Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

Inference of the Genetic Architecture Underlying BMI and Height with the Use of 20,240 Sibling Pairs

Evidence that complex traits are highly polygenic has been presented by population-based genome-wide association studies (GWASs) through the identification of many significant variants, as well as by family-based de novo sequencing studies indicating that several traits have a large mutational target size. Here, using a third study design, we show results consistent with extreme polygenicity for body mass index (BMI) and height. On a sample of 20,240 siblings (from 9,570 nuclear families), we used a within-family method to obtain narrow-sense heritability estimates of 0.42 (SE = 0.17, p = 0.01) and 0.69 (SE = 0.14, p = 6 × 10⁻⁷) for BMI and height, respectively, after adjusting for covariates. The genomic inflation factors from locus-specific linkage analysis were 1.69 (SE = 0.21, p = 0.04) for BMI and 2.18 (SE = 0.21, p = 2 × 10⁻¹⁰) for height. This inflation is free of confounding and congruent with polygenicity, consistent with observations of ever-increasing genomic-inflation factors from GWASs with large sample sizes, implying that those signals are due to true genetic signals across the genome rather than population stratification. We also demonstrate that the distribution of the observed test statistics is consistent with both rare and common variants underlying a polygenic architecture and that previous reports of linkage signals in complex traits are probably a consequence of polygenic architecture rather than the segregation of variants with large effects. The convergent empirical evidence from GWASs, de novo studies, and within-family segregation implies that family-based sequencing studies for complex traits require very large sample sizes because the effects of causal variants are small on average.     

Emergence of an unrelated highly aberrant clone in an AML patient at relapse four months after peripheral blood stem cell transplantation

We report a case of AML-M1 with 5q aberration at diagnosis. The patient was treated with high-dose chemotherapy (HDCT). After remission induction, he received allogenic peripheral blood stem cell transplantation (PBSCT) from an HLA-match donor brother. The successive follow-up conventional cytogenetics investigations in remission after HDCT and PBSCT revealed cytogenetic remission. The most interesting observation in this case is that relapsed marrow revealed the emergence of an entirely new, highly aberrant, unrelated clone with unusual translocations t(6;17)(p23;p11.2),+8,der(8)dup inv(8)(q23qter), t(10;19)(q26;q13.3) 4½ months after PBSCT. Our findings suggest the possibility of a mutagenic effect of HDCT and myeloablative intense chemotherapy before PBSCT that could have induced a genetic lesion in the recipient''s genetically unstable stem cells in an environment of immunosuppression. The highly complex nature of the clone and the rapid clonal evolution indicates the possibility of selective pressure with proliferative advantage.     

Mechanisms of Drosophila Dscam mutually exclusive splicing regulation

Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.     

Evaluating indirect genetic effects of siblings using singletons

Estimating effects of parental and sibling genotypes (indirect genetic effects) can provide insight into how the family environment influences phenotypic variation. There is growing molecular genetic evidence for effects of parental phenotypes on their offspring (e.g. parental educational attainment), but the extent to which siblings affect each other is currently unclear. Here we used data from samples of unrelated individuals, without (singletons) and with biological full-siblings (non-singletons), to investigate and estimate sibling effects. Indirect genetic effects of siblings increase (or decrease) the covariance between genetic variation and a phenotype. It follows that differences in genetic association estimates between singletons and non-singletons could indicate indirect genetic effects of siblings if there is no heterogeneity in other sources of genetic association between singletons and non-singletons. We used UK Biobank data to estimate polygenic score (PGS) associations for height, BMI and educational attainment in self-reported singletons (N = 50,143) and non-singletons (N = 328,549). The educational attainment PGS association estimate was 12% larger (95% C.I. 3%, 21%) in the non-singleton sample than in the singleton sample, but the height and BMI PGS associations were consistent. Birth order data suggested that the difference in educational attainment PGS associations was driven by individuals with older siblings rather than firstborns. The relationship between number of siblings and educational attainment PGS associations was non-linear; PGS associations were 24% smaller in individuals with 6 or more siblings compared to the rest of the sample (95% C.I. 11%, 38%). We estimate that a 1 SD increase in sibling educational attainment PGS corresponds to a 0.025 year increase in the index individual’s years in schooling (95% C.I. 0.013, 0.036). Our results suggest that older siblings may influence the educational attainment of younger siblings, adding to the growing evidence that effects of the environment on phenotypic variation partially reflect social effects of germline genetic variation in relatives.     

Systematic Review of Erythropoietin (EPO) for Neuroprotection in Human Studies

Erythropoietin (EPO) is an exciting neurotherapeutic option. Despite its potential, concerns exist regarding the potential for thrombosis and adverse events with EPO administration in normonemic adults. Systematic review of literature using PRISMA guidelines to examine the application and risks of EPO as a treatment option for neuroprotection in normonemic adults. Independent, systematic searches were performed in July 2019. PubMed (1960–2019) and the Cochrane Controlled Trials Register (1960–2019) were screened. Search terms included erythropoietin, neuroprotection, and humans. The PubMed search resulted in the following search strategy: (“erythropoietin” [MeSH Terms] OR “erythropoietin” [All Fields] OR “epoetin alfa” [MeSH Terms] OR (“epoetin” [All Fields] AND “alfa” [All Fields]) OR “epoetin alfa” [All Fields]) AND (“neuroprotection” [MeSH Terms] OR “neuroprotection” [All Fields]) AND “humans” [MeSH Terms]. PubMed, Cochrane Controlled Trials Register, and articles based on prior searches yielded 388 citations. 50 studies were included, comprising of 4351 patients. There were 13 studies that noted adverse effects from EPO. Three attributed serious adverse effects to EPO and complications were statistically significant. Two of these studies related the adverse events to the co-administration of EPO with tPA. Minor adverse effects associated with the EPO group included nausea, pyrexia, headache, generalized weakness and superficial phlebitis. Most published studies focus on spinal cord injury, peri-surgical outcomes and central effects of EPO. We found no studies to date evaluating the role of EPO in post-operative pain. Future trials could evaluate this application in persistent post-surgical pain and in the peri-operative period.

     

Dominance Genetic Variation Contributes Little to the Missing Heritability for Human Complex Traits

For human complex traits, non-additive genetic variation has been invoked to explain “missing heritability,” but its discovery is often neglected in genome-wide association studies. Here we propose a method of using SNP data to partition and estimate the proportion of phenotypic variance attributed to additive and dominance genetic variation at all SNPs (hSNP2 and δSNP2) in unrelated individuals based on an orthogonal model where the estimate of hSNP2 is independent of that of δSNP2. With this method, we analyzed 79 quantitative traits in 6,715 unrelated European Americans. The estimate of δSNP2 averaged across all the 79 quantitative traits was 0.03, approximately a fifth of that for additive variation (average hSNP2 = 0.15). There were a few traits that showed substantial estimates of δSNP2, none of which were replicated in a larger sample of 11,965 individuals. We further performed genome-wide association analyses of the 79 quantitative traits and detected SNPs with genome-wide significant dominance effects only at the ABO locus for factor VIII and von Willebrand factor. All these results suggest that dominance variation at common SNPs explains only a small fraction of phenotypic variation for human complex traits and contributes little to the missing narrow-sense heritability problem.     

Assortative mating and within-spouse pair comparisons

Spousal comparisons have been proposed as a design that can both reduce confounding and estimate effects of the shared adulthood environment. However, assortative mating, the process by which individuals select phenotypically (dis)similar mates, could distort associations when comparing spouses. We evaluated the use of spousal comparisons, as in the within-spouse pair (WSP) model, for aetiological research such as genetic association studies. We demonstrated that the WSP model can reduce confounding but may be susceptible to collider bias arising from conditioning on assorted spouse pairs. Analyses using UK Biobank spouse pairs found that WSP genetic association estimates were smaller than estimates from random pairs for height, educational attainment, and BMI variants. Within-sibling pair estimates, robust to demographic and parental effects, were also smaller than random pair estimates for height and educational attainment, but not for BMI. WSP models, like other within-family models, may reduce confounding from demographic factors in genetic association estimates, and so could be useful for triangulating evidence across study designs to assess the robustness of findings. However, WSP estimates should be interpreted with caution due to potential collider bias.