VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: interaction between both genotypes affects dose requirement.

In a prospective follow-up study of the effects of VKORC1 and CYP2C9 genotypes on the anticoagulation status of patients, we assessed the CYP2C9 and the VKORC1 C1173T genotypes of patients during the initial 6 months of phenprocoumon treatment. We used linear regression models and Cox proportional hazard models to determine the effects of the VKORC1 and CYP2C9 genotypes on phenprocoumon dose requirements, overanticoagulation, and time to achieve stability. Allele frequencies of interest within the cohort (N=281) were 40.8% VKORC1 T-1173, 12.8% CYP2C9*2, and 6.9% CYP2C9*3. In patients with the VKORC1 CC genotype, carriers of a CYP2C9 polymorphism needed dosages that were nearly 30% lower than those for CYP2C9*1/*1 patients (P<0.001). In patients with a VKORC1 polymorphism, differences between carriers of a CYP2C9 polymorphism and CYP2C9*1/*1 were far smaller and largely not statistically significant. A larger part of the variability in dose requirement was explained by the VKORC1 genotype than by the CYP2C9 genotype (28.7% and 7.2%, respectively). Carriers of a combination of a CYP2C9 polymorphism and a VKORC1 polymorphism had a strongly increased risk of severe overanticoagulation (hazard ratio (HR) 7.20, P=0.002). Only carriers of a CYP2C9*2 allele had a decreased chance to achieve stability compared to CYP2C9*1/*1 patients (HR 0.61, P=0.004). In conclusion, the VKORC1 genotype modifies the effect of the CYP2C9 genotype on phenprocoumon dose requirements. A combination of polymorphisms of both genotypes is associated with a strongly increased risk of overanticoagulation, whereas delayed stabilization is mainly associated with the CYP2C9 genotype.

Criteria for mutation analysis in MEN 1-suspected patients: MEN 1 case-finding.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal, dominantly inherited cancer syndrome, with tumours in various endocrine glands. In 1997 the responsible tumour suppressor gene was identified. MEN1 gene germ-line mutations are detected in the vast majority of MEN 1 patients, however, with regard to case-finding, unfortunately only at a very low frequency in patients with apparently sporadic MEN 1-related tumours. In order to increase the detection rate of disease gene carriers among patients with apparently sporadic MEN 1-related tumours, clinical criteria were needed. DESIGN AND RESULTS: In this study MEN1 gene germ-line mutations were revealed in 16/16 MEN 1 patients/families (100%). Based on our clinical experience with MEN 1 patients/families we formulated clinical criteria to identify disease gene carriers among patients with apparently sporadic MEN 1-related tumours. The criteria for MEN 1-suspected patients are: young age at onset (< 35 years) and/or multiple MEN 1-related lesions in a single organ or two distinct organs affected. Application of these criteria yielded MEN1 gene germ-line mutations in nine of 15 MEN 1-suspected patients (60%), thus identifying novel MEN 1 families. Follow up was also guaranteed for patients not fulfilling these criteria. CONCLUSIONS: The clinical criteria for MEN 1-suspected patients increase the detection rate of germ-line MEN1 gene mutations among patients with apparently sporadic MEN 1-related tumours. These criteria may be used for (presymptomatic) identification of MEN 1 disease gene-carriers, thus enabling early detection of tumour development and timely treatment, as well as genetic counselling.

Internally shortened menin protein as a consequence of alternative RNA splicing due to a germline deletion in the multiple endocrine neoplasia type 1 gene.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominantly inherited cancer syndrome (OMIM 131100), with tumours in several endocrine glands. In 1997 the responsible tumour suppressor gene was identified and recently it was shown that menin, its encoded protein, represses JunD-activated gene expression. Although many MEN 1 patients have been investigated both clinically and genetically, no genotype-phenotype correlation has been found yet. The vast majority of MEN1 gene mutations involve point mutations. We describe a patient in whom a 26 base pair deletion in the MEN1 gene, comprising part of exon 3 and part of intron 3, causes activation of a cryptic donor splice site at the beginning of exon 3. This germline mutation results in an in frame deletion of 105 nucleotides in MEN1 gene mRNA, i.e. an internal deletion of 35 amino acids in the menin protein. Since the deleted region of menin has been implicated in binding to JunD, this may explain the tumourigenic effect of this mutation. The knowledge of this MEN1 gene germline defect, may be used for presymptomatic identification of MEN 1 disease gene-carriers among family-members of this proband. This enables early detection of tumour development, timely treatment and genetic counseling.

Pseudohypoparathyroidism type Ia. Albright hereditary osteodystrophy: a model for research on G protein-coupled receptors and genomic imprinting.

Pseudohypoparathyroidism type Ia (PHP Ia) is a hereditary endocrine disorder, characterised by resistance to parathyroid hormone (PTH), causing disturbance of calcium homeostasis, and to several other polypeptide hormones. Patients with PHP Ia exhibit a complex of somatic abnormalities, termed Albright hereditary osteodystrophy (AHO). Treatment with vitamin D derivatives alleviates symptoms of hypocalcemia and may prevent bone demineralisation. PTH, like many polypeptide hormones, exerts its effects via a G protein-coupled cell surface receptor. PHP Ia is caused by a heterozygous, inactivating mutation in the gene for the alpha-subunit of the Gs protein, which disrupts Gs-protein-coupled signal transduction pathways. Several mutations have been described. When the mutation is inherited from the mother, the offspring will develop PHP Ia, i.e., both hormonal resistance and somatic abnormalities. When the mutation is derived from the father, children will have normal hormone responses while exhibiting the somatic features of AHO; this form of the disorder is called pseudopseudohypoparathyroidism (PPHP). A combination of tissue-specific genomic imprinting and haploinsufficiency may explain the occurrence of PPHP, and the fact that not all Gs-mediated polypeptide hormone actions are affected equally. PHP may therefore serve as a model in studying the pleiotropic consequences of impaired Gs-mediated signal transduction.

[Diverse expression of multiple endocrine neoplasia type 1]. / Pluriforme expressie van multipele endocriene neoplasie type 1.

MEN-1 is an autosomal dominantly inherited disorder, characterised by the occurrence of multiple tumours, particularly in the parathyroid glands, the pancreatic islets, the pituitary gland and the adrenal glands, as well as by neuroendocrine carcinoid tumours. Various clinical manifestations are presented by description of three patients harbouring a MEN1 gene germline mutation. A 44-year-old man had symptoms of hyperparathyroidism and in addition to parathyroid adenomas proved to have tumours in the thymus, adrenal and pituitary glands. A 48-year-old woman from a family with MEN-1 had suffered since her 40th year from headache and heartburn; she appeared to have adenomas in the parathyroid glands and gastrinomas in the pancreas, leading to a Zollinger-Ellison syndrome. One of her relatives, a man aged 29, had suffered from childhood from convulsions due to attacks of hypoglycaemia, and an insulinoma was assessed. In all patients, surgical and/or medical treatment alleviated symptoms. Clearly, the position or nature of the mutations in the MEN1 gene do not correlate with the clinical expression of the disease. Family investigation, DNA analysis and periodic examination improve quality of life and the life expectancy.

[Multiple endocrine neoplasia type 1: recent developments and guidelines for DNA diagnosis and periodic clinical monitoring]. / Multipele endocriene neoplasie type 1: recente ontwikkelingen en richtlijnen voor DNA-diagnostiek en periodiek klinisch onderzoek.

Multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominantly inherited disorder, characterised by the occurrence of multiple tumours, particularly in the parathyroid glands, the pancreatic islets, the pituitary gland, the adrenal glands, as well as neuroendocrine carcinoid tumours. Since the identification of the responsible gene in 1997, the diagnosis MEN-1 can be assessed easily, and even presymptomatically, by DNA analysis. An early diagnosis is of importance because through periodic clinical monitoring of (putative) MEN1 gene germline mutation carriers, tumour development can be detected and treated at an early stage. Eligible for DNA analysis are MEN-1 patients and their family members, as well as patients with seemingly sporadic MEN-1 related tumours in whom on clinical grounds carriership of a MEN1 gene germline mutation is suspected. Eligible for periodic clinical monitoring are putative and confirmed carriers of a MEN1 germline mutation from the age of 5.

A putative human zinc-finger gene (ZFPL1) on 11q13, highly conserved in the mouse and expressed in exocrine pancreas. The European Consortium on MEN 1.

In the process of identification of the multiple endocrine neoplasia type 1 gene, which was recently published, we isolated a novel gene in the 11q13 region. This gene (named ZFPL1, for zinc-finger protein-like 1) is expressed strongly in the exocrine pancreas as a 1.4-kb polyadenylated RNA encoding a putative protein of 310 amino acids. A mouse EST contig predicts an equally sized murine protein with 91% amino acid sequence identity to the human protein. No significant homology with known proteins could be found through database screening. However, zinc-finger-like domains and leucine-zipper-like motifs in the predicted ZFPL1 protein were identified, suggesting the presence of DNA-binding and dimerization domains possibly involved in transcription regulation. This notion is supported by the presence of a putative bipartite nuclear localization signal. This paper presents the full-length cDNA sequence for this gene, its genomic structure and chromosomal orientation, and expression studies by Northern blot hybridization and RNA in situ hybridization.

Molecular test for the detection of tumor cells in blood and sentinel nodes of melanoma patients.

Lymph node metastasis dramatically decreases the 5-year survival of melanoma patients. The so-called sentinel node surgery offers a therapeutic approach to resect the first draining lymph node. This technique enables accurate staging of melanoma patients in an early stage of the disease. Detection of a sentinel node metastasis is a strong argument for local lymphadenectomy. To improve the detection of micrometastasis in sentinel nodes of melanoma patients, molecular biological techniques were used. The exclusively melanocyte-specific tyrosinase transcript was amplified by reverse transcription followed by polymerase chain reaction (RT-PCR). In sentinel node examination, the detection of tyrosinase-positive cells by RT-PCR was compared with routine immunohistochemistry. From 16 patients, a total of 28 lymph nodes were tested. The lymph nodes were derived after lymphadenectomy (4 patients) and after sentinel node resection (12 patients with melanoma stage I). By using RT-PCR we could detect 100 tumor cells in a background of 10(8) peripheral blood mononuclear cells. The negative controls were all negative for tyrosinase. Cryostat sections of lymph nodes for mRNA isolation were alternated with sections for immunohistochemistry. By using tyrosinase RT-PCR, we detected 6 additional positive sentinel nodes in patients with melanoma stage I. Furthermore, the tyrosinase RT-PCR enabled us to design a blood test for circulating melanoma cells. Therefore, mRNA was directly isolated from whole blood of 23 blood samples, of which 3 samples were positive for tyrosinase. The present study demonstrates the possibility of a simple and rapid blood test for melanoma patients that has not been available until now. Furthermore, the detection of micrometastasis in sentinel nodes by tyrosinase RT-PCR dramatically increases the accuracy of melanoma staging.

Hereditary tyrosinemia type 1: novel missense, nonsense and splice consensus mutations in the human fumarylacetoacetate hydrolase gene; variability of the genotype-phenotype relationship.

The complete fumarylacetoacetate hydrolase (FAH) genotype of probands of thirteen unrelated families with hereditary tyrosinemia type 1 (HT 1) was established. The screening was performed by analysis of exons 2-14 of the FAH gene by using the polymerase chain reaction (PCR) and of the mRNA by reverse transcription/PCR. Nine different mutations were identified, of which six are novel. Three mutations involve consensus sequences for correct splicing, viz. IVS 6-1 (g-t), IVS 7-1 (g-a) and IVS 12 + 5 (g-a). Two missense mutations (C193R and G369V) and three nonsense mutations (R237X, E357X and E364X) were found. One silent mutation N232N was associated with the skipping of exon 8 from the FAH mRNA. Analysis of the effect of the respective mutations on the FAH mRNA showed a strong reduction of FAH mRNA levels in association with the nonsense mutations, and normal levels with the missense mutations. The splice consensus mutations give deletions of complete or small parts of exon sequences from the FAH mRNA. Data suggest a founder effect for several of the mutations, with a frequency for both the IVS 6-1 (g-t) and IVS 12 + 5 (g-a) mutations of approximately 30% in the HT 1 probands. No strict correlation between genotype and phenotype, i.e. the acute, subacute or chronic form of HT 1, was evident.