Berton Zbar

Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors, and spontaneous pneumothorax. Recombination mapping in BHD families delineated the susceptibility locus to 700 kb on chromosome 17p11.2. Protein-truncating mutations were identified in a novel candidate gene in a panel of BHD families, with a 44% frequency of insertion/deletion mutations within a hypermutable C(8) tract. Tissue expression of the 3.8 kb transcript was widespread, including kidney, lung, and skin. The full-length BHD sequence predicted a novel protein, folliculin, that was highly conserved across species. Discovery of disease-causing mutations in BHD, a novel kidney cancer gene associated with renal oncocytoma or chromophobe renal cancer, will contribute to understanding the role of folliculin in pathways common to skin, lung, and kidney development.

Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan

Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1–2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype‐phenotype correlations in VHL.

Improved detection of germline mutations in the von Hippel-Lindau disease tumor suppressor gene

von Hippel‐Lindau disease (VHL) is an inherited neoplastic disorder characterized by the development of tumors in the eyes, brain, spinal cord, inner ear, adrenal gland, pancreas, kidney, and epididymis. The VHL tumor suppressor gene was identified in 1993. Initial studies reported the detection of germline mutations in the VHL gene in 39–75% of VHL families. We used tests that detect different types of mutations to improve the frequency of detection of germline mutations in VHL families. The methods included quantitative Southern blotting to detect deletions of the entire VHL gene, Southern blotting to detect gene rearrangements, fluorescence in situ hybridization (FISH) to confirm deletions, and complete sequencing of the gene. Here we report that we have detected germline mutations in the VHL gene in 100% (93/93) of VHL families tested. In addition, we describe 13 novel intragenic VHL germline mutations. With the methodology described in this article, it is now possible to identify germline mutations in virtually all families with VHL. Hum Mutat 12:417–423, 1998.

Mutations in the Fumarate Hydratase Gene Cause Hereditary Leiomyomatosis and Renal Cell Cancer in Families in North America

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and uterus and/or renal cancer. Although the identification of germline mutations in the fumarate hydratase (FH) gene in European families supports it as the susceptibility gene for HLRCC, its role in families in North America has not been studied. We screened for germline mutations in FH in 35 families with cutaneous leiomyomas. Sequence analysis revealed mutations in FH in 31 families (89%). Twenty different mutations in FH were identified, of which 18 were novel. Of these 20 mutations, 2 were insertions, 5 were small deletions that caused frameshifts leading to premature truncation of the protein, and 13 were missense mutations. Eleven unrelated families shared a common mutation: R190H. Eighty-one individuals (47 women and 34 men) had cutaneous leiomyomas. Ninety-eight percent (46/47) of women with cutaneous leiomyomas also had uterine leiomyomas. Eighty-nine percent (41/46) of women with cutaneous and uterine leiomyomas had a total hysterectomy, 44% at age < or =30 years. We identified 13 individuals in 5 families with unilateral and solitary renal tumors. Seven individuals from four families had papillary type II renal cell carcinoma, and another individual from one of these families had collecting duct carcinoma of the kidney. The present study shows that mutations in FH are associated with HLRCC in North America. HLRCC is associated with clinically significant uterine fibroids and aggressive renal tumors. The present study also expands the histologic spectrum of renal tumors and FH mutations associated with HLRCC.

Renal tumors in the Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant genodermatosis characterized by the development of small dome-shaped papules on the face, neck, and upper trunk (fibrofolliculomas). In addition to these benign hair follicle tumors, BHD confers an increased risk of renal neoplasia and spontaneous pneumothorax. To date, there has been no systematic pathologic analysis of the renal tumors associated with this syndrome. We reviewed 130 solid renal tumors resected from 30 patients with BHD in 19 different families. Preoperative computed tomography scans demonstrated a mean of 5.3 tumors per patient (range 1–28 tumors), the largest tumors averaging 5.7 cm in diameter (± 3.4 cm, range 1.2–15 cm). Multiple and bilateral tumors were noted at an early age (mean 50.7 years). The resected tumors consisted predominantly of chromophobe renal cell carcinomas (44 of 130, 34%) or of hybrid oncocytic neoplasms that had areas reminiscent of chromophobe renal cell carcinoma and oncocytoma (65 of 130, 50%). Twelve clear cell (conventional) renal carcinomas (12 of 130, 9%) were diagnosed in nine patients. These tumors were on average larger (4.7 ± 4.2 cm) than the chromophobe (3.0 ± 2.5 cm) and hybrid tumors (2.2 ± 2.4 cm). Microscopic oncocytosis was found in the renal parenchyma of most patients, including the parenchyma of five patients with evidence of clear cell renal cell carcinoma. Our findings suggest that microscopic oncocytic lesions may be precursors of hybrid oncocytic tumors, chromophobe renal cell carcinomas, and perhaps clear cell renal cell carcinomas in patients with BHD syndrome. Recognition by the pathologist of the unusual renal tumors associated with BHD may assist in the clinical diagnosis of the syndrome.

Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling

Birt–Hogg–Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1/2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5′ AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt–Hogg–Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and/or nutrient sensing through the AMPK and mTOR signaling pathways.

Improved Identification of von Hippel-Lindau Gene Alterations in Clear Cell Renal Tumors

Purpose: To provide a comprehensive, thorough analysis of somatic mutation and promoter hypermethylation of the von Hippel-Lindau (VHL) gene in the cancer genome, unique to clear cell renal cancer (ccRCC). Identify relationships between the prevalence of VHL gene alterations and alteration subtypes with patient and tumor characteristics. Experimental Design: As part of a large kidney cancer case-control study conducted in Central Europe, we analyzed VHL mutations and promoter methylation in 205 well-characterized, histologically confirmed patient tumor biopsies using a combination of sensitive, high-throughput methods (endonuclease scanning and Sanger sequencing) and analysis of 11 CpG sites in the VHL promoter. Results: We identified mutations in 82.4% of cases, the highest VHL gene mutation prevalence reported to date. Analysis of 11 VHL promoter CpG sites revealed that 8.3% of tumors were hypermethylated and all were mutation negative. In total, 91% of ccRCCs exhibited alteration of the gene through genetic or epigenetic mechanisms. Analysis of patient and tumor characteristics revealed that certain mutation subtypes were significantly associated with Fuhrman nuclear grade, metastasis, node positivity, and self-reported family history of RCC. Conclusion: Detection of VHL gene alterations using these accurate, sensitive, and practical methods provides evidence that the vast majority of histologically confirmed ccRCC tumors possess genetic or epigenetic alteration of the VHL gene and support the hypothesis that VHL alteration is an early event in ccRCC carcinogenesis. These findings also indicate that VHL molecular subtypes can provide a sensitive marker of tumor heterogeneity among histologically similar ccRCC cases for etiologic, prognostic, and translational studies.

Molecular analysis of the short arm of chromosome 3 in small-cell and non-small-cell carcinoma of the lung.

Previous studies have suggested that the loss of DNA sequences on the short arm of chromosome 3 (3p) is associated with small-cell lung carcinoma. We therefore looked for loss of 3p alleles in tumor tissue from 42 patients with either small-cell or non-small-cell lung carcinoma. All 13 patients with small-cell lung carcinoma who were heterozygous for one or more alleles at 3p in normal tissue had the loss of at least one codominant allele in the tumor tissue. Cell lines of small-cell lung carcinoma from an additional eight patients were homozygous for 3p alleles; this result was significantly different from the predicted frequency of homozygosity. The tumor tissue studied included cell lines of small-cell lung carcinoma obtained from biopsy specimens, an autopsy sample, and an excised lymph node containing tumor cells. Loss of alleles at 3p was observed in tumor samples obtained before and after chemotherapy. Four of 15 evaluable patients with non-small-cell carcinoma of the lung had loss of 3p alleles. We conclude that loss of alleles at 3p is a change found consistently in small-cell lung carcinoma and occasionally in non-small-cell lung carcinoma.

The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the VHL gene), 3p12-p14.2 (containing the FHIT gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both VHL-caused clear RCC tumors and clear RCC without VHL inactivation. We found hypermethylation of RASSF1As GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2′-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that RASSF1A is the candidate renal TSG gene for the 3p21.3 region.

A novel germ line juxtamembrane Met mutation in human gastric cancer

Activating mutations in the Met receptor tyrosine kinase, both germline and somatic, have been identified in human papillary renal cancer. Here we report a novel germline missense Met mutation, P1009S, in a patient with primary gastric cancer. The dosage of the mutant Met DNA was elevated in the tumor when compared to its matched normal DNA. Therefore, as with hereditary renal papillary cancer, the mutant Met allele may also be selectively duplicated in the tumor. Different from previously reported Met mutations, which occur in the tyrosine kinase domain, this missense mutation is located at the juxtamembrane domain, and is not constitutively activated. However, following treatment with HGF/SF, the P1009S mutant Met protein, expressed in NIH3T3 cells, displays increased and persistent tyrosine phosphorylation compared to the wild-type Met. Importantly, these cells also form colonies in soft agar, and are highly tumorigenic in athymic nude mice. A second nucleotide change in this region of Met, T1010I, was found in a breast cancer biopsy and a large cell lung cancer cell line. Although this previously reported ‘polymorphism’ did not stimulate NIH3T3 cell growth in soft agar, it was more active than the wild-type Met in the athymic nude mice tumorigenesis assay, suggesting that it may have effects on tumorigenesis. Met has been shown to be highly expressed in human gastric carcinoma cell lines, and our results raise the possibility that activating missense Met mutations could contribute to tumorigenesis of gastric cancer.