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Background and Objectives: Mixed‐field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full‐gene haplotypes at high resolution. Materials and Methods: We used long‐read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long‐range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. Results: Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti‐A specific mixed‐field agglutination. Conclusion: We discovered a regulatory variant in the 8‐bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long‐range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes. [ABSTRACT FROM AUTHOR]

Abstract: Single nucleotide polymorphisms (SNPs) within the regulatory elements of a gene can alter gene expression, making these SNPs of prime importance for candidate gene association studies. We aimed to determine whether such regulatory variants are associated with clinical outcomes in three cohorts of patients with prostate cancer. We used RegulomeDB to identify potential regulatory variants based on in silico predictions and reviewed genome‐wide experimental findings. Overall, 131 putative regulatory SNPs with the highest confidence score on predicted functionality were investigated in two independent localized prostate cancer cohorts totalling 458 patients who underwent radical prostatectomy. The statistically significant SNPs identified in these two cohorts were then tested in an additional cohort of 504 patients with advanced prostate cancer. We identified one regulatory SNPs, rs1646724, that are consistently associated with increased risk of recurrence in localized disease (P = .003) and mortality in patients with advanced prostate cancer (P = .032) after adjusting for known clinicopathological factors. Further investigation revealed that rs1646724 may affect expression of SLC35B4, which encodes a glycosyltransferase, and that down‐regulation of SLC35B4 by transfecting short hairpin RNA in DU145 human prostate cancer cell suppressed proliferation, migration and invasion. Furthermore, we found increased SLC35B4 expression correlated with more aggressive forms of prostate cancer and poor patient prognosis. Our study provides robust evidence that regulatory genetic variants can affect clinical outcomes. [ABSTRACT FROM AUTHOR]

Introduction We hypothesized that common Alzheimer's disease (AD)-associated variants within the triggering receptor expressed on myeloid ( TREM ) gene cluster influence disease through gene expression. Methods Expression microarrays on temporal cortex and cerebellum from ∼400 neuropathologically diagnosed subjects and two independent RNAseq replication cohorts were used for expression quantitative trait locus analysis. Results A variant within a DNase hypersensitive site 5′ of TREM2 , rs9357347-C, associates with reduced AD risk and increased TREML1 and TREM2 levels (uncorrected P = 6.3 × 10 −3 and 4.6 × 10 −2 , respectively). Meta-analysis on expression quantitative trait locus results from three independent data sets ( n = 1006) confirmed these associations (uncorrected P = 3.4 × 10 −2 and 3.5 × 10 −3 , Bonferroni-corrected P = 6.7 × 10 −2 and 7.1 × 10 −3 , respectively). Discussion Our findings point to rs9357347 as a functional regulatory variant that contributes to a protective effect observed at the TREM locus in the International Genomics of Alzheimer's Project genome-wide association study meta-analysis and suggest concomitant increase in TREML1 and TREM2 brain levels as a potential mechanism for protection from AD. [ABSTRACT FROM AUTHOR]

ABSTRACT In silico methods for detecting functionally relevant genetic variants are important for identifying genetic markers of human inherited disease. Much research has focused on protein-coding variants since coding regions have well-defined physicochemical and functional properties. However, many bioinformatics tools are not applicable to variants outside coding regions. Here, we increase the classification performance of our regulatory single-nucleotide variant predictor (RSVP) for variants that cause regulatory abnormalities from an AUC of 0.90-0.97 by incorporating genomic regions identified by the ENCODE project into RSVP. RSVP is comparable to a recently published tool, Genome-Wide Annotation of Variants (GWAVA); both RSVP and GWAVA perform better on regulatory variants than a traditional variant predictor, combined annotation-dependent depletion (CADD). However, our method outperforms GWAVA on variants located at similar distances to the transcription start site as the positive set (AUC: 0.96) as compared with GWAVA (AUC: 0.71). Much of this disparity is due to RSVP's incorporation of features pertaining to the nearest gene (expression, GO terms, etc.), which are not included in GWAVA. Our findings hold out the promise of a framework for the assessment of all functional regulatory variants, providing a means to predict which rare or de novo variants are of pathogenic significance. [ABSTRACT FROM AUTHOR]

Nonsyndromic cleft lip with or without cleft palate ( NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 ( LOD = 2.98). This region contained four genes, Frizzled-6 ( FZD6), Matrilin-2 ( MATN2), Odd-skipped related 2 ( OSR2) and Solute Carrier Family 25, Member 32 ( SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway. [ABSTRACT FROM AUTHOR]

Chen JM, Férec C, Cooper DN. Revealing the human mutome. The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the ‘mutome’). With the advent of next-generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole-genome sequencing (WGS) has the potential to identify all disease-causing or disease-associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene-coding sequences for those disease-causing or disease-associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation-screening strategies but also for enhancing the interpretation of findings derived from genome-wide association studies, whole-exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology. [ABSTRACT FROM AUTHOR]