Source Control of Gram-Negative Bacteria Using Self-Disinfecting Sinks in a Swedish Burn Centre.

Several retrospective studies have identified hospital sinks as reservoirs of Gram-negative bacteria. The aim of this study was to prospectively investigate the bacterial transmission from sinks to patients and if self-disinfecting sinks could reduce this risk. Samples were collected weekly from sinks (self-disinfecting, treated with boiling water, not treated) and patients in the Burn Centre at Linköping University Hospital, Sweden. The antibiotic susceptibility of Gram-negative isolates was tested, and eight randomly chosen patient isolates and their connected sink isolates were subjected to whole genome sequencing (WGS). Of 489 sink samples, 232 (47%) showed growth. The most frequent findings were Stenotrophomonas maltophilia (n = 130), Pseudomonas aeruginosa (n = 128), and Acinetobacter spp. (n = 55). Bacterial growth was observed in 20% of the samplings from the self-disinfecting sinks and in 57% from the sinks treated with boiling water (p = 0.0029). WGS recognized one transmission of Escherichia coli sampled from an untreated sink to a patient admitted to the same room. In conclusion, the results showed that sinks can serve as reservoirs of Gram-negative bacteria and that self-disinfecting sinks can reduce the transmission risk. Installing self-disinfecting sinks in intensive care units is an important measure in preventing nosocomial infection among critically ill patients.

Clonal spread of carbapenem-resistant Klebsiella pneumoniae among patients at admission and discharge at a Vietnamese neonatal intensive care unit.

BACKGROUND: The increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is a growing problem globally, particularly in low- to middle-income countries (LMICs). Previous studies have shown high rates of CRE colonisation among patients at hospitals in LMICs, with increased risk of hospital-acquired infections. METHODS: We isolated carbapenem-resistant Klebsiella pneumoniae (CRKP) from faecal samples collected in 2017 from patients at admission and discharge at a Vietnamese neonatal intensive care unit (NICU). 126 CRKP were whole-genome sequenced. The phylogenetic relationship between the isolates and between clinical CRKP isolates collected in 2012-2018 at the same hospital were investigated. RESULTS: NDM-type carbapenemase-(61%) and KPC-2-encoding genes (41%) were the most common carbapenem resistance genes observed among the admission and discharge isolates. Most isolates (56%) belonged to three distinct clonal clusters of ST15, carrying blaKPC-2, blaNDM-1 and blaNDM-4, respectively. Each cluster also comprised clinical isolates from blood collected at the study hospital. The most dominant ST15 clone was shown to be related to isolates collected from the same hospital as far back as in 2012. CONCLUSIONS: Highly resistant CRKP were found colonising admission and discharge patients at a Vietnamese NICU, emphasising the importance of continued monitoring. Whole-genome sequencing revealed a population of CRKP consisting mostly of ST15 isolates in three clonally related clusters, each related to blood isolates collected from the same hospital. Furthermore, clinical isolates collected from previous years (dating back to 2012) were shown to likely be clonally descended from ST15 isolates in the largest cluster, suggesting a successful hospital strain which can colonise inpatients.

Colistin Dependence in Extensively Drug-Resistant Acinetobacter baumannii Strain Is Associated with ISAjo2 and ISAba13 Insertions and Multiple Cellular Responses.

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

Cluster of S. maltophilia among patients with respiratory tract infections at an intensive care unit.

BACKGROUND: Stenotrophomonas maltophilia is associated with respiratory tract infections in immunocompromised patients, and it has emerged as an important nosocomial pathogen, with admission to intensive care units (ICUs) and ventilators as recognized risk factors. AIM: To describe the investigation of a sudden increase in patients with pneumonia caused by S. maltophilia at a Swedish ICU and the control measures taken. METHODS: Lower respiratory tract cultures from patients admitted to the ICU were obtained, and environmental cultures were collected from sink drains and medical equipment. Isolates identified as S. maltophilia were subjected to antibiotic susceptibility testing and whole genome sequencing (WGS). FINDINGS: A total of 17 S. maltophilia isolates were found (four from patients and 13 from the environment). The WGS identified two outbreak clones, sequence type (ST) 361 and ST138, and seven unique ones. Most likely, the outbreak clones originated from two sinks, and transmission was enhanced by a calorimeter. After changing the sink and calorimeter routines, no more cases were registered. CONCLUSION: Acquisition of S. maltophilia from the hospital environment appears to be easy, especially if water is involved. To control this bacterium, better knowledge of its transmission routes in hospital environments is required.

Molecular and phenotypic characterization of clinical isolates belonging to a KPC-2-producing strain of ST15 Klebsiella pneumoniae from a Vietnamese pediatric hospital.

Background: Carbapenem-resistant Klebsiella pneumoniae are becoming increasingly common in hospital settings worldwide and are a source of increased morbidity, mortality and health care costs. The global epidemiology of carbapenem-resistant K. pneumoniae is characterized by different strains distributed geographically, with the strain ST258 being predominant in Europe and USA, and ST11 being most common in East Asia. ST15 is a less frequently occurring strain but has nevertheless been reported worldwide as a source of hospital outbreaks of carbapenem-resistant K. pneumoniae. Methods: In this study, whole-genome sequencing and antimicrobial susceptibility testing was used to characterize 57 clinical isolates of carbapenem-resistant K. pneumoniae belonging to a strain of ST15, which were collected at a Vietnamese pediatric hospital from February throughout September 2015. Results: Aside from the carbapenem resistance gene blaKPC-2, which was carried by all isolates, prevalence of resistance genes to other antibiotics including aminoglycosides, macrolides, quinolones, fosfomycin and trimethoprim, was also high. All isolates were multidrug-resistant. Susceptibility was highest to ceftazidime/avibactam (96%), gentamicin (91%) and tigecycline (82%). Notably, the colistin resistance rate was very high (42%). Single-nucleotide polymorphism analysis indicated that most isolates belonged to a single clone. Conclusions: The diverse variety of antibiotic resistance genes and the high antibiotic resistance rates to last-resort antibiotics such as carbapenems and colistin, is indicative of a highly adaptable strain. This emphasizes the importance of implementation of infection controls measures, continued monitoring of antibiotic resistance and prudent use of antibiotics to prevent further selection of resistant strains and the emergence of pan-resistant clones.

Characterization of extended-spectrum ß-lactamase-producing Escherichia coli harboring mcr-1 and toxin genes from human fecal samples from China.

AIM: To characterize extended-spectrum ß-lactamase-producing Escherichia coli harboring the colistin resistance gene mcr-1 from human fecal samples collected in 2012 in a rural area of Shandong province, PR China. MATERIALS & METHODS: Whole-genome sequencing and antimicrobial susceptibility testing was performed on 25 mcr-1-positive isolates to determine carriage of antibiotic resistance and virulence genes, diversity and antibiotic resistance profiles. RESULTS: The isolates were highly genetically diverse and carried a large variety of different antibiotic resistance genes. The multidrug-resistance rate was high (96%). Virulence genes associated with intestinal pathogenic E. coli were carried by 32% of the isolates. CONCLUSION: Further monitoring of the epidemiological situation is necessary to ensure a preparedness for potential emergence of novel, difficult-to-treat strains and awareness of available treatment options.

Insertion sequence transpositions and point mutations in mgrB causing colistin resistance in a clinical strain of carbapenem-resistant Klebsiella pneumoniae from Vietnam.

Resistance among Klebsiella pneumoniae to the last-resort antibiotics carbapenems and colistin is increasing worldwide. In this study, whole-genome sequencing was used to determine the colistin resistance mechanisms in clinical isolates of carbapenem- and colistin-resistant K. pneumoniae from Vietnam. Alterations in the regulatory gene mgrB, via mutations and insertion sequence transpositions, were found in 30 of 31 isolates, emphasising the importance of this resistance mechanism in colistin-resistant K. pneumoniae.

Activating FGFR1 Mutations in Sporadic Pheochromocytomas.

INTRODUCTION: Pheochromocytomas are neuroendocrine tumors of the adrenal glands. Up to 40% of the cases are caused by germline mutations in one of at least 15 susceptibility genes, making them the human neoplasms with the highest degree of heritability. Recurrent somatic alterations are found in about 50% of the more common sporadic tumors with NF1 being the most common mutated gene (20-25%). In many sporadic tumors, however, a genetic explanation is still lacking. MATERIALS AND METHODS: We investigated the genomic landscape of sporadic pheochromocytomas with whole-exome sequencing of 16 paired tumor and normal DNA samples and extended confirmation analysis in 2 additional cohorts comprising a total of 80 sporadic pheochromocytomas. RESULTS: We discovered on average 33 non-silent somatic variants per tumor. One of the recurrently mutated genes was FGFR1, encoding the fibroblast growth factor receptor 1, which was recently revealed as an oncogene in pediatric brain tumors. Including a subsequent analysis of a larger cohort, activating FGFR1 mutations were detected in three of 80 sporadic pheochromocytomas (3.8%). Gene expression microarray profiling showed that these tumors clustered with NF1-, RET,- and HRAS-mutated pheochromocytomas, indicating activation of the MAPK and PI3K-AKT signal transduction pathways. CONCLUSION: Besides RET and HRAS, FGFR1 is only the third protooncogene found to be recurrently mutated in pheochromocytomas. The results advance our biological understanding of pheochromocytoma and suggest that somatic FGFR1 activation is an important event in a subset of sporadic pheochromocytomas.

Clinical Characterization of the Pheochromocytoma and Paraganglioma Susceptibility Genes SDHA, TMEM127, MAX, and SDHAF2 for Gene-Informed Prevention.

IMPORTANCE: Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. OBJECTIVE: To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. DESIGN, SETTING, AND PATIENTS: This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. MAIN OUTCOMES AND MEASURES: Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. RESULTS: Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). CONCLUSIONS AND RELEVANCE: The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation.

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are neural-crest-derived tumours of the sympathetic or parasympathetic nervous system that are often inherited and are genetically heterogeneous. Genetic testing is recommended for patients with these tumours and for family members of patients with hereditary forms of PPGLs. Due to the large number of susceptibility genes implicated in the diagnosis of inherited PPGLs, next-generation sequencing (NGS) technology is ideally suited for carrying out genetic screening of these individuals. This Consensus Statement, formulated by a study group comprised of experts in the field, proposes specific recommendations for the use of diagnostic NGS in hereditary PPGLs. In brief, the study group recommends target gene panels for screening of germ line DNA, technical adaptations to address different modes of disease transmission, orthogonal validation of NGS findings, standardized classification of variant pathogenicity and uniform reporting of the findings. The use of supplementary assays, to aid in the interpretation of the results, and sequencing of tumour DNA, for identification of somatic mutations, is encouraged. In addition, the study group launches an initiative to develop a gene-centric curated database of PPGL variants, with annual re-evaluation of variants of unknown significance by an expert group for purposes of reclassification and clinical guidance.

HRAS mutation prevalence and associated expression patterns in pheochromocytoma.

Pheochromocytomas (PCC) and abdominal paragangliomas (PGL) display a highly diverse genetic background and recent gene expression profiling studies have shown that PCC and PGL (together PPGL) alter either kinase signaling pathways or the pseudo-hypoxia response pathway dependent of the genetic composition. Recurrent mutations in the Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been verified in sporadic PPGLs. In order to further establish the HRAS mutation frequency and to characterize the associated expression profiles of HRAS mutated tumors, 156 PPGLs for exon 2 and 3 hotspot mutations in the HRAS gene was screened, and compared with microarray-based gene expression profiles for 93 of the cases. The activating HRAS mutations G13R, Q61R, and Q61K were found in 10/142 PCC (7.0%) and a Q61L mutation was revealed in 1/14 PGL (7.1%). All HRAS mutated cases included in the mRNA expression profiling grouped in Cluster 2, and 21 transcripts were identified as altered when comparing the mutated tumors with 91 HRAS wild-type PPGL. Somatic HRAS mutations were not revealed in cases with known PPGL susceptibility gene mutations and all HRAS mutated cases were benign. The HRAS mutation prevalence of all PPGL published up to date is 5.2% (49/950), and 8.8% (48/548) among cases without a known PPGL susceptibility gene mutation. The findings support a role of HRAS mutations as a somatic driver event in benign PPGL without other known susceptibility gene mutations. HRAS mutated PPGL cluster together with NF1- and RET-mutated tumors associated with activation of kinase-signaling pathways.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole-exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis-related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well-established cancer gene lysine (K)-specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome-sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D-mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

Immunohistochemical NF1 analysis does not predict NF1 gene mutation status in pheochromocytoma.

Pheochromocytomas (PCCs) are tumors originating from the adrenal medulla displaying a diverse genetic background. While most PCCs are sporadic, about 40 % of the tumors have been associated with constitutional mutations in one of at least 14 known susceptibility genes. As 25 % of sporadic PCCs harbor somatic neurofibromin 1 gene (NF1) mutations, NF1 has been established as the most recurrently mutated gene in PCCs. To be able to pinpoint NF1-related pheochromocytoma (PCC) disease in clinical practice could facilitate the detection of familial cases, but the large size of the NF1 gene makes standard DNA sequencing methods cumbersome. The aim of this study was to examine whether mutations in the NF1 gene could be predicted by immunohistochemistry as a method to identify cases for further genetic characterization. Sixty-seven PCCs obtained from 67 unselected patients for which the somatic and constitutional mutational status of NF1 was known (49 NF1 wild type, 18 NF1 mutated) were investigated for NF1 protein immunoreactivity, and the results were correlated to clinical and genetic data. NF1 immunoreactivity was absent in the majority of the PCCs (44/67; 66 %), including 13 out of 18 cases (72 %) with a somatic or constitutional NF1 mutation. However, only a minority of the NF1 wild-type PCCs (18/49; 37 %) displayed retained NF1 immunoreactivity, thereby diminishing the specificity of the method. We conclude that NF1 immunohistochemistry alone is not a sufficient method to distinguish between NF1-mutated and non-mutated PCCs. In the clinical context, genetic screening therefore remains the most reliable tool to detect NF1-mutated PCCs.

YAP1 is a potential biomarker for cetuximab resistance in head and neck cancer.

OBJECTIVES: Targeted therapy against the epidermal growth factor receptor (EGFR) only variably represents a therapeutic advance in head and neck squamous cell carcinoma (HNSCC). This study addresses the need of biomarkers of treatment response to the EGFR-targeting antibody cetuximab (Erbitux®). MATERIALS AND METHODS: The intrinsic cetuximab sensitivity of HNSCC cell lines was assessed by a crystal violet assay. Gene copy number analysis of five resistant and five sensitive cell lines was performed using the Affymetrix SNP 6.0 platform. Quantitative real-time PCR was used for verification of selected copy number alterations and assessment of mRNA expression. The functional importance of the findings on the gene and mRNA level was investigated employing siRNA technology. The data was statistically evaluated using Mann-Whitney U-test and Spearman's correlation test. RESULTS: Analysis of the intrinsic cetuximab sensitivity of 32 HNSCC cell lines characterized five and nine lines as cetuximab sensitive or resistant, respectively. Gene copy number analysis of five resistant versus five sensitive cell lines identified 39 amplified protein-coding genes, including YAP1, in the genomic regions 11q22.1 or 5p13-15. Assessment using qPCR verified that YAP1 amplification associated with cetuximab resistance. Amplification of YAP1 correlated to higher mRNA levels, and RNA knockdown resulted in increased cetuximab sensitivity. Assessment of several independent clinical data sets in the public domain confirmed YAP1 amplifications in multiple tumor types including HNSCC, along with highly differential expression in a subset of HNSCC patients. CONCLUSION: Taken together, we provide evidence that YAP1 could represent a novel biomarker gene of cetuximab resistance in HNSCC cell lines.

Frequent EPAS1/HIF2α exons 9 and 12 mutations in non-familial pheochromocytoma.

Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in tumors, and recently EPAS1, encoding HIF2α, has been revealed to be a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterize EPAS1 alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations in EPAS1 by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF2α degradation, and also exon 2. In addition, the copy number at the EPAS1 locus as well as transcriptome-wide gene expression were studied by DNA and RNA microarray analyses, respectively. We identified six missense EPAS1 mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap in 11 cases (26%) with somatic mutations in NF1 or RET. One sample had two different EPAS1 mutations, shown by cloning to occur in cis, possibly indicating a novel mechanism of HIF2α stabilization through inactivation of both hydroxylation sites. One of the tumors with an EPAS1 mutation also had a gain in DNA copy number at the EPAS1 locus. All EPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, indicating an oncogenic role of the identified mutations.

Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes. OBJECTIVE: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. DESIGN: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bß, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. RESULTS were verified in tumor and constitutional DNA with Sanger sequencing. RESULTS: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1Bß, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1Bß, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation. CONCLUSIONS: Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bß, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.

Role of SDHAF2 and SDHD in von Hippel-Lindau associated pheochromocytomas.

BACKGROUND: Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel-Lindau (VHL) syndrome. In VHL, only about 30 % of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and are required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found both in patients with PCCs and in patients with PGLs. MATERIALS AND METHODS: Because loss of 11q is a common event in VHL-associated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In the present study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation, as well as mRNA expression of SDHAF2 and SDHD, was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted. RESULTS AND CONCLUSIONS: LOH was found in more than 50 % of the VHL-associated PCCs, and was correlated with a significant decrease (p < 0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression. In addition, the lack of mutations and promoter methylation in the investigated samples indicates that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.

A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures.

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method's robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.