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Cerebral Cavernomas in a Family with Multiple Cutaneous and Uterine Leiomyomas Associated with a New Mutation in the Fumarate Hydratase Gene

2007; Elsevier BV; Volume: 127; Issue: 9 Linguagem: Inglês

10.1038/sj.jid.5700851

1523-1747

Elena Campione, Alessandro Terrinoni, Augusto Orlandi, Andrea Codispoti, Gerry Melino, Luca Bianchi, Annamaria Mazzotta, Francesco Garaci, Andrea Ludovici, Sergio Chimenti,

Tuberous Sclerosis Complex Research

cerebral cavernoma malformation fumarate hydratase fumarate hydratase deficiency hereditary leiomyomatosis and renal cell cancer hypoxia-inducible factors multiple cutaneous and uterine leiomyomas magnetic resonance imaging succinate dehydrogenase Von Hippel Lindau TO THE EDITOR Solitary or multiple cutaneous leiomyomas are benign tumors derived from vascular, areolar, dartos, or pilar smooth muscle fibers (Holst et al., 2002Holst V.A. Junkins-Hopkins J.M. Elenitsas R. Cutaneous smooth muscle neoplasms: clinical features, histologic findings, and treatment options.J Am Acad Dermatol. 2002; 46 (quiz, 491–474): 477-490Google Scholar), which can be part of a syndrome when associated with MCUL (Garman et al., 2003Garman M.E. Blumberg M.A. Ernst R. Raimer S.S. Familial leiomyomatosis: a review and discussion of pathogenesis.Dermatology. 2003; 207: 210-213Google Scholar). Furthermore, 15–62% of MCUL patients may develop a papillary renal type II or renal duct cancer HLRCC configuring a more complex entity (MCUL/HLRCC, OMIM 150800) (Toro, 2003Toro J.R. Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America.Am J Hum Genet. 2003; 73: 95Google Scholar). This syndrome is due to heterozygous mutations in the FH gene (Kiuru et al., 2001Kiuru M. Launonen V. Hietala M. Aittomaki K. Vierimaa O. Salovaara R. et al.Familial cutaneous leiomyomatosis is a two-hit condition associated with renal cell cancer of characteristic histopathology.Am J Pathol. 2001; 159: 825-829Google Scholar; Tomlinson et al., 2002Tomlinson I.P. Alam N.A. Rowan A.J. Barclay E. Jaeger E.E. Kelsell D. et al.Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata, and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Google Scholar), which acts as a tumor-suppressor gene to protect cells from hypoxic stress (Pollard, 2005Pollard P.J. Accumulation of Krebs cycle intermediates and over-expression of HIF1[alpha] in tumors which result from germline FH and SDH mutations.Hum Mol Genet. 2005; 14: 2231Google Scholar). Homozygous mutations cause a complete FHD syndrome (FHD; OMIM 606812), which is lethal in childhood (Bourgeron et al., 1994Bourgeron T. Chretien D. Poggi-Bach J. Doonan S. Rabier D. Letouze P. et al.Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.J Clin Invest. 1994; 93: 2514-2518Google Scholar). FH enzymatic activity is absent in FHD, reduced by nearly 50% in MCUL/HLRCC, and very low or absent in tumors from MCUL patients (Tomlinson et al., 2002Tomlinson I.P. Alam N.A. Rowan A.J. Barclay E. Jaeger E.E. Kelsell D. et al.Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata, and papillary renal cell cancer.Nat Genet. 2002; 30: 406-410Google Scholar), suggesting loss of heterozygosity. Besides FH, a number of different Krebs cycle genes have also been found to be mutated in neoplasms, such as SDH in paraganglioma and pheochromocytoma (Baysal, 2000Baysal B.E. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.Science. 2000; 287: 848Google Scholar; Niemann and Muller, 2000Niemann S. Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3.Nat Genet. 2000; 26: 268-270Google Scholar); or in neo or hyper vascular conditions such as in VHL syndrome (Kim and Kaelin, 2004Kim W.Y. Kaelin W.G. Role of VHL gene mutation in human cancer.J Clin Oncol. 2004; 22: 4991Google Scholar). We performed a study in an Italian family (Figure 1a) (consanguinity was not reported). Informed consents were obtained and approved by the Institutional Review Boards according to the Declaration of Helsinki Principles. Patients I-2, II-2, and III-2 exhibited cutaneous leiomyomas, and in addition, patients I-2 and II-2 had undergone a hysterectomy for uterine fibroids. Patient II-2, a 62-year-old woman, presented multiple painful skin-colored papules and nodules on the upper limbs and trunk (Figure 1b). Patient III-2, a 35-year-old man, was affected by multiple cutaneous lesions on the abdomen; 3 months before diagnosis, he had reported a sudden loss of consciousness without subsequent neurological or ophthalmic deficit. Immunohistochemical staining of skin biopsies from patients II-2 and III-2, using muscle-specific antibodies, were positive (data not shown). To investigate the loss of consciousness in patient III-2 and the possible presence of renal neoplasia, we performed an MRI study, which revealed multiple, well-defined lesions in both cerebral and cerebellar hemispheres (Figure 1c and d). The heterogeneous signal intensity or “popcorn-like” appearance is the result of a hemorrhage in different stages of evolution. The hypointensity on gradient-echo images reflects microscopic deposits of hemosiderin and/or calcification, a typical finding of cerebral cavernomas. CT scan and MRI analysis of the mother (II-2) excluded renal neoplasia, but showed the same cerebral pattern (data not shown). Possible co-segregation of familial CCM syndrome (OMIM 116860) was excluded since molecular analysis of CCM genes was negative (data not shown). Analysis of the FH gene demonstrated, in one of the alleles, the presence of two “in frame” copies of exon 7 (Figure 2a), which, from the analysis of exon 7 flanking sequences, arises from an alteration of the FH gene at genomic level (Figure 2b). The duplication involves a portion of the gene flanking introns 6 and 7, resulting in an additional exon 7 (exon 7′), and in a new hybrid intron (Figure 2c), formed by part of intron 7 and part of intron 6 (intron 7′). As this duplication is flanked by an AluSq SINE-repeated sequence (Figure 2c), it is probably due to an ectopic crossing over involving this repeated sequence.Figure 2Molecular, genetic, and structural analysis of FH. (a), RT-PCR of the whole FH transcript (primers: +5′-AATTCTACCCAAGCTCCCTC-3′; -5′-AAATTGCTCTGCTAGAGATGC-3′), showing the extra band due to the mutated allele, approximately700 bp larger than the wild type. The extra band (asterisks) is present in all affected members. (b) Analysis of genomic DNA (primers: +5′-AGGGTTTGGCAAATGTAGATT-3′; -5′-TCAGTATGAGTGTGAGGCAATTAG-3′), showing the presence of the mutated allele (asterisks) demonstrating that the duplication detected on the mRNA is due to a duplication present in the genomic DNA. (c) Schematic representation of the mutation. The duplication of a region of 925 bp, containing exon 7 and part of the flanking introns, 6 and 7, generates an additional exon (exon 7′) and a functional hybrid intron (intron 7′) (oblique stripes). Model structure of wild-type fumarase enzyme, based on yeast template 1YFM.pdb (d) compared to the mutant enzyme (e). The models obtained were processed using DeepView/Swiss PdbViewer v 3.7 from GlaxoSmithKline. The active site (green) is formed by the tetrameric interaction of four identical molecules (not shown). The duplication of exon 7 in the mutant form creates an extra helical domain protruding from the active site. This domain also contains a duplication of a portion of the active site (white arrows), probably causing the loss of activity of the mutant enzyme.View Large Image Figure ViewerDownload (PPT) The analysis of genomic DNA of all family members revealed the presence of the duplication (Figure 2b) in both the proband and his affected mother, whereas it was absent in the unaffected family members. Proband IV-1 was a 4-year-old boy, who, although currently asymptomatic, was also carrying the mutation (Figure 2b) with a high risk of developing the disease later in his life. Structural studies suggested that the duplication of exon 7 results in the addition of a helical domain protruding from the active site (Figure 2d and e), which presumably interferes with the correct tetramerization process and/or opening of the active site to aqueous solvent. We also assayed FH activity in the peripheral lymphocytes (Hatch, 1978Hatch M.D. A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts.Anal Biochem. 1978; 85: 271-275Google Scholar); this showed that affected family members (II-2, III-2, IV-1) had an enzymatic activity that was less than half that of non-affected family members (18±4, 25±6, 32±6 nU/mg/min, respectively, compared with II-1, 76±4; III-1, 54±13), and healthy controls (C-1, 82±9; C-2, 81±25; C-3, 90±11), thus confirming the genetic analysis. The mechanism by which FH mutations promote tumorigenesis has yet to be fully clarified. The increased cytoplasmic concentrations of fumarate or succinate substrates, due to low or absent FH/SDH activity, inhibit the degradation of HIF. Persistence of HIF induces activation of sustained hypoxic response signals, which are considered to be involved in hypervascularization or neoplastic growth (Pollard, 2005Pollard P.J. Accumulation of Krebs cycle intermediates and over-expression of HIF1[alpha] in tumors which result from germline FH and SDH mutations.Hum Mol Genet. 2005; 14: 2231Google Scholar). However, some major questions still remain unanswered. Why, for example, do mutations in FH, SDH or VHL genes, that are components of the same pathway of hypoxia detection, cause different tumors? Moreover, as a unique mutation should lead to a unique phenotype, could the specific duplication of exon 7, which we detected in this family, be in some way related to cerebral cavernomas? To our knowledge this is the first time that cerebral angiomatous lesions are described in MCUL patients. We believe that this finding adds new insight into the genetic background of this syndrome, suggesting that a reassessment of the overall clinical spectrum of MCUL might be required. The authors state no conflict of interest. We thank Dr Marco Ranalli for setting up the biochemical test for FH activity; Professor Richard Knight (MRC, Toxicology Unit, Leicester, UK) for proofreading the manuscript and for helpful suggestions and discussions. We also thank Diana Saltarelli for editing this paper. This work was partially supported by EU-Grants EPISTEM (LSHB-CT-019067), FIRB-Grants RBNE01KJHT_004,RBNE01NWCH_008; MIUR/PRIN 004064744_003; AIRC rif. 1338; ISS n. 530/F-A19. The work was also supported by Grant Telethon GGPO4110 to Gerry Melino.