Leveraging network analytics to infer patient syndrome and identify causal genes in rare disease cases.

BACKGROUND: Next-generation sequencing is widely used to identify disease-causing variants in patients with rare genetic disorders. Identifying those variants from whole-genome or exome data can be both scientifically challenging and time consuming. A significant amount of time is spent on variant annotation, and interpretation. Fully or partly automated solutions are therefore needed to streamline and scale this process. RESULTS: We describe Phenotype Driven Ranking (PDR), an algorithm integrated into Ingenuity Variant Analysis, that uses observed patient phenotypes to prioritize diseases and genes in order to expedite causal-variant discovery. Our method is based on a network of phenotype-disease-gene relationships derived from the QIAGEN Knowledge Base, which allows for efficient computational association of phenotypes to implicated diseases, and also enables scoring and ranking. CONCLUSIONS: We have demonstrated the utility and performance of PDR by applying it to a number of clinical rare-disease cases, where the true causal gene was known beforehand. It is also shown that PDR compares favorably to a representative alternative tool.

Working toward precision medicine: Predicting phenotypes from exomes in the Critical Assessment of Genome Interpretation (CAGI) challenges.

Precision medicine aims to predict a patient's disease risk and best therapeutic options by using that individual's genetic sequencing data. The Critical Assessment of Genome Interpretation (CAGI) is a community experiment consisting of genotype-phenotype prediction challenges; participants build models, undergo assessment, and share key findings. For CAGI 4, three challenges involved using exome-sequencing data: Crohn's disease, bipolar disorder, and warfarin dosing. Previous CAGI challenges included prior versions of the Crohn's disease challenge. Here, we discuss the range of techniques used for phenotype prediction as well as the methods used for assessing predictive models. Additionally, we outline some of the difficulties associated with making predictions and evaluating them. The lessons learned from the exome challenges can be applied to both research and clinical efforts to improve phenotype prediction from genotype. In addition, these challenges serve as a vehicle for sharing clinical and research exome data in a secure manner with scientists who have a broad range of expertise, contributing to a collaborative effort to advance our understanding of genotype-phenotype relationships.

Lessons from the CAGI-4 Hopkins clinical panel challenge.

The CAGI-4 Hopkins clinical panel challenge was an attempt to assess state-of-the-art methods for clinical phenotype prediction from DNA sequence. Participants were provided with exonic sequences of 83 genes for 106 patients from the Johns Hopkins DNA Diagnostic Laboratory. Five groups participated in the challenge, predicting both the probability that each patient had each of the 14 possible classes of disease, as well as one or more causal variants. In cases where the Hopkins laboratory reported a variant, at least one predictor correctly identified the disease class in 36 of the 43 patients (84%). Even in cases where the Hopkins laboratory did not find a variant, at least one predictor correctly identified the class in 39 of the 63 patients (62%). Each prediction group correctly diagnosed at least one patient that was not successfully diagnosed by any other group. We discuss the causal variant predictions by different groups and their implications for further development of methods to assess variants of unknown significance. Our results suggest that clinically relevant variants may be missed when physicians order small panels targeted on a specific phenotype. We also quantify the false-positive rate of DNA-guided analysis in the absence of prior phenotypic indication.

Evaluating the association of multiple single nucleotide polymorphisms with response to gemcitabine and platinum combination chemotherapy in urothelial carcinoma of the bladder
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OBJECTIVE: To examine germline single nucleotide polymorphisms (SNPs) as markers of response to gemcitabine platinum (GP) combination chemotherapy in urothelial carcinoma (UC). METHODS: Saliva or blood was prospectively collected from 216 patients treated with GP for UC of the bladder between 1991 and 2011. Based on reported associations with gemcitabine and cisplatin response or putative mechanisms of gemcitabine or cisplatin/carboplatin activity, we selected SNPs of interest and were able to genotype 59 SNPs (using the SequenomMass ARRAYiPLEX platform) in 261 patients randomly split 2/3 into a training set (n = 174) and 1/3 into a test set (n = 87). Logistic regression was used to test the association between response to GP and SNPs. RESULTS: The median age at diagnosis was 64 years (range: 28 - 85) for the discovery set and 67 years (range: 30 - 84) for the validation set. Males composed 76% and 69%, and white non-Hispanics composed 88% and 91% of the training and test validation sets, respectively. Three SNPs on GALNTL4 (rs7937567, rs12278731, and rs9988868) and one intergenic SNP (rs1321391) were significantly associated with response to GP in the training set and were used to build a SNP score. However, when assessed in the test set, the SNP score was not significantly associated with response. CONCLUSION: Multiple SNPs selected from previous studies failed to predict response to GP in this cohort. Larger studies capable of accounting for population-based allele frequency heterogeneity may be required for replication of genetic alterations important to pharmacogenomics.
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Intrahepatic Cholestasis of Pregnancy (ICP) in U.S. Latinas and Chileans: Clinical features, Ancestry Analysis, and Admixture Mapping.

In the Americas, women with Indigenous American ancestry are at increased risk of intrahepatic cholestasis of pregnancy (ICP), relative to women of other ethnicities. We hypothesized that ancestry-related genetic factors contribute to this increased risk. We collected clinical and laboratory data, and performed biochemical assays on samples from U.S. Latinas and Chilean women, with and without ICP. The study sample included 198 women with ICP (90 from California, U.S., and 108 from Chile) and 174 pregnant control women (69 from California, U.S., and 105 from Chile). SNP genotyping was performed using Affymetrix arrays. We compared overall genetic ancestry between cases and controls, and used a genome-wide admixture mapping approach to screen for ICP susceptibility loci. We identified commonalities and differences in features of ICP between the 2 countries and determined that cases had a greater proportion of Indigenous American ancestry than did controls (p = 0.034). We performed admixture mapping, taking country of origin into account, and identified one locus for which Native American ancestry was associated with increased risk of ICP at a genome-wide level of significance (P = 3.1 x 10(-5), Pcorrected = 0.035). This locus has an odds ratio of 4.48 (95% CI: 2.21-9.06) for 2 versus zero Indigenous American chromosomes. This locus lies on chromosome 2, with a 10 Mb 95% confidence interval which does not contain any previously identified hereditary 'cholestasis genes.' Our results indicate that genetic factors contribute to the risk of developing ICP in the Americas, and support the utility of clinical and genetic studies of ethnically mixed populations for increasing our understanding of ICP.

Empowered genome community: leveraging a bioinformatics platform as a citizen-scientist collaboration tool.

There is on-going effort in the biomedical research community to leverage Next Generation Sequencing (NGS) technology to identify genetic variants that affect our health. The main challenge facing researchers is getting enough samples from individuals either sick or healthy - to be able to reliably identify the few variants that are causal for a phenotype among all other variants typically seen among individuals. At the same time, more and more individuals are having their genome sequenced either out of curiosity or to identify the cause of an illness. These individuals may benefit from of a way to view and understand their data. QIAGEN's Ingenuity Variant Analysis is an online application that allows users with and without extensive bioinformatics training to incorporate information from published experiments, genetic databases, and a variety of statistical models to identify variants, from a long list of candidates, that are most likely causal for a phenotype as well as annotate variants with what is already known about them in the literature and databases. Ingenuity Variant Analysis is also an information sharing platform where users may exchange samples and analyses. The Empowered Genome Community (EGC) is a new program in which QIAGEN is making this on-line tool freely available to any individual who wishes to analyze their own genetic sequence. EGC members are then able to make their data available to other Ingenuity Variant Analysis users to be used in research. Here we present and describe the Empowered Genome Community in detail. We also present a preliminary, proof-of-concept study that utilizes the 200 genomes currently available through the EGC. The goal of this program is to allow individuals to access and understand their own data as well as facilitate citizen-scientist collaborations that can drive research forward and spur quality scientific dialogue in the general public.

CCBE1 mutation in two siblings, one manifesting lymphedema-cholestasis syndrome, and the other, fetal hydrops.

BACKGROUND: Lymphedema-cholestasis syndrome (LCS; Aagenaes syndrome) is a rare autosomal recessive disorder, characterized by 1) neonatal intrahepatic cholestasis, often lessening and becoming intermittent with age, and 2) severe chronic lymphedema, mainly lower limb. LCS was originally described in a Norwegian kindred in which a locus, LCS1, was mapped to a 6.6cM region on chromosome 15. Mutations in CCBE1 on chromosome 18 have been reported in some cases of lymphatic dysplasia, but not in LCS. METHODS: Consanguineous parents of Mexican ancestry had a child with LCS who did not exhibit extended homozygosity in the LCS1 region. A subsequent pregnancy was electively terminated due to fetal hydrops. We performed whole-genome single nucleotide polymorphism genotyping to identify regions of homozygosity in these siblings, and sequenced promising candidate genes. RESULTS: Both siblings harbored a homozygous mutation in CCBE1, c.398 T>C, predicted to result in the missense change p.L133P. Regions containing known 'cholestasis genes' did not demonstrate homozygosity in the LCS patient. CONCLUSIONS: Mutations in CCBE1 may yield a phenotype not only of lymphatic dysplasia, but also of LCS or fetal hydrops; however, the possibility that the sibling with LCS also carries a homozygous mutation in an unidentified gene influencing cholestasis cannot be excluded.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia.

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Assessment of SLX4 Mutations in Hereditary Breast Cancers.

BACKGROUND: SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers. METHODS AND RESULTS: To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65-1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so. CONCLUSION: Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.

Prevalence of HOXB13 mutation in a population of Ashkenazi Jewish men treated for prostate cancer.

We performed a retrospective analysis of germline DNA samples from Ashkenazi Jewish men and a comparison group of non-Ashkenazi men treated for prostate cancer at our institution to determine the prevalence of HOXB13 G84E mutation in prostate cancer patients of Ashkenazi Jewish heritage, an ethnic group common to the New York City area. Patients were genotyped for G84E using a TaqMan assay (Applied Biosystems). Positive cases were confirmed using Sanger sequencing. Median age at prostate cancer diagnosis was 68 years for 889 Ashkenazi Jewish patients, 64 years for 920 non-Ashkenazi Jewish patients. The median follow up was 9 years for Ashkenazi Jewish patients and 8.8 years for non-Ashkenazi Jewish patients. Only 4 patients were found to be heterozygous carriers of G84E. They were all of non-Ashkenazi Jewish ancestry and were diagnosed at 70, 66, 78, and 49 years of age. Two of them presented with high-risk prostate cancer. The prevalence of G84E in the non-Ashkenazi sample was 0.4%. HOXB13 G84E mutation was not observed in prostate cancer patients of Ashkenazi Jewish ancestry treated at our institution. Screening for G84E, therefore, may be unnecessary in Ashkenazi Jewish men if these results are validated by other studies.

Genetic defects in bile acid conjugation cause fat-soluble vitamin deficiency.

BACKGROUND & AIMS: The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis. METHODS: We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-CoA ligase (SLC27A5). RESULTS: Levels of urinary bile acids were increased (432 ± 248 µmol/L) and predominantly excreted in unconjugated forms (79.4% ± 3.9%) and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile, and serum. Unconjugated bile acids accounted for 95.7% ± 5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4% ± 5.5% of the total. Duodenal bile acid concentrations were 12.1 ± 5.9 mmol/L, which is too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested. CONCLUSIONS: Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.

Susceptibility loci associated with specific and shared subtypes of lymphoid malignancies.

The genetics of lymphoma susceptibility reflect the marked heterogeneity of diseases that comprise this broad phenotype. However, multiple subtypes of lymphoma are observed in some families, suggesting shared pathways of genetic predisposition to these pathologically distinct entities. Using a two-stage GWAS, we tested 530,583 SNPs in 944 cases of lymphoma, including 282 familial cases, and 4,044 public shared controls, followed by genotyping of 50 SNPs in 1,245 cases and 2,596 controls. A novel region on 11q12.1 showed association with combined lymphoma (LYM) subtypes. SNPs in this region included rs12289961 near LPXN, (P(LYM) = 3.89×10(-8), OR = 1.29) and rs948562 (P(LYM) = 5.85×10(-7), OR = 1.29). A SNP in a novel non-HLA region on 6p23 (rs707824, P(NHL) = 5.72×10(-7)) was suggestive of an association conferring susceptibility to lymphoma. Four SNPs, all in a previously reported HLA region, 6p21.32, showed genome-wide significant associations with follicular lymphoma. The most significant association with follicular lymphoma was for rs4530903 (P(FL) = 2.69×10(-12), OR = 1.93). Three novel SNPs near the HLA locus, rs9268853, rs2647046, and rs2621416, demonstrated additional variation contributing toward genetic susceptibility to FL associated with this region. Genes implicated by GWAS were also found to be cis-eQTLs in lymphoblastoid cell lines; candidate genes in these regions have been implicated in hematopoiesis and immune function. These results, showing novel susceptibility regions and allelic heterogeneity, point to the existence of pathways of susceptibility to both shared as well as specific subtypes of lymphoid malignancy.

Absence of loss of heterozygosity of BRCA1 in a renal tumor from a BRCA1 germline mutation carrier.

BRCA1 functions as a tumor suppressor gene and germline and somatic mutations in this gene have been shown to be associated with many types of cancer. We report the first tumor study of renal cell carcinoma in a carrier of the deleterious BRCA1 mutation-c.68_69delAG.

Rare variants in XRCC2 as breast cancer susceptibility alleles.

BACKGROUND: Recently, rare germline variants in XRCC2 were detected in non-BRCA1/2 familial breast cancer cases, and a significant association with breast cancer was reported. However, the breast cancer risk associated with these variants needs further evaluation. METHODS: The coding regions and exon-intron boundaries of XRCC2 were scanned for mutations in an international cohort of 3548 non-BRCA1/2 familial breast cancer cases and 1435 healthy controls using various mutation scanning methods. Predictions on functional relevance of detected missense variants were obtained from three different prediction algorithms. RESULTS: The only protein-truncating variant detected was found in a control. Rare non-protein-truncating variants were detected in 20 familial cases (0.6%) and nine healthy controls (0.6%). Although the number of variants predicted to be damaging or neutral differed between prediction algorithms, in all instances these categories were evenly represented among cases and controls. CONCLUSIONS: Our data do not confirm an association between XRCC2 variants and breast cancer risk, although a relative risk smaller than two could not be excluded. Variants in XRCC2 are unlikely to explain a substantial proportion of familial breast cancer.

Rare de novo germline copy-number variation in testicular cancer.

Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer trios or controls but were indeed found in 7% of 43 testicular germ cell tumor trios; this percentage exceeds background CNV rates and suggests a rare de novo genetic paradigm for susceptibility to some human malignancies.

Strain background modifies phenotypes in the ATP8B1-deficient mouse.

BACKGROUND: Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.

Babesia microti primarily invades mature erythrocytes in mice.

Babesia microti is a tick-borne red blood cell parasite that causes babesiosis in people. Its most common vertebrate reservoir is the white-footed mouse. To determine whether B. microti invades reticulocytes, as does the canine pathogen B. gibsoni, we infected the susceptible inbred mouse strains C.B-17.scid and DBA/2 with a clinical isolate of B. microti. Staining of fixed permeabilized red blood cells with 4',6'-diamidino-2-phenylindole or YOYO-1, a sensitive nucleic acid stain, revealed parasite nuclei as large bright dots. Flow cytometric analysis indicated that parasite DNA is primarily found in mature erythrocytes that expressed Babesia antigens but not the transferrin receptor CD71. In contrast, CD71-positive reticulocytes rarely contained Babesia nuclei and failed to express Babesia antigens. Accordingly, the frequency of YOYO-1-positive, CD71-negative cells strongly correlated with parasitemia, defined as the frequency of infected red blood cells assessed on Giemsa-stained blood smears. Importantly, the absolute numbers generated by the two techniques were similar. Parasitemia was modest and transient in DBA/2 mice but intense and sustained in C.B-17.scid mice. In both strains, parasitemia preceded reticulocytosis, but reticulocytes remained refractory to B. microti. In immunocompetent C.B-17 mice, reticulocytosis developed early, despite a marginal and short-lived parasitemia. Likewise, an early reticulocytosis developed in resistant BALB/cBy and B10.D2 mice. These studies establish that B. microti has a tropism for mature erythrocytes. Although reticulocytes are rarely infected, the delayed reticulocytosis in susceptible strains may result from parasite or host activities to limit renewal of the mature erythrocyte pool, thereby preventing an overwhelming parasitemia.