HER2 Overexpression and Amplification in Uterine Carcinosarcomas With Serous Morphology.

Uterine carcinosarcoma (UCS) is an aggressive malignancy with few treatment options. A recent clinical trial has shown an increase in progression-free survival in patients with human epidermal growth factor receptor 2 (HER2)-positive serous endometrial carcinomas treated with anti-HER2-targeted therapies. Few studies have evaluated HER2 expression/amplification in UCS. Similar to serous endometrial carcinoma, the majority of UCS have TP53 mutations and a serous epithelial component, suggesting that UCS may show similar rates of HER2 positivity and therapeutic response. Therefore, we evaluated HER2 expression/amplification in a cohort of UCS over a 5-year period. HER2 immunohistochemistry (IHC) and chromogenic in situ hybridization were performed on tissue microarray and whole tissue sections and scored according to the most recent clinical trial recommendations. Three of 48 UCS (6%) had strong (3+) HER2 IHC expression, and 3 cases (6%) were equivocal (2+). Seven cases (15%) had HER2 amplification by chromogenic in situ hybridization, including all 3 with overexpression and 2 that were equivocal by IHC. Mismatch repair (MMR) protein, p53, and programmed cell death-ligand 1 (PD-L1) expression status was obtained from prior whole section analyses. All HER2-positive cases had a serous morphology and aberrant p53 expression. Only minimal PD-L1 expression was seen in the HER2-positive cases, and none had MMR loss. A subset of UCS with serous morphology have overexpression and/or amplification of HER2, which may predict response to HER2-targeted therapies. HER2-positive UCS may be less susceptible to immune checkpoint inhibition as they uncommonly show MMR deficiency and/or strong PD-L1 expression. Thus, HER2-targeted therapies could be of clinical utility in a subset of UCS without other adjuvant treatment options.

Gene of the month: FH.

Fumarate hydratase (FH), encoded by the FH gene, is an enzyme which catalyses the conversion of fumarate to L-malate as part of the tricarboxylic acid cycle. Biallelic germline mutations in FH result in fumaric aciduria, a metabolic disorder resulting in severe neurological and developmental abnormalities. Heterozygous germline mutations in FH result in hereditary leiomyomatosis and renal cell carcinoma, a cancer predisposition syndrome. FH deficiency has multiple oncogenic mechanisms including through promotion of aerobic glycolysis, induction of pseudohypoxia, post-translational protein modification and impairment of DNA damage repair by homologous recombination. FH-deficient neoplasms can present with characteristic morphological features that raise suspicion for FH alterations and also frequently demonstrate loss of FH immunoreactivity and intracellular accumulation of 2-succinocysteine, also detected by immunohistochemistry.

Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase-deficient renal cancer.

Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.

Prevalence of somatic and germline mutations of Fumarate hydratase in uterine leiomyomas from young patients.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. METHODS AND RESULTS: We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. CONCLUSION: Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

Germline mutation and aberrant transcripts of WWOX in a syndrome with multiple primary tumors.

Multiple primary tumors are defined by the presence of two or more independent primary tumors in the same or different organs of an individual patient. However, the underlying genetic cause for the development of multiple primary tumors is largely unknown. In the study, we report a rare case with four synchronous distinct histological cancer types in a 26 years old Chinese female. In the patient, whole-exome sequencing identified a homozygous germline insertion mutation in WWOX which encodes the DNA repair-related enzyme, WW domain containing oxidoreductase. The mutation was found in a heterozygous state in her parents and brother without any cancer phenotype thus far. Surprisingly, we found multiple novel aberrant WWOX transcripts in the patient's normal colon tissue. The patient's colon metastasis from clear cell adenocarcinoma of the ovary showed a nonhypermutated profile enriched for C-T transition, and harbored somatic pathogenic mutations of HRAS, BRCA2, SMAD4, CHEK2, and AKT1 genes. To our knowledge, this is the first study reporting WWOX gene aberrations in a young patient with the early occurrence of multiple primary tumors. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prospective Detection of Germline Mutation of Fumarate Hydratase in Women With Uterine Smooth Muscle Tumors Using Pathology-based Screening to Trigger Genetic Counseling for Hereditary Leiomyomatosis Renal Cell Carcinoma Syndrome: A 5-Year Single Institutional Experience.

Pathology-based screening of uterine smooth muscle tumors (uSMT) for morphology suggestive of fumarate hydratase deficiency (FH-d morphology) has been proposed as a method to identify women at increased risk for hereditary leiomyomatosis renal cell carcinoma (HLRCC) syndrome. For 5 years our clinical diagnostic practice has evaluated all women with any type of uSMT for FH-d morphology (defined, at low magnification, as staghorn shaped blood vessels and alveolar pattern edema and, at high magnification, as tumor macronucleoli surrounded by a halo and cytoplasmic eosinophilic globules) and, when present, used the pathology report to advise genetic counseling to further evaluate for HLRCC syndrome. We now report the results of this prospective screening strategy, with emphasis on the incidence and clinicopathologic features of FH-d morphology in uSMT, the rate of patient uptake of referral to genetic counseling, and the results of genetic testing for FH germline mutation. Among 2060 women with a uSMT, FH-d morphology was reported in 1.4% (30 women). Ten women elected to undergo FH genetic testing and 6 of 10 (60%) had a FH germline mutation: 5 were pathogenic mutations and 1 was a mutation variant of unknown significance. Therefore, the screening program led to a confirmed genetic diagnosis of HLRCC syndrome in 0.24% of all women with any type of uSMT. The women with a pathogenic mutation were ages 24 to 40 years. Although the majority of leiomyoma with bizarre nuclei exhibited FH-d morphology, the uSMT were conventional leiomyomas with FH-d morphology in 2 of 5 women found to have a pathogenic FH germline mutation. Relying on an abnormal FH immunostain result to trigger genetic counseling referral would have resulted in 2 of 5 (40%) cases with pathogenic FH germline mutation but normal FH immunoexpression going undetected, both of which were missense type mutations. There was no difference in the incidence of pathogenic FH germline mutation between FH-d morphology uSMT with an abnormal versus a normal FH immunostain result. Overall, this study demonstrates that prospective morphology-based screening, integrated with referral for genetic counseling, can result in the diagnosis of HLRCC syndrome in otherwise unselected women with uSMT. We conclude that this strategy should be incorporated in the routine pathologic examination of all uterine smooth muscle tumors.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

Diagnostic Investigation of Lesions Associated with Succinate Dehydrogenase Defects.

The mitochondrial enzyme succinate dehydrogenase (SDH) acts as a tumor suppressor. Biallelic inactivation of one of the genes encoding for SDH subunits (collectively named SDHx) leads to complete loss of the protein function and the development of diverse group of tumors. Pheochromocytomas-paragangliomas are the prime example of hereditary tumors caused by SDH deficiency. In this review, we discuss the roles of imaging examinations, and illustrate new insights into genotype-imaging phenotype relationships.

Succinate Dehydrogenase-Deficient Renal Cell Carcinoma: A Short Review.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme complex composed of 4 protein subunits (SDHA, SDHB, SDHC, and SDHD). Germ line mutations of the genes encoding these SDH subunits result in hereditary syndromes harboring pheochromocytomas/paragangliomas, gastrointestinal stromal tumors, renal cell carcinomas, and pituitary adenomas. SDH-deficient renal cell carcinomas are rare, with a mean age of 38 to 40 years. Histologically, these tumors show a characteristic appearance that includes a solid, nested, or tubular architecture with variable cysts. Cells are typically cuboidal, have indistinct cell borders and eosinophilic cytoplasm, and show flocculent intracytoplasmic inclusions. Loss of immunohistochemical staining for SDHB is the hallmark of these tumors. Although most SDH-deficient renal cell carcinomas are clinically indolent, some tumors may behave aggressively, particularly those with a high nuclear grade, tumor necrosis, or sarcomatoid differentiation. Accurate classification of these tumors is important for clinical follow-up, screening, and genetic evaluation of the patients and other family members for this hereditary tumor syndrome.

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH-/- ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH-/- cell line UOK262. Preferential cell death against UOK262-FH-/- was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH-/- sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH-/- , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.

Succinate dehydrogenase (SDH)-deficient neoplasia.

The succinate dehydrogenase (SDH) complex is a key respiratory enzyme composed of four subunits: SDHA, SDHB, SDHC and SDHD. Remarkably, immunohistochemistry for SDHB becomes negative whenever there is bi-alleic inactivation of any component of SDH, which is very rare in the absence of syndromic disease. Therefore, loss of SDHB immunohistochemistry serves as a marker of syndromic disease, usually germline mutation of one of the SDH subunits. Tumours which show loss of SDHB expression are termed succinate dehydrogenase-deficient. In addition to loss of SDHB, tumours associated with SDHA mutation also show loss of SDHA expression. Fifteen per cent of pheochromocytoma and paraganglioma (PHEO/PGL) are associated with germline SDH mutation, and therefore SDH-deficient. We recommend screening SDHB immunohistochemistry for all PHEO/PGL. SDH-deficient gastrointestinal stromal tumours (GISTs) show distinctive features, including absent KIT proto-oncogene receptor tyrosine kinase/platelet-derived growth factor receptor A (KIT/PDGFRA) mutations [but positive staining for cKIT and DOG1], virtually exclusive gastric location, lobulated growth, multi-focality, a prognosis not predicted by size and mitotic rate, frequent metastasis to lymph nodes and primary resistance to imatinib therapy. Thirty per cent are associated with SDHA germline mutation and 50% are associated with SDHC epimutation (post-zygotic promoter hypermethylation) - the hallmark of the syndromic but non-hereditary Carney triad (SDH- deficient GIST, SDH-deficient paraganglioma and pulmonary chondroma). SDH-deficient renal carcinoma is newly recognized under the World Health Organization (WHO) 2016 classification and shows vacuolated eosinophilic cytoplasmic and cytoplasmic inclusions. It is particularly associated with SDHB mutation, although SDHC and SDHA mutation occur. SDH-deficient pituitary adenomas are recognized, but appear to be the least common SDH-deficient neoplasm.

A case of neuroblastoma in DICER1 syndrome: Chance finding or noncanonical causation?

DICER1 syndrome is an inherited disorder associated with at least a dozen rare, mainly pediatric-onset tumors. Its characterization remains incomplete. Some studies suggested that neuroblastoma (NB) may be involved in this syndrome. Here, we describe the case of a 14-year-old female presenting with a multinodular goiter (MNG) and a collision tumor composed of NB and cystic nephroma (CN). She is a carrier of a deleterious germline mutation in exon 23 of DICER1 and harbored different somatic mutations in the CN and MNG. However, no second hit was found in the NB, questioning its status as a DICER1-related tumor.

Tropomyosin Receptor Antagonism in Cylindromatosis (TRAC), an early phase trial of a topical tropomyosin kinase inhibitor as a treatment for inherited CYLD defective skin tumours: study protocol for a randomised controlled trial.

BACKGROUND: Patients with germline mutations in a tumour suppressor gene called CYLD develop multiple, disfiguring, hair follicle tumours on the head and neck. The prognosis is poor, with up to one in four mutation carriers requiring complete surgical removal of the scalp. There are no effective medical alternatives to treat this condition. Whole genome molecular profiling experiments led to the discovery of an attractive molecular target in these skin tumour cells, named tropomyosin receptor kinase (TRK), upon which these cells demonstrate an oncogenic dependency in preclinical studies. Recently, the development of an ointment containing a TRK inhibitor (pegcantratinib - previously CT327 - from Creabilis SA) allowed for the assessment of TRK inhibition in tumours from patients with inherited CYLD mutations. METHODS/DESIGN: Tropomysin Receptor Antagonism in Cylindromatosis (TRAC) is a two-part, exploratory, early phase, single-centre trial. Cohort 1 is a phase 1b open-labelled trial, and cohort 2 is a phase 2a randomised double-blinded exploratory placebo-controlled trial. Cohort 1 will determine the safety and acceptability of applying pegcantratinib for 4 weeks to a single tumour on a CYLD mutation carrier that is scheduled for a routine lesion excision (n = 8 patients). Cohort 2 will investigate if CYLD defective tumours respond following 12 weeks of treatment with pegcantratinib. As patients have multiple tumours, we intend to treat 10 tumours in each patient, 5 with active treatment and 5 with placebo. Patients will be allocated both active and placebo treatments to be applied randomly to tumours on the left or right side. The target is to treat 150 tumours in a maximum of 20 patients. Tumour volume will be measured at baseline and at 4 and 12 weeks. The primary outcome measure is the proportion of tumours responding to treatment by 12 weeks, based on change in tumour volume, with secondary measures based on adverse event profile, treatment compliance and acceptability, changes in tumour volume and surface area, patient quality of life and pain. DISCUSSION: Interventions for rare genetic skin diseases are often difficult to assess in an unbiased way due to small patient numbers and the challenges of incorporating adequate controls into trial design. Here we present a single-centre, randomised, placebo-controlled trial design that leverages the multiplicity of tumours seen in an inherited skin tumour syndrome that may inform the design of other studies in similar genetic diseases. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Registry, ISRCTN75715723 . Registered on 22 October 2014.

Immunohistochemical Characterization of Fumarate Hydratase (FH) and Succinate Dehydrogenase (SDH) in Cutaneous Leiomyomas for Detection of Familial Cancer Syndromes.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by germline mutations in the FH gene, and is associated with increased incidence of leiomyomas and a potentially aggressive variant of renal cell carcinoma (HLRCC-associated RCC). Absent immunohistochemical expression of fumarate hydratase (FH) has previously been used to diagnose HLRCC-associated RCC, but immunohistochemical staining of leiomyomas is not standard practice. We performed immunohistochemistry (IHC) on whole sections from consecutive cutaneous leiomyomas from our archives to evaluate for both FH and succinate dehydrogenase B expression, in addition to clinicopathologic data collection and review of all hematoxylin and eosin-stained slides for blinded morphologic evaluation of features reported to be seen in HLRCC-associated uterine leiomyomas. Ninety-six cutaneous leiomyomas from 87 patients were identified; 12 of these specimens were from 7 patients with documented HLRCC. FH expression by IHC was absent in 9 specimens and retained in 85 specimens; 2 cases were equivocal with minimal FH expression. Seven of the 9 absent expression specimens were from patients with HLRCC, as were both of the equivocal specimens. The overall sensitivity and specificity of absent FH expression in leiomyomas for detection of patients with HLRCC were 70.0% and 97.6%, respectively. Inclusion of cases classified as equivocal increased sensitivity to 75.0%. Succinate dehydrogenase B expression was retained in 95 specimens and equivocal in 1 specimen. None of the evaluated morphologic features showed any association with leiomyomas in HLRCC. Loss of FH immunohistochemical expression in cutaneous leiomyomas is a sensitive and specific marker for detection of HLRCC, thus suggesting a role for prospective FH IHC in patients with these tumors to screen for HLRCC.

Rapamycin inhibits the proliferation of endothelial cells in hemangioma by blocking the mTOR-FABP4 pathway.

FABP4 is widely expressed in both normal and pathologic tissues. It promotes cell proliferation, survival and migration of endothelial cells, and therefore, angiogenesis. However, the role of FABP4 in hemangioma or hemangioma endothelial cells (HemECs) has not been explored. In this study, we investigated whether FABP4 directly regulates the proliferation of HemECs. The expression of cell cycle checkpoint genes was analyzed with the microarray data of human dermal microvascular endothelial cells (HDVECs) and infantile hemangioma endothelial cells. Real-time RT-PCR and western blotting were used to examine the expression of FABP4 in HemECs. Next, the FABP4 expression was inhibited in HemECs using siRNA or rapamycin and upregulated using retroviral transduction of HemECs to assess its influence on proliferation of HemECs. The microarray data showed that cell cycle checkpoint genes were upregulated in HemECs. Moreover, HemECs showed significantly higher proliferation rates than HDVECs. The expression of FABP4 and mTOR was increased in the HemECs. While FABP4 knockdown reduced the BrdU incorporation and cell number of HemECs as expected, cell proliferation was accelerated by FABP4 over-expression. Moreover, rapamycin (10nM) inhibited mTOR-FABP4 signaling and HemEC proliferation. Taken together, these results indicated that mTOR signaling pathway-activated FABP4 directly regulates the proliferation of endothelial cells in hemangioma. Rapamycin and inhibitors of FABP4 have therapeutic potential for treating infantile hemangiomas.

No difference in phenotype of the main Dutch SDHD founder mutations.

OBJECTIVE: SDHD mutations predispose carriers to hereditary paraganglioma syndrome. The objective of this study was to assess the genotype-phenotype correlation of a large Dutch cohort of SDHD mutation carriers and evaluate potential differences in clinical phenotypes due to specific SDHD gene mutations. DESIGN: Retrospective, descriptive single-centre study. PATIENTS: All consecutive SDHD mutation carriers followed at the Department of Endocrinology of the Leiden University Medical Center were included. MEASUREMENTS: Subjects were investigated according to structured protocols used for standard care, including repetitive biochemical and radiological screening for paragangliomas. RESULTS: Two hundred and one SDHD mutation carriers with a mean age at presentation of 42·6 ± 14·4 years and a mean follow-up of 5·8 ± 5·4 years were evaluated. Eighty-one percent carried the SDHD c.274G>T (p.Asp92Tyr) mutation and 13% the SDHD c.416T>C (p.Leu139Pro) mutation. No differences in clinical phenotype between these two specific SDHD mutations were found. Ninety-one percent developed one or multiple paragangliomas in the head and neck region (HNPGLs), of which the carotid body tumour was the most prevalent (85%). Eighteen carriers developed pheochromocytomas, fifteen sympathetic paragangliomas and nine carriers (4%) suffered from malignant paraganglioma. By end of follow-up, sixteen SDHD mutation carriers (8%) displayed no biochemical or radiological evidence of manifest disease. CONCLUSIONS: The two main Dutch SDHD founder mutations do not differ in clinical expression. SDHD mutations are associated with the development of multiple HNPGLs and predominantly benign disease.

Mast cells in infantile haemangioma possess a primitive myeloid phenotype.

AIMS: Recent reports on infantile haemangioma (IH) have demonstrated a primitive population of interstitial cells expressing the embryonic transcription factor, Nanog, with decreasing abundance during involution. In this report we investigated the expression of Nanog on mast cells in all three phases of IH progression. METHODS: Paraffin-embedded sections of six proliferating, six involuting and six involuted IH lesions were used to investigate the expression of tryptase, Nanog, CD45, CD34 and GLUT-1 by immunostaining. RESULTS: Mast cells, identified by their expression of tryptase, were located in the interstitium of IH lesions. 93%, 42% and 0% of these tryptase(+) cells also expressed Nanog, in proliferating, involuting and involuted IH, respectively. CONCLUSIONS: The identification of an abundant population of tryptase(+)/Nanog(+) cells in IH is novel. The relative loss of Nanog expression as IH involutes may be a result of maturation and/or proliferation of these cells. This report supports the primitive nature of IH.

[Familial paraganglioma syndrome: phenotype and relevance of a new SDHB mutation]. / Síndrome de paraganglioma familiar: expresividad clínica y relevancia de una nueva mutación en SDHB.

BACKGROUND AND OBJECTIVE: Advances in molecular biology have discovered new genes involved in the development of familial paraganglioma syndrome (PGL) including those encoding mitochondrial succinate dehydrogenase complex (SDH). We describe the diagnosis, clinical expression and genetic counselling in a family diagnosed of PGL due to a new SDHB mutation. PATIENTS AND METHOD: Genetic study by PCR-direct sequencing SDHB gene and biochemical determination in blood/urine fractionated catecholamine 24h, metanephrines and conventional (computed tomography/magnetic resonance imaging) and functional imaging ((123)I-MIBG) in all members of a family diagnosed of PGL. RESULT: DNA sequencing showed a non-described SDHB heterozygous mutation (c.287-3C>G intron3/exon4) in 5 of the subjects (71%). The estimated penetrance of the mutation's carriers was 40%, with a mean age of 35 years at diagnosis. All patients with active illness required surgical treatment after imaging and laboratory confirmation. CONCLUSIONS: We describe the pathogenicity, diagnostic algorithm, genetic counselling and clinical expression of a new SDHB mutation (c.287-3C>G) in a family diagnosed of PGL.