative components of genomic tests. We then illustrate how this framework can be used to evaluate the expanding landscape of elective genomic testing.

Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with "Corner Fractures".

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.

A non-mosaic PORCN mutation in a male with severe congenital anomalies overlapping focal dermal hypoplasia.

Mutations in the PORCN gene cause the X-linked dominant condition focal dermal hypoplasia (FDH). Features of FDH include striated pigmentation of the skin, ocular and skeletal malformations. FDH is generally associated with in utero lethality in non-mosaic males and most of the currently reported male patients show mosaicism due to de novo post-zygotic mutations in the PORCN gene. There is only one previous report of a surviving male with an inherited mutation in the PORCN gene. Here, we report two male siblings with multiple malformations including skeletal, ocular and renal defects overlapping with FDH. A novel PORCN mutation (p.Ser250Phe) was identified in a non-mosaic, hemizygous state in one of the siblings who survived to 8 years of age. The mother is a heterozygous carrier, has a random X-inactivation pattern and is asymptomatic. Findings unusual for FDH include dysplastic clavicles and bilateral Tessier IV facial clefts. This is the second case report of a non-mosaic PORCN mutation in a male individual with multiple congenital anomalies. While the pathogenicity of this mutation remains to be further investigated, the survival of a male with a non-mosaic mutation in PORCN is suggestive of a functionally mild mutation leading to an X-linked recessive mode of inheritance.

Heterozygous variants in ACTL6A, encoding a component of the BAF complex, are associated with intellectual disability.

Pathogenic variants in genes encoding components of the BRG1-associated factor (BAF) chromatin remodeling complex have been associated with intellectual disability syndromes. We identified heterozygous, novel variants in ACTL6A, a gene encoding a component of the BAF complex, in three subjects with varying degrees of intellectual disability. Two subjects have missense variants affecting highly conserved amino acid residues within the actin-like domain. Missense mutations in the homologous region in yeast actin were previously reported to be dominant lethal and were associated with impaired binding of the human ACTL6A to ß-actin and BRG1. A third subject has a splicing variant that creates an in-frame deletion. Our findings suggest that the variants identified in our subjects may have a deleterious effect on the function of the protein by disturbing the integrity of the BAF complex. Thus, ACTL6A gene mutation analysis should be considered in patients with intellectual disability, learning disabilities, or developmental language disorder.

An exome sequencing study of Moebius syndrome including atypical cases reveals an individual with CFEOM3A and a TUBB3 mutation.

Moebius syndrome is characterized by congenital unilateral or bilateral facial and abducens nerve palsies (sixth and seventh cranial nerves) causing facial weakness, feeding difficulties, and restricted ocular movements. Abnormalities of the chest wall such as Poland anomaly and variable limb defects are frequently associated with this syndrome. Most cases are isolated; however, rare families with autosomal dominant transmission with incomplete penetrance and variable expressivity have been described. The genetic basis of this condition remains unknown. In a cohort study of nine individuals suspected to have Moebius syndrome (six typical, three atypical), we performed whole-exome sequencing to try to identify a commonly mutated gene. Although no such gene was identified and we did not find mutations in PLXND1 and REV3L, we found a de novo heterozygous mutation, p.E410K, in the gene encoding tubulin beta 3 class III (TUBB3), in an individual with atypical Moebius syndrome. This individual was diagnosed with near-complete ophthalmoplegia, agenesis of the corpus callosum, and absence of the septum pellucidum. No substantial limb abnormalities were noted. Mutations in TUBB3 have been associated with complex cortical dysplasia and other brain malformations and congenital fibrosis of extraocular muscles type 3A (CFEOM3A). Our report highlights the overlap of genetic etiology and clinical differences between CFEOM and Moebius syndrome and describes our approach to identifying candidate genes for typical and atypical Moebius syndrome.

Loss of DDRGK1 modulates SOX9 ubiquitination in spondyloepimetaphyseal dysplasia.

Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1-/- mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.

Spondylometaphyseal dysplasia (SMD) corner fracture type (also known as SMD "Sutcliffe" type, MIM 184255) is a rare skeletal dysplasia that presents with mild to moderate short stature, developmental coxa vara, mild platyspondyly, corner fracture-like lesions, and metaphyseal abnormalities with sparing of the epiphyses. The molecular basis for this disorder has yet to be clarified. We describe two patients with SMD corner fracture type and heterozygous pathogenic variants in COL2A1. These two cases together with a third case of SMD corner fracture type with a heterozygous COL2A1 pathogenic variant previously described suggest that this disorder overlaps with type II collagenopathies. The finding of one of the pathogenic variants in a previously reported case of spondyloepimetaphyseal dysplasia (SEMD) Strudwick type and the significant clinical similarity suggest an overlap between SMD corner fracture and SEMD Strudwick types. © 2016 Wiley Periodicals, Inc.

Adult presentation of X-linked Conradi-Hünermann-Happle syndrome.

Conradi-Hünermann-Happle syndrome, or X-linked dominant chondrodysplasia punctata type 2 (CDPX2), is a genodermatosis caused by mutations in EBP. While typically lethal in males, females with CDPX2 generally manifest by infancy or childhood with variable features including congenital ichthyosiform erythroderma, chondrodysplasia punctata, asymmetric shortening of the long bones, and cataracts. We present a 36-year-old female with short stature, rhizomelic and asymmetric limb shortening, severe scoliosis, a sectorial cataract, and no family history of CDPX2. Whole exome sequencing (WES) revealed a p.Arg63del mutation in EBP, and biochemical studies confirmed a diagnosis of CDPX2. Short stature in combination with ichthyosis or alopecia, cataracts, and limb shortening in an adult should prompt consideration of a diagnosis of CDPX2. As in many genetic syndromes, the hallmark features of CDPX2 in pediatric patients are not readily identifiable in adults. This demonstrates the utility of WES as a diagnostic tool in the evaluation of adults with genetic disorders.

Genotype-phenotype correlation--promiscuity in the era of next-generation sequencing.

Newly cost-effective next-generation sequencing has led to an explosion of discoveries of novel genetic mutations that reveal the rampant "promiscuity" of genotype-phenotype relationships. Such discoveries should ultimately revolutionize clinical care.

Diagnosis of ALG12-CDG by exome sequencing in a case of severe skeletal dysplasia.

Congenital Disorder of Glycosylation type Ig (ALG12-CDG) is part of a group of autosomal recessive conditions caused by deficiency of proteins involved in the assembly of dolichol-oligosaccharides used for protein N-glycosylation. In ALG12-CDG, the enzyme affected is encoded by the ALG12 gene. Affected individuals present clinically with neurodevelopmental delay, growth retardation, immune deficiency, male genital hypoplasia, and cardiomyopathy. A total of six individuals have been reported in the literature. Here, we present an infant with rhizomelic short stature, talipes equinovarus, platyspondyly, and joint dislocations. The infant had marked underossification of the pubic bones. Exome sequencing was performed and two deletions, each resulting in frameshifts, were found in ALG12. A review of the literature revealed two infants with ALG12-CDG and a severe skeletal dysplasia, including under-ossification of cervical vertebrae, pubic bones, and knees; in addition to talipes equinovarus and rhizomelic short stature. The phenotype of the individual we describe resembles pseudodiastrophic dysplasia and we discuss similarities and differences between ALG12-CDG and pseudodiastrophic dysplasia. The differential diagnosis in selected undiagnosed skeletal dysplasias should include CDGs.

Exome sequencing identifies a novel homozygous mutation in the phosphate transporter SLC34A1 in hypophosphatemia and nephrocalcinosis.

CONTEXT: Two Argentinean siblings (a boy and a girl) from a nonconsanguineous family presented with hypercalcemia, hypercalciuria, hypophosphatemia, low parathyroid hormone (PTH), and nephrocalcinosis. OBJECTIVE: The goal of this study was to identify genetic causes of the clinical findings in the two siblings. DESIGN: Whole exome sequencing was performed to identify disease-causing mutations in the youngest sibling, and a candidate variant was screened in other family members by Sanger sequencing. In vitro experiments were conducted to determine the effects of the mutation that was identified. PATIENTS AND OTHER PARTICIPANTS: Affected siblings (2 y.o. female and 10 y.o male) and their parents were included in the study. Informed consent was obtained for genetic studies. RESULTS: A novel homozygous mutation in the gene encoding the renal sodium-dependent phosphate transporter SLC34A1 was identified in both siblings (c.1484G>A, p.Arg495His). In vitro studies showed that the p.Arg495His mutation resulted in decreased phosphate uptake when compared to wild-type SLC34A1. CONCLUSIONS: The homozygous G>A transition that results in the substitution of histidine for arginine at position 495 of the renal sodium-dependent phosphate transporter, SLC34A1, is involved in disease pathogenesis in these patients. Our report of the second family with two mutated SLC34A1 alleles expands the known phenotype of this rare condition.