Missense mutations in fumarate hydratase in multiple cutaneous and uterine leiomyomatosis and renal cell cancer

Abstract

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.

Figure 1

The E. coli FumC homotetramer showing the location of one of four FumC A/B sites. The three conserved regions within the fumarate hydratase superfamily are colored cyan (corresponding to region 1 H133 to T150 in human FH), khaki (corresponding to region 2 G189 to E204 in human FH), and magenta (corresponding to region 3 G321 to E335 in human FH).

Figure 2

Clustal alignment of FH across seven species (from top, rat, mouse, human, C. elegans, Rhizopus, S. cerevisiae, E. coli) using the default Clustal setting based on biochemical properties of residues as follows: *, positions that have a single, fully conserved residue; :, indicates that one of the following strong groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW; ., indicates that one of the following weaker groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM. We have referred to these in the text as follows: *, fully conserved residues, :, strongly conserved residues, ., weakly conserved residues. All other residues are considered to be unconserved. Missense mutations are shown. Mutations reported in FH deficiency are black, mutations reported in MCUL are blue, mutations reported in both MCUL and FHD are red.

Figure 3

The E. coli FumC (a) monomer (b) homotetramer with B site (green), active site (red), and subunit interface (black) amino acids mapped. Coordinates from 1FUO A-subunit. Illustration prepared using Chimera.

Figure 4

The E. coli FumC (a) monomer (b) homotetramer with MCUL mutations indicated in terms of function of the mutated residue with B-site (green), active A-site (red), and subunit interface (black) amino acids mapped. Mutated residues of unknown function are shown in cyan. Coordinates from 1FUO A-subunit. Illustration prepared using Chimera.

Figure 5

FH activity of lymphoblastoid lines from MCUL/HLRCC probands with heterozygote missense FH mutation with the likely function of the mutated residue shown as follows: black, A-site; white, subunit-subunit interactions; and gray, B-site. The y axis is FH activity in nmol/g/minute.

Figure 6

The E. coli FumC (a) monomer (b) homotetramer with FHD mutations indicated in terms of function of the mutated residue with B-site (green), active A-site (red), and subunit interface (black) amino acids mapped Coordinates from 1FUO A-subunit. Illustration prepared using Chimera.