BITES: balanced individual treatment effect for survival data.

MOTIVATION: Estimating the effects of interventions on patient outcome is one of the key aspects of personalized medicine. Their inference is often challenged by the fact that the training data comprises only the outcome for the administered treatment, and not for alternative treatments (the so-called counterfactual outcomes). Several methods were suggested for this scenario based on observational data, i.e. data where the intervention was not applied randomly, for both continuous and binary outcome variables. However, patient outcome is often recorded in terms of time-to-event data, comprising right-censored event times if an event does not occur within the observation period. Albeit their enormous importance, time-to-event data are rarely used for treatment optimization. We suggest an approach named BITES (Balanced Individual Treatment Effect for Survival data), which combines a treatment-specific semi-parametric Cox loss with a treatment-balanced deep neural network; i.e. we regularize differences between treated and non-treated patients using Integral Probability Metrics (IPM). RESULTS: We show in simulation studies that this approach outperforms the state of the art. Furthermore, we demonstrate in an application to a cohort of breast cancer patients that hormone treatment can be optimized based on six routine parameters. We successfully validated this finding in an independent cohort. AVAILABILITY AND IMPLEMENTATION: We provide BITES as an easy-to-use python implementation including scheduled hyper-parameter optimization (https://github.com/sschrod/BITES). The data underlying this article are available in the CRAN repository at https://rdrr.io/cran/survival/man/gbsg.html and https://rdrr.io/cran/survival/man/rotterdam.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular signatures that can be transferred across different omics platforms.

MOTIVATION: Molecular signatures for treatment recommendations are well researched. Still it is challenging to apply them to data generated by different protocols or technical platforms. RESULTS: We analyzed paired data for the same tumors (Burkitt lymphoma, diffuse large B-cell lymphoma) and features that had been generated by different experimental protocols and analytical platforms including the nanoString nCounter and Affymetrix Gene Chip transcriptomics as well as the SWATH and SRM proteomics platforms. A statistical model that assumes independent sample and feature effects accounted for 69-94% of technical variability. We analyzed how variability is propagated through linear signatures possibly affecting predictions and treatment recommendations. Linear signatures with feature weights adding to zero were substantially more robust than unbalanced signatures. They yielded consistent predictions across data from different platforms, both for transcriptomics and proteomics data. Similarly stable were their predictions across data from fresh frozen and matching formalin-fixed paraffin-embedded human tumor tissue. AVAILABILITY AND IMPLEMENTATION: The R-package 'zeroSum' can be downloaded at https://github.com/rehbergT/zeroSum . Complete data and R codes necessary to reproduce all our results can be received from the authors upon request. CONTACT: rainer.spang@ur.de.

Reference point insensitive molecular data analysis.

MOTIVATION: In biomedicine, every molecular measurement is relative to a reference point, like a fixed aliquot of RNA extracted from a tissue, a defined number of blood cells, or a defined volume of biofluid. Reference points are often chosen for practical reasons. For example, we might want to assess the metabolome of a diseased organ but can only measure metabolites in blood or urine. In this case, the observable data only indirectly reflects the disease state. The statistical implications of these discrepancies in reference points have not yet been discussed. RESULTS: Here, we show that reference point discrepancies compromise the performance of regression models like the LASSO. As an alternative, we suggest zero-sum regression for a reference point insensitive analysis. We show that zero-sum regression is superior to the LASSO in case of a poor choice of reference point both in simulations and in an application that integrates intestinal microbiome analysis with metabolomics. Moreover, we describe a novel coordinate descent based algorithm to fit zero-sum elastic nets. AVAILABILITY AND IMPLEMENTATION: The R-package "zeroSum" can be downloaded at https://github.com/rehbergT/zeroSum Moreover, we provide all R-scripts and data used to produce the results of this manuscript as Supplementary Material CONTACT: Michael.Altenbuchinger@ukr.de, Thorsten.Rehberg@ukr.de and Rainer.Spang@ukr.deSupplementary information: Supplementary material is available at Bioinformatics online.

Rifaximin preserves intestinal microbiota balance in patients undergoing allogeneic stem cell transplantation.

Intestinal dysbiosis has been associated with acute gastrointestinal GvHD and poor outcome following allogeneic stem cell transplantation (ASCT). To assess the effect of a switch in 2012 from ciprofloxacin/metronidazole to rifaximin for gut decontamination on intestinal microbiota composition and ASCT outcome, we retrospectively analyzed 394 patients receiving ASCT from September 2008 through June 2015. In 131 and 90 patients, respectively, urinary 3-indoxyl sulfate levels and intestinal enterococcal load were measured before conditioning and weekly within the first 28 days after ASCT. The use of rifaximin correlated with lower enterococcal positivity (6.9 vs 21.9%, P=0.05) and higher urinary 3-indoxyl sulfate concentrations (10.5 vs 4.6 µmoL/mmoL crea, P<0.001) after ASCT. Patients on rifaximin showed lower 1-year transplant-related mortality (P=0.04) and higher overall survival (P=0.008). Treatment of infectious complications with systemic antibiotics did not abrogate the beneficial effects of rifaximin on intestinal microbiota composition in the early course of ASCT and outcome. The data underscore the importance of maintaining a diverse population of symbiotic and mutualistic bacteria in the gut on ASCT outcome.

Increased incidence of chronic GvHD and CMV disease in patients with vitamin D deficiency before allogeneic stem cell transplantation.

Vitamin D has emerged as a central player in the immune system, with its deficiency being implicated in the pathogenesis of several autoimmune diseases, including chronic GvHD. This is a retrospective cohort analysis of 166 patients, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) at the Karolinska University Hospital, evaluating GvHD, graft failure, infectious complications and survival after HSCT in relation to pre-transplantation vitamin D levels. Most of the patients were deficient in vitamin D before HSCT (median 42 nmol/L). In multivariate analysis, vitamin D level before HSCT was identified as a significant independent risk factor for development of cGvHD. The increased incidence of cGvHD was not coupled to better disease-free survival; instead there was a trend towards lower overall survival in the vitamin D-deficient patients. In addition, we found a significant correlation between vitamin D deficiency and incidence of CMV disease, with no case of CMV disease occurring in patients with sufficient levels of vitamin D before HSCT. Our results support a role of vitamin D in immune tolerance following HSCT. These findings could be highly relevant for the care of HSCT patients, and prospective, randomized studies on the effect of vitamin D supplementation are therefore needed.

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation.

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.

Re-sequencing of multiple single nucleotide polymorphisms by liquid chromatography-electrospray ionization mass spectrometry.

Allelic discrimination of single nucleotide polymorphisms (SNPs) and, particularly, determination of the phase of multiple variations are of utmost importance in genetics. The physicochemical separation of alleles by completely denaturing ion-pair reversed-phase high-performance liquid chromatography and their on-line sequence determination by electrospray ionization mass spectrometry is demonstrated. Simultaneous genotyping of two and three simple sequence polymorphisms contained within 73-114 bp was accomplished with low femtomolar amounts of unpurified amplicons from polymerase chain reaction. Determination of allelic composition is enabled by the high accuracy (better than 0.019%) of intact mass measurements or by comparative sequencing using gas-phase fragmentation and tandem mass spectrometry in combination with fully automated, computer-aided data interpretation.

Origins and divergence of the Roma (gypsies).

The identification of a growing number of novel Mendelian disorders and private mutations in the Roma (Gypsies) points to their unique genetic heritage. Linguistic evidence suggests that they are of diverse Indian origins. Their social structure within Europe resembles that of the jatis of India, where the endogamous group, often defined by profession, is the primary unit. Genetic studies have reported dramatic differences in the frequencies of mutations and neutral polymorphisms in different Romani populations. However, these studies have not resolved ambiguities regarding the origins and relatedness of Romani populations. In this study, we examine the genetic structure of 14 well-defined Romani populations. Y-chromosome and mtDNA markers of different mutability were analyzed in a total of 275 individuals. Asian Y-chromosome haplogroup VI-68, defined by a mutation at the M82 locus, was present in all 14 populations and accounted for 44.8% of Romani Y chromosomes. Asian mtDNA-haplogroup M was also identified in all Romani populations and accounted for 26.5% of female lineages in the sample. Limited diversity within these two haplogroups, measured by the variation at eight short-tandem-repeat loci for the Y chromosome, and sequencing of the HVS1 for the mtDNA are consistent with a small group of founders splitting from a single ethnic population in the Indian subcontinent. Principal-components analysis and analysis of molecular variance indicate that genetic structure in extant endogamous Romani populations has been shaped by genetic drift and differential admixture and correlates with the migrational history of the Roma in Europe. By contrast, social organization and professional group divisions appear to be the product of a more recent restitution of the caste system of India.

Temperature-modulated array high-performance liquid chromatography.

Using novel monolithic poly(styrene-divinylbenzene) capillary columns with an internal diameter of 0.2 mm, we demonstrate for the first time the feasibility of constructing high-performance liquid chromatography arrays for the detection of mutations by heteroduplex analysis under partially denaturing conditions. In one embodiment, such an array can be used to analyze one sample simultaneously at different temperatures to maximize the detection of mutations in DNA fragments containing multiple discrete melting domains. Alternatively, one may inject different samples onto columns kept at the same effective temperature. Further improvements in throughput can be obtained by means of laser-induced fluorescence detection and the differential labeling of samples with up to four different fluorophores. Major advantages of monolithic capillary high-performance liquid chromatographic arrays over their capillary electrophoretic analogs are the chemical inertness of the poly(styrene-divinylbenzene) stationary phase, the physical robustness of the column bed due to its covalent linkage to the inner surface of the fused silica capillary, and the feasibility to modify the stationary phase thereby allowing the separation of compounds not only on the principle of size exclusion, but also adsorption, distribution, and ion exchange. Analyses times are on the order of a few minutes and turnaround time is extremely short as there is no need for the replenishment of the separation matrix between runs.

Global analysis of ATM polymorphism reveals significant functional constraint.

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.

Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection.

Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of monolithic poly(styrene-divinylbenzene) capillary columns with four-color confocal argon ion laser-induced fluorescence detection. Depending on the change in retention caused by the fluorophores, adjustments in the analysis temperature may be required to ensure the maximum mutation detection sensitivity.

Denaturing high-performance liquid chromatography: A review.

Denaturing high-performance liquid chromatography (DHPLC) compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presence of a mutation by the differential retention of homo- and heteroduplex DNA on reversed-phase chromatography supports under partial denaturation. Temperature determines sensitivity, and its optimum can be predicted by computation. Single-nucleotide substitutions, deletions, and insertions have been detected successfully by on-line UV or fluorescence monitoring within 2-3 minutes in unpurified amplicons as large as 1.5 Kb. Sensitivity and specificity of DHPLC consistently exceed 96%. These features and its low cost make DHPLC one of the most powerful tools for the re-sequencing of the human and other genomes. Aside from its application to the mutational analysis of candidate genes, DHPLC has proven instrumental in elucidating human evolution and in the mapping of genes. Employing completely denaturing conditions, the utility of DHPLC has been extended to the genotyping of known polymorphisms by utilizing the ability of poly(styrene-divinylbenzene) to resolve single-stranded DNA molecules of identical size that differ in a single base. Under completely denaturing conditions, it is thus possible to resolve all possible base substitutions with the single exception of C-->G transversions. Improvements in throughput became feasible with the recent introduction of monolithic poly(styrene-divinylbenzene) capillaries that lend themselves to the fabrication of arrays connected to a multi-color laser induced fluorescence scanner or a mass spectrometer.

The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations.

Although molecular genetic evidence continues to accumulate that is consistent with a recent common African ancestry of modern humans, its ability to illuminate regional histories remains incomplete. A set of unique event polymorphisms associated with the non-recombining portion of the Y-chromosome (NRY) addresses this issue by providing evidence concerning successful migrations originating from Africa, which can be interpreted as subsequent colonizations, differentiations and migrations overlaid upon previous population ranges. A total of 205 markers identified by denaturing high performance liquid chromatography (DHPLC), together with 13 taken from the literature, were used to construct a parsimonious genealogy. Ancestral allelic states were deduced from orthologous great ape sequences. A total of 131 unique haplotypes were defined which trace the microevolutionary trajectory of global modern human genetic diversification. The genealogy provides a detailed phylogeographic portrait of contemporary global population structure that is emblematic of human origins, divergence and population history that is consistent with climatic, paleoanthropological and other genetic knowledge.

Maori origins, Y-chromosome haplotypes and implications for human history in the Pacific.

An assessment of 28 pertinent binary genetic markers on the non-recombining portion of the Y chromosome (NRY) in New Zealand Maori and other relevant populations has revealed a diverse genetic paternal heritage of extant Maori. A maximum parsimony phylogeny was constructed in which nine of the 25 possible binary haplotypes were observed. Although approximately 40% of the samples have haplotypes of unequivocal European origin, an equivalent number of samples have a single binary haplotype that is also observed in Indonesia and New Guinea, indicative of common indigenous Melanesian ancestry. The balance of the lineages has either typical East Asian signatures or alternative compositions consistent with their affinity to Melanesia or New Guinea. Molecular analysis of mtDNA variation confirms the presence of a single predominant characteristic Southeast Asian (9-bp deletion in the Region V) lineage. The Y-chromosome results support a pattern of complex interrelationships between Southeast Asia, Melanesia, and Polynesia, in contrast to mtDNA and linguistic data, which uphold a rapid and homogeneous Austronesian expansion. The Y-chromosome data highlight a distinctive gender-modulated pattern of differential gene flow in the history of Polynesia.

High-resolution analysis of human Y-chromosome variation shows a sharp discontinuity and limited gene flow between northwestern Africa and the Iberian Peninsula.

In the present study we have analyzed 44 Y-chromosome biallelic polymorphisms in population samples from northwestern (NW) Africa and the Iberian Peninsula, which allowed us to place each chromosome unequivocally in a phylogenetic tree based on >150 polymorphisms. The most striking results are that contemporary NW African and Iberian populations were found to have originated from distinctly different patrilineages and that the Strait of Gibraltar seems to have acted as a strong (although not complete) barrier to gene flow. In NW African populations, an Upper Paleolithic colonization that probably had its origin in eastern Africa contributed 75% of the current gene pool. In comparison, approximately 78% of contemporary Iberian Y chromosomes originated in an Upper Paleolithic expansion from western Asia, along the northern rim of the Mediterranean basin. Smaller contributions to these gene pools (constituting 13% of Y chromosomes in NW Africa and 10% of Y chromosomes in Iberia) came from the Middle East during the Neolithic and, during subsequent gene flow, from Sub-Saharan to NW Africa. Finally, bidirectional gene flow across the Strait of Gibraltar has been detected: the genetic contribution of European Y chromosomes to the NW African gene pool is estimated at 4%, and NW African populations may have contributed 7% of Iberian Y chromosomes. The Islamic rule of Spain, which began in a.d. 711 and lasted almost 8 centuries, left only a minor contribution to the current Iberian Y-chromosome pool. The high-resolution analysis of the Y chromosome allows us to separate successive migratory components and to precisely quantify each historical layer.

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.