C. J. M. Lips

Extensive islet amyloid formation is induced by development of Type II diabetes mellitus and contributes to its progression: pathogenesis of diabetes in a mouse model

Aims/hypothesis. Type II (non-insulin-dependent) diabetes mellitus is a multifactorial disease in which pancreatic islet amyloid is a characteristic histopathological finding. Islet amyloid fibrils consist of the beta-cell protein “islet amyloid polypeptide” (IAPP)/“amylin”. Unlike human IAPP (hIAPP), mouse IAPP cannot form amyloid. In previously generated transgenic mice, high expression of hIAPP as such did not induce islet amyloid formation. To further explore the potential diabetogenic role of amyloidogenic IAPP, we introduced a diabetogenic trait (“ob” mutation) in hIAPP transgenic mice. Methods. Plasma concentrations of IAPP, insulin and glucose were determined at 3.5 (t1), 6 (t2), and 16–19 months of age (t3). At t3, the mice were killed and the pancreas was analysed (immuno)histochemically. Results. In non-transgenic ob/ob mice, insulin resistance caused a compensatory increase in insulin production, normalizing the initial hyperglycaemia. In transgenic ob/ob mice, concurrent increase in hIAPP production resulted in extensive islet amyloid formation (more often and more extensive than in transgenic non-ob/ob mice), insulin insufficiency and persistent hyperglycaemia: At t3, plasma insulin levels in transgenic ob/ob mice with amyloid were fourfold lower than in non-transgenic ob/ob mice (p < 0.05), and plasma glucose concentrations in transgenic ob/ob mice were almost twofold higher (p < 0.05). In addition, the degree of islet amyloid formation in ob/ob mice was positively correlated to the glucose:insulin ratio (rs = 0.53, p < 0.05). Conclusion/interpretation. Islet amyloid is a secondary diabetogenic factor which can be both a consequence of insulin resistance and a cause of insulin insufficiency. [Diabetologia (1999) 42: 427–434]

Structure and expression of the human calcitonin/CGRP genes

Recently, we have reported the isolation of cDNA encoding a second human calcitonin gene‐related peptide (hCGRP‐II) [(1985) FEBS Lett. 183, 403‐407]. In this report we describe the isolation and characterization of the gene encoding hCGRP‐II. This gene, designated CALC‐II, is structurally closely related to the known CALC‐I gene encoding human calcitonin (hCT) and hCGRP‐I. In constrast to CALC‐I, CALC‐II does not seem to be alternatively expressed. The formation of a second, hCT‐like mRNA by differential splicing of CALC‐II transcripts is unlikely in view of the structure of CALC‐II, and could not be demonstrated in tissues known to express CALC‐I and CALC‐II.

Islet amyloid polypeptide: Identification and chromosomal localization of the human gene

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy‐terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy‐terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.

Expression of insulin-like growth factor-I and -II genes in human smooth muscle tumours.

The insulin‐like growth factors I and II (IGF‐I and ‐II) are polypeptides which play an important role in growth and development of the organism. In the present report we describe the detection of human IGF‐I RNAs (both type Ia and type Ib) and IGF‐II RNAs in benign (leiomyoma) and malignant (leiomyosarcoma) tumours from smooth muscle origin, using Northern blot hybridization analysis. In normal smooth muscle tissue of the uterus we found low levels of IGF‐I RNAs only. In the tumours the same IGF‐I RNA species were detected as in adult non‐tumour tissues (uterus, liver). For transcription of the IGF‐II gene in these tumours, two promoters are used which are expressed in fetal liver, but not in adult liver. The presence of IGF‐I and IGF‐II RNAs was also established by nucleotide sequence analysis of recombinant DNA clones isolated from cDNA libraries derived from two leiomyosarcomas. The nucleotide sequences of these cDNA clones, together covering the entire coding regions of IGF‐Ia and IGF‐II var RNA, predict that IGFs encoded by the tumour RNAs do not differ in amino acid sequence from the corresponding polypeptides isolated from serum. In those tissues containing IGF‐I RNAs, IGF‐I immunoreactivity was also demonstrated.

The complete islet amyloid polypeptide precursor is encoded by two exons

Islet amyloid polypeptide (IAPP) is the 37‐amino acid peptide subunit of amyloid found in pancreatic islets of type 2 diabetic patients and in insulinomas. Recently, we isolated the human gene encoding IAPP [(1988) FEBS Lett. 239, 227–232]. We now report the nucleotide sequences of a human insulinoma cDNA encoding a complete IAPP precursor, and of the corresponding parts of the IAPP gene. Two exons, which are approx. 5 kb apart in the human genome, encode the 89‐amino acid pre‐pro‐IAPP. At least one additional exon is present further upstream in the IAPP gene. A putative signal sequence at the amino‐terminus of the precursor suggests that IAPP is a secreted protein.

The second human calcitonin/CGRP gene is located on chromosome 11

SummaryA second human calcitonin/calcitonin gene related peptide (hCT/CGRP) gene has been identified. This second hCT/CGRP gene has been shown to contain sequences highly homologous to exons 3, 5 (CGRP-encoding), and 6 of the first hCT/CGRP gene, but sequences closely related to exon 4 (CT-encoding) could not be demonstrated. Southern blot hybridization analysis of DNA from human-rodent somatic cell hybrids showed that the second hCT/CGRP gene is located in the q12-pter region of chromosome 11. The first hCT/CGRP gene has previously been assigned to the p13–p15 region of chromosome 11.

Genotype-phenotype correlations in families with deletions in the von Hippel-Lindau (VHL) gene.

Abstract. Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome characterized by predisposition for bilateral and multi-centric hemangioblastoma in the retina and central nervous system, pheochromocytoma, renal cell carcinoma, and cysts in the kidney, pancreas, and epididymis. We describe five families for which direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis revealed deletions involving the VHL gene in each of these families. In four families, partial deletions of one or more exons were detected by Southern blot analysis. In the fifth family, FISH analysis demonstrated the deletion of the entire VHL gene. Our results show that (quantitative) Southern blot analysis is a sensitive method for detecting germline deletions of the VHL gene and should be implemented in routine DNA diagnosis for VHL disease. Our data support the previously established observation that families with a germline deletion have a low risk for pheochromocytoma. Further unraveling of genotype-phenotype correlations in VHL disease has revealed that families with a full or partial deletion of the VHL gene exhibit a phenotype with a preponderance of central nervous system hemangioblastoma.

Localization of the polymorphic human calcitonin gene on chromosome 11

SummaryA molecular probe containing a 584 base pairs sequence corresponding to part of the human calcitonin mRNA was used for the chromosomal assignment of the calcitonin gene. Restriction endonuclease analysis of DNA from human-Chinese hamster and human-mouse somatic cell hybrids, including some containing a translocation of human chromosomes, placed the calcitonin gene in the p14→qter region of chromosome 11.Analysis of human DNA showed that the calcitonin gene has a polymorphic site for restriction endonuclease TaqI.

Cryptic von Hippel-Lindau disease: germline mutations in patients with haemangioblastoma only

OBJECTIVES Central nervous system haemangioblastoma (HAB) is a major feature of von Hippel-Lindau (VHL) disease, and it is estimated that about 30% of HAB patients have VHL disease. Consequently, it is widely recommended that sporadic HAB patients are screened for clinical and radiological features of VHL disease because of the risk of multiple tumours. We investigated the frequency of VHL germline mutations in patients with HAB only with no clinical or radiological evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients. METHODS Eighty four patients with a single HAB (23 Dutch, 61 UK) and four with multiple HAB (two Dutch, two UK) were studied by direct sequencing of the coding region and quantitative Southern blotting. RESULTS AVHL germline mutation was found in three of 69 (4.3%) single HAB patients aged 50 years or less (three of 84 (3.6%) total single HAB patients). A germlineVHL mutation was detected in a 44 year old woman with a solitary cerebellar HAB, as well as in four clinically unaffected close relatives, and in two single HAB cases presenting at the ages of 29 and 36 years. Germline VHL mutations were detected in two of four cases with multiple HAB. CONCLUSIONS Early detection of VHL disease is important to reduce morbidity and mortality and therefore we recommend that, in addition to conventional clinical and radiological investigations, VHL gene mutation analysis should be offered to all HAB patients younger than 50 years. HAB patients aged >50 years will have a lower a priori risk of VHL disease and further data are required to evaluate the role of routine molecular genetic investigations in late onset HAB cases. The failure to detect germline VHL mutations in some patients with multiple HAB may indicate the presence of somatic mosaicism or additional HAB susceptibility genes.

The human gene encoding insulin-like growth factor I is located on chromosome 12

SummaryA cDNA probe corresponding to mRNA encoding human somatomedin-C/insulin-like growth factor I (IGF-I) was used for the chromosomal assignment of the IGF-I gene. Southern-blot hybridization analysis of DNA from human-Chinese hamster somatic cell hybrids showed that the IGF-I gene is located on chromosome 12. Comparison of the chromosomal assignments of the IGF-I gene and two other members of the insulin gene family, with three c-ras oncogenes, reveals a remarkable association of the two gene families.