Lessons From the EThIGII Trial: Proper Putative Benefit Assessment of Low-Molecular-Weight Heparin Treatment in M2/ANXA5 Haplotype Carriers

This study presents sample size considerations derived from the Efficacy of Thromboprophylaxis as an Intervention during Gravidity (EThIGII) trial (ClinicalTrials.gov: NCT00400387) to address the question of low-molecular-weight heparin (LMWH) treatment in women with recurrent pregnancy loss (RPL) depending on the M2/ANXA5 haplotype. To evaluate the possible influence of such treatment on miscarriage rates of trial participants, a post hoc analysis of ANXA5 promoter genotypes in the light of M2/ANXA5 (RPRGL3) distribution was performed using logistic models. DNA for genotyping was available from 129 LMWH and 95 control patients, 44 (19.6%) of whom were M2/ANXA5 carriers. Miscarriages occurred in 1 (4.0%) of 25 M2/ANXA5 carriers from the LMWH group compared to 4 (21.1%) of 19 in the control group, resulting in an odds ratio (95% confidence interval) for miscarriage of 0.16 (0.016-1.5) for women treated with LMWH. In noncarriers, miscarriage rates were 6 (5.8%) of 104 versus 7 (9.2%) of 76 for the LMWH and the control groups, respectively, corresponding to an odds ratio for miscarriage of 0.60 (0.19-1.9). The apparent beneficial effects of miscarriage rate reduction in M2/ANXA5 carriers with RPL concur with biological considerations about improvement in reduced ANXA5 function through LMWH treatment in an adequate murine model. The data obtained were instrumental to design proper assessment of the existence and magnitude of this effect

Applications and data analysis of next-generation sequencing

Over the past 6 years, next-generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry, and data analysis need to be overcome. Each workflow and sequencing platform have their particular problems and caveats, which need to be addressed. Regarding NGS, there is a variety of different enrichment methods, sequencing devices, or technologies as well as a multitude of analyzing software products available. In this manuscript, the authors focus on challenges in data analysis when employing different target enrichment methods and the best applications for each of the

Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics

Strong Association of a Common Dihydropyrimidine Dehydrogenase Gene Polymorphism with Fluoropyrimidine-Related Toxicity in Cancer Patients

variations associated with enhanced drug toxicity. = 0.001; the attributable risk was 56.9%. Comparing tumor-type matched sets of samples, correlation of c.496A>G with toxicity was particularly present in patients with gastroesophageal and breast cancer, but did not reach significance in patients with colorectal malignancies. polymorphism strongly contributes to the occurrence of fluoropyrimidine-related drug adverse effects. Carriers of this variant could benefit from individual dose adjustment of the fluoropyrimidine drug or alternate therapies

Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCAT-specific activity. Implications for classification and prognosis.

We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes

Novel <i>KCNH1</i> Mutations Associated with Epilepsy: Broadening the Phenotypic Spectrum of <i>KCNH1</i>-Associated Diseases

Here, we describe four patients suffering from a rather broad spectrum of epilepsy-related disorders, ranging from developmental and epileptic encephalopathy with intellectual disability (DEE) to genetic generalized epilepsy (GGE), which all harbor novel KCNH1 mutations. In one family, we found a weak association of a novel nonsense mutation with epilepsy, suggesting reduced penetrance, and which shows, in agreement with previous findings, that gain-of-function effects rather than haploinsufficiency are important for the pathogenicity of mutations. De novo missense variants in the pore region of the channel result in severe phenotypes presenting usually with DEE with various malformations. The potential pathogenicity of a novel KCNH1 germline mutation located outside of the critical pore domain observed in a GGE patient with a milder phenotype is supported by the fact that the very same amino acid exchange was detected as a somatic mutation in the resected brain tissue of a patient suffering from a focal cortical dysplasia type IIb. Thus, our case series broadens the phenotypic spectrum of KCNH1-associated diseases