Polymorphism Linkage Research Articles

Selective allele stacking of a novel quantitative trait locus facilitates the enhancement of seed epicatechin content in soybean (Glycine max (L.) Merr.)

(-)-Epicatechin (EC), a primary form of flavan-3ol and a building block of proanthocyanidins, has health benefits as it is a potent antioxidant. So far, no quantitative trait loci (QTLs) associated with EC have yet been identified in soybean. In this study, QTLs for EC and hilum color were identified in recombinant inbred lines (RILs) derived from the varieties Jinpung and IT109098 using high-resolution single nucleotide polymorphism linkage mapping. This revealed two major QTLs for EC content, qEC06 and qEC08. qEC06 spanned the Tawny (T) locus encoding flavonoid 3′-hydroxylase. qEC08, located near the Inhibitor (I) locus on Chr08, was also a major QTL for hilum color; however, allelic stacking of qEC08 and I revealed no relationship between I and EC content. RILs with IT109098 alleles at both qEC06 and qEC08 had higher EC content than other lines. These results will enable the production of soybean varieties with high EC content via marker-assisted selection.

Conditional transcriptome-wide association study for fine-mapping candidate causal genes.

Transcriptome-wide association studies (TWASs) aim to integrate genome-wide association studies with expression-mapping studies to identify genes with genetically predicted expression (GReX) associated with a complex trait. In the present report, we develop a method, GIFT (gene-based integrative fine-mapping through conditional TWAS), that performs conditional TWAS analysis by explicitly controlling for GReX of all other genes residing in a local region to fine-map putatively causal genes. GIFT is frequentist in nature, explicitly models both expression correlation and cis-single nucleotide polymorphism linkage disequilibrium across multiple genes and uses a likelihood framework to account for expression prediction uncertainty. As a result, GIFT produces calibrated P values and is effective for fine-mapping. We apply GIFT to analyze six traits in the UK Biobank, where GIFT narrows down the set size of putatively causal genes by 32.16-91.32% compared with existing TWAS fine-mapping approaches. The genes identified by GIFT highlight the importance of vessel regulation in determining blood pressures and lipid metabolism for regulating lipid levels.

Construction of a high-density genetic map and mapping of a spike length locus for rye.

Genetic maps provide the foundation for QTL mapping of important traits of crops. As a valuable food and forage crop, rye (Secale cereale L., RR) is also one of the tertiary gene sources of wheat, especially wild rye, Secale cereale subsp. segetale, possessing remarkable stress tolerance, tillering capacity and numerous valuable traits. In this study, based on the technique of specific-locus amplified fragment sequencing (SLAF-seq), a high-density single nucleotide polymorphism (SNP) linkage map of the cross-pollinated (CP) hybrid population crossed by S. cereale L (female parent) and S. cereale subsp. segetale (male parent) was successfully constructed. Following preprocessing, the number of 1035.11 M reads were collected and 2425800 SNP were obtained, of which 409134 SNP were polymorphic. According to the screening process, 9811 SNP markers suitable for constructing linkage groups (LGs) were selected. Subsequently, all of the markers with MLOD values lower than 3 were filtered out. Finally, an integrated map was constructed with 4443 markers, including 1931 female mapping markers and 3006 male mapping markers. A major quantitative trait locus (QTL) linked with spike length (SL) was discovered at 73.882 cM on LG4, which explained 25.29% of phenotypic variation. Meanwhile two candidate genes for SL, ScWN4R01G329300 and ScWN4R01G329600, were detected. This research presents the first high-quality genetic map of rye, providing a substantial number of SNP marker loci that can be applied to marker-assisted breeding. Additionally, the finding could help to use SLAF marker mapping to identify certain QTL contributing to important agronomic traits. The QTL and the candidate genes identified through the high-density genetic map above may provide diverse potential gene resources for the genetic improvement of rye.

Application of the PGT-M strategy using single sperm and/or affected embryos as probands for linkage analysis in males with hereditary tumor syndromes without family history.

Hereditary tumor syndromes have garnered substantial attention due to their adverse effects on both the physical and psychological health of patients, as well as the elevated risk of transmission to subsequent generations. This has prompted a growing interest in exploring preimplantation genetic testing (PGT) as a treatment option to mitigate and eliminate these impacts. Several studies have demonstrated that de novo variants have become a great cause of many hereditary tumor syndromes, which introduce certain difficulties to PGT. In the absence of adequate genetic linkage information (parents and offspring), haplotype construction seems unrealizable. In the study, researchers used single sperm or affected embryos as proband to perform single-nucleotide polymorphism linkage analysis for cases with de novo variants. For complicated variants, the strategy that sperm combined with embryo detection will increase accuracy while avoiding the limitations and potential failures of using a single detection material. The study recruited 11 couples with male de novo carriers, including 3 tumor types and 4 genes. To date, 4 couples have been clinically confirmed as pregnant and three healthy babies have been born. The results of amniocentesis or umbilical cord blood verification were consistent with the results of PGT-M. The study aims to introduce the application of the PGT-M strategy in hereditary tumor syndromes.

Investigation of genetic factors regulating chlorophyll and carotenoid biosynthesis in red pepper fruit.

Chlorophylls and carotenoids are synthesized in the chloroplast and chromoplast, respectively. Even though the two pigments are generated from the same precursor, the genetic correlation between chlorophyll and carotenoid biosynthesis has not yet been fully understood. We investigated the genetic correlation of chlorophyll and carotenoid biosynthesis during fruit ripening. Two recombinant inbred lines populations, “Long Sweet” × “AC2212” (“LA”) RILs derived from a cross between Capsicum annuum “Long Sweet” with light-green and light-red fruit and C. annuum “AC2212” with dark-green and brown-fruit and “3501 (F)” × “3509 (C)” (“FC”) RILs from C. annuum “3501” with dark-green and dark-red fruit and C. annuum “3509” with intermediate green and light-red fruit, were used. As the fruit ripened, three accessions produced high levels of xanthophyll. The dark-green immature fruit accumulated more total carotenoids than the light-green fruit. This trend corresponded to the expression pattern of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and CaGLK2 genes during fruit development. The expression levels of DXS and CaGLK2 in the dark-green accession “3501” were significantly higher than those of “3509” and “Long Sweet” during the early stages of fruit development. Furthermore, the genotype analysis of the transcription factor controlling chloroplast development (CaGLK2) in LA RILs revealed that CaGLK2 expression affected both carotenoid and chlorophyll contents. The single nucleotide polymorphism (SNP) linkage maps were constructed using genotyping-by-sequencing (GBS) for the two populations, and QTL analysis was performed for green fruit color intensity and carotenoid content. The QTL (LA_BG-CST10) for capsanthin content in LA RILs located at 24.4 to 100.4 Mbp on chromosome 10 was overlapped with the QTL (FC15-Cap10) for capsanthin content in FC RILs. Three QTLs for capsanthin content, American spice trade association (ASTA) value, and immature green fruit color intensity were also overlapped from 178.2 to 204 Mbp on chromosome 10. At the location, 151.6 to 165 Mbp on chromosome 8, QTLs (FC15-tcar8, FC17-ASTA8.1, and FC17-ASTA8.2) for total carotenoid content and ASTA value were discovered, and this region contained 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), which is involved in the MEP pathway. This result is the first report to show the correlation between carotenoid and chlorophyll biosynthesis in pepper. This research will expand our understanding of the mechanism of the chloroplast-to-chromoplast transition and the development of high pigment pepper varieties.

Combination of Genomics, Transcriptomics Identifies Candidate Loci Related to Cold Tolerance in Dongxiang Wild Rice.

Rice, a cold-sensitive crop, is a staple food for more than 50% of the world’s population. Low temperature severely compromises the growth of rice and challenges China’s food safety. Dongxiang wild rice (DXWR) is the most northerly common wild rice in China and has strong cold tolerance, but the genetic basis of its cold tolerance is still unclear. Here, we report quantitative trait loci (QTLs) analysis for seedling cold tolerance (SCT) using a high-density single nucleotide polymorphism linkage map in the backcross recombinant inbred lines that were derived from a cross of DXWR, and an indica cultivar, GZX49. A total of 10 putative QTLs were identified for SCT under 4 °C cold treatment, each explaining 2.0–6.8% of the phenotypic variation in this population. Furthermore, transcriptome sequencing of DXWR seedlings before and after cold treatment was performed, and 898 and 3413 differentially expressed genes (DEGs) relative to 0 h in cold-tolerant for 4 h and 12 h were identified, respectively. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed on these DEGs. Using transcriptome data and genetic linkage analysis, combined with qRT-PCR, sequence comparison, and bioinformatics, LOC_Os08g04840 was putatively identified as a candidate gene for the major effect locus qSCT8. These findings provided insights into the genetic basis of SCT for the improvement of cold stress potential in rice breeding programs.

Fatty acid metabolism-related genes are associated with flavor-presenting aldehydes in Chinese local chicken

Aldehydes are primary volatile organic compounds (VOCs) in local Chinese chicken meat and contribute green grass, fatty, citrus, and bitter almond aromas to chicken meat. To understand the genetic basis of these aldehyde VOC aromas, we used approximately 500 Chinese Jingxing Yellow (JXY) chickens to conduct genome-wide association studies (GWAS) on the flavor traits with the data of single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs). In total, 501 association variants (253 SNPs and 248 INDELs) were found to be suggestively (SNPs: p-value < 2.77e-06 and INDELs: p-value < 3.78e-05) associated with total aldehydes (the sum of nine aldehydes), hexanal, heptanal, benzaldehyde, (E,E)-2,4-nonadienal, octanal, (E)-2-decenal, nonanal, decanal, and octadecanal. Of them, six SNPs and 23 INDELs reached a genome-wide significance level (SNPs: p-value < 1.38e-07 and INDELs: p-value < 1.89e-06). Potential candidate aldehyde genes were functionally annotated for lipid metabolism, especially fatty acid-related pathways and phospholipid-related gene ontology (GO) terms. Moreover, the GWAS analysis of total aldehydes, hexanal, and nonanal generated the most significant signals, and phenotypic content differed between different genotypes at candidate gene-related loci. For total aldehydes and hexanal traits, candidate genes were annotated based on the significant and suggestive variants on chromosomes 3 and 8 with highly polymorphic linkage blocks. The following candidate genes were also identified: GALM, MAP4K3, GPCPD1, RPS6KA2, CRLS1, ASAP1, TRMT6, SDC1, PUM2, ALDH9A1, MGST3, GMEB1, MECR, LDLRAP1, GPAM and ACSL5. We also found that polyunsaturated fatty acids (PUFAs) (C18:2n6c linoleic acid and C18:3n3 linolenic acid) were significantly correlated with total aldehydes and hexanal contents. PUFAs are important aldehyde precursors, and consistently, our results suggested that candidate genes involved in fatty acid pathways and phospholipid GO terms were identified in association loci. This work provides an understanding of the genetic basis of aldehyde formation, which is a key flavor-forming compound.

Potential regulation of linoleic acid and volatile organic compound contents in meat of chickens by PLCD1

Omega-3 (linolenic acid (ALA), docosapentaenoic acid, eicosapentaenoic acid) and omega-6 (linoleic acid (LA), arachidonic acid) polyunsaturated fatty acids are essential for health and normal physiological functioning in humans. Here we report a genome-wide association study (GWAS) on LA content in chicken meat. The 19 significant single nucleotide polymorphisms (SNPs) identified by the GWAS approach were annotated in VILL, PLCD1 and OXSR1 genes with highly polymorphic linkage blocks, and explained 4.5% of the phenotypic variation in the LA content. Specifically, the PLCD1 mRNA expression level was negatively correlated with the LA content, and significantly higher in chickens with low LA content than in those with high LA content. In addition, PLCD1 was found to be involved in metabolic pathways, etc. Furthermore, the LA content was correlated with volatile organic compounds (e.g., octanal, etc.), but no relationship was found with intramuscular fat and triglycerides in chicken meat. The results indicated that there are key SNPs in PLCD1 that regulate the content of LA, and it has no significant effect on fat deposition, but may affect the content of volatile organic compounds (VOCs).

Outbreak of sexually transmitted, extensively drug-resistant Shigella sonnei in the UK, 2021–22: a descriptive epidemiological study

Shigellosis, traditionally a foodborne and waterborne infection, causes substantial morbidity globally. It is now a leading cause of sexually transmitted gastroenteritis among gay, bisexual, and other men who have sex with men (MSM). We describe an ongoing outbreak of extensively drug-resistant (XDR) Shigella sonnei in the UK. Routine laboratory surveillance (Second Generation Surveillance System, Gastrointestinal Data Warehouse) identified an exceedance of S sonnei clade 5 in England, first detected in September, 2021. Cases within this clade were subsequently reported from Scotland, Wales, and Northern Ireland. Confirmed cases in this outbreak were defined as individuals diagnosed with S sonnei clade 5 in the UK, with a specimen date between Sept 1, 2021, and Feb 9, 2022, who were genomically confirmed as part of a ten-single nucleotide polymorphism (SNP) linkage cluster. We used whole-genome sequencing with SNP typing to identify genomic clusters and antimicrobial-resistance determinants, analysing cases across the UK. We collected demographic, epidemiological, and clinical data from people infected with S sonnei clade 5 in England using questionnaires (standard and bespoke outbreak questionnaires). We used descriptive summary statistics to characterise cases. 72 cases (70 [97%] male, median age 34 years [IQR 27-39]) belonging to the ten-SNP single linkage cluster of S sonnei clade 5 were identified between Sept 4, 2021, and Feb 9, 2022. Isolates were predominantly XDR, with 66 (92%) of 72 harbouring bla_CTX-M-27, a plasmid-mediated gene for production of extended-spectrum β-lactamases (ESBLs). Of 33 cases with clinical data, 19 (58%) received antibiotics and eight (24%) were hospitalised. 21 (78%) of 27 cases with completed bespoke outbreak questionnaires were HIV-negative MSM taking HIV pre-exposure prophylaxis (PrEP) who reported sexual contacts in the UK and Europe within the incubation period. We highlight the rapid dissemination of XDR ESBL-producing S sonnei in sexual networks of MSM. We recommend strengthening shigella testing where clinically indicated, antimicrobial-resistance surveillance, and integrated health promotion messaging among all MSM, including PrEP users, to reduce the burden of shigellosis. National Institute for Health Research Health Protection Research Unit in Gastrointestinal Infections at the University of Liverpool in partnership with the UK Health Security Agency.

Systematic trait dissection in oilseed rape provides a comprehensive view, further insight, and exact roadmap for yield determination

BackgroundYield is the most important and complex trait that is influenced by numerous relevant traits with very complicated interrelations. While there are a large number of studies on the phenotypic relationship and genetic basis of yield traits, systematic studies with further dissection focusing on yield are limited. Therefore, there is still lack of a comprehensive and in-depth understanding of the determination of yield.ResultsIn this study, yield was systematically dissected at the phenotypic, genetic to molecular levels in oilseed rape (Brassica napus L.). The analysis of correlation, network, and principal component for 21 traits in BnaZN-RIL population showed that yield was determined by a complex trait network with key contributors. The analysis of the constructed high-density single nucleotide polymorphism (SNP) linkage map revealed the concentrated distribution of distorted and heterozygous markers, likely due to selection on genes controlling the growth period and yield heterosis. A total of 134 consensus quantitative trait loci (QTL) were identified for 21 traits, of which all were incorporated into an interconnecting QTL network with dozens of hub-QTL. Four representative hub-QTL were further dissected to the target or candidate genes that governed the causal relationships between the relevant traits.ConclusionsThe highly consistent results at the phenotypic, genetic, and molecular dissecting demonstrated that yield was determined by a multilayer composite network that involved numerous traits and genes showing complex up/down-stream and positive/negative regulation. This provides a systematic view, further insight, and exact roadmap for yield determination, which represents a significant advance toward the understanding and dissection of complex traits.

RETRACTED ARTICLE: Multidisciplinary management for Peutz–Jeghers syndrome and prevention of vertical transmission to offspring using preimplantation genetic testing

BackgroundPeutz Jeghers syndrome (PJS) is an autosomal dominant genetic disorder caused by STK11 mutation with a predisposition to gastrointestinal polyposis and cancer. PJS patients suffer poor quality of life and are highly concerned about whether deleterious mutations transmit to their offspring. Therefore, this study aimed to propose feasible clinical management and provide effective preimplantation genetic testing for monogenic defect (PGT-M) strategies to protect offspring from inheriting the disease.MethodsA hospital-based clinical retrospective analysis reviewing the clinical characteristics and fertility aspects was first conducted on 51 PJS patients at the First Affiliated Hospital of Zhengzhou University between January 2016 and March 2021. Among the 51 patients, the PGT-M strategy was further carried out in 4 couples, which started with a biopsy of the trophectoderm cells of embryos and whole genome amplification using multiple displacement amplification. Thereafter, single nucleotide polymorphism linkage analyses based on karyomapping were performed with copy number variations of the embryos identified simultaneously. Finally, prenatal diagnosis was used to verify the validity of the PGT-M results.ResultsA comprehensive management flowchart adopted by the multidisciplinary team model was formulated mainly focusing on clinical genetic and gastrointestinal aspects. Under the guidelines of this management, 32 embryos from 4 PJS pedigrees were diagnosed and 2 couples successfully conceived healthy babies free of the STK11 pathogenic mutation.ConclusionsOur comprehensive management could help affected families avoid having children with PJS through preimplantation genetic testing and provide meaningful guidance for multidisciplinary clinical practice on PJS.

Major histocompatibility IIβ diversity and peptide-binding groove properties associated with red sea bream iridovirus resistance

Major histocompatibility complex (MHC) II genes are key factors in presenting antigens derived from extracellular pathogens to CD4+ T cells. These genes are known to be associated with disease resistance in livestock, including cultured fish species. Genome-wide linkage analysis of RSIV-resistant red sea bream has revealed that MHC IIβ is linked to the RSIV-resistant trait and was suggested as a probable candidate gene responsible for the resistant trait. In the present study, diversity of the MHC IIβ gene in wild and farmed red sea bream populations was evaluated by analysis of an intronic minisatellite locus, PmaMHC2b. Haplotypes of the βI domain of MHC IIβ were identified by cloning, and linkage of minisatellite polymorphisms with haplotype was confirmed in farmed populations. Large numbers of alleles were observed in the wild population (30), while the domesticated populations showed significantly lower values (13). Thirteen haplotypes of MHC IIβ were observed; most haplotypes possessed the unique PmaMHC2b allele, but some were present in more than one PmaMHC2b allele. Each haplotype possessed a distinct amino acid sequence with no synonymous mutations. Among 16 amino acid positions involved in peptide-binding grooves, four were monomorphic and 12 polymorphic. Several positions differed in the RSIV-resistant and RSIV-susceptible phenotypes: five in hydrophilic/hydrophobic character and five in polarity. These results provide insight applicable to broodstock management and contribute to understanding RSIV resistance.

Identification of favorable haplotypes/alleles and candidate genes for three plant architecture-related traits via a restricted two-stage multilocus genome-wide association study in upland cotton

Plant architecture-related traits are extremely crucial for cotton plants and are closely linked to their ability to adapt to reasonably close planting and to their suitability for mechanized harvesting. However, the genetic basis for cotton plant architecture-related traits remains poorly understood. To identify favorable haplotypes/alleles (FHs/FAs) and candidate genes for target traits, we performed a restricted two-stage multilocus genome-wide association study (RTM-GWAS) using a natural population consisting of 315 upland cotton accessions and 9244 single-nucleotide polymorphism linkage disequilibrium blocks (SNPLDBs), which were explored and established using the specific-locus amplified fragment sequencing (SLAF-seq) technique. Totals of 68, 34 and 55 significant SNPLDB loci associated with the plant height (PH), fruit branch angle (FBA) and fruit branch length (FBL), respectively, were identified via the RTM-GWAS method. We then estimated the allele effects of the significant loci associated with the target traits and further generated SNPLDB allele matrices to provide a brief inheritance constitution of the population. Among the significant SNPLDB associations, five involved in four major loci (LDB_1_21982826, LDB_9_51578589_51578819, LDB_16_37952328 and LDB_19_52309050_52309284) for the PH or FBL were concurrently detected in a multiple-environment combined model and in at least one single-environment model. A comparison of FHs/FAs frequencies for the four major SNPLDB loci revealed that the frequencies of FHs or FAs that were hidden in short and compact cotton accessions were much higher than those in tall and noncompact cotton accessions. We also observed that cotton accessions from the northern super-early-maturation region (NSER) had relatively short and compact plants compared with those from the Yangtze River region (YZRR) and northwestern inland region (NIR), and the high FA frequencies of the four major SNPLDB loci were consistent with the relatively short and compact plant architecture in five different geographic regions. In the neighboring genome fragments of the four major SNPLDB loci, ultimately, the five candidate genes that were highly expressed during the early stages of the growth and were responsible for cotton PH and FBL through the auxin signaling pathway were explored by RNA-seq data and bioinformatics analysis. This study identified FHs/FAs and candidate genes for PH and FBL, which could provide key insights into the genetic basis for the plant architecture of upland cotton and could promote the breeding of new cotton varieties with ideal plant architecture suitable for mechanized harvesting.

Linkage and association of PAX7 polymorphisms (rs742071, rs766325, and rs4920520) with the risk of non-syndromic cleft lip with/without cleft palate: A systematic review and meta-analysis

The studies have reported several additional non-syndromic cleft lip/palate (NSCL/P) susceptibility loci. This systematic review and meta-analysis aimed to evaluate the linkage and association of PAX7 polymorphisms with the NSCL/P risk. A comprehensive search was conducted in the PubMed, Cochrane Library, Web of Science, and Scopus databases until May 15, 2021. The association between PAX7 polymorphisms and NSCL/P susceptibility was analyzed by calculation of the odds ratios (ORs) and 95% confidence intervals (CIs), and the linkage was assessed by the allelic transmission disequilibrium test. Some articles included more than one study (reporting more than one polymorphism). Therefore, nine articles including 13 studies were entered into the meta-analysis. With regard to the association of rs742071 polymorphism with the risk of NSCL/P, the pooled OR was 1.33 for the allelic (P < 0.0001), 1.98 for the homozygous (P = 0.0007), 1.41 for the heterozygous (P = 0.0267), and 1.15 for the dominant (P = 0.2542) models. For the association of PAX7 rs766325 and rs4920520 polymorphisms with the risk of NSCL/P, the pooled OR was 0.96 (P = 0.5571) and 1.14 (P = 0.1020), respectively. In addition, the pooled allelic transmission disequilibrium test for s742071 and rs766325 polymorphisms did not show any allelic linkage between these polymorphisms and susceptibility to NSCL/P. The main results of the present systematic review and meta-analysis showed an association between PAX7 rs742071 polymorphism and NSCL/P susceptibility; but there was no linkage or association with rs766325 and rs4920520 polymorphisms.

Growth period QTL‐allele constitution of global soybeans and its differential evolution changes in geographic adaptation versus maturity group extension

SummarySoybean (Glycine max(L.) Merr.) has been disseminated globally as a photoperiod/temperature‐sensitive crop with extremely diverse days to flowering (DTF) and days to maturity (DTM) values. A population with 371 global varieties covering 13 geographic regions and 13 maturity groups (MGs) was analyzed for its DTF and DTM QTL‐allele constitution using restricted two‐stage multi‐locus genome‐wide association study (RTM‐GWAS). Genotypes with 20 701 genome‐wide SNPLDBs (single‐nucleotide polymorphism linkage disequilibrium blocks) containing 55 404 haplotypes were observed, and 52 DTF QTLs and 59 DTM QTLs (including 29 and 21 new ones) with 241 and 246 alleles (two to 13 per locus) were detected, explaining 84.8% and 74.4% of the phenotypic variance, respectively. The QTL‐allele matrix characterized with all QTL‐allele information of each variety in the global population was established and subsequently separated into geographic and MG set submatrices. Direct comparisons among them revealed that the genetic adaptation from the origin to geographic subpopulations was characterized by new allele/new locus emergence (mutation) but little allele exclusion (selection), while that from the primary MG set to emerged early and late MG sets was characterized by allele exclusion without allele emergence. The evolutionary changes involved mainly 72 DTF and 71 DTM alleles on 28 respective loci, 10–12 loci each with three to six alleles being most active. Further recombination potential for faster maturation (12–21 days) or slower maturation (14–56 days) supported allele convergence (recombination) as a constant genetic factor in addition to migration (inheritance). From the QTLs, 44 DTF and 36 DTM candidate genes were annotated and grouped respectively into nine biological processes, indicating multi‐functional DTF/DTM genes are involved in a complex gene network. In summary, we identified QTL‐alleles relatively thoroughly using RTM‐GWAS for direct matrix comparisons and subsequent analysis.

Transcriptional-regulatory convergence across functional MDD risk variants identified by massively parallel reporter assays

Family and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters) based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients and promote depressive-like behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, selecting SNPs based on overlap with predicted regulatory features—including expression quantitative trait loci (eQTL) and histone marks—from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs), with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional-regulatory program across loci, a component of which is unmasked by retinoids.

ADGRL3, FGF1 and DRD4: Linkage and Association with Working Memory and Perceptual Organization Candidate Endophenotypes in ADHD.

Attention deficit hyperactivity disorder (ADHD) is a highly heritable neurobehavioral disorder that affects children worldwide, with detrimental long-term consequences in affected individuals. ADHD-affected patients display visual–motor and visuospatial abilities and skills that depart from those exhibited by non-affected individuals and struggle with perceptual organization, which might partially explain impulsive responses. Endophenotypes (quantifiable or dimensional constructs that are closely related to the root cause of the disease) might provide a more powerful and objective framework for dissecting the underlying neurobiology of ADHD than that of categories offered by the syndromic classification. In here, we explore the potential presence of the linkage and association of single-nucleotide polymorphisms (SNPs), harbored in genes implicated in the etiology of ADHD (ADGRL3, DRD4, and FGF1), with cognitive endophenotypes related to working memory and perceptual organization in 113 nuclear families. These families were ascertained from a geographical area of the Caribbean coast, in the north of Colombia, where the community is characterized by its ethnic diversity and differential gene pool. We found a significant association and linkage of markers ADGRL3-rs1565902, DRD4-rs916457 and FGF1-rs2282794 to neuropsychological tasks outlining working memory and perceptual organization such as performance in the digits forward and backward, arithmetic, similarities, the completion of figures and the assembly of objects. Our results provide strong support to understand ADHD as a combination of working memory and perceptual organization deficits and highlight the importance of the genetic background shaping the neurobiology, clinical complexity, and physiopathology of ADHD. Further, this study supplements new information regarding an ethnically diverse community with a vast African American contribution, where ADHD studies are scarce.

A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis.

A novel monogenic preimplantation genetic testing strategy for sporadic polycystic kidney caused by de novo PKD1 mutation.

Autosomal dominant hereditary polycystic kidney disease (ADPKD) is the most common inherited kidney disease that causes end-stage renal disease and kidney failure. Preimplantation genetic testing for monogenic (PGT-M) can effectively prevent the transmission of genetic diseases from parents to the offspring before pregnancy. However, PGT-M currently adopts the single nucleotide polymorphism (SNP) linkage analysis for embryo's pathogenic gene carrying status and linkage analysis requires proband of the family. Here we report a new PGT-M strategy using single sperm SNP linkage analysis for male patient with sporadic ADPKD caused by de novo PKD1 mutation. We recruited five couples with male patient with ADPKD caused by de novo PKD1 mutation, and 39 embryos from six PGT-M cycles were detected. The five couples had at least one embryo that does not carry the PKD1 mutation. Within these five couples, the accuracy of carrier status of embryos was confirmed by amniotic fluid gene detection of two couples and two couples successfully delivered healthy fetuses. Therefore, the new PGT-M strategy of using single sperm SNP linkage analysis was proved to be feasible and effective for male patient with ADPKD caused by de novo PKD1 mutation.

Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross

BackgroundLumefantrine and mefloquine are used worldwide in artemisinin-based combination therapy (ACT) of malaria. Better understanding of drug susceptibility and resistance is needed and can be obtained from studies of genetic crosses. MethodsDrug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC50s) and days to recovery (DTR) after 24 h exposure to 500 nM lumefantrine. EC50s of mefloquine, halofantrine, chloroquine, and dihydroartemisinin were also determined. Quantitative trait loci (QTL) analysis and statistical tests with candidate genes were used to identify polymorphisms associated with response phenotypes. ResultsLumefantrine EC50s averaged 5.8-fold higher for the 803 than GB4 parent, and DTR results were 3–5 and 16–18 days, respectively. In 803 × GB4 progeny, outcomes of these two lumefantrine assays showed strong inverse correlation; these phenotypes also correlated strongly with mefloquine and halofantrine EC50s. By QTL analysis, lumefantrine and mefloquine phenotypes mapped to a chromosome 5 region containing codon polymorphisms N86Y and Y184F in the P. falciparum multidrug resistance 1 protein (PfMDR1). Statistical tests of candidate genes identified correlations between inheritance of PfK13 Kelch protein polymorphism C580Y (and possibly K189T) and lumefantrine and mefloquine susceptibilities. Correlations were detected between lumefantrine and chloroquine EC50s and polymorphisms N326S and I356T in the CVIET-type P. falciparum chloroquine resistance transporter (PfCRT) common to 803 and GB4. ConclusionsCorrelations in this study suggest common mechanisms of action in lumefantrine, mefloquine, and halofantrine responses. PfK13 as well as PfMDR1 and PfCRT polymorphisms may affect access and/or action of these arylaminoalcohol drugs at locations of hemoglobin digestion and heme metabolism. In endemic regions, pressure from use of lumefantrine or mefloquine in ACTs may drive selection of PfK13 polymorphisms along with versions of PfMDR1 and PfCRT associated with lower susceptibility to these drugs.