DHPLC screening of cystic fibrosis gene mutations

Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.     

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.     

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long‐term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p‐Nitrophenylpalmitate and 5‐bromo‐4‐chloro‐3‐indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva‐ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large‐leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large‐leaved lime are a promising source for developing functional foods or isolating active compounds. Copyright © 2008 John Wiley & Sons, Ltd.     

VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation

To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT1, pT2, and pT3 CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT3 CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.     

The importance of non-invasive genetic analysis in the initial diagnostics of Alport syndrome in young patients

Alport syndrome is an important hereditary disorder characterized by nephritis and sometimes accompanied by impairment or loss of vision and hearing. The most common form of Alport syndrome is an X-linked dominant trait that has been associated with the gene COL4A5, one of the six types of IV collagen genes. More than 300 different mutations have been identified in the COL4A5 gene, and appear randomly along the whole gene. Three novel mutations, G198E, G3189D and G669R, were found in 5 young patients from 3 different Slovenian families. On the basis of the results of our study and the existing national register of Alport syndrome patients, we demonstrated that non-invasive methods, such as the genetic analysis of collagen genes, particularly in the case of young patients with undefined clinical features, may be of great importance and could diminish the need for invasive skin and renal biopsy. Our study showed the importance of molecular genetic data for the purpose of providing quick and precise diagnoses for affected family members and their offspring, particularly small children.M. lajpah and A. Megli contributed equally to this work     

Identification of novel genes with somatic frameshift mutations within coding mononucleotide repeats in colorectal tumors with high microsatellite instability

We have systematically retrieved genes with coding mononucleotide repeats from sequence databases and analyzed them for mutations in tumors with high levels of microsatellite instability (MSI-H). We found somatic frameshift mutations in 7/13 genes previously not analyzed in MSI-H tumors. According to the frequency of mutations in MSI-H tumors, these genes could be divided into genes with high coding mononucleotide repeat instability (CMRI-H) and genes with low coding mononucleotide instability (CMRI-L). CMR-H genes were mutated in more than 9/38 and CMRI-L in less than 4/38 of MSI-H tumors. Four genes in our study were CMRI-H and could thus possibly play a role in the development of MSI-H tumors: TFE3 (9/38), TEF4 (12/38), RGS12 (11/38), and TCF1 (12/38). Our results suggest that systematic identification of genes with CMR in the sequence databases and determination of mutation frequency in MSI-H tumors might be a powerful tool for identification of new molecular targets in the development of MSI-H tumors.     

The Fertility Problem Inventory: measuring perceived infertility-related stress.

OBJECTIVE: To develop a reliable, valid instrument to evaluate perceived infertility-related stress. DESIGN: Prospective study. SETTING: University-affiliated teaching hospital. PATIENT(S): Consecutively referred patients (1,153 women and 1,149 men) seen for infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Participants' infertility-related stress was assessed by written questionnaire using the Fertility Problem Inventory. Current levels of anxiety, depression, and marital satisfaction also were determined. RESULT(S): Women described greater global stress than men and higher specific stress in terms of social concerns, sexual concerns, and need for parenthood. Both men and women facing male infertility reported higher global stress and more social and sexual concerns than men and women experiencing female infertility. Social, sexual, and relationship concerns related to infertility were more effective predictors of depression and marital dissatisfaction than expressed needs for parenthood or attitudes toward child-free living. CONCLUSION(S): The Fertility Problem Inventory provides a reliable measure of perceived infertility-related stress and specific information on five separate domains of patient concern. Patterns of infertility-related stress differed depending on gender, fertility history, and infertility diagnosis. Among patients receiving treatment, social, sexual, and relationship concerns appear central to current distress. Counseling interventions that target these domains appear likely to offer maximal therapeutic benefit.     

MicroRNA Microarray Expression Profiling in Human Myocardial Infarction

MicroRNAs (miRNAs), small non-coding RNA molecules, are negative regulators of gene expression. Recent studies have indicated their role in various forms of cardiovascular disease. In spite of the number of miRNA microarray analyses performed, little is known about the genome-wide miRNA expression pattern in human myocardial infarction (MI). Using miRNA microarrays and bioinformatic analysis, miRNA expression was analyzed on human MI and foetal hearts compared to healthy adult hearts, to determine whether there is any similar expression pattern between MI and foetal hearts, and to identified miRNAs that have not previously been described as dysregulated in cardiovascular diseases. Of 719 miRNAs analyzed, ∼ 50% were expressed in human hearts, 77 miRNAs were absent from all tested tissues and 57 were confidently dysregulated in at least one tested group. Some expression patterns appeared to be similar in MI and foetal hearts. Bioinformatic analysis revealed 10 miRNAs as dysregulated in MI not yet related to cardiovascular disease, and 5 miRNAs previously described only in animal models of cardiovascular diseases. Finally, qRT-PCR analysis confirmed dysregulation of 7 miRNAs, miR-150, miR-186, miR-210, miR-451, and muscle-specific, miR-1 and miR-133a/b; all of these are believed to be involved in various physiological and pathological processes.     

Vipera ammodytes bites treated with antivenom ViperaTAb: a case series with pharmacokinetic evaluation

Context: In clinical practice it is difficult to differentiate between V. berus and V. ammodytes venomous bites. In the past this was not a concern, but due to the current shortage in Viperfav? and European viper venom antiserum availability, V. a. ammodytes venomous bites have recently been treated with ViperaTAb^®, which is a pharmaceutical formulation containing a monospecific ovine Fab fragments against the venom of V. berus.

Objective: To evaluate ViperaTAb^® in V. a. ammodytes envenomations.

Materials and methods: This is a prospective case series of three consecutive patients envenomed by V. a. ammodytes snakebite treated with ViperaTAb^®. V. ammodytes venom, neurotoxic ammodytoxins, and Fab fragment levels were determined in serum samples and a pharmacokinetic analysis of the antivenom Fab fragments was carried out.

Results: Three patients bitten by V. a. ammodytes with extensive local swelling, neurological symptoms and recurrent thrombocytopenia were treated with ViperaTAb^®. V. ammodytes venom was detected in serum of all three patients. Ammodytoxins were detected in the serum of only the most severely envenomed patient who developed neurological symptoms. In the presented moderate cases, a dose of 8?mL of ViperaTAb^® reduced swelling and improved systemic effects, such as thrombocytopenia. However, this dose of ViperaTAb^® was not effective in the most severely envenomed patient with the highest serum values of V. ammodytes venom. In this case ViperaTAb^® did not stop local swelling and it had no effect on neurological signs. ViperaTAb^®’s systemic clearance, distribution and elimination half-lives were 4.3–13.4?mL/h/kg, 1.2–3.2?h and 14.1–55.4?h, respectively.

Conclusions: In patients envenomed by V. a. ammodytes venom, ViperaTAb^® reduces moderate swelling and temporarily improves systemic effects, except neurological symptoms. ViperaTAb^® application induces a decrement of V. ammodytes venom level in the blood, but did not affect serum concentration of neurotoxic ammodytoxins in the one patient with measurable concentrations.