Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Intrahepatic cholestasis of pregnancy (ICP): case report and review of the literature.

Intrahepatic cholestasis of pregnancy (ICP) represents the most common pregnancy-related liver disease in women. Women frequently present in the third trimester with pruritus and elevated serum bile acid and/or alanine transaminase levels. Clinical symptoms quickly resolve after delivery; however, recurrence in subsequent pregnancies has to be expected. Intrahepatic cholestasis of pregnancy is associated with increased perinatal complications, such as premature delivery, meconium staining of the amniotic fluid, respiratory distress, low Apgar scores, and even stillbirth. The risk for the fetus is significantly increased with maternal serum bile acid levels above 40 µmol/L, which characterize severe ICP. An important factor for ICP development is a rise of gestational hormones leading to cholestasis in genetically predisposed women. Variants in the bile salt export pump (BSEP) and the multidrug resistance protein 3 (MDR3) are most often identified in ICP. Here, we give an overview of the current literature on ICP and present the case of a woman with recurrent severe ICP. A common BSEP polymorphism as well as a rare MDR3 mutation may underlie the development of ICP in our patient. She had a premature delivery with meconium staining of the amniotic fluid. The neonate showed signs of respiratory distress with a low Apgar score. This case emphasizes that women with severe ICP have an increased risk for perinatal complications. Furthermore, severe ICP was associated with a MDR3 mutation, which has already been described in adult patients with liver cirrhosis. Thus, ICP may unmask an underlying MDR3 defect, which may predispose to development of hepatobiliary diseases such as gallstone disease, liver fibrosis/cirrhosis, as well as hepatobiliary malignancies. Therefore, genetic testing should be considered in women with severe as well as early onset ICP. Furthermore, regular follow-up should be discussed for women with genetic variants.

Synthetic gestagens exert differential effects on arterial thrombosis and aortic gene expression in ovariectomized apolipoprotein E-deficient mice.

BACKGROUND AND PURPOSE: Combined hormone replacement therapy with oestrogens plus the synthetic progestin medroxyprogesterone acetate (MPA) is associated with an increased risk of thrombosis. However, the mechanisms of this pro-thrombotic effect are largely unknown. The purpose of this study was to: (i) compare the pro-thrombotic effect of MPA with another synthetic progestin, norethisterone acetate (NET-A), (ii) determine if MPA's pro-thrombotic effect can be antagonized by the progesterone and glucocorticoid receptor antagonist mifepristone and (iii) elucidate underlying mechanisms by comparing aortic gene expression after chronic MPA with that after NET-A treatment. EXPERIMENTAL APPROACH: Female apolipoprotein E-deficient mice were ovariectomized and treated with placebo, MPA, a combination of MPA + mifepristone or NET-A for 90 days on a Western-type diet. Arterial thrombosis was measured in vivo in a photothrombosis model. Aortic gene expression was analysed using microarrays; GeneOntology and KEGG pathway analyses were conducted. KEY RESULTS: MPA's pro-thrombotic effects were prevented by mifepristone, while NET-A did not affect arterial thrombosis. Aortic gene expression analysis showed, for the first time, that gestagens induce similar effects on a set of genes potentially promoting thrombosis. However, in NET-A-treated mice other genes with potentially anti-thrombotic effects were also affected, which might counterbalance the effects of the pro-thrombotic genes. CONCLUSIONS AND IMPLICATIONS: The pro-thrombotic effects of synthetic progestins appear to be compound-specific, rather than representing a class effect of gestagens. Furthermore, the different thrombotic responses elicited by MPA and NET-A might be attributed to a more balanced, 'homeostatic' gene expression induced in NET-A- as compared with MPA-treated mice.

Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases.

The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

Increased prevalence of class I integrons in Escherichia coli, Klebsiella species, and Enterobacter species isolates over a 7-year period in a German university hospital.

The prevalence of integrons in five enterobacterial species was analyzed in 900 blood culture isolates from 1993, 1996, and 1999. Remarkably, the prevalence increased from 4.7% in 1993 to 9.7% in 1996 and finally to 17.4% in 1999 (P < 0.01). Within 7 years the combined percentage of P1 strong promoters and P1 weak plus P2 active promoters with high transcription efficacies has increased from 23.1 to 33.3 and finally 60% (P < 0.05).

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking.

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.

Lysophosphatidic acid, a novel lipid growth factor for human thyroid cells: over-expression of the high-affinity receptor edg4 in differentiated thyroid cancer.

Lysophosphatidic acid (LPA) is a small lipid mediator with pleiotropic biological activities, e.g., the regulation of cellular proliferation and various aspects of cellular physiology. Signal transduction is achieved by binding to 2 high-affinity receptors, EDG2 and EDG4, and a group of low-affinity receptors, EDG1-7, all belonging to the superfamily of G protein-coupled receptors. We examined the growth-regulatory effects of LPA in primary cultures of 8 goiters and 1 papillary thyroid cancer. We further assessed mRNA expression of high-affinity receptors EDG2 and EDG4 in 14 normal thyroids, 29 papillary thyroid cancers, 7 follicular thyroid cancers and 13 goiters by quantitative RT-PCR. We also identified mRNA expression of phospholipase A(2) and LPA acyltransferase in fresh thyroid tissues derived from various sources. At concentrations of 10, 50 and 150 microM, LPA induced a 2-fold rise of proliferation (p < 0.001) and acted as strongly as thyrotropin. The combination of LPA and TSH produced significant synergistic effects compared with each substance alone (p < 0.05). Normal thyroid, goiter and papillary or follicular thyroid cancer expressed 2 high-affinity cognate LPA receptors, EDG2 and EDG4. EDG4 receptor mRNA expression was increased 3-fold in differentiated thyroid cancer (p < 0.01), both papillary (p < 0.01) and follicular (p < 0.05), compared to normal thyroid or goiter. Overall expression of EDG2 receptor was unchanged in malignancy; however, increased EDG2 expression in individual samples correlated with lymphonodular metastasis (p = 0.01). Thus, lipid mediators are a novel class of factors involved in the control of proliferation in the human thyroid. Altered mRNA expression of the high-affinity LPA receptor EDG4 suggests a role in the pathogenesis of differentiated thyroid cancer.

Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs.

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus.

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

Multiplex PCR for the Rapid Simultaneous Speciation and Detection of Methicillin-Resistance and Genes Encoding Toxin Production in Staphylococcus aureus.

The use of polymerase chain reaction (PCR) to rapidly amplify target DNA molecules has evolved a place within diagnostic microbiology allowing the sensitive and specific identification of microorganisms concurrent with the detection of specific genes involved in resistance or virulence. This is particularly helpful when dealing with problem pathogens such as Staphylococcus aureus, where the rapid diagnosis and detection of methicillin-resistance can assist in the fast implementation of appropriate treatment and infection control measures (1-4). In addition, the rapid assessment of toxin production can ascertain whether toxin-related symptoms need to be considered (5). The protocol described here enables the simultaneous differentiation between S. aureus (and coagulase negative staphylococci) from other eubacterial organisms. Concomitant with species identification this protocol allows for the detection of specific staphylococcal resistance genes, such as mecA, encoding methicillin resistance, or staphylococcal virulence genes, such as seb, sec1 and tst, encoding enterotoxin B, enterotoxin C, and toxic shock syndrome toxin-1, respectively. In S. aureus the detection of methicillin-resistance using agar diffusion or broth micro-dilution techniques, or toxin production using immunodiffusion, agglutination, or ELISA is often difficult. This is because the mecA gene and the genes involved in toxin production may be poorly expressed and influenced significantly by culture conditions (5-10). Other methods, in particular various hybridization techniques have been used as diagnostic tools. However.

Development of resistance to ciprofloxacin, rifampin, and mupirocin in methicillin-susceptible and -resistant Staphylococcus aureus isolates.

A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described for Staphylococcus aureus. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant S. aureus (MRSA). No differences in MR, DIFF, and mutations in grlA and gyrA were detected between methicillin-susceptible S. aureus and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.

Prevalence of macrolide-resistance genes in Staphylococcus aureus and Enterococcus faecium isolates from 24 European university hospitals.

The polymerase chain reaction (PCR) was used to study the prevalence of the macrolide resistance genes ermA, ermB, ermC, msrA/msrB, ereA and ereB, in 851 clinical isolates of Staphylococcus aureus and 75 clinical isolates of Enterococcus faecium that were erythromycin resistant. The isolates were from 24 European university hospitals. In S. aureus, the ermA gene was more common in methicillin-resistant S. aureus (MRSA) isolates (88%) than in methicillin-susceptible S. aureus (MSSA) isolates (38%), and occurred mainly in strains with constitutive MLS(B) expression. In contrast, ermC was more common in MSSA (47%) than in MRSA (5%), occurring mainly in strains with inducible expression. The ereB gene was only found in MRSA isolates expressing a constitutive MLS(B) phenotype (1%). The ereA gene was not detected. Macrolide resistance by efflux due to the msrA/msrB gene was only detected in MSSA isolates (13%). In contrast to S. aureus, erythromycin resistance in E. faecium was almost exclusively due to the presence of the ermB gene (93%).

Complete sequencing and mRNA expression analysis of the MEN-I gene in adrenal myelolipoma.

The molecular pathogenesis of adrenal myelolipoma is unclear. Endocrine activity of these tumors and association with other endocrine tumors have stimulated the hypothesis that it may belong to the group of sporadic tumors caused by defects of the gene responsible for multiple endocrine neoplasia type I (MEN-I). DNA of blood and tumoral sections from two patients with adrenal myelolipoma were analyzed by examination of variable number of tandem repeats (VNTR) loci PYGM, D11S987, D11S480, and D11S449 on chromosome 11q13 and by complete direct DNA sequencing of all coding exons and splice junctions of the MEN-I gene. Menin expression was examined by RT-PCR. RT-PCR did not detect menin expression in one adrenal myelolipoma. No loss of heterozygozity on chromosome 11q13 was identified. Intragenic heterozygozity was retained in codon 418 of the menin gene in both patients. No mutation was identified in the coding exons of the menin gene. Complete DNA sequencing yielded no hint that defects of the MEN-I gene are responsible for the formation of adrenal myelolipomas. Adrenal myelolipomas do not share the loss of heterozygozity on chromosome 11q13 observed in some benign adenomatous and many malignant adrenocortical tumors.

Multiple mutations conferring ciprofloxacin resistance in Staphylococcus aureus demonstrate long-term stability in an antibiotic-free environment.

Two unrelated strains of Staphylococcus aureus, one with a single mutation in grlA, the other with multiple mutations in gyrA, gyrB, grlA, grlB, norA and the norA promoter region, encoding low-level and high-level ciprofloxacin resistance, respectively, were studied. The characterized mutations in these genes were conserved when both strains were passaged for at least 500 generations in an antibiotic-free environment. New, rapidly stabilized mutations and higher MICs were detected for strains passaged in sub-MIC ciprofloxacin concentrations. The seeming irreversibility of quinolone resistance may affect the long-term success of this drug class.

Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors.

The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors.

Complete sequencing and messenger ribonucleic acid expression analysis of the MEN I gene in adrenal cancer.

Adrenal cancer is a rare sporadic disease that has also been observed in the context of multiple endocrine neoplasia type I (MEN I). Adrenal lesions occur in up to 40% of MEN I patients. Loss of heterozygosity of the 11q13 band harboring the menin gene has been reported in more than 50% of patients with adrenal cancer. Despite this high index of suspicion, former screening studies did not reveal mutations of the MEN I gene in 28 patients. We identified loss of heterozygosity of 11q13 microsatellites in five of five patients (100%). In 40%, heterozygosity was retained in codon 418 of the MEN I gene. Complete direct DNA sequencing data of the entire coding region and adjacent splice sites of the MEN I gene were obtained in 14 patients with sporadic adrenal cancer. In only one of them a heterozygous missense mutation, R176Q (exon 3), was identified. Due to the heterozygous pattern and unknown biological effect of this mutation, it is not clear whether there is a causal relationship with adrenal cancer. The total mutation frequency in sporadic adrenal cancer is 1 of 14 (7%). Menin messenger RNA expression was identified in 14 of 14 patients (100%). Transcriptional inactivation of the menin gene is, hence, unlikely to cause loss of its tumor suppressor function in adrenal cancer. Furthermore, we examined three patients who presented adrenal cancer in the context of sporadic multiglandular endocrine tumor disease previously diagnosed on clinical grounds to be MEN I syndrome. An opal stop codon mutation was identified in codon 126 (exon 2) in the adrenal cancer of one of these patients. Formation of the adrenal cancer in this patient may be rather coincidental because the mutation was present in a heterozygous pattern. There was no mutation of the menin gene in the two other patients. This may mean that formation of adrenal cancer in the context of multiglandular endocrine disease denotes an entity different from MEN I in some patients.

Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from Worldwide Surveillance Studies during the 1997-1998 respiratory season.

From 8,419 worldwide clinical isolates of Streptococcus pneumoniae obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, only parC (Ser-79-->Tyr) and gyrA (Ser-81-->Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxaci n/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.