Our study can be divided into two groups: 37 cases of sporadic head and neck paraganglioma from the Leiden University Medical Center; and 5 index cases with pheochromocytoma and/or paraganglioma and familial antecedents, collected from several other national and international clinical centers.
Of the 37 cases with sporadic head and neck paraganglioma, 2 individuals were identified as carrying heterozygous germline mutations of SDHB (5%).
Two patients carried a splice site mutation in intron 4, c.423+1G>A (Table ). Patient S-003, a 50 year old male, presented with elevated catecholamine levels and single jugular paraganglioma, and died at the age of 58 due to complications resulting from tumor recurrence. A second male patient, S-020, presented at the age of 55 with a single carotid body tumor which was successfully removed, and a subsequent MRI scan three years later showed no abnormalities. This patient was described briefly in a previous report that examined SDH activity in paraganglioma, using the former mutation nomenclature of IVS4+1G>A [20
]. The tumor from this patient was negative for SDH activity.
Mutations of SDHB and SDHC found in this study.
The splice site mutation in intron 4 abolishes the consensus splice donor sequence and thus may result in splicing abnormalities. RNA was available from patient S-020, and RT-PCR analysis confirmed that splicing was shifted 54 nucleotides upstream of the normal splice site, into the exon 4 coding sequence, leading to an in frame deletion of 18 amino acids (Figure ). Normal control RNA did not reveal any alternative splicing. This variant was not found in the control population of 300 chromosomes. This mutation has recently been identified in two other patients of Dutch origin. As these patients are all of ethnic Dutch origin and are apparently unrelated, this mutation may represent a SDHB founder mutation in The Netherlands.
Figure 1 The SDHB mutation c.423+1G>A disrupts normal splicing. Primers in exon 3 and exon 5 were used to amplify cDNA. Lane 1: Patient S-020: In addition to a product of ~250 bp, a fragment missing 54 bp was also amplified (arrow). Sequencing showed an (more ...)
All sporadic patients were also analyzed for mutations in the six exons of SDHC. A single patient, a 36-year-old male of Turkish origin, was found to carry a germline mutation of SDHC. This patient was diagnosed with a single carotid body tumor, and had no known family history. A transition of C to T at base pair 214 in exon 4, resulted in the missense mutation of a highly conserved arginine residue (p.Arg72Cys), located in the putative quinone-binding site of complex II. A C. elegans
, (SDHC) that results in oxidative stress and premature ageing also affects a residue of the quinone binding site, at position 69 of the human sequence [21
]. This mutation is only the fifth identified in SDHC to date. The variant was not found in a Dutch control population of 328 chromosomes (although it should be noted that such a group is not an appropriate ethnic control to exclude the possibility of a non-functional Turkish polymorphism).
The functionality of all missense mutations should be interpreted cautiously, and particularly in a case such as this as this is only the second report of a missense mutation of SDHC and the patient presented without a family history.
In none of the sporadic cases was DNA available from the parents, so we were not able to establish whether these were de novo mutations.
In addition to the cases with sporadic head and neck paraganglioma, 5 index cases with pheochromocytoma and/or paraganglioma were collected, all of which were known or subsequently found to have a family history (Table ).
The first index case, a 36-year-old Dutch female patient (FGT 61.1) presented with a suspected extra-adrenal paraganglioma, and two paragangliomas (close to left kidney) were identified. After surgical removal no additional clinical signs of paraganglioma could be detected. The sister of the index patient (FGT61.2) presented at the age of 27 with symptoms of dizziness and elevated noradrenalin. An extra-adrenal paraganglioma, located between the pancreas and left kidney was identified. A female cousin had previously been diagnosed with paraganglioma but was not available for testing. Both patients were found to carry a germline nonsense mutation of SDHB, c.343C>T (p.Arg115X) which leads to a truncation of the protein at under half its normal length of 280 amino acids.
A second Dutch index patient (FGT66), presented at the age of 67 (2004) with pain in the upper abdomen, and a CT-scan revealed a left-sided retroperitoneal mass. Normetanephrine was highly elevated and were no indications of localisations or metastases elsewhere. The patient's daughter had previously been diagnosed with paraganglioma of the carotid body, that was removed at the age of 29.
No other family members have clinical signs of either paraganglioma or pheochromocytoma. Analysis of DNA from the index patient revealed a missense mutation c. 653G>C (p.Trp218Ser) in exon 7 of SDHB. This missense change affects an amino acid that is conserved in both mammals and in D. melanogaster, C. elegans, S. cerevisiae and A. thaliana. This variant was not found in 318 chromosomes from healthy blood donors. The daughter of the index patient was subsequently tested and also carries the mutation. Other family members are currently receiving genetic counseling.
A 35-year-old Italian female (FGT62.1) presented with a family history of retroperitoneal paraganglioma, severe hypertension, and elevated urinary excretion of catecholamine metabolites. An extra-adrenal paraganglioma, infiltrating the Glissonian of the liver, was surgically removed. At follow-up, MIBG scintiscan showed 3 areas of intense uptake including the site of the previous surgery and two paravertebral lesions in the abdomen and chest. It is not known if these were metastases or primary tumors, as the patient refused further investigation. One year later the patient was suffering from widespread disease with bone metastases.
A sister (FGT62.2) of the index case presented at the age of 13 with an abdominal mass below the aortic bifurcation and increased urinary excretion of catecholamine metabolites. At surgery a 10 × 7 cm retroperitoneal paraganglioma was removed. At the age of 29, a left-sided cervical paraganglioma was diagnosed and removed. After two further relapses the patient presently shows bone metastases and a retroperitoneal mass, possibly a new tumor.
At the age of 60, a paternal aunt (FGT62.3) of the index case was found to have a paraganglioma of the posterior mediastinum. The patient had no further symptoms, and both she and her sons have refused further follow-up.
The index patient was found to carry a c.141G>A germline variant of SDHB resulting in a stop codon at codon 47 (p.Trp47X). Analysis of the DNA of the sister and aunt confirmed their carrier status for the mutation and demonstrated that the mutation segregated with disease. Surprisingly, this mutation was also identified in one sample from the Dutch blood donors screened as controls (1/314 chromosomes). As these donors are anonymous no information is available on the clinical status of this individual.
FGT57.6, a male of Pakistani descent, presented with a retroperitoneal paraganglioma, and had three male relatives (from a large extended family) with similar clinical profiles. A preoperative CT scan revealed a retroperitoneal mass adherent to inferior vena cava and extending up to the left renal vein. Pathology revealed a 9 × 8 × 4 cm encapsulated mass. This patient is currently doing well at age 61.
The brother (FGT57.8) of the index case was first seen at the age 47 with a paraganglioma arising in the diaphragm. The patient is currently alive. A second brother (FGT57.9) presented with adrenal cell carcinoma with bone and lung metastases. An arteriogram revealed an 11 cm highly vascularised adrenal neoplasm, most likely a primary adrenal, with vascular metastases to the left ileum. No remarkable hormonal changes were found. The patient died at age 41.
The nephew of the index patient (FGT57.7) presented at the age of 27 with a recent history of diminished hearing, pulsatile tinnitus and positional vertigo. A CT scan revealed a classic glomus jugulare tumour. The patient completed radiation therapy and remains stable.
The index case and his nephew were available for testing and both were found to be carrying a missense variant of SDHB, c.281G>A, resulting in the substitution of arginine for lysine at codon 94 (p.Arg94Lys). Arginine 94 is highly conserved and adjacent to a cysteine that constitutes one of the highly conserved cysteine regions that are the putative non-heme iron-sulfur clusters of SDHB [22
Finally, a 32-year-old English woman (FGT68) presented with neck pain, swelling at the jaw, left vocal cord palsy and some wasting of the trapezius muscle. An MRI scan showed a para-pharyngeal mass, most likely a vagal paraganglioma. At surgery a highly vascular tumor virtually encircling the internal carotid artery and intimately connected to the hypoglossal nerve was revealed. A sub-total resection was carried out but the tumor progressed and the patient eventually died at the age of 39. The sister of the index patient underwent surgery for a jugulotympanic paraganglioma at the age of 33. The father of the index patient was still alive at the age of 75 and had a vague history of surgery for lumps on the face and neck. Other members of the family currently have no symptoms. The index patient was found to carry a previously described mutation in exon 2 of SDHB, c.136C>T, p.Arg46X [9
]. The family is currently being offered genetic counseling.