A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome).

Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients.

Fine mapping of the 9q31 Hirschsprung's disease locus.

Hirschsprung's disease (HSCR) is a congenital disorder characterised by the absence of ganglia along variable lengths of the intestine. The RET gene is the major HSCR gene. Reduced penetrance of RET mutations and phenotypic variability suggest the involvement of additional modifying genes in the disease. A RET-dependent modifier locus was mapped to 9q31 in families bearing no coding sequence (CDS) RET mutations. Yet, the 9q31 causative locus is to be identified. To fine-map the 9q31 region, we genotyped 301 tag-SNPs spanning 7 Mb on 137 HSCR Dutch trios. This revealed two HSCR-associated regions that were further investigated in 173 Chinese HSCR patients and 436 controls using the genotype data obtained from a genome-wide association study recently conducted. Within one of the two identified regions SVEP1 SNPs were found associated with Dutch HSCR patients in the absence of RET mutations. This ratifies the reported linkage to the 9q31 region in HSCR families with no RET CDS mutations. However, this finding could not be replicated. In Chinese, HSCR was found associated with IKBKAP. In contrast, this association was stronger in patients carrying RET CDS mutations with p = 5.10 x 10(-6) [OR = 3.32 (1.99, 5.59)] after replication. The HSCR-association found for IKBKAP in Chinese suggests population specificity and implies that RET mutation carriers may have an additional risk. Our finding is supported by the role of IKBKAP in the development of the nervous system.

Conversion from tacrolimus to everolimus with complete and early glucocorticoid withdrawal after kidney transplantation: a randomised trial.

BACKGROUND: While conversion from cyclosporine to everolimus is well documented, conversion from tacrolimus has been poorly studied. In this randomised, controlled trial the safety and tolerability of switching from tacrolimus to everolimus with glucocorticoid withdrawal after living-donor kidney transplantation was studied. METHODS: A total of 194 patients were planned to be randomised 1:1 to either continue tacrolimus or to convert to everolimus at month 3 after transplantation. At randomisation, all patients received tacrolimus, mycophenolate mofetil and prednisolone. Everolimus was started in a dose of 1.5 mg twice daily, aiming for predose concentrations of 4-7 ng/ml. Prednisolone was gradually withdrawn in both groups. RESULTS: The trial was stopped prematurely after the inclusion of 60 patients. The interim analysis showed an unacceptably high rejection rate in the everolimus group as compared with the control group: 30.0% vs. 6.7% (95% CI: 0.047-0.420; p = 0.045). An additional 8 patients stopped everolimus because of toxicity. At the end of follow-up (month 12) only 12 (40%) patients assigned to everolimus were still on the study drug. CONCLUSIONS: Conversion from tacrolimus to everolimusbased immunosuppression with withdrawal of prednisolone three months after kidney transplantation results in an unacceptably high risk of acute rejection and causes considerable toxicity. Based on our findings, such a switch strategy cannot be recommended.

Reliability of sentinel lymph-node extirpation as a diagnostic method for malignant melanoma of the head and neck region.

A series of 106 patients with malignant melanoma of the head and neck and clinically negative local lymph-node status were included in a multimodal therapy programme and underwent sentinel lymph-node extirpation in 1999-2003. Out of 246 preoperatively marked lymph nodes, only 172 (70%) were identified intraoperatively and removed. In 89% of all patients at least one sentinel lymph node was removed. Histological examination revealed metastases in the sentinel lymph nodes of 17 patients. In the mean follow-up period of 47 months (range 4-76 months), regional lymph-node metastases were found in another eight patients. The non-marked lymph nodes that were often removed at the same time, in an elective cervical lymph-node dissection, did not reveal any metastasis in any of the cases where the sentinel lymph nodes were negative. The sensitivity of sentinel lymph-node extirpation was influenced by the length of the follow-up period and the detection rate, and was 68% (17/17+8), a result superior to that of any other diagnostic tool. Sentinel lymph-node extirpation is a valuable method in addition to elective lymph-node dissection.

Cellular effects of imatinib on medullary thyroid cancer cells harboring multiple endocrine neoplasia Type 2A and 2B associated RET mutations.

BACKGROUND: Activating mutations in the RET gene, which encodes a tyrosine kinase receptor, often cause medullary thyroid carcinoma (MTC). Surgical resection is the only curative treatment; no effective systemic treatment is available. We evaluated imatinib, a tyrosine kinase inhibitor currently used to treat chronic myelogenous leukemia and gastrointestinal stromal tumors, as a potential drug for systemic treatment of MTC, in 2 MTC-derived cell lines expressing multiple endocrine neoplasia-associated mutant RET receptors. METHODS: We determined RET expression and Y1062 phosphorylation using Western blot analysis and quantitative polymerase chain reaction. We determined the effects on cell proliferation by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and we used fluorescence-activated cell sorter analysis with annexin V/propidium iodide staining to study imatinib-induced cell-cycle arrest, apoptosis, and cell death. RESULTS: Imatinib inhibited RET Y1062 phosphorylation in a dose-dependent manner after 1.5 hours of exposure. After 16 hours both RET Y1062 phosphorylation and protein expression levels were affected. Dose-dependent decreases in cell proliferation of both cell lines after exposure to imatinib with inhibitory concentration of 50% levels of 23 +/- 2 micromol/L and 25 +/- 4 micromol/L were seen. These values are high, compared with those for chronic myelogenous leukemia and gastrointestinal stromal tumors. We further could show that imatinib induced cell-cycle arrest, and apoptotic and nonapoptotic cell death. CONCLUSIONS: Imatinib inhibits RET-mediated MTC cell growth affecting RET protein levels in vitro in a dose-dependent manner. The concentration of imatinib necessary to inhibit RET in vitro, however, makes it impossible to conclude that imatinib monotherapy will be a good option for systemic therapy of MTC.

Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE.

Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants. If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection. To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods. Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments.

Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is believed to be the most powerful pre-screening method for mutation detection currently available, being used mostly on an exon-by-exon basis. Broad-range DGGE for the analysis of multiple fragments or an entire gene is rarely applied. We and others have already shown that one or two DGGE conditions are usually sufficient to analyse an entire gene. Conditions, however, have never been profoundly tested and compared with alternative methods suggested in the literature. Trying to do so in this study, we found significant differences between the various gel systems. The optimal conditions we found for broad-range DGGE include 9% polyacrylamide for the gel, a denaturing gradient with a difference of 30-50% between the lowest and the highest concentration of denaturant, and electrophoresis in 0.5x TAE buffer at a voltage >100 V and <200 V.

A gene from human chromosome region 3p21 with reduced expression in small cell lung cancer.

A combination of cytogenetic and molecular studies has implicated the p21 region of human chromosome 3 as the probable site of a gene the loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative polymerase chain reaction assay showed that their concentration in small cell lung cancer cell lines is less than 3% of that in normal lung.

Time-action profiles of novel premixed preparations of insulin lispro and NPL insulin.

OBJECTIVE: To study the pharmacodynamic properties of three premixed formulations of the rapid-acting insulin analog insulin lispro and its protamine-retarded preparation, neutral protamine lispro (NPL) insulin. RESEARCH DESIGN AND METHODS: In this open, single-center, euglycemic glucose clamp study, 30 healthy volunteers (12 women, 18 men) aged 27 +/- 2 years (mean +/- SD), whose BMI was 23.0 +/- 2.3 kg/m2, received subcutaneous injections of 0.3 U/kg body wt of insulin mixture (high-mixture 75/25, mid-mixture 50/50, or low-mixture 25/75 insulin lispro/NPL insulin), insulin lispro, or NPL insulin on one of the five study days in randomized order. Glucose infusion rates were determined over a period of 24 h after administration. RESULTS: Maximal metabolic activity decreased after subcutaneous injection of the mixtures with lower insulin lispro content; however, the time point of maximal and of early half-maximal metabolic activity was comparable among the three mixtures. Higher proportions of insulin lispro resulted in higher values for area under the curve within the first 360 min after injection and a more rapid decline to late half-maximal activity. Serum insulin concentrations showed a similar pattern. CONCLUSIONS: This study shows that the pharmacodynamic and pharmacokinetic properties of insulin lispro are preserved in stable mixtures with NPL insulin.

Mutations in Hirschsprung disease: when does a mutation contribute to the phenotype.

Hirschsprung disease is a congenital disorder clinically characterized by the absence of colonic ganglia and genetically by extensive heterogeneity. Genes involved include RET, GDNF, EDNRB and EDN3. Mutations of these genes may give dominant, recessive, or polygenic patterns of inheritance. In particular in the case of missense mutations, it is therefore far from easy to assess whether a given mutation will contribute to the phenotype. We discuss criteria for such an assessment and pay special attention to functional assays. The interpretation of mutations as contributing to a disease phenotype or as merely representing a rare polymorphism has direct clinical consequences. Hirschsprung disease with major and modifying sequence variants in a variety of genes might well serve as a model for the many complex disorders for which the search for genes involved has only just been initiated.

Prenatal prediction of spinal muscular atrophy. Experience with linkage studies and consequences of present SMN deletion analysis.

With the localisation of the gene for the autosomal recessive forms of proximal spinal muscular atrophies (SMA) to the chromosomal region 5q13 and the later detection of homozygous deletions of the SMN gene located in this region, prenatal prediction of SMA has become feasible and is widely applied now. In our experience with 77 prenatal predictions of SMA, follow-up of the 39 liveborn children from these pregnancies never led to a false-negative result. Application of SMN deletion analysis has consequences for prenatal prediction of SMA. When the index patient has a homozygously deleted exon 7 of the SMN gene, prenatal prediction and interpretation of results are straightforward. In families in which no DNA from the index patient is available, prenatal detection of a homozygous SMN deletion may be considered almost proof of SMA in the fetus. Absence of a deletion, however, will not guarantee an unaffected child. A real problem exists if the index patient does not show a homozygous deletion of SMN exon 7. In such cases with non-homozygous SMN deletions, one cannot be certain of 5q linkage and autosomal recessive inheritance until other SMN mutations are detected. This is an argument to abstain from prenatal diagnosis by linkage analysis in these families.

A provisional transcript map of the spinal muscular atrophy (SMA) critical region.

YACs from the region containing the spinal muscular atrophy (SMA) locus at 5q12 have been used as probes in a direct screening of cDNA libraries to isolate 8 cDNAs, mapped to different YAC fragments. Three clones showed complete identity to the genes for cyclin B1 (CCNB1), the p44 subunit of the transcription factor BTF2 (BTF2p44), and cofilin (CFL). Two clones showed partial identity to the beta-glucuronidase gene (GLCB) and a rat integral membrane glycoprotein gene (RNINMEGLA). CFL turned out to have been identified by a pseudogene sequence. Related sequences occurred on other chromosomes. CCNB1 and BTF2p44 were given an exact location. The GLCB-like gene and the RNINMEGLA-like gene detected loci on both 5q and 5p. The remaining three cDNA clones were localized to the SMA region only. Their sequences did not show identity to any gene for which a function is already known. Two of them have now turned out to be identical to recently reported candidate genes for SMA.

Age-dependent variability of ribosomal RNA-gene activity in man as determined from frequencies of silver staining nucleolus organizing regions on metaphase chromosomes of lymphocytes and fibroblasts.

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80--89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69--83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromsomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss tcehnical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Linkage and apparent heterogeneity in proximal spinal muscular atrophies.

Linkage studies with 9 highly informative DNA markers on the long arm of chromosome 5 were performed in 12 multiplex families (29 patients) with spinal muscular atrophy (SMA) from The Netherlands. The results of the linkage analysis were compatible with localization of a major SMA gene in the chromosomal region 5q12-13. By minimum recombinant analysis the most likely position of the SMA locus was between loci D5S6/D5S125 and D5S112/MAP1B, which is in agreement with several linkage studies from other countries. In four families, however, more than one crossover between SMA and a flanking DNA marker appeared, and in one family the observed hybridization phenotype for the markers closely flanking the SMA locus was identical for an unaffected individual and for his two affected sibs with SMA type III. For this latter family, among several explanations the most likely are either the presence of a double crossover or linkage heterogeneity.

Loss of heterozygosity for a chromosome 3 sequence presumably at 3p21 in small cell lung cancer.

A recombinant DNA fragment detecting a chromosome #3 restriction fragment length polymorphism presumably at p21 was hybridized to HindIII-digested DNA isolated from the leukocytes of 12 patients of small cell lung cancer. Four of them appeared to be heterozygous. Analysis of tumor material from these four patients revealed homozygosity for either one or the other restriction fragment in every case. Our findings suggest the presence on the short arm of chromosome #3 of a recessive mutant cancer gene contributing to the development of small cell lung cancer.

Localization of amplified c-myc and n-myc in small cell lung cancer cell lines.

In this study 12 small cell lung cancer cell lines were tested for amplification of myc oncogenes, the location of amplified sequences, and the possible correlation between number of dmin and degree of amplification in dmin-containing lines. C-myc appeared to be amplified in four cell lines, and N-myc amplification was detected in two cell lines. No amplification of L-myc was found. The degree of amplification in the different cell lines varied between 20X and 100X. The cell lines with myc amplification appeared to contain numerous dmin, although in one cell line they occurred in only 10% of the cells. The other cells in this line contained a homogeneously staining region (HSR). In situ hybridization was carried out to find the location of the amplification. In four cell llines the amplified myc genes were found to be located on the dmin. In the cell line with the HSR in most cells and dmin in a minority of its cells, amplification was found both at the HSR and on the dmin. In one cell line the myc sequences seemed to be dispersed through the genome. The ratio between the average number of dmin per cell and the degree of amplification did not vary considerably between the cell lines, with one exception. In that cell line the number of dmin exceeded the number of myc sequences by about one order of magnitude. Apparently, the population of dmin in this cell was heterogeneous and amplified myc genes were only present on a subpopulation.

Complete association of loss of heterozygosity of chromosomes 13 and 17 in osteosarcoma.

Mutations of the retinoblastoma (RB1) gene are not confined to retinoblastoma, but are also involved in the development of osteosarcoma. Structural aberrations within the RB1 gene have been studied in fresh samples of eleven cases of osteosarcoma. In five cases a rearrangement was detected, one of which was best explained as a partial duplication. The chromosomal mechanisms by which the nonmutated RB1 allele was lost appeared to be similar in frequency to those that have been reported for retinoblastoma. Loss of heterozygosity was observed for chromosomes 3, 11, 13, 17, and 22. However, when no loss of heterozygosity of chromosome 13 was detected, the other chromosomes retained their heterozygosity as well. A complete association of loss of heterozygosity of chromosomes 13 and 17 was observed. This can be taken as an indication of the involvement of another tumor suppressor gene at chromosome 17 in the initiation of osteosarcoma.

[Sentinel lymph node dissection in patients with malignant melanoma. Diagnostic and therapeutic standards]. / Sentinel-Lymphknoten-Dissektion beim malignen Melanom. Ein diagnostischer und therapeutischer Standard.

INTRODUCTION: In patients with cutaneous malignant melanoma, the sentinel lymph node (SLN) reflects the histopathological features of the lymphatic basin with high accuracy. MATERIAL AND METHODS: Three hundred eighty-one melanoma patients at the Hornheide clinic with an overall follow-up of 36 months (November 1998 to October 2001) underwent sentinel lymph node dissection (SLND). RESULTS: The SLNs were successfully found in 93% of truncal melanoma ( n=136), 97% of melanoma of the extremities ( n=184), and 86% of melanoma of the head and neck region ( n=61). Of truncal midline melanomas, 84% ( n=43) showed two or more regional basins, in contrast to 18% of nonmidline melanoma ( n=93). Histopathological analysis revealed occult nodal disease in 25% of all patients. Completion lymphadenectomy revealed residual nodal disease in 8% of all patients with low risk melanoma with a tumor thickness of 0-1.5 mm (two of 26 patients with positive SLN) and in 11% of all patients with high risk melanoma with tumor thickness above 1.5 mm (eight of 70 patients with positive SLN). Tumor relapse was noted in 5% of negative SLN patients and 14% of positive SLN patients. The results of the method were false negative in 2% with a sensitivity of 98%. CONCLUSION: Sentinel lymph node dissection is a reliable and accurate method of staging regional lymph nodes for all primary tumor sites. It can localize occult metastases in unexpected lymphatic basins and provides critical indications for completion lymphadenectomy. It represents an essential method of establishing stratification criteria for future adjuvant trials. Further long-term follow-up is needed to investigate its prognostic relevance to recurrence and overall survival.

Abundance of protein-bound sulfhydryl and disulfide groups at chromosomal nucleolus organizing regions: a cytochemical study on the selective silver staining of NORs.

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.