Fumarate hydratase is an enzyme that functions in the mitochondrial citric acid cycle, catalyzing the reversible hydration/dehydration reaction in which fumarate is converted to malate.
The human gene encoding fumarate hydratase is located in the chromosome segment 1q42.3-q43. It consists of ten exons that span over 20 kb of genomic DNA. The transcript is approximately 1.8 kb long and is predicted to encode a 510 amino acid polypeptide. The first exon of FH encodes a signal peptide that directs the protein to the mitochondrion. There the signal peptide is cleaved, and the remaining mature FH protein forms a functional homotetramer in the mitochondrial matrix. Some processed FH is also present in the cytosol, although the function of this cytosolic FH is unclear. In addition to the mitochondrial signal, the processed FH contains other domains such as alpha-helical and lyase domains. The alpha-helixes form a superhelical core for the tetramer. The functionally active FH enzyme converts fumarate to malate. This hydration reaction is a part of the citric acid cycle (also known as the tricarboxylic acid cycle or the Krebs cycle) which is an essential component of cellular carbohydrate metabolism. In the cytosol, fumarate is produced in the urea cycle and therefore FH is connected to protein metabolism as well. FH is well conserved, human FH sharing a 57% amino acid identity with the Escherichia coli FumC protein, and it belongs to a protein superfamily which includes mostly other enzymes such as aspartase, adenylosuccinate lyase, and arginosuccinate lyase.
The first clues as to the role of FH in human disease came from the identification of two siblings that presented with progressive encephalopathy, dystonia, leucopenia, and neutropenia. They had elevated levels of lactate in their cerebrospinal fluid and high fumarate excretion in their urine. A Homozygous mutation was discovered in a conserved region of the FH gene in both of these patients. Also, FH deficiency was shown to be present in all tissues studied in the patients, and their healthy parents were shown to carry the mutation in a heterozygous form. Since then, about 20 families with FH deficiency have been reported in the literature. The symptoms are severe and the affected individuals usually die within a few months of birth.
Evidence for yet another role of FH in human disease came from quite a different line of research. The genomic locus harboring the FH gene was independently mapped by genetic linkage analysis to segregate with inherited predisposition to leiomyoma and renal carcinoma; Hereditary leiomyomatosis and renal cell cancer (HLRCC), and to multiple cutaneous and uterine leiomyomatosis (MCUL). Soon after, these two conditions were shown to be variants of the same syndrome and the underlying gene was identified as FH. The tumors showed loss of heterozygosity (LOH) and retention of the mutated allele, therefore suggesting that the gene acted as a tumor suppressor. Also, FH enzyme activity was shown to be reduced in the leukocytes and absent in the tumors of mutation carriers.
Clinical features of HLRCC/MCUL
Since the first reports indicating the involvement of FH in tumorigenesis, more than 100 families with the HLRCC/MCUL phenotype have been reported in the literature (Fig. 1). Although no population-based studies have been carried out, it seems clear that the prevalence of HLRCC/MCUL is very low. There are reports of HLRCC/MCUL from all around the world, and there seem to be population differences in the phenotype. For example, HLRCC seems to be more common in Finland and North America, whereas in the UK, renal cell cancer is rarely detected in the families segregating heterozygous FH mutations. The most common manifestation of HLRCC/ MCUL is cutaneous and/or uterine leiomyomas. Early-onset renal cell cancer is significantly rarer and is typically of the papillary type II histology.
Cutaneous leiomyomas are small benign tumors of the skin that show as multiple 0.5–2 cm skin-colored nodules, and their tissue of origin is thought to be the arrectores pili muscle of the hair follicle. They can manifest clinically as pain and paresthesias already in childhood, and the age of onset ranges from 10 to 50 in the HLRCC/MCUL families.
Uterine leiomyomas (also known as myomas or fibroids) are smooth muscle cell tumors that arise within the smooth muscle lining of the uterus, the myometrium. They are some of the most common neoplastic tumors of women, and estimates of affected individuals range from 25% to up to 77% depending on the methods used for the diagnosis. Even though they are benign, they can cause severe morbidity such as aberrant bleeding, abdominal pain, and even infertility. In families affected by HLRCC/MCUL, the onset of leiomyomas seems to be earlier than in the general population. In these families, leiomyomas are also more often symptomatic and therefore they more commonly result in hysterectomy (i.e., the surgical removal of the uterus). In addition to typical leiomyomas, some HLRCC/MCUL patients develop atypical leiomyomas. These are rare variants of leiomyomas which are sometimes hard to discern from uterine leiomyosarcomas. Whether HLRCC/ MCUL predisposes to malignant leiomyosarcoma is still a somewhat open question, although some studies suggest that this might indeed be the case.
Familial clustering of renal cell cancer was the key finding in the identification of the syndrome HLRCC. In the first family reported, four patients aged 33–48 were identified. Since then, additional cases of renal cell cancer have been detected in some HLRCC/MCUL families, the median age of onset being around 40. The natural history of HLRCC/ MCUL renal tumors is malignant with early metastasis often leading to the demise of the patient. In the early reports, all renal cell cancers related to HLRCC/ MCUL displayed a distinctive papillary type II histology, although other types of renal tumors, such as collecting duct and clear-cell carcinoma, have been later associated with HLRCC/MCUL as well. HLRCC/MCUL renal tumors are typically unilateral, which is in contrast to other inherited forms of renal cell cancer such as von Hippel–Lindau Syndrome (VHL), Hereditary Papillary Renal Carcinoma, and Birt–Hogg–Dubeґ Syndrome (BHD), in which tumors often affect both kidneys.
FH-mutation carriers might be at risk of developing Leydig cell tumors and ovarian cystadenomas. Incidental cases of other tumors, such as breast and prostate cancer and some hematological malignancies, have also been reported in HLRCC/MCUL families, although it remains unclear whether any of these are true manifestations of the germline FH mutations.
Mutations in the FH gene
The syndrome HLRCC/MCUL is transmitted in an autosomal dominant manner, and germline FH mutations have been detected in ~85% of all the families displaying the HLRCC/MCUL phenotype. Altogether, ~60 different mutations have been identified. The vast majority (_70%) are single base pair substitutions, of which missense mutations comprise about 60%; the rest are nonsense mutations. Small deletions and insertions as well as splice site changes have been reported. In addition, whole gene FH deletions have been detected in some families. Mutations occur throughout the gene. The mutation R58X has been detected in four families of diverse ethnic and geographical backgrounds in North America. Haplotype analysis has suggested that the mutation has occurred independently in these families, indicating that this might represent a mutational hot spot. The same mutation has also been detected in families from the United Kingdom and Australia. Other mutations that have been detected in several families of different geographical backgrounds are, for example, N64T and R190H, and these may represent mutational hot spots as well. The mutations in families with the renal cell cancer phenotype do not differ from those seen in families without these malignant tumors and, in fact, the same mutations have been detected in families with either of the two phenotypes. This has raised the question of whether an additional genomic locus could act as a modifier together with FH mutations.
A founder effect has been detected at least in populations of the Finnish and Iranian origin. Two mutations, H153R and a 2-bp deletion in codon 181, have both been identified in three different families in Finland. Similarly, a splice site mutation IVS6-1G>A has been detected in a common haplotype in several families of Iranian origin. Most tumors arising in HLRCC/MCUL families have a second somatic inactivating hit in the FH gene. This is often acquired through the loss of the wild-type FH by a partial or whole genomic deletion of chromosome arm 1q.
FH mutations are also rarely seen in sporadic tumors. Inactivation of the FH gene has been detected in three tumors from the Finnish population, one soft-tissue sarcoma of the lower limb, and two uterine leiomyomas, all showing loss of the wild-type FH. However, despite mutation screens comprising hundreds of tumor specimen, no other somatic changes in FH have been detected in various tumor types including prostate, breast, colorectal, lung, ovarian, thyroid, head and neck cancers, pheochromocytomas, gliomas, and melanomas. Therefore, it is safe to say that, in general, somatic inactivation of the FH gene is a very rare occurrence in human tumors.
As determined by microarray-based gene expression analysis, as well as by traditional immunohistochemical methods, uterine fibroids carrying FH mutations have distinct biological properties which seem to require two hits in the FH gene. The molecular mechanisms through which mutations in FH lead to tumorigenesis are still far from being well understood. Some evidence suggests that the disruption of FH activity would lead to the stabilization of the hypoxia inducible factor-1 (HIF1) under normoxic conditions, thus activating several growth-promoting signaling cascades.
The mutations detected in the recessively inherited developmental disorder FH deficiency are also mostly missense mutations, and they occur throughout the FH gene. The mutational spectrum of FH deficiency does not seem to be different from that of HLRCC/MCUL and, indeed, a phenotype compatible with HLRCC/ MCUL has been reported in some of the parents of the children affected by FH deficiency.
Fig. 1. Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant tumor predisposition syndrome caused by germline mutations in the fumarate hydratase gene. The most common features are cutaneous and/or uterine leiomyomas. Predisposition to renal cell carcinoma is present in a subset of families. Typically, HLRCC renal cell carcinomas display papillary type 2 histology