Isolation and characterization of genes within the region for multiple endocrine neoplasia type 1 on chromosome 11q13

Abstract

This thesis summarizes the investigations of three genes isolated from the locus for multiple endocrine neoplasia type 1 (MEN1) within the chromosomal region 11q13. As a common strategy for all three genes, the cDNA was sequenced, the genomic structure characterized, and the expression pattern analyzed by Northern blotting on human tissues and by in situ RNA hybridization on slides from rodent embryos. The phospholipase C beta3 gene (PLCB3) encodes for a key enzyme in signal transduction. Due to its loss of expression in some endocrine tumors we had considered PLCB3 as a promising MEN1 candidate.

In paper I the genomic organization and complete cDNA sequence is presented. The previously published sequence of PLCB3 was extended with 876 bp in the 5' direction giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids, the same size as detected by Northern blot analysis. The complete transcription unit of PLCB3 is approximately 15 kb, contains 31 exons, and is preceded by a highly GC rich 5'flanking region without TATA and CAAT boxes. This analysis was a prerequisite for the investigation in paper III in which PLCB3 is excluded as a MEN1 candidate, by mutation analysis both on cDNA and on genomic level.

Paper II describes the expression in rat. High transcript levels were mainly seen in the dorsal root ganglia (DRGs) and the trigeminal ganglion of the nervous system. Slightly lower levels were seen in the skin, the mucous lining of the airways, the gastrointestinal canal, the lung and the thymus. The pituitary, pancreas and thyroid gland did not give any positive signals. The analysis supported the idea that PLCB3 does not play a role in endocrine tissues. The PLCB3 neighboring gene (PNG) which was identified only a few kb upstream of PLCB3 had not been described before.

In paper IV the cDNA sequence, Northern blot analysis, and genomic structure of PNG including a complete sequence of the region between the 5' ends of PLCB3 and PNG is presented. PNG is expressed in most tissues as a 1kb transcript, comparable to the size of the cDNA clone. On somatic cell hybrid panels cross-hybridization to chromosomes 2, 4, 7 and 22 was detected, possibly indicating a gene family. The highly GC-rich sequence did not show any significant similarities with other known genes. The predicted protein of 199 amino acids has a high content of glycine, alanine, and proline which may result in a collagenous structure. PNG spans over 2.5 kb and contains 4 exons. The intergenic region between PNG and PLCB3 revealed two separate CpG islands without TATA or CAAT boxes, one in front of each gene.

In paper V the cDNA sequence, and expression during development, of the mouse homologue (png) is described. In the developing mouse embryo expression was seen in most organs, with higher levels over parts of the brain, spinal cord, heart, liver, intestines and tongue. In the adult mouse, thymus and testis showed the strongest expression while most tissues including those related to MEN1 exhibited low or no specific expression. The third cDNA corresponds to a novel gene highly similar to the gene for vascular endothelial growth factor (VEGF), a potent mitogen for endothelial cells and an inducer of blood vessel permeability.

Paper VI describes the sequence, the genomic structure, the expression by Northern blot analysis, and the splice pattern of the novel gene which we named the VEGF Related Factor gene (VRF). Two alternate splicing forms were detected. The shorter of the two was created by splicing of the 6th of 8 exons. This version preserves similarity with VEGF for the whole predicted protein while maintenance of exon 6 results in a frame shift for the terminal third part of the gene. Northern blot analysis detected bands of 5.5 kb and 2.0 kb. Paper VII describes the cDNA sequence, genomic structure and expression of the mouse VRF gene (vrf). vrf is highly homologous to its human counterpart, in sequence and genomic organization, and also generates the two alternative splicing forms. Northern blots showed only one band of 1.3 kb in size. Paper VIII describes the expression of vrf in the prenatal mouse. Expression was primarily seen over heart and in the nervous system. Just prior to and after birth strong expression was seen in heart, brown fat and in some locations in the central nervous system.