Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.

Genomic profile analysis of leiomyomas with bizarre nuclei and fumarate hydratase deficient leiomyomas: Strengths, weaknesses, and limitations of array-CGH interpretation.

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.

Conditional expression of FumA in Aspergillus niger enhances synthesis of L-malic acid.

Currently, the L-malic acid titer achieved through Aspergillus niger fermentation reaches 201 g/L, meeting industrial demands satisfactorily. However, the co-presence of structurally similar fumaric acid and succinic acid in fermentation products suggests a theoretical potential for further improvement in L-malic acid production. In the tricarboxylic acid cycle, fumarate reductase mediates the conversion of succinic acid to fumaric acid. Subsequently, fumarase catalyzes the conversion of fumaric acid to L-malic acid. Notably, both enzymatic reactions are reversible. Our investigation revealed that A. niger contains only one mitochondria-located fumarase FumA. Employing CRISPR-Cas9 technology, we performed a replacement of the fumA promoter with a doxycycline-induced promoter Tet. Under non-inducing condition, the conditional strain exhibited increased levels of fumaric acid and succinic acid. It strongly suggests that FumA mainly promotes the flow of fumaric acid to L-malic acid. Furthermore, a promoter PmfsA that is exclusively activated in a fermentation medium by calcium carbonate was identified through RNA-sequencing screening. Utilizing PmfsA to regulate fumA expression led to a 9.0% increase in L-malic acid titer, an 8.75% increase in yield (glucose to L-malic acid), and an 8.86% enhancement in productivity. This research serves as a significant step toward expediting the industrialization of L-malic acid synthesis via biological fermentation. Additionally, it offers valuable insights for the biosynthesis of other organic acids.IMPORTANCEThis study focuses on enhancing L-malic acid synthesis by modifying the tricarboxylic acid cycle within the mitochondria of Aspergillus niger. We emphasize the significant role of fumarase in converting fumaric acid into L-malic acid, enhancing our understanding of metabolic pathways in A. niger. The precise regulation of fumA is highlighted as a key factor in enhancing L-malic acid production. Furthermore, this research introduces a stringent conditional promoter (PmfsA), exclusively activated by CaCO3. The utilization of PmfsA for fumA expression resulted in heightened L-malic acid titers. The progress in metabolic engineering and bioprocess optimization holds promise for expediting industrial L-malic acid synthesis via biological fermentation. Moreover, it carries implications for the biosynthesis of various other organic acids.

The role of promoter methylation of the genes encoding the enzymes metabolizing di- and tricarboxylic acids in the regulation of plant respiration by light.

We discuss the role of epigenetic changes at the level of promoter methylation of the key enzymes of carbon metabolism in the regulation of respiration by light. While the direct regulation of enzymes via modulation of their activity and post-translational modifications is fast and readily reversible, the role of cytosine methylation is important for providing a prolonged response to environmental changes. In addition, adenine methylation can play a role in the regulation of transcription of genes. The mitochondrial and extramitochondrial forms of several enzymes participating in the tricarboxylic acid cycle and associated reactions are regulated via promoter methylation in opposite ways. The mitochondrial forms of citrate synthase, aconitase, fumarase, NAD-malate dehydrogenase are inhibited while the cytosolic forms of aconitase, fumarase, NAD-malate dehydrogenase, and the peroxisomal form of citrate synthase are activated. It is concluded that promoter methylation represents a universal mechanism of the regulation of activity of respiratory enzymes in plant cells by light. The role of the regulation of the mitochondrial and cytosolic forms of respiratory enzymes in the operation of malate and citrate valves and in controlling the redox state and balancing the energy level of photosynthesizing plant cells is discussed.

The clinicopathologic and molecular features, and treatment outcome of fumarate hydratase-deficient renal cell carcinoma: a retrospective comparison with type 2 papillary renal cell carcinoma.

BACKGROUND: To compare clinicopathologic, molecular features, and treatment outcome between fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and type 2 papillary renal cell carcinoma (T2 pRCC). METHODS: Data of T2 pRCC patients and FH-dRCC patients with additional next-generation sequencing information were retrospectively analyzed. The cancer-specific survival (CSS) and disease-free survival (DFS) were primary endpoint. RESULTS: A combination of FH and 2-succino-cysteine (2-SC) increased the rate of negative predictive value of FH-dRCC. Compared with T2 pRCC cases, FH-dRCC cases displayed a greater prevalence in young patients, a higher frequency of radical nephrectomy. Seven FH-dRCC and two T2 pRCC cases received systemic therapy. The VEGF treatment was prescribed most frequently, with an objective response rate (ORR) of 22.2% and a disease control rate (DCR) of 30%. A combined therapy with VEGF and checkpoint inhibitor reported an ORR of 40% and a DCR of 100%. FH-dRCC cases showed a shortened CSS (P = 0.042) and DFS (P < 0.001). The genomic sequencing revealed 9 novel mutations. CONCLUSIONS: Coupled with genetic detection, immunohistochemical biomarkers (FH and 2-SC) can distinguish the aggressive FH-dRCC from T2 pRCC. Future research is awaited to illuminate the association between the novel mutations and the clinical phenotypes of FH-dRCC in the disease progression.

Multiple cutaneous and uterine leiomyomatosis: Reed's syndrome.

Reed's syndrome (RS) is a rare autosomal-dominant disorder characterised by multiple cutaneous and uterine leiomyomas, with a strong tendency for renal cell carcinoma (RCC) development. A woman in her 50s, who had previously undergone total abdominal hysterectomy due to multiple uterine leiomyomas, presented with painful nodules on her trunk and right arm for the past 6 years. These nodules were confirmed as leiomyomas through histopathology. Diagnosis of RS was established through clinicopathological correlation and positive family history, particularly her mother's. Early-onset uterine leiomyomas in patients with a similar family history should raise suspicion for RS, necessitating vigilant long-term follow-up. RCC detection requires mandatory renal imaging. Screening family members and providing genetic counselling are crucial.

Aggressive renal cell carcinoma with biphasic papillary and solid clear cell features harboring IDH2 (R172M) mutation.

Renal cell carcinoma (RCC) is fundamentally a metabolic disease, and RCC associated with mutation of the Krebs cycle enzyme genes include fumarate hydratase-deficient and succinate dehydrogenase-deficient RCC. Most recently, the mutation of isocitrate dehydrogenase 2 (IDH2) has been suggested as the third Krebs cycle enzyme alteration to be associated with oncometabolite-induced RCC tumorigenesis. Herein, we report the second case of RCC harboring an IDH2 (R127M) mutation identified by targeted next-generation sequencing and further confirmed by reverse transcription polymerase chain reaction and Sanger sequencing. This tumor demonstrated a distinctive biphasic morphology, characterized by mixture of a clear cells solid component and an eosinophilic papillary component. These two components were intermingled and formed variably sized nodular or nested structures. Unfavorable histologic features, including infiltration into the perirenal and renal sinus adipose tissues, high nuclei grade, rhabdoid tumor cells, and focal tumor necrosis, were observed. The patient had local lymph nodes metastases at diagnosis and developed brain metastases 3 months after the surgery. This peculiar case provides further insights into RCCs harboring IDH2 mutations.

Diffuse Carbonic Anhydrase 9 and GATA3 Expression in Fumarate Hydratase Deficient Renal Cell Carcinoma - A Case Report and Immunoprofile Review.

Fumarate hydratase deficient renal cell carcinoma (FHRCC) can exhibit a heterogenous immunoprofile. In the present case, a solitary 10.5 cm mixed cystic and solid left kidney tumor showed various growth patterns, involving renal sinus adipose tissue and the renal pelvis. Tumor cells showed prominent nucleoli and perinucleolar halos. Aberrant diffuse (>90%), strong, and membranous carbonic anhydrase 9 and variable GATA3 expression were present. Diagnostic loss of fumarate hydratase expression and 2-succinyl cysteine overexpression (cytoplasmic and nuclear) were identified. Carbonic anhydrase 9 and GATA3 expression in FHRCC is rarely reported in the literature, and may cause misdiagnosis of clear cell RCC and/or urothelial carcinoma.

The use of Clinicopathological, immunohistochemistry and molecular detection in the diagnosis of fumarate hydratase-deficient uterine leiomyomas.

BACKGROUND: Fumarate hydratase-deficient uterine leiomyomas (FH-dUL) are rare, accounting for only 0.4-1.6% of uterine leiomyomas. FH germline mutation gene is associated with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC). METHODS: In this study, we aim to investigate Clinicopathological features and FH mutation in FH-dUL. We performed a retrospective analysis of 300 cases of uterine leiomyoma, diagnosed from January 2017 to December 2021, within the archives of the Department of Pathology at Peking University People's Hospital. In our review of the immunohistochemical(IHC) staining was performed on 300 uSMTs to detect FH deficiency. RESULTS: We identified 21cases (21/300,7%) of FH-dUL. Nineteen cases (6.33%) displayed negative FH. Twenty-one cases (7%) displayed 2SC diffuse plasma and nuclear staining. The most common FH-d morphological features included staghorn vasculature ( 100%,21/21), alveolar-pattern oedema (71.43%, 15/21), scattered bizarre nuclei (23.81%, 5/21), eosinophilic cytoplasmic (rhabdoid) inclusions (47.62%, 10/21), significant eosinophilic nucleolus with peri-nucleolus hollowing (23.81%, 5/21), ovoid nuclei sometimes arranged in chains (9.52%, 2/21). DNA sequencing for the 21 cases was performed using Next Generation Sequencing (NGS). 6 cases were detected significant variations for the FH gene, 11 cases were detected FH gene mutation forvariants of uncertain significance (VUS), and 2 cases were detected a TP53 gene mutation. No related mutations were detected in the other two cases. CONCLUSIONS: FH-dUL is rare. The combination of predictive Clinicopathological evaluation,FH and 2SC IHC test, and molecular test were helpful for the screening of FH-dUL from uSMTs,or even the screening of HLRCC.

Malignant glomus tumor arising in association with a fumarate hydratase-deficient leiomyoma: An unusual collision tumor.

Collision tumors, defined as "two independent neoplasms that occur in close proximity to one another but maintain distinct boundaries," are quite rare. We report an exceptional collision tumor composed of a genetically confirmed malignant glomus tumor and a fumarate hydratase (FH)-deficient leiomyoma, presenting as a subcutaneous thigh mass in a 38-year-old male who was known to have hereditary leiomyomatosis and renal cell carcinoma syndrome. Microscopic examination identified a biphasic subcutaneous mass comprising sheets and nodules of glomus cells, with nuclear atypia and mitotic activity, and fascicles of mitotically inactive smooth muscle with variably pleomorphic nuclei and intracytoplasmic eosinophilic inclusions, features of FH-deficient leiomyoma. Immunohistochemistry demonstrated loss of FH and robust 2-succinocysteine expression in the smooth muscle, with a normal (FH-retained) expression pattern in the glomus tumor. Next-generation sequencing, performed on the glomus tumor component, identified CARMN::NOTCH2 fusion, characteristic of malignant glomus tumors. Awareness of the distinctive morphologic, immunohistochemical, and molecular genetic features of glomus tumors and FH-deficient leiomyomas is important for correct clinical management of patients with exceptional collision tumors of this type.

Interchangeability of class I and II fumarases in an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z is an industrially promising candidate for bioconversion of methane into value-added chemicals. Here, we have study the metabolic consequences of the breaking in the tricarboxylic acid (TCA) cycle by fumarase knockout. Two fumarases belonging to non-homologous class I and II fumarases were obtained from the bacterium by heterologous expression in Escherichia coli. Class I fumarase (FumI) is a homodimeric enzyme catalyzing the reversible hydration of fumarate and mesaconate with activities of ~94 and ~81 U mg-1 protein, respectively. The enzyme exhibited high activity under aerobic conditions, which is a non-typical property for class I fumarases characterized to date. The calculation of kcat/S0.5 showed that the enzyme works effectively with either fumarate or mesaconate, but it is almost four times less specific to malate. Class II fumarase (FumC) has a tetrameric structure and equal activities of both fumarate hydration and malate dehydration (~45 U mg-1 protein). Using mutational analysis, it was shown that both forms of the enzyme are functionally interchangeable. The triple mutant strain 20Z-3E (ΔfumIΔfumCΔmae) deficient in the genes encoding the both fumarases and the malic enzyme accumulated 2.6 and 1.1 mmol g-1 DCW fumarate in the medium when growing on methane and methanol, respectively. Our data suggest the redundancy of the metabolic node in the TCA cycle making methanotroph attractive targets for modification, including generation of strains producing the valuable metabolites.

Fumarate hydratase (FH) and cancer: a paradigm of oncometabolism.

Fumarate hydratase (FH) is an enzyme of the Tricarboxylic Acid (TCA) cycle whose mutations lead to hereditary and sporadic forms of cancer. Although more than twenty years have passed since its discovery as the leading cause of the cancer syndrome Hereditary leiomyomatosis and Renal Cell Carcinoma (HLRCC), it is still unclear how the loss of FH causes cancer in a tissue-specific manner and with such aggressive behaviour. It has been shown that FH loss, via the accumulation of FH substrate fumarate, activates a series of oncogenic cascades whose contribution to transformation is still under investigation. In this review, we will summarise these recent findings in an integrated fashion and put forward the case that understanding the biology of FH and how its mutations promote transformation will be vital to establish novel paradigms of oncometabolism.

The Role of Morphology in Predicting Fumarate Hydratase-deficient Uterine Leiomyomas in Young Women.

Hereditary leiomyomatosis and renal cell carcinoma is caused by germline mutations in the fumarate hydratase (FH) gene and is associated with an increased incidence of leiomyomas and a potentially aggressive variant of renal cell carcinoma. Pathologic evaluation of uterine leiomyoma can provide an opportunity for early recognition of the syndrome. We reviewed all archived slides of the cases to identify the characteristic morphologic features described for FH-deficient leiomyomas. We performed immunohistochemistry on whole sections of patients with uterine leiomyoma to evaluate for both FH and 2-succinocysteine (2SC) expression. Of the 106 cases, 19 showed the characteristic eosinophilic nucleoli with perinuclear halos, and 24 revealed a characteristic eosinophilic cytoplasmic inclusion consisting of pink globules present within the cytoplasm. Both of these morphologic findings were present together in 15 cases, and hemangiopericytomatous vessels were detected in 23 cases. The loss of FH protein expression was detected in 14 out of 106 cases (13%), and 13 out of 106 cases (12%) were positive for 2SC. We detected 10 cases with both 2SC-positive and FH expression loss. The presence of eosinophilic nucleoli with perinuclear halos and eosinophilic cytoplasmic inclusion was associated with both loss of FH protein expression and 2SC positivity ( P < 0.001). These findings underscore the importance of hematoxylin and eosin-based predictive morphology in FH-deficient uterine leiomyomas. Therefore, morphologic assessment of uterine leiomyomas for features of FH deficiency can serve as a screening tool for hereditary leiomyomatosis and renal cell carcinoma syndrome, allowing patients to be divided according to their hereditary risk assessment.

Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases.

Malate (2-hydroxysuccinic acid) and tartrate (2,3-dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and antielimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class-I fumarate hydratase (FH) and L-tartrate dehydratase (L-TTD) are two highly conserved enzymes belonging to the iron-sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only (S)-malate and (S,S)-tartrate as substrates while the structurally similar L-TTD from Escherichia coli accepts only (R)-malate and (R,R)-tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L-TTDs and two-subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L-TTD. The switch in enantiomer preference may be rationalized by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme-substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity.

AKR1B10 Is a New Sensitive and Specific Marker for Fumarate Hydratase-Deficient Renal Cell Carcinoma.

Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare and distinct subtype of renal cancer caused by FH gene mutations. FH negativity and s-2-succinocysteine (2SC) positivity on immunohistochemistry can be used to screen for FH-deficient RCC, but their sensitivity and specificity are not perfect. The expression of AKR1B10, an aldo-keto reductase that catalyzes cofactor-dependent oxidation-reduction reactions, in RCC is unclear. We compared AKR1B10, 2SC, and FH as diagnostic biomarkers for FH-deficient RCC. We included genetically confirmed FH-deficient RCCs (n = 58), genetically confirmed TFE3 translocation RCCs (TFE3-tRCC) (n = 83), clear cell RCCs (n = 188), chromophobe RCCs (n = 128), and papillary RCCs (pRCC) (n = 97). AKR1B10, 2SC, and FH were informative diagnostic markers. AKR1B10 had 100% sensitivity and 91.4% specificity for FH-deficient RCC. The nonspecificity of AKR1B10 was shown in 26.5% of TFE3-tRCCs and 21.6% of pRCCs. 2SC showed 100% sensitivity and 88.9% specificity. However, nonspecificity for 2SC was evident in multiple RCCs, including pRCC, TFE3-tRCC, clear cell RCCs, and chromophobe RCCs. FH was 100% specific but 84.5% sensitive. AKR1B10 served as a highly sensitive and specific diagnostic biomarker. Our findings suggest the value of combining AKR1B10 and 2SC to screen for FH-deficient RCC. AKR1B10+/2SC+/FH- cases can be diagnosed as FH-deficient RCC. Patients with AKR1B10+/2SC+/FH+ are highly suspicious of FH-deficient RCC and should be referred for FH genetic tests.

Fumarase activity in NAD-dependent malic enzyme, MaeA, from Escherichia coli.

NAD-dependent malic enzymes catalyze NAD reduction to NADH while converting malate to pyruvate and CO2. In this study, NAD was reduced to NADH by MaeA, NAD-dependent malic enzyme from Escherichia coli, when fumarate was used as substrate. This suggested that MaeA catalyzed the conversion of fumarate to malate and then malate to pyruvate. The K0.5 value for fumarate was determined as 13 mM, different from previously characterized fumarases in Escherichia coli. Fumarate inhibited the malic enzyme activity of MaeA where NAD reduction to NADH was examined in the presence of malate as substrate. Human ME2, an NAD-dependent malic enzyme, also converted NAD to NADH in the presence of fumarate, suggesting that the duplex activity as fumarase and malic enzyme might be conserved in various NAD-dependent malic enzymes. MaeB, NADP-dependent malic enzyme from Escherichia coli, did not reduce NADP to NADPH in the presence of fumarate, suggesting the fumarase activities of MaeA and ME2 were specific.

Fumarate hydratase functions as a tumor suppressor in endometrial cancer by inactivating EGFR signaling.

Fumarase hydratase (FH) is an enzyme that catalyzes the reversible hydration and dehydration of fumarate to malate in the tricarboxylic acid cycle. The present study addressed the role of FH in endometrial cancer and clinically observed that the expression of FH was significantly lower in endometrial cancer tissues compared with normal endometrial tissues and, furthermore, that the decreased FH expression in endometrial cancer tissues was significantly associated with increased tumor size and lymph node metastasis. Further analysis in in vitro study showed that cell proliferation, migration and invasion abilities were increased when the expression of FH in the endometrial cancer cells was knocked down, but, by contrast, overexpression of FH in endometrial cancer cells decreased cell proliferative, migratory and invasive abilities. Mechanistic studies showed that the expression of vimentin and twist, being two well-studied mesenchymal markers in endometrial cancer cells, were upregulated in fumarate hydratase-knockdowned cells. In addition, phosphokinase array analysis demonstrated that the expression of phospho-EGFR (Y1086), which promotes carcinogenesis in cancers, was increased in endometrial cancer cells when FH was knocked down. In conclusion, the present study suggested that FH is a tumor suppressor and inhibits endometrial cancer cell proliferation and metastasis by inactivation of EGFR. Further studies are required to clarify its role as a prognostic biomarker and therapeutic target for endometrial cancer.

Cryptic splice mutation in the fumarate hydratase gene in patients with clinical manifestations of Hereditary Leiomyomatosis and Renal Cell Cancer.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.