In this study we characterise the clinical and genetic features of 21 new families with HLRCC. In addition, we present the first two African‐American families reported to have HLRCC. Sequence analysis revealed a total of 14 different FH germline mutations. We identified nine novel mutations in FH. Sixty two per cent (13/21) of the new families with HLRCC had renal tumours. Some families with renal tumours shared the same mutation, that is, R58X (three families) and R190H (three families). FH mutations were associated with a spectrum of renal tumours.
In combination with our previous report, to date we have identified 31 different germline FH mutations consisting of 20 missense, eight frameshifts (three insertions and five deletions), two nonsense, and one splice site mutation (fig 4). Mutations were distributed throughout the gene except for exon 5. Exon 4 (seven different mutations) and exon 6 (eight different mutations) had the most mutations. Forty two per cent (13/31) of FH mutations were associated with kidney tumours and were also distributed throughout the gene. There was no association between the location or type of mutation and kidney cancer. Eighty seven per cent (27/31) of FH mutations were associated with skin leiomyomas (fig 4). Four FH mutations identified in families without skin leiomyomas were associated with kidney cancer. However, no specific type of mutation was associated with absence of skin lesions. In addition, 96% (27/28) of FH mutations were associated with uterine fibroids (fig 4). Even though no clear genotype‐phenotype correlations could be identified in this study, our data suggest that families with R190H and R58X mutations tend to have a high frequency of individuals with kidney tumours. This early finding needs to be investigated in a larger group of families.
Figure 4Distribution of FH mutations and the genotype‐phenotype in HLRCC. The lower vertical arrows denote FH mutations. The upper vertical bars show the phenotype corresponding to the specific FH mutation. The colours in the vertical (more ...)
Recently, we started measuring FH enzyme activity in cell lines of patients with HLRCC. In agreement with a previous report, we found significantly lower FH enzyme activity in lymphoblastoid cells from individuals with missense (L89S) and nonsense (S102X) mutations compared with normal controls. Previously, Tomlinson and co‐workers also measured FH enzyme activity in lymphoblastoid cell lines from patients with cutaneous leiomyomas and controls.2
All lymphoblastoid cell lines with FH
mutations examined had decreased fumarase enzyme activity. However, lymphoblastoid cell lines with FH
missense mutations had significantly lower enzyme activity than lymphoblastoid cell lines with FH
truncating or large gene deletions. In addition, it is of interest that some patients had very reduced FH activity despite having one normal copy of the gene. A plausible explanation of these findings is that the missense mutants have a dominant negative action. This is conceivable as fumarase functions as a homotetramer. Thus, four wild type monomers are needed for the formation of functional fumarase. A missense mutation in one allele would result in only one out of 16 fully functional wild type tetramers and would prevent fully functional tetramers from forming. Another possible explanation for the functional effects of missense mutations is that they may alter important protein‐protein interactions.
To date, we have evaluated 56 families with HLRCC. Using direct sequencing, we detected germline mutations in FH
in 93% (52/56) of our families. The FH
mutation detection rate reported by the Leiomyoma Consortium was 60% in their cohort of European families with leiomyomatosis.2
Thirty three per cent (17/52) of families had a mutation at the R190 residue. It remains to be determined whether R190 represents a founder mutation. Eight per cent (4/52) of HLRCC families had the R58X mutation. The R58X appears to be a hot spot since haplotype analysis excluded a founder effect. Furthermore, R58X has also been reported in three European families with HLRCC.2,8
Alam and co‐workers found that the three probands with the R58X mutation shared an allele (frequency ~0.2) at only one microsatellite, (CA)13
, which is located in intron 2 immediately after the R58X mutation.8
Therefore, the possibility of a founding mutation could not be excluded. The most frequent FH
germline mutations reported in series of patients with HLRCC in Europe were N64T and G354R, which were found in six families each.8
Haplotype analysis of the six families with N64T was consistent with a founder mutation. It is of interest that, to our knowledge, the G354R mutation has been identified in families with HLRCC in North America.
Previously, using direct sequencing we were not able to identify mutations in four families with HLRCC.3
Southern analysis did not show large deletions of the entire FH
gene in two of these four HLRCC families (data not shown). However, large deletions of approximately 2.4 and 1.9 Mb including the entire FH
gene have been reported in families with HLRCC.2
These families were not phenotypically different from our 52 families with FH
To date, we have identified renal tumours in 32% (18/56) of our cohort of HLRCC families at NCI. To our knowledge, this constitutes the largest collection of reported HLRCC families with renal tumours; however, many families were ascertained through renal cancer screening. In our previous study, families were recruited based on the affected status of multiple cutaneous leiomyomas.3
In that cohort of families, the frequency of families with renal cancer was only 15%. In contrast, in the current study the frequency of families with renal tumours increased to 62% (13/21). In the current study, families were recruited based on cutaneous leiomyomas or a diagnosis of kidney tumours with cytological features characteristic of HLRCC. The increase in frequency of renal tumours in this study compared to our previous study may be due to differences in recruitment approaches. Therefore, there may be a selection bias for families with renal cancer. In addition, as our histological diagnostic acumen improved, we identified more cases of kidney cancer likely to be HLRCC.
To our knowledge, only four HLRCC families with renal cancer have been reported by other groups.1,8,9
Germline mutations in FH
have been reported in three Finnish kindreds with papillary type II renal cell carcinoma. Two families shared a 2 bp deletion in codon 181, and the other had the R300X mutation.2
In addition, an FH
missense mutation (N318K) was reported in an HLRCC British patient described as having CDC of the kidney. Some potential factors may explain the lower frequency of renal tumours previously reported in families with leiomyomatosis and/or FH
First, families were not extensively screened for renal tumours and occult kidney tumours may not have been detected. Optimal screening for renal tumours involves CT scans of the abdomen and pelvis. Alternatively, MRI of the abdomen and pelvis is sometimes used if needed. Papillary renal tumours maybe difficult to detect if patients are screened with renal ultrasound only as they are often isoechoic and can be missed.11
Second, it is also possible that some FH
mutations have low penetrance for renal tumours or specific FH
mutations are not associated with renal tumours. Third, some pathologists may lack experience to recognise the histological features associated with HLRCC renal tumours. HLRCC associated renal tumours are rare and have been only recently described.1,3
Therefore, many pathologists may not be familiar with their features.
In this study, we identified the first family with CDC of the kidney with FH
germline mutations consisting of a daughter and father, both affected with CDC of the kidney and FH
mutations carriers. Previously, two cases described as CDC of the kidney among patients with HLRCC were reported.3,8
Interestingly, this is consistent with reports in the literature of cytogenetic abnormalities found in CDC of the kidney. Monosomy of chromosome 1 and LOH of 1q is reported to occur in 60–80% of cases of CDC.12,13
Furthermore, our patients with HLRCC share some clinical features typical of the clinical presentation of CDC of the kidney characterised by an aggressive course with evidence of metastatic disease at the time of presentation and poor prognosis.14
The most significant finding in our collection of cases with renal tumours was that the cytological features present in renal tumours were very consistent even though the architectural morphology of the tumours varied. Renal tumours associated with HLRCC were characterised by the presence of cells that had an abundant amphophilic cytoplasm and large nuclei with large inclusion‐like eosinophilic nucleoli. These cytological features were originally attributed to type II papillary tumours in the original description1
; however, they can be present in other histological types of renal tumours associated with HLRCC. HLRCC is associated with a histological spectrum of renal tumours. Most renal tumours in this study share some features with type II papillary renal cell carcinoma. However, renal tumours associated with HLRCC are difficult to classify under the existing renal tumour classification schemes since they have distinct clinical and histological features. Therefore, renal tumours associated with HLRCC may in the future constitute a new renal pathological entity.
One hundred per cent of our new cohort of 22 women who were identified as FH
mutation carriers had uterine fibroids. This is similar to our previous report, in which 100% of women with cutaneous leiomyomas had uterine fibroids.3
Therefore, cutaneous leiomyomas are a good marker of affection status for uterine fibroids. In the general population, the reported prevalence rates of uterine fibroids ranged from 22 to 77% with the highest prevalence in women aged 40–44 years.15,16
In this study, 68% (15/22) of women FH
mutation carriers were diagnosed with uterine fibroids at age 30 or younger. Thus, women with HLRCC had a higher prevalence of uterine fibroids and younger age at diagnosis of uterine fibroids than women in the general population.
In agreement with our previous report, uterine fibroids associated with HLRCC are associated with increased morbidity and increased secondary infertility. Seventy three per cent of our cohort of women had a gynaecological procedure including hysterectomy or myomectomy for symptomatic uterine fibroids. Hysterectomy surveillance in the United States from 1994 to 1999 showed that hysterectomy occurred most frequently in women aged 40–44 years. Furthermore, in the United States 52% of women who have a hysterectomy have the surgery at 44 years of age or younger.17
In contrast, 50% of our cohort of women with HLRCC who had a hysterectomy or myomectomy had it at 30 years of age or younger. In conclusion, HLRCC is associated with early onset of uterine fibroids and early hysterectomy when compared with women in the general population in the United States. The young age of onset of symptomatic uterine fibroids significantly impacts the childbearing years of women with HLRCC.
Sixty five per cent (20/31) of the mutations resulted in the substitutions of single amino acid residues that were highly conserved throughout evolution. Missense mutations are important in that they may indicate residues in the fumarate hydratase protein that are functionally important. In fact, we identified two FH mutations (S144L and N145S) corresponding to residues that form the active site and two germline FH mutations (S322G and S323N) that are located in the signature sequence motif.
most likely acts as a tumour suppressor in familial leiomyoma since LOH studies have shown loss of the wild type allele in cutaneous, uterine, and renal tumours, and FH enzyme activity is low or absent in tumours from individuals with leiomyomas.2
However, the mechanisms by which FH defects promote tumourigenesis are unknown. Possible mechanisms include hypoxia, apoptosis, and oxidative stress. It is of interest that germline mutation in another mitochondrial enzyme in the Krebs cycle, succinate dehydrogenase (SDH), leads to the predisposition to develop paragangliomas and pheochromocytoma.18
Furthermore, three cases with kidney cancer and germline mutations in SHD‐B
have been reported.19
In one family with a germline SDH‐B
mutation (c.847‐50delTCTC), two members had renal cell carcinoma and paraganglioma, and in another family, a son with clear cell renal cell carcinoma and his mother with a cardiac paraganglioma both had a germline SDH‐B
R27X mutation. Furthermore, all three of these renal cell carcinomas showed LOH at SDH‐B
. Taken all together, the literature suggests that mitochondria dysfunction through various mechanisms may lead to the formation of kidney tumours. Hypoxia mediated pathways have been shown to be implicated in tumourigenesis, especially in kidney cancer. In von Hippel‐Lindau (VHL) syndrome, the over accumulation of hypoxia inducible factor (HIF) leads to increased transcription of anti‐apoptotic and proliferative genes such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and epidermal growth factor receptor (EGFR). The VHL protein (pVHL) forms a complex with elongin B and C, and cullin 2 to form the VHL complex VCB.20,21,22
When normal oxygen levels are present, this complex binds to HIF1‐alpha and HIF2‐alpha for ubiquitin mediated degradation.23
is mutated, the complex cannot bind HIF, and HIF accumulates along with the associated increase in multiple factors promoting tumourigenesis. Inactivation of VHL
leads to the development of highly vascular tumours.24
This is the mechanism that has been shown in VHL and it is possible that HLRCC and VHL share a common pathway or share some part of the pathway that is key for tumourigenesis. Recently, we showed that accumulation of fumarate and succinate following pharmacological inhibition of FH and SDH leads directly to inhibition of HIF prolyl hydroxylase by competing with 2‐oxoglutarate, a required co‐factor of the enzyme.25
These treatments resulted in accumulation of transcriptionally active HIF, as evidenced by an increased level of both Glut‐1 and VEGF transcripts. Treating cells with siRNA specific for FH has a similar effect. Similarly, Selak et al26
reported that succinate accumulation secondary to loss of SDH leads to HIF accumulation via inhibition of HIF prolyl hydroxylase in 293 cells transfected with siRNA to various SDH subunits. ROS generation does not appear to be involved in either fumarate or succinate dependent HIF induction.25,26
Recently, it has been shown that VEGF and PDGF are over expressed in leiomyomas.27,28
Taken together, these observations suggest that HIF may play a role in tumourigenesis in HLRCC.
It has been shown that mitochondria play a key major role in apoptosis. Bax and Bcl‐2 form pores in the mitochondrial membranes through which cytochrome c
can escape to the cytoplasm to form the apoptosome with Apaf‐1 and caspase 9.29
It is also possible that the mitochondrial dysfunction associated with mutation in FH
can alter the integrity of the mitochondrial membranes, can prevent apoptosis, and/or can lead to accumulation of anti‐apoptotic metabolites. Glutamine has been shown to have anti‐apoptotic effects in Jurkat cells via up regulation of glutathione and Bcl‐2.30
In addition, glutamate can rescue carcinoma cell lines from apoptosis and can have a proliferative effect in different cell types.31
Therefore, it is possible that tumourigenesis associated with HLRCC can lead to anti‐apoptosis.
In conclusion, in this study we characterise the clinical and genetic features of 21 new families and expand the spectrum of phenotypes expressed in families with HLRCC. Patients with HLRCC can have a range of clinical presentations including multiple cutaneous leiomyomas, a single skin leiomyoma, no cutaneous lesions, multiple renal tumours, a single renal tumour, absence of renal tumours, uterine fibroids, and/or various combinations of these phenotypes. Furthermore, this variability of expression is present within and among families with HLRCC. Although individuals with HLRCC have diverse racial and ethnic backgrounds including African‐American, patients with Eastern European heritage are over‐represented. However, HLRCC can occur in patients without a typical ethnic background. In this study, we did not find apparent genotype‐phenotype correlations in HLRCC. HLRCC is associated with clinically significant uterine fibroids and a spectrum of aggressive renal tumours. Appropriate surveillance and genetic counselling is needed in the clinical evaluation of patients with HLRCC.