Background Pulmonary hypertension (PH) is a common and morbid complication of left heart disease with 2 subtypes: isolated post-capillary PH (Ipc-PH) and combined post-capillary and pre-capillary PH (Cpc-PH). Little is known about the clinical or physiological characteristics that distinguish these 2 subphenotypes, and if Cpc-PH shares molecular similarities to pulmonary arterial hypertension (PAH). Objectives We sought to test the hypothesis that the hemodynamic and genetic profile of Cpc-PH would more closely resemble PAH than Ipc-PH. Methods We used Vanderbilt’s electronic medical record linked to a DNA biorepository to extract demographics, clinical data, invasive hemodynamics, echocardiography, and vital status for all patients referred for right heart catheterization between 1998 and 2014. We identified shared genetic variants between PAH and Cpc-PH compared with Ipc-PH using pre-existing single-nucleotide polymorphism data. Results We identified 2,817 patients with PH (13% Cpc-PH, 52% Ipc-PH, and 20% PAH). Cpc-PH patients were on average 6 years younger, with more severe pulmonary vascular disease than Ipc-PH patients, despite similar comorbidities and prevalence, severity, and chronicity of left heart disease. After adjusting for relevant covariates, the risk of death was similar between Cpc-PH and Ipc-PH (HR: 1.14, 95% CI: 0.96 to 1.35, p = 0.15) when defined by diastolic pressure gradient. We identified 75 shared exonic single-nucleotide polymorphisms between Cpc-PH and PAH enriched in pathways involving cell structure, extracellular matrix, and immune function. These genes are expressed, on average, 32% higher in lungs relative to other tissues. Conclusions Cpc-PH patients develop pulmonary vascular disease similar to PAH patients, despite younger age and similar prevalence of obesity, diabetes mellitus, and left heart disease compared with Ipc-PH patients. An exploratory genetic analysis in Cpc-PH identified genes and biological pathways in the lung known to contribute to PAH pathophysiology, suggesting that Cpc-PH may be a distinct and highly morbid PH subphenotype.

Heat maps and clustering are used frequently in expression analysis studies for data visualization and quality control. Simple clustering and heat maps can be produced from the “heatmap” function in R. However, the “heatmap” function lacks certain functionalities and customizability, preventing it from generating advanced heat maps and dendrograms. To tackle the limitations of the “heatmap” function, we have developed an R package “heatmap3” which significantly improves the original “heatmap” function by adding several more powerful and convenient features. The “heatmap3” package allows users to produce highly customizable state of the art heat maps and dendrograms. The “heatmap3” package is developed based on the “heatmap” function in R, and it is completely compatible with it. The new features of “heatmap3” include highly customizable legends and side annotation, a wider range of color selections, new labeling features which allow users to define multiple layers of phenotype variables, and automatically conducted association tests based on the phenotypes provided. Additional features such as different agglomeration methods for estimating distance between two samples are also added for clustering.

With the rise of high-throughput sequencing technology, traditional genotyping arrays are gradually being replaced by sequencing technology. Against this trend, Illumina has introduced an exome genotyping array that provides an alternative approach to sequencing, especially suited to large-scale genome-wide association studies (GWASs). the exome genotyping array targets the exome plus rare single-nucleotide polymorphisms (SNPs), a feature that makes it substantially more challenging to process than previous genotyping arrays that targeted common SNPs. Researchers have struggled to generate a reliable protocol for processing exome genotyping array data. The Vanderbilt epidemiology center, in cooperation with Vanderbilt Technologies for Advanced Genomics Analysis and Research Design (VANGARD), has developed a thorough exome chip–processing protocol. The protocol was developed during the processing of several large exome genotyping array-based studies, which included over 60,000 participants combined. The protocol described herein contains detailed clustering techniques and robust quality control procedures, and it can benefit future exome genotyping array–based GWASs.

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reversephase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.