Full Sequencing Research Articles

Prevalence and genetic variants of G6PD deficiency among two Malagasy populations living in Plasmodium vivax-endemic areas

BackgroundThe prevalence and variants of G6PD deficiency in the Plasmodium vivax-endemic zones of Madagascar remain unknown. The admixed African-Austronesian origins of the Malagasy population make it probable that a heterogeneous mix of genetic variants with a spectrum of clinical severity will be circulating. This would have implications for the widespread use of P. vivax radical cure therapy. Two study populations in the P. vivax-endemic western foothills region of Madagascar were selected for G6PD screening. Both the qualitative fluorescent spot test and G6PD genotyping were used to screen all participants.ResultsA total of 365 unrelated male volunteers from the Tsiroanomandidy, Mandoto, and Miandrivazo districts of Madagascar were screened and 12.9% were found to be phenotypically G6PD deficient. Full gene sequencing of 95 samples identified 16 single nucleotide polymorphisms, which were integrated into a genotyping assay. Genotyping (n = 291) found one individual diagnosed with the severe G6PD MediterraneanC563T mutation, while the remaining G6PD deficient samples had mutations of African origin, G6PD A- and G6PD A.ConclusionsDeployment of P. vivax radical cure in Madagascar must be considerate of the risks presented by the observed prevalence of G6PDd prevalence. The potential morbidity associated with cumulative episodes of P. vivax clinical relapses requires a strategy for increasing access to safe radical cure. The observed dominance of African G6PDd haplotypes is surprising given the known mixed African-Austronesian origins of the Malagasy population; more widespread surveying of G6PDd epidemiology across the island would be required to characterize the distribution of G6PD haplotypes across Madagascar.

Next-generation sequencing of non-small cell lung cancer using a customized, targeted sequencing panel: Emphasis on small biopsy and cytology.

Background:Next-generation sequencing (NGS) with a multi-gene panel is now available for patients with lung adenocarcinoma, but the performance characteristics and clinical utility of this testing are not well-described. We present the results of an extended 467 gene panel in a series of advanced, highly selected nonsmall cell lung cancer (NSCLC) patients using a range of specimens, including predominantly small biopsy and cytology specimens.Materials and Methods:A retrospective review of 22 NSCLC biopsies sent for NGS using an extended gene panel from January 2014 to July 2015. The customized NGS panel sequences 467 cancer-associated genes with exonic and intronic sequences obtained from purified tumor DNA. Genomic alterations, patient characteristics, and success of testing were determined.Results:The majority of samples tested were metastatic lung adenocarcinoma on final pathology. Of the 22 specimens tested, 5 (22.7%) were surgical resections and 17 (77.3%) were small biopsy and cytology specimens. Twenty-one (95%) of the specimens were adequate for full sequencing and yielded a total of 204 genomic alterations (average 8.9 per tumor), of which 17 (average 0.81 per tumor) were actionable and/or clinically relevant. Genomic alterations were found most commonly in the TP53, EGFR, EPHB1, MLL3, APC, SETD2, KRAS, DNMT3A, RB1, CDKN2A, ARID1A, EP300, KDM6B, RAD50, STK11, and BRCA2 genes.Conclusions:NGS using a comprehensive gene panel was performed successfully in 95% of all NSCLC cases in this series, including 94% small biopsy and cytology specimens and 100% surgical resections. This custom assay was performed on a range of tumor specimens and demonstrates that small specimens are able to provide a similar depth of information as larger ones. As many patients present at an advanced stage and only small specimens are obtained, the information these provide has the potential for guiding treatment in highly selected patients with advanced lung adenocarcinoma.

The genome nucleotide sequence of herpes simplex virus 1 strain L2

The genome nucleotide sequence of the reference strain of herpes simplex virus type 1 was obtained using the technique of full size sequencing. For the virus genome structure determination, 402444 reads with an average length of 202 bp were performed, which corresponded to the 542-fold genome coverage. The data were collected to 52 contigs with N50-4518 and the total contig length of 120929 bp. The sequence obtained was deposited into the GenBank database.

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing.

In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottleneck as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations.

Clinical UGT1A1 Genetic Analysis in Pediatric Patients: Experience of a Reference Laboratory.

Neonatal hyperbilirubinemia can be severe or prolonged and warrant exploration into the underlying etiology, which may include genetic assessment of UGT1A1 for inherited disorders (i.e. Crigler-Najjar syndrome or Gilbert syndrome). In our reference laboratory, we performed UGT1A1 gene sequencing analysis on 346 pediatric patients referred for a clinical indication of hyperbilirubinemia. Males (n=241) had significantly higher mean total bilirubin concentration compared to females (n=105) (9.7 and 7.3mg/dL, respectively, p=0.042); however, no sex-based difference was observed in frequency of known or suspected reduced function UGT1A1 variants. The presence of two UGT1A1 variants (consistent with Gilbert or Crigler-Najjar syndrome) occurred less frequently in neonates (aged ≤28days) than older children (aged 1-18years) (31.3% in neonates vs. 85.1%, p<0.0001), and among neonates there was no significant difference in mean total bilirubin between those with two UGT1A1 variants and those without (p=0.47). Three novel variants, including c.337T>G (p.Y113D), c.1037C>A (p.A346E), and c.1469A>C (p.D490A) were identified. Among older children, the most common reason for referral was Gilbert syndrome (83.8%) and UGT1A1 genetic analysis confirmed a diagnosis of Gilbert syndrome in 79.0% of those children. Among neonates, a population in which hyperbilirubinemia is common and often of multifactorial etiology, UGT1A1 genetic testing served as a useful clinical tool in ruling in or ruling out inherited hyperbilirubinemia. Here we describe our experience as a reference laboratory in clinical UGT1A1 full gene sequencing. Our results highlight the challenges in predicting the contribution of genetic variation in UGT1A1 to hyperbilirubinemia based on clinical parameters alone, particularly in neonates, and the utility of UGT1A1 full gene sequencing in the evaluation of neonatal and pediatric hyperbilirubinemia.

Aerobic anoxygenic phototrophs in gold mine tailings in Nopiming Provincial Park, Manitoba, Canada.

A sampling trip to Central Gold Mine, Nopiming Provincial Park, Canada, was taken in September 2011. Abundance, distribution, and physiology of aerobic anoxygenic phototrophs (AAP) from 4 locations were studied. Enumeration revealed 14.6% of culturable microbes were AAP. Five strains (NM4.16, NM4.18, C4, C9, C11) were chosen for analysis. All grow best on complex media without vitamin requirements and with an optimal pH 7.0-8.0, with strain C4 preferring pH 6.0. Strain NM4.18 tolerates the highest pH 11.0. Optimal temperature for all is 28 °C (range of 2-37 °C except NM4.16, which survives 45 °C). Strains C9, C11, and NM4.18 grew in 1.0%, 2.0%, and 5.0% NaCl, respectively, while NM4.16 and C4 grew only without NaCl. Isolates were all highly resistant to toxic metal(oid) oxides: tellurite (1500 μg/mL, all), tellurate (1500 μg/mL, C11), selenite (5000 μg/mL, C9, C11, and NM4.18), selenate (1000 μg/mL, C9 and C11), and orthometavanadate and metavanadate (5000 μg/mL, C11 and NM4.18). They could reduce tellurite to the less toxic elemental tellurium. Full 16S rRNA gene sequencing revealed all strains are Alphaproteobacteria, with C4 and NM4.16 closely related to Porphyrobacter colymbi (99.4% and 99.7% sequence similarity, respectively), C9 to Brevundimonas variabilis (99.1%), C11 to Brevundimonas bacteroides (98.6%), and NM4.18 to Erythromonas ursincola (98.5%).

Abstract P1-06-06: Evidence for tumor heterogeneity and clonal evolution during invasive progression of breast cancer

Abstract Purpose: Intratumoral heterogeneity is well recognized to be an important driver of treatment resistance and metastasis. We undertook this N of one study to measure the degree of heterogeneity in a single large preinvasive lesion with an invasive component to determine the relationship between tumor heterogeneity, spatial distribution, clonal evolution, and invasive progression. Methods: We identified a patient with extensive DCIS measuring 7.5 cm, associated with 1.5 cm of an invasive component. We segregated the tumor sample into 32 unique blocks with precise geospatial localization; invasive cancer was identified in 3 of 32 blocks. NGS libraries were made from FFPE derived DNA (20-40ng) for full exome sequencing and hybridization to a 4.8 million element SNP array. All data were analyzed and a phylogentic tree was constructed. Results: The sequence data was analyzed with Platypus[1] and 3922 somatic mutation sites were found in total. These sites were concatenated into one sequence for each sample. Then a Neighbor-Joining tree was built with a Jukes-Cantor model and 1000 bootstrap replicates using FastME 2.0[2], to assess the reliability of the tree (Figure 1). Phylogenetic analysis revealed that invasive cancer evolved twice, independently. Dense sampling allowed reconstruction of the temporal order of mutations that accumulated in the cell lineage of the invasive cancers. Furthermore, the phylogeny revealed that distant regions may be closely genetically related, the oldest parts of the tumor were in the interior of the tumor, and that the invasive tumors evolved near the oldest parts of the tumor, rather than at the expanding front. Conclusions: Extensive sampling and sequencing of a single tumor yields important insights about tumor heterogeneity and tumor progression for DCIS to invasive cancer. Foci of invasion were geospatially associated with preinvasive regions of progressively higher mutational load. 1. Rimmer A, Phan H, Mathieson I, Iqbal Z, Twigg SR, Wilkie AO, McVean G, Lunter G, Consortium W: Integrating mapping-, assembly-and haplotype-based approaches for calling variants in clinical sequencing applications. Nature genetics 2014, 46(8):912-918. 2. Lefort V, Desper R, Gascuel O: FastME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Molecular biology and evolution 2015:msv150. Citation Format: Ding Y, Marks JR, King LM, Hall AH, Mardis ER, Rodrigo AG, Maley CC, Hwang E-S. Evidence for tumor heterogeneity and clonal evolution during invasive progression of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-06.

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails.

Single-nucleotide variant proportion in genes: a new concept to explore major depression based on DNA sequencing data.

Major depressive disorder (MDD) is a common psychiatric illness with significant medical and socioeconomic impact. Genetic factors are likely to play important roles in the development of this condition. DNA sequencing technology has the ability to identify all private genetic mutations and provides new channels for studying the biology of MDD. In this proof-of-concept study we proposed a novel concept, single-nucleotide variant proportion (SNVP), to investigate MDD based on whole-genome sequencing (WGS) data. Our SNVP-based approach can be used to test newly found candidate genes as a complement to genome-wide genotyping analysis. Furthermore, we performed cluster analysis for MDD patients and ethnically matched healthy controls, and found that clusters based on SNVP may predict MDD diagnosis. Our results suggest that SNVP may be used as a potential biomarker associated with major depression. Our methodology could be a valuable predictive/diagnostic tool as one can test whether a new subject falls within or close to an existing MDD cluster. Advances in this study design have the potential to personalized treatments and could include the ability to diagnose patients based on their full or part DNA sequencing data.

Phylogeography of human Y-chromosome haplogroup Q3-L275 from an academic/citizen science collaboration

BackgroundThe Y-chromosome haplogroup Q has three major branches: Q1, Q2, and Q3. Q1 is found in both Asia and the Americas where it accounts for about 90% of indigenous Native American Y-chromosomes; Q2 is found in North and Central Asia; but little is known about the third branch, Q3, also named Q1b-L275. Here, we combined the efforts of population geneticists and genetic genealogists to use the potential of full Y-chromosome sequencing for reconstructing haplogroup Q3 phylogeography and suggest possible linkages to events in population history.ResultsWe analyzed 47 fully sequenced Y-chromosomes and reconstructed the haplogroup Q3 phylogenetic tree in detail. Haplogroup Q3-L275, derived from the oldest known split within Eurasian/American haplogroup Q, most likely occurred in West or Central Asia in the Upper Paleolithic period. During the Mesolithic and Neolithic epochs, Q3 remained a minor component of the West Asian Y-chromosome pool and gave rise to five branches (Q3a to Q3e), which spread across West, Central and parts of South Asia. Around 3–4 millennia ago (Bronze Age), the Q3a branch underwent a rapid expansion, splitting into seven branches, some of which entered Europe. One of these branches, Q3a1, was acquired by a population ancestral to Ashkenazi Jews and grew within this population during the 1st millennium AD, reaching up to 5% in present day Ashkenazi.ConclusionsThis study dataset was generated by a massive Y-chromosome genotyping effort in the genetic genealogy community, and phylogeographic patterns were revealed by a collaboration of population geneticists and genetic genealogists. This positive experience of collaboration between academic and citizen science provides a model for further joint projects. Merging data and skills of academic and citizen science promises to combine, respectively, quality and quantity, generalization and specialization, and achieve a well-balanced and careful interpretation of the paternal-side history of human populations.

A novel mutation in TREM2 gene causing Nasu-Hakola disease and review of the literature

Nasu-hakola disease (NHD) is a rare disease characterized by bone cysts and fractures, frontal lobe syndrome, and progressive presenile dementia. NHD may be the prototype of primary microglial disorders of the CNS or, as they have been coined, “microgliopathies”. Mutations in TREM2 and TYROBP genes are known to cause NHD. Interestingly, recent evidence-associated rare genetic variants of TREM2 gene with increased risk of Alzheimer's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Parkinson's disease. Here, we report a 33-year-old Greek female with phenotype suggestive of NHD. Full gene sequencing of the TREM2 and TYROBP genes revealed a novel mutation in exon 2 of TREM2 gene, namely c.244G>T (p.W50C) and heterozygosity in the parents and her brother. This report extends the range of TREM2 mutations that cause NHD phenotype. In addition, we provide a comprehensive review of all reported in the literature TREM2 gene mutations and the respective wide spectrum of clinical manifestations that highlights the importance of considering TREM2 gene mutations in a variety of neurodegenerative phenotypes.

Bacteriophage Genome Sequencing: A New Alternative to Understand Biochemical Interactions between Prokaryotic Cells and Phages

Bacteriophages are viruses or semi-autonomous genetic entities that depend on prokaryotic cell’s metabolism to multiply. The use of lytic phages as biocontrol agents in many fields such as food preservation, disease control, agriculture production signifies the need of most appropriate and standard methods to insure application safety. Bacteriophage full genetic material sequencing is a new alternative to better understand phage encoded proteins and biomolecules (especially phage lytic enzymes) involved in the process of bacterial cell lysis and death. Thus, this short review aimed to discuss sequencing importance applied to bacteria-phage interactions in society commercial sectors, focusing in the food industry.

A Newborn with a Blueberry Muffin Rash, Hepatosplenomegaly, Thrombocytopenia and Severe Cholestatic Jaundice at Birth

Neonatal cholestasis is characterized by an elevation of conjugated bilirubin. It occurs in approximately 1 in 2500 term infants. Gaucher Disease (GD) is an exceptionally rare cause of neonatal cholestasis. We report a male term newborn with a blueberry muffin rash, hepatomegaly and massive splenomegaly at birth. His total serum bilirubin (TSB)/direct was 13.5/11.9 mg/dL, and platelet count 20,000 bil/L. A leukocyte beta-glucosidase level was 0 and full sequencing of the GBA gene showed that he was homozygous for the pathogenic complex allele p.D409H and a second variant p.H255Q, consistent with the diagnosis of the severe phenotype GD type 2.

Chromosomal findings and sequence analysis of target exons of calcium-sensingreceptor (CaSR) gene in patients with Sagliker syndrome.

Sagliker syndrome (SS) develops as a continuation of chronic kidney disease and secondary hyperparathyroidism conditions. It was thought that there are some genetic predisposition factors leading to SS. The calcium-sensing receptor (CaSR) is essential for calcium homeostasis in the body. We aimed to examine SS patients for chromosome aberrations (CAs) and CaSR gene abnormalities in exons 2 and 3. Twenty-three patients and 23 control subjects were admitted to Balcalı Hospital of the Medical Faculty of Çukurova University in Turkey between 2009 and 2011. Chromosomal analysis was performed according to standard cytogenetic methods. Full sequencing of exons 2 and 3 of the CaSR gene was done. We found base alterations and deletions in exons 2 and 3 of the CaSR gene. We also found a statistically significant increase in the rate of CAs in patients compared to controls. In total we evaluated 639 metaphase plaques in 23 patients and found 241 CAs, of which 88% were structural and 12% were numerical abnormalities. There is no relation between the etiology of SS and nucleotide alterations that we could find in exons 2 and 3 of the CaSR gene. Our data suggest that there may be a correlation between CAs and the progression of SS.

A polymorphism upstream MIR1279 gene is associated with pericarditis development in Systemic Lupus Erythematosus and contributes to definition of a genetic risk profile for this complication.

Recently, a study has shown that a polymorphism in the region of MIR1279 modulates the expression of the TRAF3IP2 gene. Since polymorphisms in the TRAF3IP2 gene have been described in association with systemic lupus erithematosus (SLE) susceptibility and with the development of pericarditis, our aim is to verify if the MIR1279 gene variability could also be involved. The rs1463335 SNP, located upstream MIR1279 gene, was analyzed by allelic discrimination assay in 315 Italian SLE patients and 201 healthy controls. Moreover, the MIR1279 gene was full sequenced in 50 patients. A case/control association study and a genotype/phenotype correlation analysis were performed. We also constructed a pericarditis genetic risk profile for patients with SLE. The full sequencing of the MIR1279 gene in patients with SLE did not reveal any novel or known variation. The variant allele of the rs1463335 SNP was significantly associated with susceptibility to pericarditis ( P = 0.017 and OR = 1.67). A risk profile model for pericarditis considering the risk alleles of MIR1279 and three other genes (STAT4, PTPN2 and TRAF3IP2) showed that patients with 4 or 5 risk alleles have a higher risk of developing pericarditis ( OR = 4.09 with P = 0.001 and OR = 6.04 with P = 0.04 respectively). In conclusion, we describe for the first time the contribution of a MIR1279 SNP in pericarditis development in patients with SLE and a genetic risk profile model that could be useful to identify patients more susceptible to developing pericarditis in SLE. This approach could help to improve the prediction and the management of this complication.

Oncogenic mechanisms of HOXB13 missense mutations in prostate carcinogenesis.

The recurrent germline mutation HOXB13 p.(Gly84Glu) (G84E) has recently been identified as a risk factor for prostate cancer. In a recent study, we have performed full sequencing of the HOXB13 gene in 462 Portuguese prostate cancer patients with early-onset and/or familial/hereditary disease, and identified two novel missense mutations, p.(Ala128Asp) (A128D) and p.(Phe240Leu) (F240L), that were predicted to be damaging to protein function. In the present work we aimed to investigate the potential oncogenic role of these mutations, comparing to that of the recurrent G84E mutation and wild-type HOXB13. We induced site-directed mutagenesis in a HOXB13 expression vector and established in vitro cell models of prostate carcinogenesis with stable overexpression of either the wild-type or the mutated HOXB13 variants. By performing in vitro assays we observed that, while the wild-type promotes proliferation, also observed with the F240L variant along with a decrease in apoptosis, the A128D mutation decreases apoptosis and promotes anchorage independent growth. No phenotypic impact was observed for the G84E mutation in the cell line model used. Our data show that specific HOXB13 mutations are involved in the acquisition of different cancer-associated capabilities and further support an oncogenic role for HOXB13 in prostate carcinogenesis.

874P - Outcomes of clinical testing for tumor BRAC1 and BRCA2 gene analysis for 354 patients: first experience with tumor companion diagnostic for PARP inhibitors

Ovarian cancer patients with deficiencies in the DNA repair pathway have shown to have a higher probability to respond to PARP inhibitors. Pathogenic mutations in the genes BRCA1 and BRCA2 lead to defects in the DNA repair pathway. It has been shown that the prevalence of mutations in the BRCA genes is higher in the tumor cells than in germline. Thus, a tumor-based test could identify a higher number of potential responders than a germline test. This study assessed 354 consecutive individuals undergoing BRCA1 and BRCA2 full sequencing and large rearrangement analysis of DNA derived from FFPE tumor tissue in a DAkkS accredited laboratory in Munich, Germany from Jan 2015 through April 2016. The tumor-based BRCA test consists of sequencing and large rearrangement analyses of the BRCA1 and BRCA2 genes using next generation sequencing (NGS). The large rearrangements are detected by NGS dosage analysis to determine copy number abnormalities indicative of deletion or duplication mutations. Out of the 354 analyzed samples, 93 (26,5%) tested positive for a laboratory classified pathogenic mutation; 57 were found in BRCA1 and 37 in BRCA2. One specimen contained a pathogenic mutation in both BRCA1 and BRCA2. Due to different quality and age of the tumor samples, large rearrangement analysis could not be completed in 24 cases (6,8%). VUS rate in the analyzed tumors was 4.2%. Of the pathogenic mutations detected, 93,6% were sequencing variants and 6,4% were large rearrangements. Overall cancellation rate due to insufficient tumor, insufficient DNA, incorrect tumor type or other cancellation reasons is less than 9% in the observed timespan of 16 months. The current study demonstrates that a robust diagnostic platform can detect BRCA-related mutations in ovarian tumors. While the quality of specimens received into a commercial laboratory is quite variable, a positive rate of over 26% and an overall success rate (result generated) of over 91% indicates that tumor BRCA testing should be considered for ovarian cancer patients. In addition, a small but significant number of large rearrangements indicates that tumor BRCA testing should include dosage analysis for large rearrangements.

Distribution of HLA-G extended haplotypes and one HLA-E polymorphism in a large-scale study of mother-child dyads with and without severe preeclampsia and eclampsia.

The etiological pathways and pathogenesis of preeclampsia have rendered difficult to disentangle. Accumulating evidence points toward a maladapted maternal immune system, which may involve aberrant placental expression of immunomodulatory human leukocyte antigen (HLA) class Ib molecules during pregnancy. Several studies have shown aberrant or reduced expression of HLA-G in the placenta and in maternal blood in cases of preeclampsia compared with controls. Unlike classical HLA class Ia loci, the nonclassical HLA-G has limited polymorphic variants. Most nucleotide variations are clustered in the 5'-upstream regulatory region (5'URR) and 3'-untranslated regulatory region (3'UTR) of HLA-G and reflect a stringent expressional control. Based on genotyping and full gene sequencing of HLA-G in a large number of cases and controls (n > 900), the present study, which to our knowledge is the largest and most comprehensive performed, investigated the association between the HLA-G 14-bp ins/del (rs66554220) and HLA-E polymorphisms in mother and newborn dyads from pregnancies complicated by severe preeclampsia/eclampsia and from uncomplicated pregnancies. Furthermore, results from extended HLA-G haplotyping in the newborns are presented in order to assess whether a combined contribution of nucleotide variations spanning the 5'URR, coding region, and 3'UTR of HLA-G describes the genetic association with severe preeclampsia more closely. In contrast to earlier findings, the HLA-G 14-bp ins/del polymorphism was not associated with severe preeclampsia. Furthermore, the polymorphism (rs1264457) defining the two nonsynonymous HLA-E alleles, HLA-E*01:01:xx:xx and HLA-E*01:03:xx:xx, were not associated with severe preeclampsia. Finally, no specific HLA-G haplotypes were significantly associated with increased risk of developing severe preeclampsia/eclampsia.

OR18 Full gene sequencing reveals very limited variation in the regions outside of the antigen recognition domains (ARD) of 360 unrelated hematopoietic stem cell transplant donor-recipient pairs matched for 10/10 at high resolution

Aim Traditional DNA-based typing focuses primarily on interrogating the exons of HLA genes that form the ARD (peptide-binding region that interacts with T cell and killer immunoglobulin-like receptors). The relevance of mismatching outside the ARD on hematopoietic stem cell transplantation (HCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in well matched transplant pairs. Methods 360 HLA-A, B, C, DRB1, and DQB1 high resolution matched donor (D)-recipient (R) pairs were selected based on availability of retrospective Sanger-based sequencing and DNA from the CIBMTR Repository. The majority of the population was self-identified Caucasian (80%). Next generation DNA sequencing was performed on the Illumina MiSeq platform and interpreted with Connexio Assign MPS. Class I gene sequences covered 5 ′ UTR-3 ′ UTR; DRB1, intron 1-intron 3; DQA1 5 ′ UTR-exon 4; DQB1, intron 1-3 ′ UTR. DQ noncoding regions were not evaluated. Results The majority (98.1%) of the HLA-A, B, C, DRB1 D-R allele pairs are matched for sequences outside the ARD exons: 0.5% differ in non-ARD exons, 1.9% differ in noncoding regions. A small number (0.2%) differ within ARD exons. Mismatches in non-ARD exons varies from 0.7% for HLA-C and DQA1 to 0% DQB1; noncoding variation ranges from 2.8% for HLA-C to 1.3%, HLA-B and DRB1. Within non-ARD exons, both nonsynonymous (16 allele pairs) and silent (2) variation is present. Intron variation is minor, usually a single nucleotide. Conclusions This is the first study to evaluate the genetic variation and characterize mismatching outside of the ARD in a cohort of HLA-matched donor-recipient pairs. The paucity of exonic mismatches outside of the ARD is striking. Intronic variation is more common but would not contribute to an alloreactive mismatch. At present, it does not appear to be necessary to increase the resolution of HLA typing beyond the ARD in selecting a matched donor except in cases of common non-expressed variants within G-group assignments. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D–R pairs will be difficult to assess in HCT outcome studies since it does not occur very frequently. Further study is warranted to confirm these findings in larger and more diverse cohorts.

Microbe-microbe interactions trigger Mn(II)-oxidizing gene expression.

Manganese (Mn) is an important metal in geochemical cycles. Some microorganisms can oxidize Mn(II) to Mn oxides, which can, in turn, affect the global cycles of other elements by strong sorption and oxidation effects. Microbe-microbe interactions have important roles in a number of biological processes. However, how microbial interactions affect Mn(II) oxidation still remains unknown. Here, we investigated the interactions between two bacteria (Arthrobacter sp. and Sphingopyxis sp.) in a co-culture, which exhibited Mn(II)-oxidizing activity, although neither were able to oxidize Mn(II) in isolation. We demonstrated that the Mn(II)-oxidizing activity in co-culture was most likely induced via contact-dependent interactions. The expressed Mn(II)-oxidizing protein in the co-culture was purified and identified as a bilirubin oxidase belonging to strain Arthrobacter. Full sequencing of the bilirubin oxidase-encoding gene (boxA) was performed. The Mn(II)-oxidizing protein and the transcripts of boxA were detected in the co-culture, but not in either of the isolated cultures. This indicate that boxA was silent in Arthrobacter monoculture, and was activated in response to presence of Sphingopyxis in the co-culture. Further, transcriptomic analysis by RNA-Seq, extracellular superoxide detection and cell density quantification by flow cytometry indicate induction of boxA gene expression in Arthrobacter was co-incident with a stress response triggered by co-cultivation with Sphingopyxis. Our findings suggest the potential roles of microbial physiological responses to stress induced by other microbes in Mn(II) oxidation and extracellular superoxide production.