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.     

Cells lacking the fumarase tumor suppressor are protected from apoptosis through a hypoxia-inducible factor-independent, AMPK-dependent mechanism

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.     

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxiceffects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumour growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumour regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.     

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxic-effects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumor growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumor regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.Key words: HLRCC, bortezomib, cisplatin, ROS, kidney cancer, FH     

Multiplex ligation-dependent probe amplification of LDLR enhances molecular diagnosis of familial hypercholesterolemia

Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] typically results from mutations in the LDL receptor gene (LDLR), which are now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of genomic DNA (gDNA). However, many patients with FH have no LDLR mutation identified by this method. Part of the diagnostic gap is attributable to the genetic heterogeneity of AD FH, but another possible explanation is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. Multiplex ligation-dependent probe amplification (MLPA) is a new method that detects larger gDNA alterations that are overlooked by EBESA. We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormal LDLR MLPA pattern. In 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations, and 5 had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and then demonstrated the deleted sequence in 5 of these. These findings indicate that MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH.     

Multistep renal carcinogenesis in the Eker (Tsc 2 gene mutant) rat model

Cancer is a heritable disorder of somatic cells. Environment and heredity both contribute to the origin of human cancer. The Eker (Tsc 2 gene mutant) rat model of hereditary renal carcinoma (RC) is an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. To the best of our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in a naturally occurring animal model. Carcinogenesis looks like an opened Japanese fan, because initiated cells growing in several directions will develop into tumors having many gene abnormalities, and this is suggested by the edge of the fan. To search for such genetic alterations, we identified genes (Niban and Erc) that were expressed more abundantly in renal tumors than in the normal kidney.I will review this unique model for the study of multistep renal carcinogenesis and discuss cancer prevention and delay of carcinogenesis.     

The Eker rat: establishing a genetic paradigm linking renal cell carcinoma and uterine leiomyoma

Renal Cell Carcinoma (RCC) and uterine leiomyoma (often referred to as fibroids) are tumors arising from tubular epithelium and myometrial compartments of the kidney and uterus, respectively. These tumors have a very different clinical presentation, with RCC being one of the less common cancers, having a very poor prognosis, and occurring predominantly in men, whereas uterine leiomyoma are the most common tumor of women and are benign. Although they are distinct histologically, with RCC arising from epithelial cells and leiomyoma arising from smooth muscle cells, they share a common embryological origin. Renal tubular epithelial cells arise during nephrogenesis as a result of the mesenchymal-epithelial transition of condensed mesenchyme induced by the developing ureteric bud, and have a shared mesenchymal lineage with smooth muscle cells of the uterus. In addition to a common embryological origin, RCC and leiomyoma have been demonstrated to share a common genetic etiology. The Eker rat model was the first demonstration of a specific genetic linkage between RCC and uterine leiomyoma. Eker rats carry a germline defect in the rat homologue of the tuberous sclerosis complex 2 (TSC-2) tumor suppressor gene and develop spontaneous RCC and uterine leiomyoma with a high frequency. TSC patients are also at risk for RCC, and sporadic human uterine leiomyomas exhibit loss of function of the TSC-2 gene product, tuberin. Individuals with the inherited cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC) that have germline defects in the fumarate hydratase (FH) gene develop papillary RCC and uterine and skin leiomyomas. Benign cutaneous lesions and uterine leiomyoma also arise in German Shepherd dogs with germline mutations in the Birt-Hogg-Dube (BHD) gene, and these animals develop RCC and uterine leiomyoma with a high frequency. Identification of the tumor suppressor genes involved in these diseases, TSC, FH and BHD, and the elucidation of the function of their protein products, tuberin, fumarate hydratase and folliculin, respectively, opens new avenues for understanding the pathogenesis of both RCC and uterine leiomyoma.     

Molecular genetic testing for familial hypercholesterolemia in the Netherlands: a stepwise screening strategy enhances the mutation detection rate

Familial hypercholesterolemia (FH) has been identified as a major risk factor for coronary vascular disease and is associated with mutations in the low-density liporotein receptor (LDLR) and apolipoprotein B (APOB) gene. The molecular basis of FH in the Dutch population is well understood. Approximately 160 different LDLR and APOB gene defects have been identified with a panel of 9 LDLR gene and 1 APOB gene frequently occurring mutations accounting for approximately 30% of all clinically diagnosed FH cases. As molecular diagnosis of FH is becoming increasingly widely applied, a variety of mutation detection rates is reported, ranging from as low as 30% and up to 80%. This variability appears to depend on the clinical criteria applied to identify patients with FH and on the strategies and methodologies used for mutation screening. In this study we describe the application of a stepwise screening approach, combining different methodologies, to detect mutations of the LDLR gene and APOB gene in 1465 patients with FH. A mutation was found in approximately 44% of the patients, which demonstrates that this is an effective strategy for the molecular diagnosis of FH.     

Mouse MIM,a tissue-specific regulator of cytoskeletal dynamics,interacts with ATP-actin monomers through its C-terminal WH2 domain

The WH2 (WASP homology domain-2) is a small actin monomer-binding motif and is found in many proteins that regulate the actin cytoskeleton, including the beta-thymosins, ciboulot, WASP, and verprolin/WIP (WASP-interacting protein). In sequence database searches we identified a novel mouse protein containing a WH2 domain in its C-terminal region. This mouse gene also shows strong sequence homology to human MIM (Missing in Metastasis), a cDNA fragment that is present in non-metastatic but absent in metastatic bladder cancer cell lines. Northern blot and in situ hybridizations show that MIM is strongly expressed in the developing neurons and skeletal and cardiac muscles in mouse embryos. In adult mice, the strongest expression of MIM mRNA is in liver, outer layers of the kidney, and in the Purkinje cells of the brain. Recombinant MIM protein interacts with actin monomers and inhibits actin filament nucleation in vitro. However, the MIM/ATP-G-actin complex can participate in actin filament assembly at the barbed end. MIM binds ATP-G-actin with a higher affinity (K(D) = 0.06 microm) than ADP-G-actin (K(D) = 0.3 microm) and inhibits the nucleotide exchange on actin monomers. Site-directed mutagenesis demonstrates that the actin monomer-binding site resides in the C-terminal WH2 domain of MIM. Overexpression of mouse MIM in NIH 3T3 cells results in the disappearance of actin stress fibers and appearance of abnormal actin filament structures. These data show that MIM is an ATP-G-actin binding protein that regulates cytoskeletal dynamics in specialized mammalian cell-types.     

Birt-Hogg-Dubé syndrome, a genodermatosis that increases risk for renal carcinoma

Over the past decade cancer-causing genes have been identified for the most common histologic types of renal cancer, specifically clear cell, papillary type 1 and papillary type 2. Genes predisposing to the more rare chromophobe renal carcinoma and renal oncocytoma were unknown until the recent discovery of a novel gene, BHD, on chromosome 17p that was found to be mutated in the germline of affected family members with the Birt-Hogg-Dubé (BHD) syndrome. These patients develop the hallmark BHD skin lesions (fibrofolliculomas), lung cysts and spontaneous pneumothorax. Importantly, BHD patients have an increased risk for developing a variety of renal neoplasia, most commonly chromophobe and oncocytic hybrid tumors. This review will describe the phenotypic manifestations of BHD including the histologic features of BHD-associated renal tumors, the identification of this novel renal cancer-predisposing gene, the BHD mutation spectrum found in BHD patients, and will discuss the potential role of BHD as a tumor suppressor gene.     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

激光显微切割分选少量胃癌细胞中PSCA基因的SNP分析

随着基因组关联分析方法的应用,越来越多与胃癌相关的易感基因被发现．易感基因的多态性检测已逐步进入胃癌临床诊断和研究．然而,利用少量胃粘膜细胞开展单核苷酸多态性（SNP）分析对胃癌进行早期诊断常遇下述困难,一是少量胃癌细胞混杂在多种细胞中,异常信号常易被淹没,二是细胞量极少,因此获得的基因组DNA量微,进行多位点或全基因组分析存在困难. 本文利用激光显微切割技术分选少量胃癌细胞,结合全基因组放大技术,进行胃癌相关的前列腺干细胞抗原基因(PSCA)的SNP分析．通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和克隆测序方法分析,在分选的胃癌细胞中检测到PSCA的rs2976392位点胃癌相关的“A”等位与rs2294008位点胃癌相关的“T”等位．研究结果表明,所采用的全基因组放大方法保真性高,经过分选的胃癌细胞中SNP位点的检测灵敏度和可靠性大为提高．所建立的少量细胞基因多位点检测方法将同样应用于其它肿瘤和组织的少量细胞研究中,全基因组放大产物也可进行高通量的基因芯片和第二代测序研究．     

Gene expression analysis of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common cause of a variety of cardiovascular diseases. The aim of this study was to uncover the underlying mechanism of FH and provide a possible treatment project for FH. We tried to identify the differently expressed genes (DEGs) involved in FH by comparing the gene expression profiles between FH and normal cells. We performed GO and biological pathway analysis of differently expressed genes with DAVID. We searched for candidates for FH treatment by analyzing DEGs between normal cells and FH cells and compared the differences with the DEGs caused by the small interfering molecules in The Connectivity Map (cmap). Using a bioinformatics method, we identified the abnormal metabolic processes in the cells of FH patients, including cell adhesion, material transport, signal transduction and gene expression, and found that the small molecule trazodone could be a potential drug in restoring the dysregulated metabolic pathway. In conclusion, candidates for further evaluation as possible therapeutic agents for FH have been identified using bioinformatics analysis of differentially expressed genes. Phenotype targeting using genomic profiling is a rational approach to drug discovery, which provides a new guideline in treatment of FH and a potential new clinical drug for FH patients.     

Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.     

Familial non-syndromic clear cell renal cell carcinoma

The diagnosis of familial non-syndromic clear cell renal cell carcinoma is one of exclusion. In families presenting with clear cell RCC a germline VHL mutation and a constitutional translocation of chromosome 3 must be excluded before familial non-syndromic clear cell RCC can be diagnosed. Large familial non-syndromic clear cell RCC kindreds are uncommon and a predisposing gene has not been identified. However inheritance is autosomal dominant in most cases and age at onset is earlier than in sporadic cases. Recognition and appropriate screening of familial non-syndromic clear cell RCC cases will reduce morbidity and mortality. Large scale collaborative linkage studies may provide a basis for the identification of familial non-syndromic clear cell RCC susceptibility gene(s).