A transcript map for the 2.8-Mb region containing the multiple endocrine neoplasia type 1 locus

Abstract

Multiple endocrine neoplasia type 1 (MEN 1) is an inherited cancer syndrome in which affected individuals develop multiple parathyroid, enteropancreatic, and pituitary tumors. The locus for MEN1 is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis places the MEN1 gene within a 2-Mb interval flanked by the markers D11S1883 and D11S449. Loss of heterozygosity studies in MEN 1 and sporadic tumors suggest that the MEN1 gene encodes a tumor suppressor and have helped to narrow the location of the gene to a 600-kb interval between PYGM and D11S449. Focusing on this smaller MEN1 interval, we have identified and mapped 12 transcripts to this 600-kb region. A precise ordered map of 33 transcripts, including 12 genes known to map to this region, was generated for the 2.8-Mb D11S480-D11S913 interval. Fifteen candidate genes (of which 10 were examined exhaustively) were evaluated by Southern blot and/or dideoxy fingerprinting analysis to identify the gene harboring disease-causing mutations.

Figure 1

Transcript map of 2.8-Mb region containing the MEN1 locus in the interval D11S480–D11S913. (A) The MEN1 interval identified by linkage analysis spanning from D11S1883 to D11S449 is shown, with the region from PYGM to D11S4936 marked by arrows as the minimal interval inferred from LOH data. The arrow with a dotted line represents the boundary determined from a sporadic gastrinoma. (B) The 1-Mb region from PYGM to D11S4933 is expanded for clarity. Some relevant markers are indicated above the map and the transcripts are identified below. Sizes of BAC clones (b137C7, b79G17, and b18H3) are given. Sequence contigs from cosmids C1, C2, and C3 are 14 kb, 46 kb, and 6.8 kb, respectively. (*) Genes that have been eliminated as MEN1 candidates by mutation detection analysis. Mutation analysis is incomplete for the Kappa and Eta (**). Identification and mapping of OVO will be reported independently (A. Chidambaram, R. Allikmets, S.C. Chandrasekharappa, S.C. Guru, W. Modi, B. Gerrard, and M. Dean, in prep.).

Figure 2

Comparison of GRAIL-predicted exons vs the cDNA sequence. GRAIL-predicted exons of b137C7 sequence contigs 194, 168, and 200 are shown in open boxes and compared with the corresponding exons identified by amplifying and sequencing cDNA, represented by solid boxes. (*) Two exons missed by GRAIL in contigs 168 and 200. A vertical arrow shows a 33-nucleotide intron predicted by GRAIL that is part of the exon in cDNA. Arrows indicate the primers used for amplifying the corresponding cDNA. All three contigs identified the same 8-kb transcript, denoted Kappa, on Northern blots.

Figure 3

Representative Northern blots of new transcripts. New transcripts were analyzed for message size and expression levels by probing one or more of the three commercially available multiple tissue Northern blots. (A, left) Endocrine blot probed with a 1.8-kb Mu cDNA from the Mu transcript; (middle) blot probed with a 220-bp Mu cDNA. In both cases a 2.8-kb message is identified that is expressed in all tissues. (A, right) Blot containing total RNA from EBV-transformed lymphocytes from seven FMEN1 cases, probed with the 8.8-kb Epsilon/Beta probe. Two messages are seen: a more abundant 4.4-kb Epsilon transcript and a less abundant 19-kb message named Beta. (B) Mu cDNA and location of probes used for blots in A left and middle. (C) Epsilon/Beta cDNA and probes used in A right. Probes used in A are in B and C. The 2.8-kb probe in C independently confirmed the 19-kb Beta transcript, and did not detect Epsilon (data not shown).