An efficient genotyper and star-allele caller for pharmacogenomics.

High-throughput sequencing provides sufficient means for determining genotypes of clinically important pharmacogenes that can be used to tailor medical decisions to individual patients. However, pharmacogene genotyping, also known as star-allele calling, is a challenging problem that requires accurate copy number calling, structural variation identification, variant calling, and phasing within each pharmacogene copy present in the sample. Here we introduce Aldy 4, a fast and efficient tool for genotyping pharmacogenes that uses combinatorial optimization for accurate star-allele calling across different sequencing technologies. Aldy 4 adds support for long reads and uses a novel phasing model and improved copy number and variant calling models. We compare Aldy 4 against the current state-of-the-art star-allele callers on a large and diverse set of samples and genes sequenced by various sequencing technologies, such as whole-genome and targeted Illumina sequencing, barcoded 10x Genomics, and Pacific Biosciences (PacBio) HiFi. We show that Aldy 4 is the most accurate star-allele caller with near-perfect accuracy in all evaluated contexts, and hope that Aldy remains an invaluable tool in the clinical toolbox even with the advent of long-read sequencing technologies.

Two epilepsy-associated variants in KCNA2 (KV 1.2) at position H310 oppositely affect channel functional expression.

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV 1.2 by KV ß1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of KV 1.2 voltage-activated, K+ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels. The p.H310Y variant caused 'dual gain of function', increasing both KV 1.2-channel activity and the number of KV 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of KV 1.2 by KV ß1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of KV 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.

Human Mutation special issue on innovations in genomic diagnostics.

This special issue of Human Mutation focuses on Innovations in Genomic Diagnostics. The increasing interest in genomic medicine, and the growing possibilities for treatment and management of genetic disease, make complete and accurate diagnosis mission critical. This issue describes leading-edge technologies with emerging utility for genomic diagnostics. Genomic testing has dramatically evolved as a result of advances in technology, data analytics, and the continuing pace of disease gene discovery. Since 2011, clinical laboratories have increasingly employed next-generation sequencing-based tests in addition to historical techniques to identify a spectrum of germline and somatic variants implicated in human disease. However, common testing platforms have known limitations, including failure to detect disease-causing variants in certain regions, inability to identify all variant types, variant phasing, measuring epigenetic changes, and ongoing challenges with variant interpretation. Innovative solutions are emerging, including increasingly rapid genome sequencing, long-read sequencing, clinical RNA sequencing, epigenomic profiling, facial phenotyping, and an array of computational tools for variant identification and interpretation.

Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Attitudes on pharmacogenomic results as secondary findings among medical geneticists.

OBJECTIVES: As evidence mounts supporting the utility of pharmacogenomic-guided medication management, incorporating pharmacogenomic genes into secondary finding results from sequencing panels is increasingly under consideration. We studied medical geneticists' attitudes on receiving pharmacogenomic results as secondary finding. METHODS: Four focus groups with 16 medical geneticists total were conducted followed by thematic analysis. RESULTS: All participants ordered genetic sequencing tests; however, the majority had rarely or never ordered pharmacogenomic tests (10/16) or prescribed medications with established response variability (11/16). In total 81.3% expressed low comfort interpreting pharmacogenomic results without appropriate clinical resources (13/16). The positives of receiving pharmacogenomic results as secondary finding included prevention of adverse drug reactions in adults, grateful information-seeking patients, the ability to rapidly prescribe more effective treatments and appreciation of the recent advances in both pharmacogenomic knowledge and available guidelines. Negatives included laboratory reporting issues, exclusivity of pharmacogenomic results to certain populations, lengthy reports concealing pharmacogenomic results in patient charts and laboratories marketing to individuals without prior pharmacogenomic knowledge or targeting inappropriate populations. The most desirable pharmacogenomic resources included a universal electronic health record clinical decision support tool to assist identifying and implementing pharmacogenomic results, a specialized pharmacist as part of the care team, additional pharmacogenomic training during medical/graduate school, and a succinct interpretation of pharmacogenomic results included on laboratory reports. CONCLUSIONS: The majority of participants agreed that adding certain actionable pharmacogenomic genes to the American College of Medical Genetics and Genomics SF list is reasonable; however, this was qualified with a need for additional resources to support implementation.

Clinical pharmacogenomic testing and reporting: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Pharmacogenomic testing interrogates germline sequence variants implicated in interindividual drug response variability to infer a drug response phenotype and to guide medication management for certain drugs. Specifically, discrete aspects of pharmacokinetics, such as drug metabolism, and pharmacodynamics, as well as drug sensitivity, can be predicted by genes that code for proteins involved in these pathways. Pharmacogenomics is unique and differs from inherited disease genetics because the drug response phenotype can be drug-dependent and is often unrecognized until an unexpected drug reaction occurs or a patient fails to respond to a medication. Genes and variants with sufficiently high levels of evidence and consensus may be included in a clinical pharmacogenomic test; however, result interpretation and phenotype prediction can be challenging for some genes and medications. This document provides a resource for laboratories to develop and implement clinical pharmacogenomic testing by summarizing publicly available resources and detailing best practices for pharmacogenomic nomenclature, testing, result interpretation, and reporting.

Pharmacogenomic education among genetic counseling training programs in North America.

The increasing number of genetic counselors participating directly in clinical pharmacogenomic post-test counseling prompted our evaluation of pharmacogenomic education across genetic counseling training programs in North America. Thirty-one program leadership participants from both the United States (U.S.) and Canada responded to a survey assessing pharmacogenomics education and the role of genetic counselors. Eighty-five percent of respondents agreed pharmacogenomics is currently within the scope of genetic counseling practice, and 96.3% indicated their training programs currently provide education on pharmacogenomics, with the majority reporting < 7 hr of education. Lectures on pharmacogenomics were the most common method for didactics; however, some programs also included practical modalities (e.g., case studies, clinical rotations) and online resources. Barriers to expanding pharmacogenomic education included the constrained timeline of training, and lack of resources and local expertise. Moreover, participants suggested that genetic counselors ideally should be able to order pharmacogenomic tests and counsel patients on pharmacogenomics, including result interpretation, as they believe pharmacogenomics does fall within the scope of practice of genetic counseling. Our novel results also confirm that training program leadership support a pharmacogenomic service delivery model that includes a combined effort between genetic counselors and pharmacists to utilize their synergistic expertise. However, this model likely still necessitates expanding pharmacogenomic didactics in genetic counseling training programs through more practical training and/or by leveraging online pharmacogenomic courses dedicated to supporting clinical implementation.

Knowledge and attitudes on pharmacogenetics among pediatricians.

Increasing enthusiasm for clinical pharmacogenetic testing and the availability of pharmacogenetic-based guidelines indicate that pediatricians will increasingly be expected to interpret and apply pharmacogenetic test results into medical care. Previous studies have identified a lack of knowledge on pharmacogenetics across many physician specialties; however, this has not been systematically assessed among pediatricians. To evaluate pediatrician knowledge, attitude, and educational interest in pharmacogenetics, we surveyed physician cohorts from both the United States (U.S.) and Japan. A total of 282 pediatricians (210 from the U.S. and 72 from Japan) participated in an anonymous survey (online or hardcopy) on pharmacogenetics knowledge, perception, and education. Over 50% of all respondents had >10 years of clinical experience and >75% had some prior education in genetics. However, <10% felt they were familiar with pharmacogenetics, which was very consistent with <20% of the U.S. pediatricians correctly responding to a codeine/CYP2D6 pharmacogenetics knowledge question and <10% of U.S. pediatricians being aware of the Clinical Pharmacogenetics Implementation Consortium (CPIC). Despite being generally unfamiliar with pharmacogenetics, >80% of all respondents indicated that implementation of clinical pharmacogenetic testing will improve efficacy and safety, and that pediatricians should be capable of applying this testing to their practice. Moreover, the majority (83.1%) were interested in educational opportunities on pharmacogenetics, particularly on result interpretation and therapeutic recommendations. Taken together, these data indicate that although practical knowledge of pharmacogenetics among pediatricians in the U.S. and Japan is currently very low, their interest in clinical pharmacogenetics and related education is high, which will likely facilitate future implementation.

Haploinsufficiency of the basic helix-loop-helix transcription factor HAND2 causes congenital heart defects.

Congenital heart defects (CHDs) are caused by a disruption in heart morphogenesis, which is dependent, in part, on a network of transcription factors (TFs) that regulate myocardial development. Heterozygous sequence variants in the basic helix-loop-helix TF gene heart and neural crest derivatives expressed 2 (HAND2) have been reported among some patients with CHDs; however, HAND2 has not yet been established as a Mendelian disease gene. We report a 31-month-old male with unicommissural unicuspid aortic valve, moderate aortic stenosis, and mild pulmonic stenosis. Chromosome analysis revealed a normal 46,XY karyotype, and a CHD sequencing panel was negative for pathogenic variants in NKX2.5, GATA4, TBX5, and CHD7. However, chromosomal microarray (CMA) testing identified a heterozygous 546.0-kb deletion on chromosome 4q34.1 (174364195_174910239[GRCh37/hg19]) that included exons 1 and 2 of SCRG1, HAND2, and HAND2-AS1. Familial CMA testing determined that the deletion was paternally inherited, which supported a likely pathogenic classification as the proband's father had previously undergone surgery for Tetralogy of Fallot. The family history was also notable for a paternal uncle who had previously died from complications related to an unknown heart defect. Taken together, this first report of a HAND2 and HAND2-AS1 deletion in a family with CHDs strongly supports haploinsufficiency of HAND2 as an autosomal dominant cause of CHD.

Structural variation at the CYP2C locus: Characterization of deletion and duplication alleles.

The human CYP2C locus harbors the polymorphic CYP2C18, CYP2C19, CYP2C9, and CYP2C8 genes, and of these, CYP2C19 and CYP2C9 are directly involved in the metabolism of ~15% of all medications. All variant CYP2C19 and CYP2C9 star (*) allele haplotypes currently cataloged by the Pharmacogene Variation (PharmVar) Consortium are defined by sequence variants. To determine if structural variation also occurs at the CYP2C locus, the 10q23.33 region was interrogated across deidentified clinical chromosomal microarray (CMA) data from 20,642 patients tested at two academic medical centers. Fourteen copy number variants that affected the coding region of CYP2C genes were detected in the clinical CMA cohorts, which ranged in size from 39.2 to 1,043.3 kb. Selected deletions and duplications were confirmed by MLPA or ddPCR. Analysis of the clinical CMA and an additional 78,839 cases from the Database of Genomic Variants (DGV) and ClinGen (total n = 99,481) indicated that the carrier frequency of a CYP2C structural variant is ~1 in 1,000, with ~1 in 2,000 being a CYP2C19 full gene or partial-gene deletion carrier, designated by PharmVar as CYP2C19*36 and *37, respectively. Although these structural variants are rare in the general population, their detection will likely improve metabolizer phenotype prediction when interrogated for research and/or clinical testing.

Multi-site investigation of strategies for the clinical implementation of CYP2D6 genotyping to guide drug prescribing.

PURPOSE: A number of institutions have clinically implemented CYP2D6 genotyping to guide drug prescribing. We compared implementation strategies of early adopters of CYP2D6 testing, barriers faced by both early adopters and institutions in the process of implementing CYP2D6 testing, and approaches taken to overcome these barriers. METHODS: We surveyed eight early adopters of CYP2D6 genotyping and eight institutions in the process of adoption. Data were collected on testing approaches, return of results procedures, applications of genotype results, challenges faced, and lessons learned. RESULTS: Among early adopters, CYP2D6 testing was most commonly ordered to assist with opioid and antidepressant prescribing. Key differences among programs included test ordering and genotyping approaches, result reporting, and clinical decision support. However, all sites tested for copy-number variation and nine common variants, and reported results in the medical record. Most sites provided automatic consultation and had designated personnel to assist with genotype-informed therapy recommendations. Primary challenges were related to stakeholder support, CYP2D6 gene complexity, phenotype assignment, and sustainability. CONCLUSION: There are specific challenges unique to CYP2D6 testing given the complexity of the gene and its relevance to multiple medications. Consensus lessons learned may guide those interested in pursuing similar clinical pharmacogenetic programs.

Standardizing terms for clinical pharmacogenetic test results: consensus terms from the Clinical Pharmacogenetics Implementation Consortium (CPIC).

INTRODUCTION: Reporting and sharing pharmacogenetic test results across clinical laboratories and electronic health records is a crucial step toward the implementation of clinical pharmacogenetics, but allele function and phenotype terms are not standardized. Our goal was to develop terms that can be broadly applied to characterize pharmacogenetic allele function and inferred phenotypes. MATERIALS AND METHODS: Terms currently used by genetic testing laboratories and in the literature were identified. The Clinical Pharmacogenetics Implementation Consortium (CPIC) used the Delphi method to obtain a consensus and agree on uniform terms among pharmacogenetic experts. RESULTS: Experts with diverse involvement in at least one area of pharmacogenetics (clinicians, researchers, genetic testing laboratorians, pharmacogenetics implementers, and clinical informaticians; n = 58) participated. After completion of five surveys, a consensus (>70%) was reached with 90% of experts agreeing to the final sets of pharmacogenetic terms. DISCUSSION: The proposed standardized pharmacogenetic terms will improve the understanding and interpretation of pharmacogenetic tests and reduce confusion by maintaining consistent nomenclature. These standard terms can also facilitate pharmacogenetic data sharing across diverse electronic health care record systems with clinical decision support.Genet Med 19 2, 215-223.

Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6.

The cytochrome P450-2D6 (CYP2D6) enzyme metabolizes ∼25% of common medications, yet homologous pseudogenes and copy number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant-calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run nonreference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement. Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis, and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

In situ studies of materials for high temperature CO2 capture and storage.

Carbon capture and storage (CCS) offers a possible solution to curb the CO2 emissions from stationary sources in the coming decades, considering the delays in shifting energy generation to carbon neutral sources such as wind, solar and biomass. The most mature technology for post-combustion capture uses a liquid sorbent, amine scrubbing. However, with the existing technology, a large amount of heat is required for the regeneration of the liquid sorbent, which introduces a substantial energy penalty. The use of alternative sorbents for CO2 capture, such as the CaO-CaCO3 system, has been investigated extensively in recent years. However there are significant problems associated with the use of CaO based sorbents, the most challenging one being the deactivation of the sorbent material. When sorbents such as natural limestone are used, the capture capacity of the solid sorbent can fall by as much as 90 mol% after the first 20 carbonation-regeneration cycles. In this study a variety of techniques were employed to understand better the cause of this deterioration from both a structural and morphological standpoint. X-ray and neutron PDF studies were employed to understand better the local surface and interfacial structures formed upon reaction, finding that after carbonation the surface roughness is decreased for CaO. In situ synchrotron X-ray diffraction studies showed that carbonation with added steam leads to a faster and more complete conversion of CaO than under conditions without steam, as evidenced by the phases seen at different depths within the sample. Finally, in situ X-ray tomography experiments were employed to track the morphological changes in the sorbents during carbonation, observing directly the reduction in porosity and increase in tortuosity of the pore network over multiple calcination reactions.

Association of Arrhythmia-Related Genetic Variants With Phenotypes Documented in Electronic Medical Records.

IMPORTANCE: Large-scale DNA sequencing identifies incidental rare variants in established Mendelian disease genes, but the frequency of related clinical phenotypes in unselected patient populations is not well established. Phenotype data from electronic medical records (EMRs) may provide a resource to assess the clinical relevance of rare variants. OBJECTIVE: To determine the clinical phenotypes from EMRs for individuals with variants designated as pathogenic by expert review in arrhythmia susceptibility genes. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study included 2022 individuals recruited for nonantiarrhythmic drug exposure phenotypes from October 5, 2012, to September 30, 2013, for the Electronic Medical Records and Genomics Network Pharmacogenomics project from 7 US academic medical centers. Variants in SCN5A and KCNH2, disease genes for long QT and Brugada syndromes, were assessed for potential pathogenicity by 3 laboratories with ion channel expertise and by comparison with the ClinVar database. Relevant phenotypes were determined from EMRs, with data available from 2002 (or earlier for some sites) through September 10, 2014. EXPOSURES: One or more variants designated as pathogenic in SCN5A or KCNH2. MAIN OUTCOMES AND MEASURES: Arrhythmia or electrocardiographic (ECG) phenotypes defined by International Classification of Diseases, Ninth Revision (ICD-9) codes, ECG data, and manual EMR review. RESULTS: Among 2022 study participants (median age, 61 years [interquartile range, 56-65 years]; 1118 [55%] female; 1491 [74%] white), a total of 122 rare (minor allele frequency <0.5%) nonsynonymous and splice-site variants in 2 arrhythmia susceptibility genes were identified in 223 individuals (11% of the study cohort). Forty-two variants in 63 participants were designated potentially pathogenic by at least 1 laboratory or ClinVar, with low concordance across laboratories (Cohen κ = 0.26). An ICD-9 code for arrhythmia was found in 11 of 63 (17%) variant carriers vs 264 of 1959 (13%) of those without variants (difference, +4%; 95% CI, -5% to +13%; P = .35). In the 1270 (63%) with ECGs, corrected QT intervals were not different in variant carriers vs those without (median, 429 vs 439 milliseconds; difference, -10 milliseconds; 95% CI, -16 to +3 milliseconds; P = .17). After manual review, 22 of 63 participants (35%) with designated variants had any ECG or arrhythmia phenotype, and only 2 had corrected QT interval longer than 500 milliseconds. CONCLUSIONS AND RELEVANCE: Among laboratories experienced in genetic testing for cardiac arrhythmia disorders, there was low concordance in designating SCN5A and KCNH2 variants as pathogenic. In an unselected population, the putatively pathogenic genetic variants were not associated with an abnormal phenotype. These findings raise questions about the implications of notifying patients of incidental genetic findings.

Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

BACKGROUND: DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS). RESULTS: Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625-1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r=0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r=0.906; 42 CpG sites) and second generation sequencing (r=0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r=0.836-0.897 and r=0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r=0.940-0.951 and r=0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702-866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines. CONCLUSIONS: SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards unmethylated DNA should be considered when measuring intermediately methylated regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.

Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.

Reducing excessive light harvesting in photosynthetic organisms may increase biomass yields by limiting photoinhibition and increasing light penetration in dense cultures. The cyanobacterium Synechocystis sp. PCC 6803 harvests light via the phycobilisome, which consists of an allophycocyanin core and six radiating rods, each with three phycocyanin (PC) discs. Via targeted gene disruption and alterations to the promoter region, three mutants with two (pcpcT→C) and one (ΔCpcC1C2:pcpcT→C) PC discs per rod or lacking PC (olive) were generated. Photoinhibition and chlorophyll levels decreased upon phycobilisome reduction, although greater penetration of white light was observed only in the PC-deficient mutant. In all strains cultured at high cell densities, most light was absorbed by the first 2 cm of the culture. Photosynthesis and respiration rates were also reduced in the ΔCpcC1C2:pcpcT→C and olive mutants. Cell size was smaller in the pcpcT→C and olive strains. Growth and biomass accumulation were similar between the wild-type and pcpcT→C under a variety of conditions. Growth and biomass accumulation of the olive mutant were poorer in carbon-saturated cultures but improved in carbon-limited cultures at higher light intensities, as they did in the ΔCpcC1C2:pcpcT→C mutant. This study shows that one PC disc per rod is sufficient for maximal light harvesting and biomass accumulation, except under conditions of high light and carbon limitation, and two or more are sufficient for maximal oxygen evolution. To our knowledge, this study is the first to measure light penetration in bulk cultures of cyanobacteria and offers important insights into photobioreactor design.

Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study.

BACKGROUND: VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans. METHODS: We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 -1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10(-8) in the discovery cohort and p<0·0038 in the replication cohort. FINDINGS: The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10(-8)). This association was confirmed in the replication cohort (p=5·04×10(-5)); analysis of the two cohorts together produced a p value of 4·5×10(-12). Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement). INTERPRETATION: A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population. FUNDING: National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.

An Ashkenazi Jewish SMN1 haplotype specific to duplication alleles improves pan-ethnic carrier screening for spinal muscular atrophy.

PURPOSE: Spinal muscular atrophy is a common autosomal-recessive disorder caused by mutations of the SMN1 gene. Spinal muscular atrophy carrier screening uses dosage-sensitive methods that determine SMN1 copy number, and the frequency of carriers varies by ethnicity, with detection rates ranging from 71 to 94% due to the inability to identify silent (2 + 0) carriers with two copies of SMN1 on one chromosome 5 and deletion on the other. We hypothesized that identification of deletion and/or duplication founder alleles might provide an approach to identify silent carriers in various ethnic groups. METHODS: SMN1 founder alleles were investigated in the Ashkenazi Jewish population by microsatellite analysis and next-generation sequencing. RESULTS: An extended haplotype block, specific to Ashkenazi Jewish SMN1 duplications, was identified by microsatellite analysis, and next-generation sequencing of SMN1 further defined a more localized haplotype. Of note, six novel SMN1 sequence variants were identified that were specific to duplications and not present on single-copy alleles. The haplotype was also identified on SMN1 duplication alleles in additional ethnic groups. CONCLUSION: Identification of these novel variants in an individual with two copies of SMN1 significantly improves the accuracy of residual risk estimates and has important implications for spinal muscular atrophy carrier screening.

Frequency of the cholesteryl ester storage disease common LIPA E8SJM mutation (c.894G>A) in various racial and ethnic groups.

UNLABELLED: Cholesteryl ester storage disease (CESD) and Wolman disease are autosomal recessive later-onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ∼3%-5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African-American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ∼1 in 300, respectively. No African-American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% confidence interval [CI]: 51%-69%) of reported mutations among multiethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ∼0.8 per 100,000 (∼1 in 130,000; 95% CI: ∼1 in 90,000 to 1 in 170,000). CONCLUSION: These data indicate that CESD may be underdiagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full-gene LIPA sequencing to determine heterozygote frequencies, since c.894G>A is not common in these racial groups.