Blood-derived Genomic DNA Research Articles

#2918 Genetic background of kidney graft cancer

Abstract Background and Aims Renal cell carcinoma (RCC) is one of the most prevalent cancer in kidney transplant recipient (KTR). Majority of RCC occurs in native kidney as opposed to kidney graft. The genetic background of RCC in native kidneys has been determined implicating the clinical importance. Method This single-center cohort study aimed to identify genetic background of kidney graft RCC and outcome in KTR who underwent single kidney transplantation between January 2000 and December 2020 and manifested kidney graft RCC. Genetic analysis (NGS) from peripheral blood-derived genomic DNA (gDNA) was performed in both the recipient and donor using cancer-predisposition panel CZECANCA (CZEch CAncer paNel for Clinical Application). Results The calculated incidence of kidney graft RCC was 0.43%. Of the eighteen KTR with manifested kidney graft RCC, fifteen KTR and donors underwent NGS. The average patient age at transplantation was 50.3 years (median 54; 5-67 years) and 66 years (median 66; 24-79 years) at the diagnosis of kidney graft RCC. The mean donor age at transplantation was 39.7 years (median 42; 7-68 years) and graft age at kidney graft RCC diagnosis 50.2 years (median 46; 20-83 years). The mean follow-up after RCC diagnosis was 47 months (median 39.1; 0-112 months). The most prevalent was papillary RCC (n=8) followed by clear cell RCC (n=6) and renal pelvis urothelial RCC (n=1). Thirteen RCC were low stage (pT1a/b) disease, one was pT3 and one was unknown stage. Majority of RCC were higher graded. No cancer-predisposition gene was found in KTR or his donor. Conclusion Kidney graft RCC is very rare. NGS cancer-predisposition panel CZECANCA has not detected any genetic predisposition for kidney graft RCC. Further research is needed to assess clinical relevance of genetic testing for cancer risk in KTR.

Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses

BackgroundWhole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols.MethodsThe genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood–saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results.ResultsThe quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood–saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030–0.9998 for SNVs and between 0.8883–0.9991 for small-indels in the case of the WGS protocol, and between 0.8643–0.999 for SNVs and between 0.7781–1.000 for small-indels in the case of the WES protocol.ConclusionSaliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

Multiplex Detection of Copy Number Alterations in Multiple Myeloma Including Emerging Therapeutic Targets By Digitalmlpa

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability.

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay’s conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

Telomere length: population epidemiology and concordance in Australian children aged 11-12 years and their parents.

ObjectivesTo (1) describe the epidemiology of child and adult telomere length, and (2) investigate parent–child telomere length concordance.DesignPopulation-based cross-sectional study within the Longitudinal Study of Australian Children.SettingAssessment centres in seven major Australian cities and eight selected regional towns; February 2015 to March 2016.ParticipantsOf 1874 participating families, telomere data were available for analysis for 1206 children and 1343 parents, of whom 1143 were parent–child pairs. There were 589 boys and 617 girls; 175 fathers and 1168 mothers.Outcome measuresRelative telomere length (T/S ratio), calculated by comparing telomeric DNA (T) level with the single copy (S) beta-globin gene in venous blood-derived genomic DNA by quantitative real-time PCR.ResultsMean T/S ratio for all children, boys and girls was 1.09 (SD 0.56), 1.05 (SD 0.53) and 1.13 (SD 0.59), respectively. Mean T/S ratio for all parents, fathers and mothers was 0.81 (SD 0.37), 0.82 (SD 0.36) and 0.81 (SD 0.38), respectively. Parent–child T/S ratio concordance was moderate (correlation 0.24). In adjusted regression models, one unit higher parent T/S ratio was associated with 0.36 (estimated linear regression coefficient (β); 95% CI 0.28 to 0.45) higher child T/S ratio. Concordance was higher in the youngest parent-age tertile (β 0.49; 95% CI 0.34 to 0.64) compared with the middle (β 0.35; 95% CI 0.21 to 0.48) and oldest tertile (β 0.26; 95% CI 0.11 to 0.41; p-trend 0.04). Father–child concordance was 0.34 (95% CI 0.18 to 0.48), while mother–child was 0.22 (95% CI 0.17 to 0.28).ConclusionsWe provide telomere length population values for children aged 11–12 years and their mid-life parents. Relative telomere length was shorter in adults than children, as expected. There was modest evidence of parent–child concordance, which diminished with increasing parent age.

Clinical relevance of screening checklists for detecting cancer predisposition syndromes in Asian childhood tumours

Assessment of cancer predisposition syndromes (CPS) in childhood tumours is challenging to paediatric oncologists due to inconsistent recognizable clinical phenotypes and family histories, especially in cohorts with unknown prevalence of germline mutations. Screening checklists were developed to facilitate CPS detection in paediatric patients; however, their clinical value have yet been validated. Our study aims to assess the utility of clinical screening checklists validated by genetic sequencing in an Asian cohort of childhood tumours. We evaluated 102 patients under age 18 years recruited over a period of 31 months. Patient records were reviewed against two published checklists and germline mutations in 100 cancer-associated genes were profiled through a combination of whole-exome sequencing and multiplex ligation-dependent probe amplification on blood-derived genomic DNA. Pathogenic germline mutations were identified in ten (10%) patients across six known cancer predisposition genes: TP53, DICER1, NF1, FH, SDHD and VHL. Fifty-four (53%) patients screened positive on both checklists, including all ten pathogenic germline carriers. TP53 was most frequently mutated, affecting five children with adrenocortical carcinoma, sarcomas and diffuse astrocytoma. Disparity in prevalence of germline mutations across tumour types suggested variable genetic susceptibility and implied potential contribution of novel susceptibility genes. Only five (50%) children with pathogenic germline mutations had a family history of cancer. We conclude that CPS screening checklists are adequately sensitive to detect at-risk children and are relevant for clinical application. In addition, our study showed that 10% of Asian paediatric solid tumours have a heritable component, consistent with other populations.

Sample Identification Capacity of 20 Single Nucleotide Polymorphisms in Blood-Derived Genomic DNA Samples of Korean Individuals.

Sample Identification Capacity of 20 Single Nucleotide Polymorphisms in Blood-Derived Genomic DNA Samples of Korean Individuals.

Mutation in noncoding RNA RNU12 causes early onset cerebellar ataxia.

Exome sequences account for only 2% of the genome and may overlook mutations causing disease. To obtain a more complete view, whole genome sequencing (WGS) was analyzed in a large consanguineous family in which members displayed autosomal recessively inherited cerebellar ataxia manifesting before 2 years of age. WGS from blood-derived genomic DNA was used for homozygosity mapping and a rare variant search. RNA from isolated blood leukocytes was used for quantitative polymerase chain reaction (PCR), RNA sequencing, and comparison of the transcriptomes of affected and unaffected family members. WGS revealed a point mutation in noncoding RNA RNU12 that was associated with early onset cerebellar ataxia. The U12-dependent minor spliceosome edits 879 known transcripts. Reverse transcriptase PCR demonstrated minor intron retention in all of 9 randomly selected RNAs from this group, and RNAseq showed splicing disruption specific to all U12-type introns detected in blood monocytes from affected individuals. Moreover, 144 minor intron-containing RNAs were differentially expressed, including transcripts for 3 genes previously associated with cerebellar neurodegeneration. Interference with particular spliceosome components, including small nuclear RNAs, cause reproducible uniquely distributed phenotypic and transcript-specific effects, making this an important category of disease-associated mutation. Our approach to differential expression analysis of minor intron-containing genes is applicable to other diseases involving altered transcriptome processing. ANN NEUROL 2017;81:68-78.

Human Papillomavirus Type 16 L2 DNA Methylation in Exfoliated Cervical Cells From College-Age Women.

The Carolina Women's Care Study (CWCS) at the University of South Carolina followed 467 young women with the goal of identifying biomarkers of human papillomavirus (HPV) persistence. In this study, we analyzed the methylation of HPV16 DNA. The aims of this study were to determine the methylation status of the HPV16 L2 gene in DNA isolated from exfoliated cervical cells collected longitudinally as part of the CWCS and to determine the prevalence of polymorphisms (single nucleotide polymorphisms [SNPs]) in folate metabolizing enzymes and DNA repair enzymes known to affect DNA methylation in blood-derived genomic DNA from CWCS participants. For methylation studies, DNA samples were bisulfite converted and amplified with the EpiTect Whole Bisulfitome kit. Polymerase chain reaction was performed for amplicons containing 5 CpG sites in L2. Pyrosequencing was carried out using EpigenDx and analyzed with PyroMark Software. Taqman genotyping assays were performed to determine selected SNP alleles in the CWCS cohort. Methylation data were obtained for 82 samples from 27 participants. Of these, 22 participants were positive for HPV16 for 3 or more visits (≥12 months). Methylation in L2 was detectable, but methylation levels varied and were not associated with HPV16 persistence. No linearity of methylation levels over time was observed in participants for whom longitudinal data could be analyzed. Analysis of 9 selected SNPs did not reveal an association with persistence. We conclude that at early stages of infection methylation of HPV16 L2 DNA in Pap test samples is not a predictive biomarker of HPV persistence.

Rhodopsin mutations are scarcely implicated in autosomal recessive retinitis pigmentosa: A preliminary study of Egyptian retinitis pigmentosa patients

BackgroundRetinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations that is estimated to affect more than 1.5 million people worldwide. RP is characterized by retinal pigment deposits visible on fundus examination, abnormal electroretinogram and progressive retinal dysfunction. AimThe present work aimed to identify the possible mutations in the rhodopsin gene (RHO) among Egyptian RP patients as well as identifying the different inheritance patterns of those patients. Subjects and methodsThirty diagnosed retinitis pigmentosa patients were enrolled in the study. Inheritance forms of RP were identified by recording full family history, the coding regions of the rhodopsin gene were sequenced using blood-derived genomic DNA samples donated by patients and fifteen healthy controls. ResultsA high percentage of autosomal recessive cases was reported. Also a high parental consanguinity rates were evident. Sequencing of rhodopsin gene revealed no mutations among the study population. ConclusionRhodopsin mutations are scarcely associated with the autosomal recessive RP, suggesting that wide scale studies are needed to determine the genetic variations involved in RP and particularly in the autosomal recessive inheritance.

Confirmation of Cause and Manner of Death Via a Comprehensive Cardiac Autopsy Including Whole Exome Next-Generation Sequencing

Annually, the sudden death of thousands of young people remains inadequately explained despite medicolegal investigation. Postmortem genetic testing for channelopathies/cardiomyopathies may illuminate a potential cardiac mechanism and establish a more accurate cause and manner of death and provide an actionable genetic marker to test surviving family members who may be at risk for a fatal arrhythmia. Whole exome sequencing allows for simultaneous genetic interrogation of an individual's entire estimated library of approximately 30000 genes. Following an inconclusive autopsy, whole exome sequencing and gene-specific surveillance of all known major cardiac channelopathy/cardiomyopathy genes (90 total) were performed on autopsy blood-derived genomic DNA from a previously healthy 16-year-old adolescent female found deceased in her bedroom. Whole exome sequencing analysis revealed a R249Q-MYH7 mutation associated previously with familial hypertrophic cardiomyopathy, sudden death, and impaired β-myosin heavy chain (MHC-β) actin-translocating and actin-activated ATPase (adenosine triphosphatase) activity. Whole exome sequencing may be an efficient and cost-effective approach to incorporate molecular studies into the conventional postmortem examination.

2093 A ROLE FOR THE GLUCOSE TRANSPORTER GLUT3 IN MALE FERTILITY

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20122093 A ROLE FOR THE GLUCOSE TRANSPORTER GLUT3 IN MALE FERTILITY Alexander W. Pastuszak, Carolina J. Jorgez, Larry I. Lipshultz, and Dolores J. Lamb Alexander W. PastuszakAlexander W. Pastuszak Houston, TX More articles by this author , Carolina J. JorgezCarolina J. Jorgez Houston, TX More articles by this author , Larry I. LipshultzLarry I. Lipshultz Houston, TX More articles by this author , and Dolores J. LambDolores J. Lamb Houston, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2260AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Approximately 15% of couples encounter fertility problems, with a 50% male factor contribution. In many of these men, a genetic cause is suspected. While numerous known genetic alterations cause male infertility, it is likely that many other genetic etiologies exist. Glucose transporters (GLUTs) may be involved in sperm motility, though their functional role in this process is unclear and no genetic studies exist supporting a role for GLUTs in fertility. Here, we present genomic data implicating the glucose transporter GLUT3 in male fertility. METHODS Blood-derived genomic DNA from 16 men with non-obstructive azoospermia (NOA) and normal Y-chromosome microdeletion and karyotype analyses, as well as 4 fertile male controls, was hybridized to microarrays and comparative genomic hybridization (aCGH) performed (3x720k Whole-Genome Tiling Array, Roche Nimblegen, Inc.) to assess for copy number variations (CNVs). Copy number gains and losses were identified after baseline correction using Nexus Copy Number software (BioDiscovery, Inc.) and candidate fertility genes selected based on 1) the frequency of CNVs in the gene, 2) the magnitude of the gain / loss, 3) evidence supporting a potential role in male fertility, and 4) available data on gene expression. Gene copy numbers were validated using Taqman qPCR, and Sanger DNA sequencing performed to assess for single nucleotide polymorphisms (SNPs) within genes of interest. Immunohistochemical staining of testis sections utilized commercially available antibodies and standard protocols. RESULTS Copy number gains were identified using aCGH in GLUT3 in 2 of 16 patients, and in no controls. These gains were confirmed in both patients using Taqman qPCR copy number assays. Subsequent Taqman assays of DNA from 43 infertile men yielded 5 more men with GLUT3 gains. Of note, the frequency of GLUT3 CNVs in the general population (which includes infertile as well as fertile individuals) is approximately 5%, whereas our observed gain frequency is 16%. Sequencing of GLUT3 yielded SNPs in exons 2 (2465 A>C, 36 K>T), 6 (6505 A>G, 258 L>L), and 10 (14680 C>T, 436 T>T), none of which were predicted to be damaging. Staining for GLUT3 in testis sections of a male with 3 copies of GLUT3 and hypospermatogenesis demonstrated cytoplasmic Leydig cell and spermatocyte staining. CONCLUSIONS Copy number gains in GLUT3 were identified in several infertile males. Future work in the form of functional assays analyzing the impact of GLUT3 expression on fertility-related cell signaling pathways, as well as the study of animal models, will further elucidate the role of GLUT3 in male fertility. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e844 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander W. Pastuszak Houston, TX More articles by this author Carolina J. Jorgez Houston, TX More articles by this author Larry I. Lipshultz Houston, TX More articles by this author Dolores J. Lamb Houston, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 2749: Mitochondrial DNA variants independently predict survival in invasive breast cancer: Results from the Carolina Breast Cancer Study

Abstract Background Mitochondria possess a distinct DNA genome (mtDNA) that encodes proteins of critical importance to cellular energy metabolism, free radical production, and apoptosis. These processes are central to cancer-cell proliferation, angiogenesis, and metastasis, as well as to treatment response; hence, the rich variation present in mtDNA may impact cancer prognosis. We previously reported the association of mtDNA variant G10398A with risk of invasive breast cancer (BC) in African American (AA) women from the Carolina Breast Cancer Study (CBCS), a large, population-based BC case control study. This study evaluated the role of two homoplasmic, mtDNA germline variants, G10398A and T4216C, on BC outcome. Methods The CBCS enrolled 1,557 women with incident, primary invasive BC, aged 20-74, between 1993 and 2001 (Phases I and II). In-person interviews were conducted to collect a blood sample and ascertain demographic, lifestyle, and risk-factor information; tumor histology and clinical data were confirmed by pathology and medical-chart review, respectively. Median active follow-up of enrolled BC cases is currently >10 years. G10398A and T4216C genotypes were determined from blood-derived genomic DNA, using a fluorogenic 5’ nuclease allelic discrimination TaqMan PCR assay. Stratified Cox regression models were used to estimate hazard ratios (HRs) and their 95% confidence intervals (95% CIs) for associations of mtDNA variants with BC-specific mortality, adjusting for potential confounders. Results Genotyping was successful at both mtDNA loci in >98% of subjects. Survival in the cohort overall was significantly superior among women with the 4216C allele compared to 4216T (HR 0.55; 95% CI 0.36-0.83, P<0.01) adjusting for age, race, and tumor stage (I-IV). Women with 4216C and 10398A alleles had the highest survival (adjusted HR 0.27, compared to the 4216T/10398G haplotype; 95% CI 0.11-0.67, P<0.01). The same prognostic pattern was noted in white women (N=899): HR 0.46 for 4216C vs. T (95% CI 0.28-0.77, P<0.01), adjusting for age and tumor stage; adjusted HR 0.26 for 4216C+10398A vs. T+G haplotype (95% CI 0.09-0.80, P<0.05). Similar but statistically insignificant results were observed in 658 AA cases. Finally, although T4216C and T4216C/G10398A genotypes correlated with tumor hormone-expression status and subtypes, and G10398A genotype also correlated with tumor stage and mitotic index (P<0.05 for all), the 4216C allele and 4216C/10398A haplotype remained independently predictive of survival even after adjustment for recognized prognostic factors. Conclusions These findings, which require replication in other cohorts, suggest that mtDNA variant T4216C, together with G10398A, carries significant, independent prognostic impact in BC and that the role of mtDNA germline variants should be comprehensively explored in BC outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2011-2749

On genomic DNA paradigms, research publications, and scholarly inquiry

An article published in Human Mutation by Gottlieb et al. [2009] received much attention for the reported observation of tissue-specific polymorphic differences in the BAK1 genotype among abdominal aortic anerurysm (AAA) patients and some controls. In particular, sequencing revealed a statistically significant number of instances in which single nucleotide polymorphisms (SNPs) present in tissue samples from patient abdominal aorta (AA) were absent from matching blood samples. The authors proposed a novel hypothesis “postulating that multiple variants of genes may preexist in ‘minority’ forms within specific non-diseased tissues and be selected for, when intra- and/or extra-cellular conditions change” [Gottlieb et al., 2009]. The implications of this unusual finding are of potential significance as regards the role somatic mutation and intraorganismal selection might play in genetic disease, not to mention the suitability of using blood-derived genomic DNA to evaluate patient mutational status of genetic diseases that affect various tissues. The work was challenged in a Letter to the Editors by Hatchwell [2010], who argued that the Gottlieb et al.'s [2009] findings were likely artifactual due to pseudogene interference, genomic DNA contamination of RNA samples, and inaccurate reference sequences in GenBank. Gottlieb et al. [2010a] responded to this criticism with additional information about the methods they used, both in vitro and in silico, to reduce the possibility for such artifacts. In the current issue, Kury et al. [2010] discuss additional concerns that they have about the original article in another letter. These concerns have, in turn, been responded to by Gottlieb et al. [2010b]. These four letters were all carefully peer reviewed and evaluated editorially, but within the limitations of the rather constrained “Letter to the Editors” format. As the Managing Editor and Co-Editors of Human Mutation, we feel that discussion of the original article by Gottlieb et al. [2009] and its implications is valuable for the scientific process. At this juncture, however, we believe that further comment on this study must be accompanied with thorough, original research aimed to either validate or refute the original findings. We welcome such submissions for editorial consideration and peer review.

Epigenetic and inflammatory marker profiles associated with depression in a community-based epidemiologic sample

BackgroundRecent work suggests that epigenetic differences may be associated with psychiatric disorders. Here we investigate, in a community-based sample, whether methylation profiles distinguish between individuals with and without lifetime depression. We also investigate the physiologic consequences that may be associated with these profiles.MethodUsing whole blood-derived genomic DNA from a subset of participants in the Detroit Neighborhood Health Study (DNHS), we applied methylation microarrays to assess genome-wide methylation profiles for over 14 000 genes in 33 persons who reported a lifetime history of depression and 67 non-depressed adults. Bioinformatic functional analyses were performed on the genes uniquely methylated and unmethylated in each group, and inflammatory biomarkers [interleukin (IL)-6 and C-reactive protein (CRP)] were measured to investigate the possible functional significance of the methylation profiles observed.ResultsUniquely unmethylated gene sets distinguished between those with versus without lifetime depression. In particular, some processes (e.g. brain development, tryptophan metabolism) showed patterns suggestive of increased methylation among individuals with depression whereas others (e.g. lipoprotein) showed patterns suggestive of decreased methylation among individuals with depression. IL-6 and CRP levels were elevated among those with lifetime depression and, among those with depression only, IL-6 methylation showed an inverse correlation with circulating IL-6 and CRP.ConclusionsGenome-wide methylation profiles distinguish individuals with versus without lifetime depression in a community-based setting, and show coordinated signals with pathophysiological mechanisms previously implicated in the etiology of this disorder. Examining epigenetic mechanisms in concert with other dynamic markers of physiologic functioning should improve our understanding of the neurobiology of depression.

Genetic polymorphism of cytochrome P450s and P‐glycoprotein in the Finnish population

The objective of this study was to investigate the genetic polymorphism of selected cytochrome P450 (CYP) enzymes and ABCB1 (encoding P-glycoprotein) of central importance with regard to the disposition of clinically used drugs in the Finnish population and to compare the results to pre-existing data from Caucasian populations. A random sample of 449 Finns was studied. Single nucleotide polymorphisms (SNPs) were genotyped using blood-derived genomic DNA and 5'-nuclease assays. We found that the allele frequencies of CYP1A2 SNP g.-163C>A, CYP2C8*3, CYP2C9*2, CYP2C9*3 and CYP2C19*2 were similar to those seen in other Caucasian populations. However, the allelic frequency of the variant ABCB1 SNP c.3435C>T allele was lower than previously reported. The frequency of the homozygous CYP3A5*1 expression was significantly higher than expected based on Hardy-Weinberg calculations (observed n = 8 vs. expected n = 3, P = 0.01). Other genotype frequencies corresponded to the expected values. The strong linkage between the CYP2C8*3 and the CYP2C9*2 alleles was confirmed in this study and the number of individuals with the rare haplotype CYP2C8*3*3/CYP2C9*2*2 was higher than expected. We conclude that the frequency of mutated CYP alleles in Finns were in agreement with earlier findings in Caucasian populations, but a lower frequency of the ABCB1 variant allele 3435T corresponding to that reported in Asian populations was found. The higher than expected frequency of the CYP3A5*1*1 genotype and the CYP2C8*3*3/CYP2C9*2*2 haplotype may influence the response to treatment with drugs metabolized by these enzymes.

Response to systemic endotoxemia among humans bearing polymorphisms of the Toll-like receptor 4 (hTLR4)

Mutations (Asp299Gly and Thr399Ile) in the human Toll-like receptor 4 (hTLR4) gene are reportedly associated with hyporesponsiveness to inhaled LPS in humans. It was hypothesized that normal volunteers with these hTLR4 mutations would manifest altered physiological responses to intravenous LPS administration. Human subjects ( n = 57) were administered LPS (2 ng/kg) intravenously and monitored for vital signs (temperature, heart rate, mean arterial pressure). Blood-derived genomic DNA samples were evaluated using PCR to detect hTLR4 and TLR2 mutations. Heterozygous hTLR4 mutations were identified in 8 of the 57 subjects studied. Subjects with hTLR4 mutations demonstrated similar responses to LPS administration. The moderate systemic inflammatory response produced by intravenous LPS administration in human subjects is not modulated by the presence of heterozygous mutations in the hTLR4 gene.