Evaluation of cross-platform and interlaboratory concordance via consensus modelling of genomic measurements.

MOTIVATION: A synoptic view of the human genome benefits chiefly from the application of nucleic acid sequencing and microarray technologies. These platforms allow interrogation of patterns such as gene expression and DNA methylation at the vast majority of canonical loci, allowing granular insights and opportunities for validation of original findings. However, problems arise when validating against a "gold standard" measurement, since this immediately biases all subsequent measurements towards that particular technology or protocol. Since all genomic measurements are estimates, in the absence of a "gold standard" we instead empirically assess the measurement precision and sensitivity of a large suite of genomic technologies via a consensus modelling method called the row-linear model. This method is an application of the American Society for Testing and Materials Standard E691 for assessing interlaboratory precision and sources of variability across multiple testing sites. Both cross-platform and cross-locus comparisons can be made across all common loci, allowing identification of technology- and locus-specific tendencies. RESULTS: We assess technologies including the Infinium MethylationEPIC BeadChip, whole genome bisulfite sequencing (WGBS), two different RNA-Seq protocols (PolyA+ and Ribo-Zero) and five different gene expression array platforms. Each technology thus is characterised herein, relative to the consensus. We showcase a number of applications of the row-linear model, including correlation with known interfering traits. We demonstrate a clear effect of cross-hybridisation on the sensitivity of Infinium methylation arrays. Additionally, we perform a true interlaboratory test on a set of samples interrogated on the same platform across twenty-one separate testing laboratories. AVAILABILITY AND IMPLEMENTATION: A full implementation of the row-linear model, plus extra functions for visualisation, are found in the R package consensus at https://github.com/timpeters82/consensus. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Changing interpretation of chromosomal microarray over time in a community cohort with intellectual disability.

Chromosomal microarray (CMA) is the first-line diagnostic test for individuals with intellectual disability, autism, or multiple congenital anomalies, with a 10-20% diagnostic yield. An ongoing challenge for the clinician and laboratory scientist is the interpretation of variants of uncertain significance (VOUS)-usually rare, unreported genetic variants. Laboratories differ in their threshold for reporting VOUS, and clinical practice varies in how this information is conveyed to the family and what follow-up is arranged. Workflows, websites, and databases are constantly being updated to aid the interpretation of VOUS. There is a growing literature reporting new microdeletion and duplication syndromes, susceptibility, and modifier copy number variants (CNVs). Diagnostic methods are also evolving with new array platforms and genome builds. In 2010, high-resolution arrays (Affymetrix 2.7 M Oligo and SNP, 50 kB resolution) were performed on a community cohort of 67 individuals with intellectual disability of unknown aetiology. Three hundred and one CNVs were detected and analyzed using contemporary resources and a simple scoring system. Thirteen (19%) of the arrays were assessed as potentially pathogenic, 4 (6%) as benign and 50 (75%) of uncertain clinical significance. The CNV data were re-analyzed in 2012 using the contemporary interpretative resources. There was a statistically significant difference in the assessment of individual CNVs (P < 0.0001). An additional eight patients were reassessed as having a potentially pathogenic array (n = 21, 31%) and several additional susceptibility or modifier CNVs were identified. This study highlights the complexity involved in the interpretation of CMA and uniquely demonstrates how, even on the same array platform, it can be subject to change over time.

PI3K(p110 alpha) protects against myocardial infarction-induced heart failure: identification of PI3K-regulated miRNA and mRNA.

OBJECTIVE: Myocardial infarction (MI) is a serious complication of atherosclerosis associated with increasing mortality attributable to heart failure. Activation of phosphoinositide 3-kinase [PI3K(p110 alpha)] is considered a new strategy for the treatment of heart failure. However, whether PI3K(p110 alpha) provides protection in a setting of MI is unknown, and PI3K(p110 alpha) is difficult to target because it has multiple actions in numerous cell types. The goal of this study was to assess whether PI3K(p110 alpha) is beneficial in a setting of MI and, if so, to identify cardiac-selective microRNA and mRNA that mediate the protective properties of PI3K(p110 alpha). METHODS AND RESULTS: Cardiomyocyte-specific transgenic mice with increased or decreased PI3K(p110 alpha) activity (caPI3K-Tg and dnPI3K-Tg, respectively) were subjected to MI for 8 weeks. The caPI3K-Tg subjected to MI had better cardiac function than nontransgenic mice, whereas dnPI3K-Tg had worse function. Using microarray analysis, we identified PI3K-regulated miRNA and mRNA that were correlated with cardiac function, including growth factor receptor-bound 14. Growth factor receptor-bound 14 is highly expressed in the heart and positively correlated with PI3K(p110 alpha) activity and cardiac function. Mice deficient in growth factor receptor-bound 14 have cardiac dysfunction. CONCLUSIONS: Activation of PI3K(p110 alpha) protects the heart against MI-induced heart failure. Cardiac-selective targets that mediate the protective effects of PI3K(p110 alpha) represent new drug targets for heart failure.

Xylosyltransferase gene variants and their role in essential hypertension.

BACKGROUND: An accumulation of extracellular matrix molecules, such as proteoglycans, is observed in the vascular wall of hypertensive patients. Xylosyltransferases I and II (XT-I and XT-II), the chain-initiating enzymes in the biosynthesis of proteoglycans, catalyze the transfer of D-xylose from UDP-D-xylose to specific serine residues of the core protein. Because associations between XYLT polymorphisms and an altered blood pressure have been observed, genetic variations in the XYLT genes might predispose to essential hypertension. The localization of the XYLT2 gene on chromosome 17q increases its attractiveness as this region has been reported to be a potential candidate locus for essential hypertension. METHODS: Genotyping of four polymorphisms in the genes XYLT1 and XYLT2 was performed in 150 unrelated essential hypertension patients and 150 age- and sex-matched normotensive controls using restriction fragment length polymorphism analysis. RESULTS: The allele and genotype frequencies of the XYLT variants investigated did not show any significant differences between patients and controls, among allele-carriers and nonallele-carriers and among recessive and nonrecessive allele-carriers comparing patients and controls. Systolic and diastolic blood pressures did not differ significantly between the genotypes concerning all XYLT variants analyzed. Two XYLT2 variants deviated from Hardy-Weinberg equilibrium (HWE) in the hypertensive group. CONCLUSIONS: No statistically significant association was found between four XYLT variants and hypertension or blood pressure, suggesting that they do not play a significant role in the development of essential hypertension. The deviation from HWE of two XYLT2 variants might be due to gene-phenotype associations which remain to be explored, as well as the possibility of gene-gene interactions.

XE7: a novel splicing factor that interacts with ASF/SF2 and ZNF265.

Pre-mRNA splicing is performed by the spliceosome. SR proteins in this macromolecular complex are essential for both constitutive and alternative splicing. By using the SR-related protein ZNF265 as bait in a yeast two-hybrid screen, we pulled out the uncharacterized human protein XE7, which is encoded by a pseudoautosomal gene. XE7 had been identified in a large-scale proteomic analysis of the human spliceosome. It consists of two different isoforms produced by alternative splicing. The arginine/serine (RS)-rich region in the larger of these suggests a role in mRNA processing. Herein we show for the first time that XE7 is an alternative splicing regulator. XE7 interacts with ZNF265, as well as with the essential SR protein ASF/SF2. The RS-rich region of XE7 dictates both interactions. We show that XE7 localizes in the nucleus of human cells, where it colocalizes with both ZNF265 and ASF/SF2, as well as with other SR proteins, in speckles. We also demonstrate that XE7 influences alternative splice site selection of pre-mRNAs from CD44, Tra2-beta1 and SRp20 minigenes. We have thus shown that the spliceosomal component XE7 resembles an SR-related splicing protein, and can influence alternative splicing.

No association with hypertension of CLCNKB and TNFRSF1B polymorphisms at a hypertension locus on chromosome 1p36.

OBJECTIVE: Chromosome 1p36 has been linked to essential hypertension and systolic blood pressure. This locus contains the chloride channel-Kb gene (CLCNKB) and the tumour necrosis factor receptor 2 gene (TNFRSF1B). Polymorphisms of each of these have shown association with hypertension, and a CLCNKB T481S variant alters receptor function. Here we performed association studies in a well-characterized cohort of hypertensives and normotensives whose blood pressure status matched that of both their parents. METHODS: The study involved 196 essential hypertensives and 321 normotensives. These were genotyped for TNFRSF1B variants T-1710A upstream, A257G in exon 2, a CA-repeat polymorphism in intron 4, E232K and M196R in exon 6, and T1668G and T1690C in the 3'-untranslated region, and the T481S variant of CLCNKB. RESULTS: The CLCNKB T481S variant showed no association with hypertension. Thermodynamic modelling of the 3'-untranslated region of TNFRSF1B mRNA predicted that the T1668G variant alters the stem-loop structure and thus the mRNA stability and expression. However, neither this nor the other TNFRSF1B polymorphisms, either alone or after haplotype analysis, were associated with hypertension. Moreover, for each gene the blood pressure, body mass index, plasma sodium and plasma lipid concentrations were generally similar across genotypes. CONCLUSION: Our data fail to support previous association findings for TNFRSF1B and CLCNKB at the chromosome 1p36 locus implicated in hypertension.

Genome-wide scan for hypertension in Sydney Sibships: the GENIHUSS study.

We report here the results of the GENIHUSS study (GENetic Investigation of Hypertension Undertaken in Sydney Sibships)-a genome-wide scan to identify loci linked to essential hypertension (HT). Subjects were Anglo-Celtic Australian sibpairs resident in or near Sydney, Australia, with onset of HT before age 60 years (mean, 44 +/- 13 SD years). A 10-cM scan involving 400 microsatellite markers and 252 HT sibpairs was followed by fine mapping of the most promising locus using 296 HT sibpairs (481 individuals from 200 families). Multipoint and two-point nonparametric linkage analyses were performed using MAPMAKER/SIBS, GENEHUNTER II, and SPLINK. Suggestive loci were found on chromosomes 1 (4 cM) and 4 (129 cM). The chromosome 4 locus coincided with a QTL for systolic blood pressure (BP) in the Australian Victorian Family Heart Study, and the locus on chromosome 1 contains the chloride channel gene CLCNKB and tumor necrosis factor receptor 2 gene TNFRSF1B, which have each shown association with HT. Our study adds to findings of HT loci emanating from genome scans.

Association of G-protein-coupled receptor kinase 4 haplotypes, but not HSD3B1 or PTP1B polymorphisms, with essential hypertension.

OBJECTIVE: To perform association studies of polymorphisms of the potential candidate essential hypertension (HT) genes GRK4, PTP1B and HSD3B1. METHODS: Subjects consisted of 168 unrelated, Caucasian essential hypertensive (HT) patients and 312 normotensive (NT) controls. Biological power was increased by ensuring subjects in each group had parents with the same blood pressure (BP) status as theirs. Three GRK4gamma variants (R65L, A142V and A486V), one HSD3B1 variant (T<---C Leu) and one PTP1B variant (1484insG) were genotyped by polymerase chain reaction and restriction enzyme digestion or by homogenous MassEXTEND Assay. RESULTS: The V allele of the A486V variant of GRK4gamma, but not the R65L or A142V variants, showed an association with HT (P = 0.02). The V allele was also associated with an elevation in systolic blood pressure (SBP) (P = 0.002). Although the L65 and the V142 alleles tracked with elevation in diastolic (DBP), this was seen only in male HTs (P = 0.009; P = 0.002, respectively). Haplotype frequencies differed between the HT and NT groups, particularly for the R, V, V haplotype combination of R65L, A142V and A486V, respectively. Neither of the HSD3B1 or PTP1B variants were associated with HT. CONCLUSION: Genetic variation in GRK4gamma was associated with HT in the subjects studied.

WNK4 intron 10 polymorphism is not associated with hypertension.

A polymorphism in intron 10 of the serine-threonine kinase with no lysine (K) 4 gene WNK4 (G-->A, base 1156666 on chromosome 17) has recently been associated with essential hypertension in a white American population. We have attempted to replicate this finding in a well characterized cohort of 184 unrelated hypertensive Australians of British extraction in which biological power was enhanced by them each having 2 hypertensive parents. Controls were 219 normotensive ethnically matched subjects whose parents were both normotensive. Genotyping was performed using the homogeneous MassEXTEND Assay. This showed a frequency of 0.10 for the minor allele in each group (P=0.88). Moreover, blood pressure, body mass index, sex, and plasma lipid levels were similar across genotypes. In conclusion, our study provides no support for an association of the intron 10 variant of WNK4 with essential hypertension in the Anglo-Australian population studied.

WNK1, a gene within a novel blood pressure control pathway, tissue-specifically generates radically different isoforms with and without a kinase domain.

WNK1 is a member of a novel serine/threonine kinase family, With-No-K, (lysine). Intronic deletions in the encoding gene cause Gordon syndrome, an autosomal dominant, hypertensive, hyperkalemic disorder particularly responsive to thiazide diuretics, a first-line treatment in essential hypertension. To elucidate the novel WNK1 BP control pathway active in distal nephron, WNK1 expression in mouse was studied. It was found that WNK1 is highly expressed in testis > heart, lung, kidney, placenta > skeletal muscle, brain, and widely at low levels. Several WNK1 transcript classes are demonstrated, showing tissue-, developmental-, and nephron-segment-specific expression. Importantly, in kidney, the most prominent transcripts are smaller than elsewhere, having the first four exons replaced by an alternative 5'-exon, deleting the kinase domain, and showing strong distal nephron expression, whereas larger transcripts show low-level widespread distribution. Alternative splicing of exons 11 and 12 is prominent-for example, transcripts containing exon 11 are abundant in neural tissues, testis, and secondary renal transcripts but are predominantly absent in placenta. The transcriptional diversity generated by these events would produce proteins greatly differing in both structure and function. These findings help further define and clarify the role of WNK1 and the thiazide-responsive pathway relevant to essential hypertension in which it participates.

Sgk1 gene expression in kidney and its regulation by aldosterone: spatio-temporal heterogeneity and quantitative analysis.

The serine-threonine kinase sgk1 was recently identified as a gene rapidly induced by mineralocorticoids, resulting in increased sodium transport in vitro. To carefully localize and quantify the renal sgk1 expression response to aldosterone, in situ hybridization was performed on kidneys of mice having aldosterone excess over a range of doses and durations. In control and adrenalectomized animals, the glomeruli and inner medullary collecting ducts were the major sites of sgk1 expression, which was maintained independent of aldosterone. Sgk1 upregulation induced by aldosterone excess exhibited spatio-temporal heterogeneity. Both acute (3-h) and chronic (6-d) aldosterone excess stimulated sgk1 expression in the distal nephron, i.e., from the distal convoluted tubules through to the outer medullary collecting ducts. Treatments for 6 d with low sodium diet (0.03% [I]) and aldosterone infusions (50 microg/kg per d [II], 150 microg/kg per d [III], and 750 microg/kg per d [IV]) generated elevation of circulating aldosterone. Across these treatments (I through IV), the circulating level correlated with the progressive induction of sgk1 expression, with highly stimulated tubules first appearing in cortex (I) and continuing downward (II) until there was a strong stimulation throughout outer medulla (III and IV). Interestingly, chronic but not acute aldosterone excess caused a slight increase of sgk1 expression in glomerulus (30 to 50%; P < 0.01) and a dramatic downregulation in the initial portion of inner medulla, which could result from diminished interstitial osmolarity. Relative quantification (versus control) of sgk1 upregulation in individual tubules revealed: (1) a 1.8-fold increase of sgk1 mRNA at 3 h (150 microg/kg injection) and (2) a dose-dependence of chronic upregulation reaching a ceiling of eightfold elevation.