A germ-line insertion in the Birt-Hogg-Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer

Abstract

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan and named the "Nihon" rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (Tsc2 gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 gene (human 16p13.3)-Il3 gene-Nihon gene-Llgl1 locus- Myhse gene. We now describe a germ-line mutation in the Birt-Hogg-Dubé gene (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudson's "two-hit" model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome.

Fig. 1.

Genetic mapping and genomic structure of the rat Bhd homologue. (A) Linkage analysis of rat (Rattus norvegicus) chromosome 10 (RNO10). Gene names and genetic distances from the Nihon gene are indicated. GLR1, Q9DCE9, COPS3, and LLGL1 indicate polymorphic markers (8). MMU11, mouse (Mus musculus) chromosome 11; HSA, human (Homo sapiens) chromosome loci. (B) The genomic structure of rat Bhd homologue. Exons are denoted by shaded boxes with numbers. Below the overall exon–intron structure, a CpG island found around exon 1 is shown in an enlarged view. Each vertical line marks a CpG sequence.

Fig. 2.

LOH and expression of the Bhd gene in Nihon rat RCs. (A) Representative Southern blot analysis of RCs with a Bhd probe. DNAs from normal tissues (N) and primary RCs (T) from two Nihon rats and Nihon rat-derived RC cell lines were analyzed by HincII digestion. Positions of Nihon rat-associated band (Mut) and wild-type band (Wild) are shown on the right. (B) Northern blot analysis of Nihon rat-derived RC cell lines (NR). Total RNAs from two NR with LOH at the Bhd locus (lanes 1 and 2) and normal kidney (lane N) were analyzed by Bhd probe. (C) RT-PCR analysis of Nihon rat-derived RC cell lines. Total RNAs from three cell lines with LOH of Bhd (lanes 1–3) and rat and mouse embryos (lanes R and M) were subjected to amplification of Bhd cDNA. Lane Mr, molecular weight markers; lane –, negative control without first-strand cDNA template.

Fig. 3.

A germ-line mutation of Bhd homologue found in the Nihon rat. (A) A cytosine (C) insertion in the Bhd cDNA found in RC cells in exon 3. The Bhd cDNA from the Nihon rat-associated allele in RC cells is schematically represented. The coding region of Bhd is shown by an open box. Regions encoding evolutionarily conserved domain and coiled-coil motif are denoted by shaded and filled boxes, respectively. The position of the cytosine insertion is marked by an arrow (insC). A representative chromatograph (reverse sequence) of direct sequence analysis of the amplified Bhd cDNA product is shown. A C/G₆ tract replaces wild-type C/G₅ tract in the sequence (bar). Below the chromatograph, reading frames of wild-type and mutant cDNAs are compared. As a result of the C insertion, a frameshift occurs after codon 17. Italics denote incorrect amino acid sequence generated by insertion. (B) Genomic sequence analysis of Bhd gene in the Nihon rat. Genomic DNAs of wild-type rat, RC cell with LOH of Bhd and Nihon rat heterozygote are analyzed for exon 3 sequence. Representative chromatographs (reverse sequence) are shown. In the Nihon rat, peaks are overlapped after the insertion site.

Fig. 4.

An intragenic Bhd mutation found in an LOH-negative primary RC (T2 in Fig. 2 A) in the Nihon rat. (Left) Chromatographs of direct sequence analysis for exon 6. Reverse sequences of exon 6 in a LOH-negative primary RC and control tissue are shown. Near the splicing acceptor site, C/A overlapped peak was detected in the RC sample (arrow), indicating that a mutation was introduced at this site. (Right) A nonsense mutation found in exon 6. First G of codon 209 (GAG for Glu) in the wild-type sequence is replaced by T in the RC. This G → T transversion creates a premature stop codon (asterisk).

Fig. 5.

Loss of expression of the folliculin proteins in Nihon rat RC cell lines. (A) Northern blot analysis of Bhd expression in RC cells. Total RNAs from the Eker rat RC cell line (LK) and two Nihon rat RC cell lines (NR 32 and 45) were analyzed by a Bhd probe. 28S RNA bands stained with ethidium bromide are shown below. (B) Western blot analysis of folliculin expression in RC cell lines. Total extracts from cells shown in A were analyzed by Western blotting with anti-folliculin antibody. Bands of ≈66-kDa folliculin are absent in Nihon rat RC cells (NR 32 and 45). Results of Western blot analysis for β-actin are shown below as a control.