Artigo Acesso aberto Revisado por pares

Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

2005; Elsevier BV; Volume: 7; Issue: 4 Linguagem: Inglês

10.1016/s1525-1578(10)60574-0

ISSN

1943-7811

Autores

Neyaz Alam, S. E. Olpin, Andrew Rowan, David P. Kelsell, Irene M. Leigh, Ian Tomlinson, Todd Weaver,

Tópico(s)

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Resumo

Heterozygous germline mutations in fumarate hydratase (FH) predispose to the multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), which, when co-existing with renal cancer, is also known as hereditary leiomyomatosis and renal cell cancer. Twenty-seven distinct missense mutations represent 68% of FH mutations reported in MCUL. Here we show that FH missense mutations significantly occurred in fully conserved residues and in residues functioning in the FH A-site, B-site, or subunit-interacting region. Of 24 distinct missense mutations, 13 (54%) occurred in the substrate-binding A-site, 4 (17%) in the substrate-binding B-site, and 7 (29%) in the subunit-interacting region. Clustering of missense mutations suggested the presence of possible mutational hotspots. FH functional assay of lymphoblastoid cell lines from 23 individuals with heterozygous FH missense mutations showed that A-site mutants had significantly less residual activity than B-site mutants, supporting data from Escherichia coli that the A-site is the main catalytic site. Missense FH mutations predisposing to renal cancer had no unusual features, and identical mutations were found in families without renal cancer, suggesting a role for genetic or environmental factors in renal cancer development in MCUL. That all missense FH mutations associating with MCUL/hereditary leiomyomatosis and renal cell cancer showed diminished FH enzymatic activity suggests that the tumor suppressor role of fumarate hydratase may relate to its enzymatic function. Heterozygous germline mutations in fumarate hydratase (FH) predispose to the multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), which, when co-existing with renal cancer, is also known as hereditary leiomyomatosis and renal cell cancer. Twenty-seven distinct missense mutations represent 68% of FH mutations reported in MCUL. Here we show that FH missense mutations significantly occurred in fully conserved residues and in residues functioning in the FH A-site, B-site, or subunit-interacting region. Of 24 distinct missense mutations, 13 (54%) occurred in the substrate-binding A-site, 4 (17%) in the substrate-binding B-site, and 7 (29%) in the subunit-interacting region. Clustering of missense mutations suggested the presence of possible mutational hotspots. FH functional assay of lymphoblastoid cell lines from 23 individuals with heterozygous FH missense mutations showed that A-site mutants had significantly less residual activity than B-site mutants, supporting data from Escherichia coli that the A-site is the main catalytic site. Missense FH mutations predisposing to renal cancer had no unusual features, and identical mutations were found in families without renal cancer, suggesting a role for genetic or environmental factors in renal cancer development in MCUL. That all missense FH mutations associating with MCUL/hereditary leiomyomatosis and renal cell cancer showed diminished FH enzymatic activity suggests that the tumor suppressor role of fumarate hydratase may relate to its enzymatic function. In the autosomal dominant syndrome of multiple cutaneous and uterine leiomyomatosis (MCUL, Reed syndrome, leiomyomatosis cutis et uteri, multiple leiomyomatosis; OMIM 150800), affected females develop uterine leiomyomas and affected individuals of both sexes develop cutaneous leiomyomas.1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar Cutaneous leiomyomas are believed to be derived from the smooth muscle of the pilo-arrector apparatus. They generally present in the second, third, or fourth decades, typically as grouped papules or nodules on the trunk or limbs and are characteristically painful particularly in response to low temperatures or touch. Uterine leiomyomas or fibroids in MCUL are severely symptomatic with a large proportion of patients requiring symptom control by hysterectomy.2Alam NA Barclay E Rowan AJ Tyrer JP Calonje E Manek S Kelsell D Leigh I Olpin S Tomlinson IP Clinical features of multiple cutaneous and uterine leiomyomatosis: an underdiagnosed tumor syndrome.Arch Dermatol. 2005; 141: 199-206Crossref PubMed Scopus (164) Google Scholar A small proportion of families with MCUL also cluster renal cancer, either papillary renal type II cancer or renal collecting duct cancer.1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar, 3Kiuru M Launonen V Hietala M Aittomaki K Vierimaa O Salovaara R Arola J Pukkala E Sistonen P Herva R Aaltonen LA Familial cutaneous leiomyomatosis is a two-hit condition associated with renal cell cancer of characteristic histopathology.Am J Pathol. 2001; 159: 825-829Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 4Launonen V Vierimaa O Kiuru M Isola J Roth S Pukkala E Sistonen P Herva R Aaltonen LA Inherited susceptibility to uterine leiomyomas and renal cell cancer.Proc Natl Acad Sci USA. 2001; 98: 3387-3392Crossref PubMed Scopus (491) Google Scholar, 5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 6Toro JR Nickerson ML Wei MH Warren MB Glenn GM Turner ML Stewart L Duray P Tourre O Sharma N Choyke P Stratton P Merino M Walther MM Linehan WM Schmidt LS Zbar B Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95-106Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar This disease variant has been referred to as hereditary leiomyomatosis and renal cancer (HLRCC, OMIM 605839). MCUL/HLRCC has been found to be caused by germline mutations in fumarate hydratase (FH) in the majority of screened cases.1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar, 5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 6Toro JR Nickerson ML Wei MH Warren MB Glenn GM Turner ML Stewart L Duray P Tourre O Sharma N Choyke P Stratton P Merino M Walther MM Linehan WM Schmidt LS Zbar B Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95-106Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar Forty-six distinct FH mutations have been reported to date in MCUL/HLRCC. Twenty-seven of these are missense mutations of 26 different residues (one residue has two reported mutations, R190H and R190L). These 27 distinct missense mutations represent 55 of 81 (68%) of the FH mutations reported in MCUL probands.1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar, 5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 6Toro JR Nickerson ML Wei MH Warren MB Glenn GM Turner ML Stewart L Duray P Tourre O Sharma N Choyke P Stratton P Merino M Walther MM Linehan WM Schmidt LS Zbar B Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95-106Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 7Martinez-Mir A Glaser B Chuang GS Horev L Waldman A Engler DE Gordon D Spelman LJ Hatzibougias I Green J Christiano AM Zlotogorski A Germline fumarate hydratase mutations in families with multiple cutaneous and uterine leiomyomata.J Invest Dermatol. 2003; 121: 741-744Crossref PubMed Scopus (56) Google ScholarThe FH locus encodes two isoforms of fumarate hydratase, cytosolic and mitochondrial, which differ only in that the latter has an initial mitochondrial signal peptide. Fumarate hydratase catalyzes the stereospecific reversible hydration of fumarate to l-malate. The mitochondrial isoform performs this reaction as part of the Krebs cycle and as such is central to aerobic respiration. The cytosolic isoform is thought to be involved in the metabolism of fumarate, which is produced in the cytosol by a number of reactions.8Pines O Even-Ram S Elnathan N Battat E Aharonov O Gibson D Goldberg I The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase.Appl Microbiol Biotechnol. 1996; 46: 393-399PubMed Google Scholar FH is relatively highly evolutionarily conserved. The crystal structures of the E. coli fumarase C and Saccharomyces cerevisiae fumarase have been elucidated.9Weaver TM Levitt DG Donnelly MI Stevens PP Banaszak LJ The multisubunit active site of fumarase C from Escherichia coli.Nat Struct Biol. 1995; 2: 654-662Crossref PubMed Scopus (115) Google Scholar, 10Weaver T Lees M Zaitsev V Zaitseva I Duke E Lindley P McSweeny S Svensson A Keruchenko J Keruchenko I Gladilin K Banaszak L Crystal structures of native and recombinant yeast fumarase.J Mol Biol. 1998; 280: 431-442Crossref PubMed Scopus (48) Google Scholar The high degree of homology of these proteins to human FH allows their use as models for predicting the effect of missense mutations on FH function. The FH protein exists as a homotetramer with two substrate-binding sites (Figure 1), designated the A-site and the B-site. The A-site is made up of residues from three of four chains in the homotetramer whereas the B-site is made up of residues from one chain. Thus any change affecting subunit-subunit interaction would be expected to affect the function of the A-site. Studies in E. coli have shown that mutating a histidine to an asparagine at the A-site results in a large decrease in enzymatic activity, but at the B-site, has little effect suggesting that the A-site may be the main catalytically active site.11Weaver T Lees M Banaszak L Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.Protein Sci. 1997; 6: 834-842Crossref PubMed Scopus (45) Google Scholar In contrast, the B-site is thought to be a substrate-binding activation site but may not be catalytically active. We have previously shown that lymphoblastoid cell lines with heterozygous mutations in FH have diminished in vitro FH activity.1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar, 5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google ScholarMutations in another Krebs cycle enzyme have also been shown to cause predisposition to an inherited tumor syndrome. Germline heterozygous mutations in subunits of succinate dehydrogenase (SDH), SDHB, SDHC, and SDHD, are associated with hereditary predisposition to pheochromocytoma and paraganglioma (OMIM 168000, 115310, 605373). A major question arising from the finding of a tumor suppressor function for genes encoding Krebs cycle enzymes is whether the tumor suppressor function is related to the metabolic/bioenergetic functions of the enzymes or whether a separate as yet unknown function of these enzymes is involved.In this study, we investigated the missense FH mutations identified in MCUL. We examined the degree of evolutionary conservation of the mutated residues. We looked for evidence of mutational hotspots and examined the likely functional effects of the mutations. We then performed FH functional assay on available lymphoblastoid cell lines to assess correlations between these features and residual FH activity. We also assessed the missense mutations associated with renal cancer for any common features.Homozygous/compound heterozygous germline FH mutations are known to underlie an autosomal recessive inborn error of metabolism characterized by fumarate hydratase deficiency (FHD, fumarase deficiency, fumaric aciduria; OMIM 606812). Clinical features of FHD include progressive encephalopathy, developmental delay, hypotonia, cerebral malformation and atrophy, and lactic and pyruvic acidemia with death usually occurring in infancy or by the first decade.12Gellera C Uziel G Rimoldi M Zeviani M Laverda A Carrara F DiDonato S Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.Neurology. 1990; 40: 495-499Crossref PubMed Google Scholar, 13Bourgeron T Chretien D Poggi-Bach J Doonan S Rabier D Letouze P Munnich A Rotig A Landrieu P Rustin P Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.J Clin Invest. 1994; 93: 2514-2518Crossref PubMed Scopus (121) Google Scholar, 14Rustin P Bourgeron T Parfait B Chretien D Munnich A Rotig A Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.Biochim Biophys Acta. 1997; 1361: 185-197Crossref PubMed Scopus (100) Google Scholar, 15Coughlin EM Christensen E Kunz PL Krishnamoorthy KS Walker V Dennis NR Chalmers RA Elpeleg ON Whelan D Pollitt RJ Ramesh V Mandell R Shih VE Molecular analysis and prenatal diagnosis of human fumarase deficiency.Mol Genet Metab. 1998; 63: 254-262Crossref PubMed Scopus (61) Google Scholar Thus this is an uncommon example of a situation in which heterozygous and homozygous mutations of a single gene give rise to very different phenotypes. The germline mutations of 11 FHD patients have been reported (Table 4).5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 12Gellera C Uziel G Rimoldi M Zeviani M Laverda A Carrara F DiDonato S Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.Neurology. 1990; 40: 495-499Crossref PubMed Google Scholar, 13Bourgeron T Chretien D Poggi-Bach J Doonan S Rabier D Letouze P Munnich A Rotig A Landrieu P Rustin P Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.J Clin Invest. 1994; 93: 2514-2518Crossref PubMed Scopus (121) Google Scholar, 14Rustin P Bourgeron T Parfait B Chretien D Munnich A Rotig A Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.Biochim Biophys Acta. 1997; 1361: 185-197Crossref PubMed Scopus (100) Google Scholar, 15Coughlin EM Christensen E Kunz PL Krishnamoorthy KS Walker V Dennis NR Chalmers RA Elpeleg ON Whelan D Pollitt RJ Ramesh V Mandell R Shih VE Molecular analysis and prenatal diagnosis of human fumarase deficiency.Mol Genet Metab. 1998; 63: 254-262Crossref PubMed Scopus (61) Google Scholar In all of these cases, and in some patients who have not had mutation screening, affected individuals have been shown to have low FH activity, ranging from <1 to 20% of that of controls.5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 12Gellera C Uziel G Rimoldi M Zeviani M Laverda A Carrara F DiDonato S Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.Neurology. 1990; 40: 495-499Crossref PubMed Google Scholar, 13Bourgeron T Chretien D Poggi-Bach J Doonan S Rabier D Letouze P Munnich A Rotig A Landrieu P Rustin P Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.J Clin Invest. 1994; 93: 2514-2518Crossref PubMed Scopus (121) Google Scholar, 14Rustin P Bourgeron T Parfait B Chretien D Munnich A Rotig A Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.Biochim Biophys Acta. 1997; 1361: 185-197Crossref PubMed Scopus (100) Google Scholar, 15Coughlin EM Christensen E Kunz PL Krishnamoorthy KS Walker V Dennis NR Chalmers RA Elpeleg ON Whelan D Pollitt RJ Ramesh V Mandell R Shih VE Molecular analysis and prenatal diagnosis of human fumarase deficiency.Mol Genet Metab. 1998; 63: 254-262Crossref PubMed Scopus (61) Google Scholar Two missense mutations, R190H and K187R, have been reported in both MCUL/HLRCC and FHD and MCUL/FHD, respectively.5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar In this study, we also examined features of the missense FH mutations reported in FHD.Table 4Missense FH Mutations in FH Deficiency PatientsReferenceMutationReported activity (as percent of control activity)15A265T/A265T2015D382V/D382V G1315F269C/435insK1015K187R/K187R3.75R190H/R190H313E319Q/E319Q<1 Open table in a new tab Materials and MethodsMissense FH mutations reported in the literature to date were examined using Clustal X PPC (1.64 b) to align human FH with the equivalent mouse, rat, Caenorhabditis elegans, Rhizopus, S. cerevisiae, and E. coli enzymes. The distribution of mutations and the degree of conservation of mutated residues were examined. Mutations were mapped onto the E. coli crystal structure 1FUO and the probable effect of the mutations examined. FH functional assay was performed on lymphoblastoid cell lines from 23 UK MCUL probands with identified heterozygous missense mutations in FH. Lymphoblastoid cell lines from 31 UK individuals without cancer were used as controls. Fumarate hydratase activity was assayed according to a modification of the method described by Hatch.16Hatch MD A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts.Anal Biochem. 1978; 85: 271-275Crossref PubMed Scopus (104) Google Scholar Briefly, the assay monitors the increase in absorbance at 340 nm due to NADPH formation in a linked assay of lymphoblast sonicate, with a final reaction medium consisting of 10 mmol/L fumarate, 25 mmol/L Hepes-KOH buffer, pH7.5, *0.2 U malic enzyme/ml, 0.27 mmol/L NADP, 4 mmol/L MgCl2, and 5 mmol/L potassium phosphate. The assay was run at least twice for each sample and the final sample activity was the mean activity of these as a percentage of the mean control activity. Inter- and intra-assay variability gave a coefficient of variation of 2.6% and 5.8%, respectively.ResultsMissense mutations in FH in MCUL/HLRCC are summarized in Table 1, Table 2. There have been 27 distinct missense mutations of 26 different residues reported (one residue has two reported mutations R190H and R190L as mentioned above).1Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Crossref PubMed Scopus (1169) Google Scholar, 5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google Scholar, 6Toro JR Nickerson ML Wei MH Warren MB Glenn GM Turner ML Stewart L Duray P Tourre O Sharma N Choyke P Stratton P Merino M Walther MM Linehan WM Schmidt LS Zbar B Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95-106Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 7Martinez-Mir A Glaser B Chuang GS Horev L Waldman A Engler DE Gordon D Spelman LJ Hatzibougias I Green J Christiano AM Zlotogorski A Germline fumarate hydratase mutations in families with multiple cutaneous and uterine leiomyomata.J Invest Dermatol. 2003; 121: 741-744Crossref PubMed Scopus (56) Google Scholar, 17Kiuru M Launonen V Hereditary leiomyomatosis and renal cell cancer (HLRCC).Curr Mol Med. 2004; 4: 869-875Crossref PubMed Scopus (50) Google Scholar Missense mutations in MCUL appear to cluster in certain regions in the protein (Figure 2). These include the regions: P131 to M152 (P131R, H137R and Q142R, S144L, N145S, M152T, H153R the first two mapping to the B-site and the remaining five to the A-site); I186 to R190 (delI186, I186T, K187R, R190H, and R190L, all at the A-site); A265 to N297(A265T, N267Y, F269C, H275Y, V279D, L292P, and N297D all involved in subunit-subunit interaction); and E312 to S323 (E312K, N318K, E319Q, S322G, and S323N, all at the A-site). The following residues have been the sites of more than one mutation in MCUL: Q142R, Q142X; I186T, delI186; R190H, R190L; and N318X, N318K. Eleven different US R190H families apparently did not share an ancestral haplotype suggesting that they may have arisen independently.6Toro JR Nickerson ML Wei MH Warren MB Glenn GM Turner ML Stewart L Duray P Tourre O Sharma N Choyke P Stratton P Merino M Walther MM Linehan WM Schmidt LS Zbar B Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95-106Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar However, all families in our UK study group with identical mutations, including two Spanish origin families with R190H mutations, appeared to share ancestral haplotypes suggesting the likelihood of a founder mutation in these cases.5Alam NA Rowan AJ Wortham NC Pollard PJ Mitchell M Tyrer JP Barclay E Calonje E Manek S Adams SJ Bowers PW Burrows NP Charles-Holmes R Cook LJ Daly BM Ford GP Fuller LC Hadfield-Jones SE Hardwick N Highet AS Keefe M MacDonald-Hull SP Potts ED Crone M Wilkinson S Camacho-Martinez F Jablonska S Ratnavel R MacDonald A Mann RJ Grice K Guillet G Lewis-Jones MS McGrath H Seukeran DC Morrison PJ Fleming S Rahman S Kelsell D Leigh I Olpin S Tomlinson IP Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.Hum Mol Genet. 2003; 12: 1241-1252Crossref PubMed Scopus (265) Google ScholarTable 1Missense FH Mutations in MCUL PatientsMutationNumber of probandsFirst report of mutationN64T61A74P11S115I17H137R11Q142R11S144L16N145S16M152T16H153R13I186T15K187R31R190L16R190H131G239V11N267Y1Our UK group, previously unpublishedH275Y26V279D16L292P16N297D16E312K25N318K15S322G16S323N25V351L17G354R65Y422C26L464P15 Open table in a new tab Table 2Features of Reported Distinct Missense FH Mutations in MCULMutated residueEvolutionary conservation of residueHomologous residue in E. coli fumarase CLikely function of mutated residue based on studies in E. coliMean FH activity of heterozygote as percentage of control activityN64TFullyN59B-site47.2A74PWeaklyA70B-site53S115IWeaklyA111B-siteH137RWeaklyD133B-site51.5Q142RFullyQ138A-site46.1S144LFullyS140A-siteN145SFullyN141A-siteM152TFullyM148A-siteH153RFullyH149A-siteI186TStronglyI181A-site22.8K187RFullyK183A-site28.2R190H R190LFullyR186A-site26.9 (R190H)G239VFullyG235Unknown39.1N267YFullyN263Subunit-subunit interactionH275YUnconserved (fully conserved except in E. coli)C271Subunit-subunit interactionV279DFullyV275Subunit-subunit interactionL292PStronglyL288Subunit-subunit interactionN297DStronglyN293Subunit-subunit interactionE312KFullyE308A-site17.4N318KFullyN314A-siteS322GFullyS318A-siteS323NFullyS319A-siteV351LStronglyI347Subunit-subunit interactionG354RWeaklyG350Subunit-subunit interaction33.9Y422CFullyY418A-siteL464PStronglyV460Not resolved in E. coli structure16.9

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