Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R

The identification of common genetic variants such as single nucleotide polymorphisms (SNPs) in the human genome has become central in human population genetics and evolution studies, as well as in the study of the genetic basis of complex traits and diseases. Crucial for the accurate identification of genetic variants is the availability of high quality genomic DNA (gDNA). Since popular sources of gDNA (buccal cells, lymphocytes, hair bulb) often do not yield sufficient quantities of DNA for molecular genetic applications, whole genome amplification methods have recently been introduced to generate a renewable source of double-stranded linear DNA. Here, we assess the fidelity of one method, multiple displacement amplification (MDA), which utilizes bacteriophage Phi29 DNA polymerase to generate amplified DNA from an original source of gDNA, in a representative SNP discovery and genetic association study at the melanocortin 1 receptor (MC1R) locus, a highly polymorphic gene in humans involved in skin and hair pigmentation. We observed that MDA has high fidelity for novel SNP discovery and can be a valuable tool in generating a potentially indefinite source of DNA. However, we observed an allele amplification bias that causes genotype miscalls at heterozygous sites. At loci with multiple polymorphic sites in linkage disequilibrium, such as at MC1R, this bias can create a significant number of heterozygote genotype errors that subsequently misrepresents haplotypes.     

Abciximab therapy is associated with increased platelet activation and decreased heparin dosage in patients with acute myocardial infarction

The inhibition of the glycoprotein (GP) IIb/IIIa receptor for reducing periprocedural ischemic events in patients undergoing coronary intervention is known to influence platelet reactivity. Suboptimal doses of GP IIb/IIIa antagonists have been suggested to be prothrombotic and proinflammatory. This study was performed to observe platelet activation markers, whole blood aggregation and the dosage of unfractionated heparin (UFH) in the presence or absence of the GP IIb/IIIa inhibitor abciximab. Patients with acute myocardial infarction undergoing percutaneous coronary intervention were treated with (n = 15) or without (n = 15) abciximab. Platelet activation markers were flow cytometrically measured before and after PCI. Whole blood platelet aggregation was tested by a platelet function assay. The patients with abciximab showed a significant increase in platelet activation markers (P-selectin: 7.12 +/- 0.36 AU vs 11.05 +/- 0.79 AU) and a lower requirement of UFH to prolong aPTT > 60 sec during the infusion. 12 hours after infusion P-selectin level decreased (7.20 +/- 0.58 AU), whereas whole blood aggregation was increasing again. After stopping abciximab, requirement of UFH to prolong aPTT increased in the treated group to a greater extent to a level similar to the untreated group even when most of the platelets were still inhibited. The increased platelet activation found at the end of abciximab treatment points to a procoaguable condition that should be carefully monitored and treated by adapting anticoagulation and antiplatelet drugs.     

Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome

BACKGROUND: Nephrotic syndrome (NS) represents the association of proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Steroid-resistant nephrotic syndrome (SRNS) is defined by primary resistance to standard steroid therapy. It remains one of the most intractable causes for end-stage renal disease (ESRD) in the first two decades of life. Sporadic mutations in the Wilms' tumor suppressor gene WT1 have been found to be present in patients with SRNS in association with Wilms' tumor (WT) and urinary or genital malformations, as well as in patients with isolated SRNS. METHODS: To further evaluate the incidence of WT1 mutations in patients with NS we performed mutational analysis in 115 sporadic cases of SRNS and in 110 sporadic cases of steroid-sensitive nephrotic syndrome (SSNS) as a control group. Sixty out of 115 (52%) patients with sporadic SRNS were male, 55/115 (48%) were female. Sex genotype was verified by haplotype analysis. Mutational analysis was performed by direct sequencing and by denaturing high-performance liquid chromatography (DHPLC). RESULTS: Mutations in WT1 were found in 3/60 (5%) male (sex genotype) cases and 5/55 (9%) female (sex genotype) cases of sporadic SRNS, and 0/110 (0%) sporadic cases of SSNS. One out of five female patients with mutations in WT1 developed a WT, 2/3 male patients presented with the association of urinary and genital malformations, 1/3 male patients presented with sexual reversal (female phenotype) and bilateral gonadoblastoma, and 4/5 female patients presented with isolated SRNS. CONCLUSION: According to the data acquired in this study, patients presenting with a female phenotype and SRNS and male patients presenting with genital abnormalities should especially be screened to take advantage of the important genetic information on potential Wilms' tumor risk and differential therapy. This will also help to provide more data on the phenotype/genotype correlation in this patient population.     

Der Vaterschaftsnachweis

For centuries, difficulties have occurred in determining unresolved paternities. In addition to the modern standard methods, such as the examination of DNA or serological proof, expert opinion on fertility once played an important role. The andrological difference between incapability to fertilise and the inability to participate in sexual intercourse was also distinguished historically. Of special significance was the discovery of spermatozoa by the medical student Johan Ham in 1677 and their further investigation by Antoni van Leeuwenhoek.Recently, modern DNA methods have also been applied for historical investigations. Illustrious examples are the DNA analysis in the case of Kaspar Hauser of Ansbach and the dispute about Thomas Jefferson, President of the U.S., fathering a child by one of his slaves.In this discourse, a medicinal-forensic review of the development of expert opinion, illustrated with historical case studies, is given.     

Die Relevanz der Harnblasenfüllung bei der suprapubischen Blasenkatheterisierung

Bei der Einlage eines suprapubischen Harnblasenkatheters (Zystostomie) kann eine Fehlpunktion aufgrund der engen anatomischen Lagebeziehungen der Beckenorgane, typische, in Einzelfällen auch letale Verletzungsmuster hervorrufen. Dieses ist insbesondere bei einem unzureichenden Blasenfüllungsvolumen der Fall. Derartige Probleme sind kasuistisch auch in der rechtsmedizinischen Literatur beschrieben worden. Um die kraniale Verdrängung gefährdeter Darmanteile durch eine gefüllte Harnblase zu verifizieren, wurden in dieser Studie bei 76 Fällen im Rahmen einer gerichtlichen Autopsie der Abstand zwischen Symphysenoberkante und Apex vesicae in situ vermessen und mit dem Blasenfüllungsvolumen korreliert. Bei einem Volumen von 200 ml wurde eine suprapubische Distanz von 4 cm in 69,2% der Fälle ermittelt. Diese kollidiert jedoch mit dem üblichen klinischen Interventionspunkt, 2 fingerbreit oberhalb der Symphyse. Alle Fälle mit einer Blasenfüllung 300 ml wiesen eine Distanz von 5 cm auf. Basierend auf unseren Ergebnissen halten wir eine präoperative Harnblasenfüllung von 300 ml für die komplikationsarme Anlage eines suprapubischen Blasenkatheters für erforderlich.     

Thoracolumbar fracture stabilization: comparative biomechanical evaluation of a new video-assisted implantable system

Minimally invasive techniques for spinal surgery are becoming more widespread as improved technologies are developed. Stabilization plays an important role in fracture treatment, but appropriate instrumentation systems for endoscopic circumstances are lacking. Therefore a new thoracoscopically implantable stabilization system for thoracolumbar fracture treatment was developed and its biomechanical in vitro properties were compared. In a biomechanical in vitro study, burst fracture stabilization was simulated and anterior short fixation devices were tested under load with pure moments to evaluate the biomechanical stabilizing characteristics of the new system in comparison with a currently available system. With interbody graft and fixation the new system demonstrated higher stabilizing effects in flexion/extension and lateral bending and restored axial stability beyond the intact spine, as well as having comparable or improved effects compared with the current system. Because of this biomechanical characterization a clinical trial is warranted; the usefulness of the new system has already been demonstrated in 45 patients in our department and more than 300 cases in a multicenter study which is currently under way.     

Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of nephrotic syndrome

Nephrotic syndrome (NS) represents the association of proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Steroid-resistant NS (SRNS) is defined by primary resistance to standard steroid therapy. It remains one of the most intractable causes of ESRD in the first two decades of life. Mutations in the NPHS2 gene represent a frequent cause of SRNS, occurring in approximately 20 to 30% of sporadic cases of SRNS. On the basis of a very small number of patients, it was suspected that children with homozygous or compound heterozygous mutations in NPHS2 might exhibit primary steroid resistance and a decreased risk of FSGS recurrence after kidney transplantation. To test this hypothesis, NPHS2 mutational analysis was performed with direct sequencing for 190 patients with SRNS from 165 different families and, as a control sample, 124 patients with steroid-sensitive NS from 120 families. Homozygous or compound heterozygous mutations in NPHS2 were detected for 43 of 165 SRNS families (26%). Conversely, no homozygous or compound heterozygous mutations in NPHS2 were observed for the 120 steroid-sensitive NS families. Recurrence of FSGS in a renal transplant was noted for seven of 20 patients with SRNS (35%) without NPHS2 mutations, whereas it occurred for only two of 24 patients with SRNS (8%) with homozygous or compound heterozygous mutations in NPHS2. None of 29 patients with homozygous or compound heterozygous mutations in NPHS2 who were treated with cyclosporine A or cyclophosphamide demonstrated complete remission of NS. It was concluded that patients with SRNS with homozygous or compound heterozygous mutations in NPHS2 do not respond to standard steroid treatment and have a reduced risk for recurrence of FSGS in a renal transplant. Because these findings might affect the treatment plan for childhood SRNS, it might be advisable to perform mutational analysis of NPHS2, if the patient consents, in parallel with the start of the first course of standard steroid therapy.     

Lung function,asthma symptoms,and quality of life for children in public housing in Boston: a case-series analysis

Background  

Children in urban public housing are at high risk for asthma, given elevated environmental and social exposures and suboptimal medical care. For a multifactorial disease like asthma, design of intervention studies can be influenced by the relative prevalence of key risk factors. To better understand risk factors for asthma morbidity in the context of an environmental intervention study, we conducted a detailed baseline evaluation of 78 children (aged 4–17 years) from three public housing developments in Boston.