Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1. 17; UGT1A1). Present methods for genotyping this polymorphism are laborious.Hybridization probes were designed complementary to the wild type (TA)(6) and to alleles with (TA)(7) and (TA)(8) repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)(5) to (TA)(8) repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis.Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)(7) or (TA)(8) genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)(7) allele.The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.
Aristaless-like homeobox 4 (ALX4) gene is an important transcription regulator in skull and limb development. In humans and mice ALX4 mutations or loss of function result in a number of skeletal and organ malformations, including polydactyly, tibial hemimelia, omphalocele, biparietal foramina, impaired mammary epithelial morphogenesis, alopecia, coronal craniosynostosis, hypertelorism, depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, and body agenesis. Here we show that a complex skeletal malformation of the hind limb in Galloway cattle together with other developmental anomalies is a recessive autosomal disorder most likely caused by a duplication of 20 bp in exon 2 of the bovine ALX4 gene. A second duplication of 34 bp in exon 4 of the same gene has no known effect, although both duplications result in a frameshift and premature stop codon leading to a truncated protein. Genotyping of 1,688 Black/Red/Belted/Riggit Galloway (GA) and 289 White Galloway (WGA) cattle showed that the duplication in exon 2 has allele frequencies of 1% in GA and 6% in WGA and the duplication in exon 4 has frequencies of 23% in GA and 38% in WGA. Both duplications were not detected in 876 randomly selected German Holstein Friesian and 86 cattle of 21 other breeds. Hence, we have identified a candidate causative mutation for tibial hemimelia syndrome in Galloway cattle and selection against this mutation can be used to eliminate the mutant allele from the breed.
Einleitung Zur Verbesserung der Diagnostik des kaninen Prostatakarzinoms und der limitierten therapeutischen Ansätze ist ein besseres Verständnis auf molekularer Ebene essenziell. Hochdurchsatz-RNA-Sequenzierungen (RNA-Seq) ermöglichen die Generierung komplexer molekularer Signaturen auch aus geringen Probenvolumina. Ziel der Studie war, Prostatakarzinom-Proben aus ultraschallgeführten Feinnadelaspirationsbiopsien (US-FNA) und Gewebe (PG) der kaninen Prostata gegenüber nichtmalignen Proben auf molekularer Ebene zu charakterisieren. Methoden Elf maligne (PG n = 9, US-FNA n = 2) und 14 nichtmaligne (PG=9, US-FNA n = 5) Proben wurden über RNA-Seq mittels Illumina NextSeq500 charakterisiert und bioinformatisch ausgewertet. Ergebnisse Die funktionelle Analyse der deregulierten Gene in malignen Proben zeigt Veränderungen in Zellzyklus- und Entzündungspathways. Verschiedene Tumorsuppressor- und Onkogene konnten im cancer related pathway sowie PI3K-Akt, VEGF, ErbB signaling pathway identifiziert werden. Schlussfolgerung RNA-Seq-Daten identifizierten deregulierte Gene, auf deren Basis diagnostische und therapeutische Ansätze für weitere Studien zum kaninen Prostatakarzinom erarbeitet werden können.
Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is being used increasingly in immunosuppressant therapy after solid organ transplantation (1)(2)(3). Its immunosuppressive activity is thought to reside in the inhibition of inosine monophosphate dehydrogenase (IMPDH), leading to a suppression of purine nucleotide synthesis in lymphocytes, thereby suppressing cell mitosis (3)(4). MPA is converted in the liver mainly to the 7- O -glucuronide metabolite, mycophenolic acid glucuronide (MPAG), which does not have immunosuppressant activity (3)(5). During MMF therapy, MPAG peak concentrations observed in plasma of patients (5–600 mg/L) can be up to 300-fold higher than those of MPA (0.1–50 mg/L). Whether drug monitoring of MPA or MPAG is required to improve the therapeutic efficacy or to minimize adverse side effects is still under investigation (3)(6). For monitoring of MPA concentrations in human plasma, several HPLC procedures as well as one immunoassay (Emit; Behring-Syva) are available (3)(6).
In the present investigation we addressed the question as to whether MPA and MPAG are stable in human plasma because it is possible that deglucuronidation of MPAG might take place in vitro during shipping and sample storage, thus leading to false high MPA concentrations. For this purpose, we added three different MPA and MPAG concentrations to three different pools of human plasma and followed both MPA and MPAG plasma concentrations for 7 days at three different storage temperatures. MPA and MPAG concentrations were measured using an HPLC method developed recently in our laboratory (7).
For this study, MPA, MPAG, and the carboxy butoxy ether of MPA (MPAC) were a gift of Hoffmann-La Roche (Grenzach-Wyhlen, Germany). Sodium tungstate dihydrate, potassium dihydrogen phosphate, sodium hydroxide, phosphoric acid, and perchloric acid were from Merck. Acetonitrile (HPLC grade) was obtained from S.T. Baker …
IntroductionThe WHO classification of pulmonary neuroendocrine tumors (PNETs) is also used to classify thymic NETs (TNETs) into typical and atypical carcinoid (TC and AC), large cell neuroendocrine carcinoma (LCNEC), and small cell carcinoma (SCC), but little is known about the usability of alternative classification systems.MethodsOne hundred seven TNET (22 TC, 51 AC, 28 LCNEC, and 6 SCC) from 103 patients were classified according to the WHO, the European Neuroendocrine Tumor Society, and a grading-related PNET classification. Low coverage whole-genome sequencing and immunohistochemical studies were performed in 63 cases. A copy number instability (CNI) score was applied to compare tumors. Eleven LCNEC were further analyzed using targeted next-generation sequencing. Morphologic classifications were tested against molecular features.ResultsWhole-genome sequencing data fell into three clusters: CNIlow, CNIint, and CNIhigh. CNIlow and CNIint comprised not only TC and AC, but also six LCNECs. CNIhigh contained all SCC and nine LCNEC, but also three AC. No morphologic classification was able to predict the CNI cluster. Cases where primary tumors and metastases were available showed progression from low-grade to higher-grade histologies. Analysis of LCNEC revealed a subgroup of intermediate NET G3 tumors that differed from LCNEC by carcinoid morphology, expression of chromogranin, and negativity for enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2).ConclusionsTNETs fall into three molecular subgroups that are not reflected by the current WHO classification. Given the large overlap between TC and AC on the one hand, and AC and LCNEC on the other, we propose a morphomolecular grading system, Thy-NET G1-G3, instead of histologic classification for patient stratification and prognostication.
Metoclopramide (Paspertin) was administered to 30 patients with duodenal ulcer at an average dosage of 2 tablets three times daily (= 60 mg/day). An immediate alleviation of pain was noted in all cases, and complete freedom from complaints has usually occurred after 3 to 8 days. Radiological examinations revealed a good relaxation of the terminal antrum, elimination of spasms in the pyloric region, quick emptying of the stomach and a normal relief of the duodenal mucosa. The excellent efficacy was noted particularly in chronic ulcers resistant to conventional medication.
