17alpha-hydroxylase/17,20-lyase deficiency as a model to study enzymatic activity regulation: role of phosphorylation.

Cytochrome P450 17alpha-hydroxylase (CYP17) is a single gene-encoded protein with two activities: 17alpha-hydroxylase and 17,20-lyase. The two catalytic activities are differentially regulated in health and disease. We took advantage of naturally occurring human mutations to understand the molecular bases of this differential regulation. We identified eight novel mutations in the CYP17 gene, different in nature and spread throughout the gene. As posttranslational modifications appear to be important for activity control, we investigated the phosphorylation state of wild-type and mutant CYP17 proteins. Although phospholabeled protein was seen when the wild-type and most mutant proteins were expressed, no phosphorylation was detected for the F417C mutant. F417C is the only 17,20-lyase deficiency case confirmed at the molecular level and represents the first phosphorylation CYP17-deficient mutant. In search of the physiological agents involved in this process, the effect of cAMP was tested on activity and phosphorylation state of our mutant CYP17 proteins. cAMP stimulates activity and phosphorylation in all cases, except in the F417C and R35L mutants. The lack of response to the physiological second messenger might explain the different phenotypes. The F417C mutant protein, which is already shown to be associated with the lack of electron transfer, provides for the first time a link between the electron transfer system and the phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase activity.

A childhood fibrolamellar hepatocellular carcinoma with increased aromatase activity and a near triploid karyotype.

We report a 15-year-old boy with hepatocellular carcinoma (HCC) of the fibrolamellar type. He presented with advanced disease and a non-resectable tumor. Clinical features included marked gynecomastia which had been present for 3 years, failure to enter puberty, and failure to thrive. These features might have been due to a high aromatase activity of the tumor. The course of the illness suggested that the tumor had been present for at least 3 years prior to diagnosis. At diagnosis the patient had multiple metastases which included infiltrated ascites. Cytogenetic analysis of the ascites revealed a near triploid karyotype with cell-to-cell variation and an abnormality of chromosome 1 q. This to our knowledge is the first karyotype report of fibrolamellar HCC in a child.

[Detection of RET-proto-oncogene mutations in the diagnosis of Type 2 endocrine neoplasia (MEN 2)]. / Nachweis von RET-Proto-Onkogen-Mutationen zur Diagnostik der multiplen endokrinen Neoplasie Typ 2 (MEN 2).

We have analyzed 95 blood- and 25 paraffin-derived DNA samples of 120 individuals from Switzerland (MEN 2 family members and patients with medullary thyroid carcinoma or pheochromocytoma) for the presence of RET protooncogene mutations in exons 10, 11, 13, 14 and 16, where recently germline point mutations have been identified in more than 95% of patients with MEN 2A, familial medullary thyroid carcinoma (FMTC) and MEN 2B. Molecular DNA screening of samples was performed by non-radioactive single strand conformation polymorphism (SSCP) and heteroduplex gel electrophoresis method followed by mutation analysis of PCR products by direct cycle sequencing using an automated DNA sequencer. We identified 12 MEN 2A/FMSC and 6 MEN 2B families with 29 gene carriers. Ten different types of mutations were identified in the MEN 2A/FMTC families (620 Cys-->Arg, 618 Cys-->Ser, Gly, 611 Cys-->Tyr; 634 Cys-->Arg, Tyr, Trp, Phe, Ser, Gly) and all 6 MEN 2B families had a 918 Met-->Thr point mutation. Our results indicate that PCR-based DNA testing for RET point mutations is a rapid, accurate and reproducible method of identifying MEN 2 gene carriers using blood or tissue DNA. Early detection of gene carriers allows preventive thyroidectomy without neck dissection or parathyroid transplantation, and non-gene carriers can be released from biochemical testing. Furthermore, it is shown that the distribution and localization of RET mutations in MEN 2 families from Switzerland concur with combined results of larger series and that a "founder effect" of MEN 2 can be excluded for this country.

[Immunosuppression in Type I diabetes mellitus]. / Immunosuppression beim Typ-I-Diabetes mellitus.

The etiopathogenesis of type I or insulin-dependent diabetes mellitus is not known in details. It is now generally accepted, that type I diabetes is the result of an autoimmune disease. Type I diabetes begins after the destruction of 80-90% of insulin-producing beta cells in the pancreas. This knowledge let to immune intervention trials with various immunosuppressive drugs, especially cyclosporine A. These studies are still in an experimental stage. Because of inefficiency and severe side effects of these drugs, the med.-scientific section of the Swiss Diabetes Association recommends not to use immunosuppressive drugs in newly diagnosed patients with type I diabetes.

[Current aspects in therapy of insulin-dependent diabetes mellitus in children and adolescents]. / Moderne Aspekte der Therapie der insulinabhängigen Diabetes mellitus bei Kindern und Jugendlichen.

It has been shown in recent years that optimal treatment delays or prevents diabetic late complications. The goal for therapy is therefore near norm-glycemia. The precondition is intensive education of the patient and the parents, as well as exact diet with known amounts of carbohydrates, multiple insulin injections and home blood glucose monitoring. We propose two different schemes for insulin therapy according to age: conventional insulin therapy in the younger child, and intensified insulin therapy with the insulin pen in adolescents. Conventional insulin therapy consists of two daily injections--before breakfast and before supper--of a mixture of short acting and long acting insulin. With the conventional insulin regime a rigid schedule of insulin injections and meals is required for optimal treatment of type I diabetes. This, however, is not accepted by adolescents since it conflicts with their age-related tendency for self-autonomy. In consequence, glucose metabolism deteriorates and HbA1 levels increase. Puberty is a period of high risk for the development of diabetic angiopathy, which determines quality of life and life expectancy. An approach to resolution of this dilemma is to adjust the insulin regime to the life-style of an adolescent rather than vice versa. Up to now, more than 30 adolescents with type I diabetes have been switched to intensified insulin therapy using the insulin pen. During a short hospital stay (5 days) they underwent an intensive teaching program of self control, insulin dose adjustment and nutrition with special attention to the management of unexpected situations such as additional or omitted meals, traveling and sports. Compliance improved markedly.(ABSTRACT TRUNCATED AT 250 WORDS)

[The pathogenesis of type 1 diabetes mellitus]. / Pathogenese des Typ-1-Diabetes-mellitus.

It is now widely accepted, that the development of type 1 diabetes is based on an autoimmune destruction of the insulin-producing betacells of the pancreas; however, a genetic predisposition is required. 90 to 95% of patients with type 1 diabetes are HLA-DR3 positive and/or HLA-DR4 positive. In addition, recent results from molecular-genetic research suggest an aspartate on position 57 of the HLA-DQ-beta protein, which seems to protect against the autoimmune destruction of the betacell, since it is not found in patients with type 1 diabetes in contrast to control persons. Trigger substances inducing the initial betacell destruction are not known, but might not be necessary according to an actual hypothesis. Finally, all insulin-producing cells are destroyed, and lifelong insulin replacement therapy cannot be circumvented. Prevention of the autoimmune destruction is not possible today; especially, not using immunosuppressive drugs because of side effects.

[Increased expression of IGF-2 in tumor tissue of nephroblastoma]. / Nachweis erhöhter Expression von IGF 2 im Tumorgewebe von Nephroblastomen.

The incidence of nephroblastoma is increased in a number of syndromes with abnormal growth pattern. Elevated IGF 2-expression has been documented in various Wilms' tumors and a defect in the IGF 2 gene has been noted in 1 case. Southern blot analysis of genomic DNA of nephroblastomas in 5 additional patients after restriction enzyme digest with EcoRI, Pvu II, Pst I and Taq I did not reveal any defect in the IGF 2 gene. Slot blot analysis however showed marked overexpression of IGF 2-mRNA reaching up to more than 25 times the level expressed in unaffected kidney tissue adjacent to the tumor and in normal kidney tissue used as controls.

[Intensive insulin therapy in adolescents with type 1 diabetes mellitus: initial experiences with a semiautomatic insulin injection device (the insulin pen)]. / Intensivierte Insulin-Therapie bei Adoleszenten mit Diabetes mellitus Typ I: Erste Erfahrungen mit dem halbautomatischen Insulin-Injektionsgerät (Insulin-Pen).

Recently new pen-like technical devices (Insulin-Pen) for the administration of insulin have become available. With the new device, short acting insulin is injected before meals and, in addition, a long acting insulin before bedtime (basis-bolus insulin regime). A group of 19 adolescents with type I diabetes mellitus (7 girls, 12 boys, age 12-18 years) have been trained during a short hospital stay (4-5 days with intensive teaching) in the new insulin regime. Experience, now amounting to 131 patients-months, has been favourable without exception. Acceptance is good, and none of the adolescents would prefer to go back to the conventional insulin regime with two injections. Regained flexibility with the amount and timing of meals, and the possibility of reducing the number of meals, have been mentioned as the principal advantages of the new system. The intensified self-monitoring of blood glucose usually was the most cited disadvantage. However, several adolescents felt more assured in daily life with a knowledge of blood glucose levels. Compliance in the management of diabetes during puberty increased markedly; as a result, longterm metabolic control improved significantly. The glycosylated hemoglobin A1 was 9.9 +/- 0.8% before and 7.7 +/- 0.5% (normal less than 8%) with the new insulin regime (p less than 0.001). It is concluded that the use of new pen-like insulin injectors helps to improve metabolic control as well as life quality in insulin-dependent adolescent diabetics. However, of equal importance is instruction by the diabetologist with respect to new possibilities and limitations, particularly the new rules of diet.

[A patient with 46 XX/46 XY chimerism without hermaphroditism. The problem of prepuberal diagnosis of the Klinefelter's syndrome]. / Ein Patient mit 46 XX/46 XY-Chimärismus ohne Hermaphroditismus. Zur Problematik der vorpubertären Diagnosestellung des Klinefelter-Syndroms.

In a 12-year-old obese boy whose parents had asked for determination of sex chromatin the result was positive and Klinefelter's syndrome was diagnosed. Subsequently, the boy developed normally, went through puberty and presented with normal primary and secondary sex characteristics at the age of 22. The diagnosis was revised and on the basis of a karyotype the diagnosis of 46 XX/46 XY chimerism was made. In contrast to our patient, most dispermic chimeras are hermaphrodites. This case exemplifies the fact that Klinefelter's syndrome should not be diagnosed in prepubertal boys on the basis of positive sex chromatin.