Genome-wide association study of multiethnic non-syndromic orofacial cleft families identifies novel loci specific to family and phenotypic subtypes

Orofacial clefts (OFCs) are among the most common craniofacial birth defects and constitute a high public health burden around the world. OFCs are phenotypically heterogeneous, affecting only the lip, only the palate, or involving both the lip and palate. Cleft palate alone is demonstrably a genetically distinct abnormality from OFCs that involve the lip, therefore, it is common to study cleft lip (CL) in combination with cleft lip plus cleft palate (CLP) as a phenotypic group (i.e. cleft lip with or without cleft palate, CL/P), usually considering CLP to be a clinically more severe form of CL. However, even within CL/P, important genetic differences among subtypes may be present. The Pittsburgh Orofacial Cleft (Pitt-OFC) multiethnic study is a rich resource for the study of non-syndromic OFC, comprising a large number of families (~12,000 individuals) from multiple populations worldwide: US and Europe (whites), Central and South America (mixed Native American, European and African), Asia, and Africa. In this study we focused on the CL/P families from this resource grouped into three non-overlapping family types: those with only CL affected members, only CLP affected members, or both CL and CLP. In all, seven total subtypes besides the combined CL/P phenotype, were defined based on the cleft type(s) that were present within pedigree members. The full sample for these analyses includes 2,218 CL and CLP cases along with 4,537 unaffected relatives, as well as 2,673 pure controls with no family history of OFC. Genome-wide association analyses were conducted within each subset, as well as the combined sample. Five novel genome-wide significant associations were observed: 3q29 (rs62284390, p=2.70E-08), 5p13.2 (rs609659, p= 4.57E-08), 7q22.1 (rs6465810, p= 1.25E-08), 19p13.3 (rs628271, p=1.90E-08) and 20q13.33 (rs2427238, p=1.51E-09). In addition, five significant and four suggestive associations confirmed regions previously published as OFC risk loci - PAX7 , IRF6 , FAM49A , DCAF4L2 , 8q24.21, ARID3B, NTN1 , TANC2 and the WNT9B:WNT3 gene cluster. At each of these loci, we compared effect sizes of associated SNPs observed across subtypes and the full sample, and found that certain loci were associated with a specific cleft type, and/or specific family types. Our findings indicate that risk factors differ between cleft and family types, but each cleft type also exhibits some degree of genetic heterogeneity.

Subspecies Variation of Daucus carota Coastal (“Gummifer”) Morphotypes (Apiaceae) Using Genotyping-by-Sequencing

Abstract—The genus Daucus is widely distributed worldwide, but with a concentration of diversity in the Mediterranean Region. The D. carota complex presents the greatest taxonomic problems in the genus. We focus on a distinctive phenotypic group of coastal morphotypes of D. carota, strictly confined to the margins to within about 0.5 km of the Mediterranean Sea and the Atlantic Ocean, which we here refer to as coastal morphotypes or D. carota subsp. “gummifer” complex. They are loosely morphologically coherent, sharing a relatively short stature, thick, broad, sometimes highly glossy leaf segments, and usually flat or convex fruiting umbels. We analyzed 288 accessions obtained from genebanks in England, France, and the USA, and an expedition to Spain in 2016, covering the Mediterranean and Atlantic coasts and Balearic Islands, where much of the gummifer complex variation occurs. Our study includes 112 accessions not examined before in this context. Genotyping-by-sequencing identified 29,041 filtered SNPs. Based on high bootstrap support from maximum likelihood and Structure analysis we highlight three main clades. The gummifer morphotypes are intercalated with members of Daucus carota subspecies carota and subspecies maximus in two of these main clades, including a clade containing accessions from Tunisia (also including D. carota subsp. capillifolius) and a clade containing accessions from western Europe (including the British Isles), southern Europe (including the Balearic Islands and the Iberian Peninsula) and Morocco. These results support five independent selections of the gummifer morphotypes in these restricted maritime environments in the Mediterranean and nearby Atlantic coasts. Daucus annuus (=Tornabenea annua) and Daucus tenuissimus (=Tornabenea tenuissima) also fall firmly within D. carota, supporting their classification as morphologically well-defined subspecies of D. carota, which are accepted here under the new combinations Daucus carota subsp. annuus and D. carota subsp. tenuissimus, respectively. Types are indicated for most of treated names, including designation of four lectotypes and three epitypes, which fix their further use.

Postembryonic screen for mutations affecting spine development in zebrafish

The spinal vertebral column gives structural support for the adult body plan, protects the spinal cord, and provides muscle attachment and stability, which allows the animal to move within its environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant mutant zebrafish, displaying overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1,229 F1 animals, and subsequently bred to homozygosity in F3 families, from these, 314 haploid genomes were screened for recessive phenotypes. We cumulatively found 39 adult-viable (3 dominant and 36 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing within this phenotypic group revealed at least 16 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report several recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Many of the alleles characterized thus far are non-synonymous mutations in known essential scospondin and adamts9 genes. Our results provide evidence of monogenic traits that are critical for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

Optimized grouping to increase accuracy of prediction of breeding values based on group records in genomic selection breeding programs

Background Phenotypic records of group means or group sums are a good alternative to individual records for some difficult to measure, but economically important traits such as feed efficiency or egg production. Accuracy of predicted breeding values based on group records increases with increasing relationships between group members. The classical way to form groups with more closely-related animals is based on pedigree information. When genotyping information is available before phenotyping, its use to form groups may further increase the accuracy of prediction from group records. This study analyzed two grouping methods based on genomic information: (1) unsupervised clustering implemented in the STRUCTURE software and (2) supervised clustering that models genomic relationships. Results Using genomic best linear unbiased prediction (GBLUP) models, estimates of the genetic variance based on group records were consistent with those based on individual records. When genomic information was available to constitute the groups, genomic relationship coefficients between group members were higher than when random grouping of paternal half-sibs and of full-sibs was applied. Grouping methods that are based on genomic information resulted in higher accuracy of genomic estimated breeding values (GEBV) prediction compared to random grouping. The increase was ~ 1.5% for full-sibs and ~ 11.5% for paternal half-sibs. In addition, grouping methods that are based on genomic information led to lower coancestry coefficients between the top animals ranked by GEBV. Of the two proposed methods, supervised clustering was superior in terms of accuracy, computation requirements and applicability. By adding surplus genotyped offspring (more genotyped offspring than required to fill the groups), the advantage of supervised clustering increased by up to 4.5% compared to random grouping of full-sibs, and by 14.7% compared to random grouping of paternal half-sibs. This advantage also increased with increasing family sizes or decreasing genome sizes. Conclusions The use of genotyping information for grouping animals increases the accuracy of selection when phenotypic group records are used in genomic selection breeding programs.

Absolute Blood Eosinophil Counts to Guide Inhaled Corticosteroids Therapy Among Patients with COPD: Systematic Review and Meta-analysis

Introduction: The Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2019 recommends the use of absolute blood eosinophil count as a guide for the escalation and de-escalation of inhaled corticosteroids (ICS) in the pharmacological management of patients with chronic obstructive pulmonary disease (COPD). We evaluated the risk of moderate or severe exacerbations among patients escalating and de-escalating ICS therapy by absolute blood eosinophil thresholds in this systematic review. Methods: Through a comprehensive literature search of Pubmed/MEDLINE, EMBASE, and clinical trial sites up to April 2019, we identified relevant studies. We used generic inverse variance method with fixed-effects estimates to compare the risk of moderate or severe exacerbations among COPD patients with elevated blood eosinophil counts exposed to inhaled corticosteroids (ICS) versus non-ICS treatments groups expressed as risk ratios. Results: Ten studies (8 randomised control trials and 2 observational studies) were included, with a total of 85,059 COPD patients. In our pooled analysis, we found an overall reduction in risk of moderate or severe exacerbations in patients with absolute blood eosinophil thresholds ranging from ≥ 100 to ≥ 340 cells/µL among patients escalating ICS (RR, 0.77, 95% CI, 0.73-0.81). For studies evaluating the effects of de-escalation of ICS on moderate to severe exacerbations using blood eosinophil thresholds of ≥ 300 to ≥ 340 cells/µL had an increased risk of moderate or severe exacerbations following the de-escalation of ICS (RR, 1.66, 95% CI, 1.31-2.10). Conclusion: This study confirms the validity of the recommended absolute blood eosinophil count thresholds for the escalation and de-escalation of ICS among COPD patients. However, this recommendation is for COPD patients with prior exacerbations rather than among newly diagnosed COPD patients as observed in this study. COPD patients with current or past history of asthma represent a unique phenotypic group which should be further evaluated.

iSeqQC: A Tool for Expression-Based Quality Control in RNA Sequencing

ABSTRACTQuality Control in any high-throughput sequencing technology is a critical step, which if overlooked can compromise the data. A number of methods exist to identify biases during sequencing or alignment, yet not many tools exist to interpret biases due to outliers or batch effects. Hence, we developed iSeqQC, an expression-based QC tool that detects outliers either produced by batch effects due to laboratory conditions or due to dissimilarity within a phenotypic group. iSeqQC implements various statistical approaches including unsupervised clustering, agglomerative hierarchical clustering and correlation coefficients to provide insight into outliers. It can be utilized either through command-line (Github: https://github.com/gkumar09/iSeqQC) or web-interface (http://cancerwebpa.jefferson.edu/iSeqQC). iSeqQC is a fast, light-weight, expression-based QC tool that detects outliers by implementing various statistical approaches.

Establishing the phenotypic basis of adherent-invasive Escherichia coli (AIEC) pathogenicity in intestinal inflammation

Background & AimsAdherent-invasive Escherichia coli (AIEC) are enriched in ileal Crohn’s disease patients and implicated in disease etiology. However, AIEC pathogenesis is poorly understood, and it is unclear if the expansion of these organisms contributes to inflammatory bowel disease (IBD). Questions also remain as to what extent the various in vitro phenotypes used to classify AIEC are pathologically relevant.MethodsWe utilized a combination of in vitro phenotyping and a murine model of intestinal inflammation to systematically relate AIEC phenotypes to pathogenicity for 30 mucosa-associated human-derived E. coli strains. In vitro assays used included survival/replication in and TNF-α production by J774 macrophages as well as invasion/replication in Caco2 intestinal epithelial cells.ResultsAIEC do not form a phenotypic group that is clearly separated from non-AIEC. However, E. coli strains displaying in vitro AIEC phenotypes caused, on average, more severe intestinal inflammation. Survival/replication of strains in J774 and Caco2 cells were positively correlated with disease in vivo, while adherence to Caco2 cells and TNF-α production by J774 cells were not. Importantly, co-colonization with adherent non-AIEC strains ameliorated AIEC-mediated disease.ConclusionOur findings do not support the existence of an AIEC pathovar that can be clearly separated from commensal E. coli. However, intracellular survival/replication phenotypes do contribute to murine intestinal inflammation, suggesting that the AIEC overgrowth observed in human IBD makes a causal contribution to disease. The ability to differentiate pathologically-relevant AIEC phenotypes from those that are not provides an important foundation for developing strategies to predict, diagnose and treat human IBD through characterizing and modulating patient E. coli populations.

Efficacy of antiparasitic drugs in control of gastrointestinal helminthiasis in naturally colored and white lambs from southern Brazil

Parasitic infections and the efficiency of different drugs were evaluated considering efficacy percentages, reduction in count of eggs per gram of feces (EPG) and larval culture in 60, 30 white and 30 naturally colored Corriedale lambs. Each phenotypic group was subdivided into six groups of five animals, each of which was administered one of the active ingredients: albendazole, levamisole hydrochloride, dysophenol, nitroxynil and moxidectin, in addition to a control group, unmedicated. Helminths were considered susceptible to nitroxinil and resistant to albendazole, levamisole, moxidectin (moderately effective) and to dysophenol (insufficiently active). In the culture of larvae, levamisole, nitroxinil and moxidectin were totally efficient (100%) against Oesophagostomum spp., moxidectin was totally efficient against Trichostrongylus spp. and nitroxinil was efficient (97%) against Haemonchus spp. Evaluating the EPG means of the phenotypic groups, it was observed higher parasitic infections in white ewes (p<0.05), in pre- and post-treatment. Therefore, it was concluded that the drug nitroxinil was the only effective for the reduction of the total parasite load of both phenotypic groups, being indicated for use in the herd in question. The genus Haemonchus spp. was the most prevalent and resistant to the drug action. Naturally colored Corriedale lambs, from the herd, showed increased resistance to gastrointestinal nematode infections.

Effect of communicating phenotypic and genetic risk of coronary heart disease alongside web-based lifestyle advice: the INFORM Randomised Controlled Trial

ObjectiveTo determine whether provision of web-based lifestyle advice and coronary heart disease risk information either based on phenotypic characteristics or phenotypic plus genetic characteristics affects changes in objectively measured health behaviours.MethodsA parallel-group, open randomised trial including 956 male and female blood donors with no history of cardiovascular disease (mean [SD] age=56.7 [8.8] years) randomised to four study groups: control group (no information provided); web-based lifestyle advice only (lifestyle group); lifestyle advice plus information on estimated 10-year coronary heart disease risk based on phenotypic characteristics (phenotypic risk estimate) (phenotypic group) and lifestyle advice plus information on estimated 10-year coronary heart disease risk based on phenotypic (phenotypic risk estimate) and genetic characteristics (genetic risk estimate) (genetic group). The primary outcome was change in physical activity from baseline to 12 weeks assessed by wrist-worn accelerometer.Results928 (97.1%) participants completed the trial. There was no evidence of intervention effects on physical activity (difference in adjusted mean change from baseline): lifestyle group vs control group 0.09 milligravity (mg) (95% CI −1.15 to 1.33); genetic group vs phenotypic group −0.33 mg (95% CI −1.55 to 0.90); phenotypic group and genetic group vs control group −0.52 mg (95% CI −1.59 to 0.55) and vs lifestyle group −0.61 mg (95% CI −1.67 to 0.46). There was no evidence of intervention effects on secondary biological, emotional and health-related behavioural outcomes except self-reported fruit and vegetable intake.ConclusionsProvision of risk information, whether based on phenotypic or genotypic characteristics, alongside web-based lifestyle advice did not importantly affect objectively measured levels of physical activity, other health-related behaviours, biological risk factors or emotional well-being.Trial registration numberISRCTN17721237; Pre-results.

pathVar:a new method for pathway-based interpretation of gene expression variability

Identifying the pathways that control a cellular phenotype is the first step to building a mechanistic model. Recent examples in developmental biology, cancer genomics, and neurological disease have demonstrated how changes in the variability of gene expression can highlight important genes that are under different degrees of regulatory control. Simple statistical tests exist to identify differentially-variable genes; however, methods for investigating how changes in gene expression variability in the context of pathways and gene sets are under-explored. Here we presentpathVar,a new method that provides functional interpretation of gene expression variability changes at the level of pathways and gene sets.pathVaris based on a multinomial exact test, or an asymptotic Chi-squared test as a more computationally-efficient alternative. The method can be used for gene expression studies from any technology platform in all biological settings either with a single phenotypic group, or two-group comparisons. To demonstrate its utility, we applied the method to a diverse set of diseases, species and samples. Results frompathVarare benchmarked against analyses based on average expression and two methods of GSEA, and demonstrate that analyses using both statistics are useful for understanding transcriptional regulation. We also provide recommendations for the choice of variability statistic that have been informed through analyses on simulations and real data. Based on the datasets selected, we show howpathVarcan be used to gain insight into expression variability of single cell versus bulk samples, different stem cell populations, and cancer versus normal tissue comparisons.