Matthias Schreiber

Association between c135G/A genotype and RET proto-oncogene germline mutations and phenotype of Hirschsprung's disease.

BACKGROUND Several genes, including the major susceptibility gene RET, have roles in development of Hirschsprungs disease. Results of genetic-linkage analysis of patients with familial disease with both long-segment and short-segment phenotypes have shown close linkage with the RET locus. We aimed to investigate whether both RET mutations and polymorphisms contribute to phenotype of Hirschsprungs disease. METHODS We looked at the coding region of all 21 exons of the RET proto-oncogene, including the flanking intronic sequences, by direct DNA sequencing in 76 caucasians from Germany with Hirschsprungs disease. FINDINGS 20 different mutations were detected in 18 patients. Mutations were under-represented in patients with a homozygous RET c135A/A genotype in association with short-segment phenotype. Short-segment phenotype also arose if the RET mutation was on the c135A allele; conversely, a RET germline mutation on the c135G allele resulted in long-segment phenotype, particularly in heterozygous c135G/A patients. INTERPRETATION These observations lend support to the idea that both RET alleles have a role in pathogenesis of Hirschsprungs disease, in a dose-dependent fashion. We also showed that the c135G/A polymorphism modifies the phenotype by a within-gene interaction between the c135A variant and a mutation.

The quality of life in end stage renal disease care

Abstract The improved prognosis and survival statistics of both renal transplantation and dialysis have focused attention on the quality of life offered by these treatments. Using a standardized questionnaire, we assessed the quality of life of 612 patients undergoing renal replacement therapy at our center. Of these patients, 359 had been transplanted and 253 patients were on dialysis. Concerning the sociodemographic data, only the time on specific treatment was longer in dialysis patients than in transplanted patients (49.2 versus 55.6 months, P < 0.05). Most complaints were more common in dialysis patients than in transplanted patients. Only the side effects of medication were seen more in transplanted patients (P < 0.005). Life satisfaction was higher in transplanted patients than in dialysis patients. Dialysis patients were more anxious (P < 0.05) and more depressed (P < 0.001) than transplanted patients. Transplanted patients also felt that they had more social support than did dialysis patients. Overall life quality was almost equal between patients on hemodialysis and patients on peritoneal dialysis, and between patients on the waiting list for transplantation and those not on the waiting list. Despite a significantly better quality of life after renal transplantation, the percentage of patients working remained unchanged. (57.5% versus 57.8%, P= n.s.). We conclude that despite an improved quality of life after renal transplantation, these patients are economically not more productive than patients on dialysis.

Protective Effects of Diltiazem and the Prostazycline Analogue Iloprost in Human Renal Transplantation

To test the hypothesis that calcium antagonists decrease the incidence and severity of delayed graft function, we conducted three separate, prospective, randomized trials. In these trials, we investigated the effects of diltiazem and those of the prostacycline analogue iloprost. In the first study, 22 control patients and 20 diltiazem patients received grafts perfused with either vehicle or diltiazem 20 mg/L in the Euro-Collins solution. Subsequently, the diltiazem subjects were given the drug as a bolus of 0.28 mg/kg, followed by a continuous infusion of 0.002 mg/min/kg for the following 2 days. Thereafter, diltiazem 60 mg was given to the treated subjects orally for up to 4 years. In the second study, 11 control subjects and 10 diltiazem subjects received the same postoperative regimen, but all grafts were harvested without addition of diltiazem to the perfusion solution. In the third protocol, four groups were studied as follows: 19 control subjects who received no specific treatment, 16 subjects who received diltiazem, 16 subjects who were given iloprost, and 14 subjects who received both iliprost and diltiazem. The donor kidney of treated patients was perfused with either diltiazem, iloprost, or both drugs. Primary graft function occurred more commonly in the groups receiving diltiazem. Further, in the first study the number of hemodialyses per patient was reduced in those patients with delayed graft function. Fewer rejection episodes occurred in patients receiving diltiazem. Plasma levels of soluble interleukin-2 receptors decreased significantly during diltiazem treatment. Moreover, renal biopsies showed less severe signs of cyclosporin-A (CyA) nephrotoxicity in diltiazem-treated patients compared to controls, even though these patients also exhibited higher CyA trough levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of RET proto-oncogene codon 609 germline mutations with RET haplotypes characterized by c.135G>A alleles modifying MEN 2A or HSCR phenotypes

Germline mutations of the RET proto-oncogene have been identified in patients with the inherited neoplastic syndrome ‘‘multiple endocrine neoplasia type 2’’ (MEN 2, [MIM no. 171400]), which comprises themedullary thyroid carcinoma as an obligatory feature, and with Hirschsprung disease (HSCR [MIM no. 142623]), a developmental disorder of the enteric nervous system characterized by the absence of intramural ganglia in the hindgut. Highly penetrant germline missense mutations of RET proto-oncogene have been detected in each one of six cysteine codons within exon 10 (codons 609, 611, 618, and 620) and exon 11 (codons 630 and 634) in about 95% of MEN 2A and in up to 85% of familial medullary thyroid carcinoma (FMTC) families [Donis-Keller et al., 1993; Mulligan et al., 1993; Eng andMulligan, 1997]. In contrast, between 10 and 40% of the patients with HSCR harbor germline mutations of the RET proto-oncogene, which are primarily point mutations scattered throughout the extracellular domain and within the intracellular tyrosine kinase domain of RET [Edery et al., 1994; Romeo et al., 1994; Angrist et al., 1995; Attie et al., 1995; Seri et al., 1997]. The phenotypic association of MEN 2A/FMTC and HSCR was previously reported in several kindreds. With one exception, this combined phenotype segregates in the involved families with a single RET haplotype harboring a cysteine germline mutation of the codons 609, 618, or 620 [Mulligan et al., 1994]. Since all of these RET germline mutations are described in various families in association withMEN 2A/FMTC alone, and assuming a genetically determined HSCR phenotype, additional genetic alterations resulting in a combination of the MEN 2 and HSCR phenotype can be hypothesized. The present study describes two unrelated kindreds with codon 609 RET mutations associated with different phenotypes and different alleles at an intragenic single nucleotide polymorphic site in exon 2. We analyzed the RET proto-oncogene in two families both harboring a codon 609 RET germline mutation. The only affected individual of the first family (Fig. 1a) has a de novo mutation in association with HSCR, without any of the expected MEN2 features. In the second family, the mutation segregates with the more characteristic MEN2A phenotype (Fig. 1b). Genomic DNA was extracted from leukocytes from peripheral venous blood samples by standard protocols. The amplification and sequencing of all 21 RET exons was performed using primers and reaction conditions described previously [Ceccherini et al., 1994;Mulligan et al., 1994;Fitze et al., 2002]. Furthermore, we determined the genotypes of seven polymorphisms of the coding region of the RET proto-oncogene in all family members [Ceccherini et al., 1994; Fitze et al., 1999]. Direct DNA sequencing of all 21 coding exons of the RET proto-oncogene, including the adjacent intronic boundaries, detected a heterozygous germline mutation of codon 609 (TGC!AGC) in the HSCR affected patient (II-1, Fig. 1a) of the first family. This mutation results in the substitution of a cysteine amino acid residue by serine. In this patient, an additional germline mutation was not found and all clinically unaffected family members were excluded as gene carriers. In the index case, the homozygous c.135A genotype, the heterozygous c.2307T/G genotype, and for all others (c.375C, c.1296G, c.2071G, c.2508C, c.2712C) the homozygous wildtype genotypes were found in the analysis of the coding region polymorphisms. Hence, the C609S mutation was located on a c.135A allele. The genotypes in this family are shown in Figure 1a. In contrast, the index case of the second family (II-2, Fig. 1b) also harbored a heterozygous germline mutation of the codon 609 (TGC!GGC), resulting in a substitution of cysteine by glycine. In this family theC609Gmutation segregatedwith the MEN 2A phenotype. Six of seven identified gene carriers underwent total thyroidectomy, and five were diagnosed with MTC. Only a 5-year-old boy (IV-4) showed a normal thyroid gland histology after prophylactic thyroidectomy. Two of the gene carriers (II-2, II-4) also had a pheochromocytoma. Moreover, the polymorphisms, particularly c.135G>A and c.2307T/G, showed the homozygous wild-type genotype (c.135G, c.375C, c.2071G, c.2307T, c.2508C, c.2712C), with exception of c.1296G>A polymorphism, which was heterozygous in five of seven C609G gene carriers (Fig. 1b). Hence, the C609G mutation was located on a c.135G allele. Whereas the family harboring the C609G (TGC!GGC) mutation showed a typical MEN 2A syndrome without clinical signs ofHSCRor chronic constipation over three generations, a novel de novo C609S (TGC!AGC)mutation in one individual was associated with isolated short-segment HSCR. A prophylactic thyroidectomy at age 30 revealed neitherMTCnor C-cell hyperplasia. Isolated HSCR in association with a common MEN 2 cysteine codon mutation involving either codon 609 (three cases) or codon620 (one case) is relatively rare [Mulligan etal., 1994;Angrist et al., 1995;Hofstra, 1998].Yet, coexistence of MEN 2 and HSCR phenotypes segregating with a specific RET allele harboring RET mutations of codons 609, 618, and 620 have been shown in 21 reported families [Chretien et al., 1994;Mulligan et al., 1994;Borst et al., 1995;Blanket al., 1996; Caron et al., 1996; Peretz et al., 1997; Seri et al., 1997; Borrego et al., 1998; Decker et al., 1998; Fitze et al., 2002; Pasini et al., 2002]. Because of the contrasting character of RETmutations in theMEN2 or theHSCRphenotype (gain of function inMEN 2-associated RET mutations versus loss-of-function in HSCRassociated RET mutations), it may be assumed that the combined HSCR/MEN 2 phenotype derives from two different RET germline mutations. However, second RET mutations *Correspondence to: Dr. Guido Fitze, M.D., Department of Pediatric Surgery, University of Technology Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail: Guido.Fitze@mailbox.tu-dresden.de

Drug-coated balloons for small coronary artery disease (BASKET-SMALL 2): an open-label randomised non-inferiority trial

BACKGROUND Drug-coated balloons (DCB) are a novel therapeutic strategy for small native coronary artery disease. However, their safety and efficacy is poorly defined in comparison with drug-eluting stents (DES). METHODS BASKET-SMALL 2 was a multicentre, open-label, randomised non-inferiority trial. 758 patients with de-novo lesions (<3 mm in diameter) in coronary vessels and an indication for percutaneous coronary intervention were randomly allocated (1:1) to receive angioplasty with DCB versus implantation of a second-generation DES after successful predilatation via an interactive internet-based response system. Dual antiplatelet therapy was given according to current guidelines. The primary objective was to show non-inferiority of DCB versus DES regarding major adverse cardiac events (MACE; ie, cardiac death, non-fatal myocardial infarction, and target-vessel revascularisation) after 12 months. The non-inferiority margin was an absolute difference of 4% in MACE. This trial is registered with ClinicalTrials.gov, number NCT01574534. FINDINGS Between April 10, 2012, and February 1, 2017, 382 patients were randomly assigned to the DCB group and 376 to DES group. Non-inferiority of DCB versus DES was shown because the 95% CI of the absolute difference in MACE in the per-protocol population was below the predefined margin (-3·83 to 3·93%, p=0·0217). After 12 months, the proportions of MACE were similar in both groups of the full-analysis population (MACE was 7·5% for the DCB group vs 7·3% for the DES group; hazard ratio [HR] 0·97 [95% CI 0·58-1·64], p=0·9180). There were five (1·3%) cardiac-related deaths in the DES group and 12 (3·1%) in the DCB group (full analysis population). Probable or definite stent thrombosis (three [0·8%] in the DCB group vs four [1·1%] in the DES group; HR 0·73 [0·16-3·26]) and major bleeding (four [1·1%] in the DCB group vs nine [2·4%] in the DES group; HR 0·45 [0·14-1·46]) were the most common adverse events. INTERPRETATION In small native coronary artery disease, DCB was non-inferior to DES regarding MACE up to 12 months, with similar event rates for both treatment groups. FUNDING Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Basel Cardiovascular Research Foundation, and B Braun Medical AG.