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Molecular characterisation of a common SDHB deletion in paraganglioma patients
  1. A Cascón1,2,
  2. Í Landa1,
  3. E López-Jiménez1,
  4. A Díez-Hernández3,
  5. M Buchta4,
  6. C Montero-Conde1,
  7. S Leskelä1,
  8. L J Leandro-García1,
  9. R Letón1,
  10. C Rodríguez-Antona1,
  11. C Eng5,
  12. H P H Neumann4,
  13. M Robledo1,2
  1. 1
    Hereditary Endocrine Cancer Group; Spanish National Cancer Research Centre (CNIO), Madrid, Spain
  2. 2
    ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Spain
  3. 3
    Endocrinology Service, Bierzo Hospital, Ponferrada, Spain
  4. 4
    Department of Nephrology and General Medicine, Albert-Ludwigs-University, Freiburg, Germany
  5. 5
    Genomic Medicine Institute, Lerner Research Institute, and Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio, USA
  1. Dr M Robledo, Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, 28029 Madrid, Spain; mrobledo{at}cnio.es

Abstract

Background: Hereditary susceptibility to familial paraganglioma syndromes is mainly due to mutations in one of six genes, including three of the four genes encoding the subunits of the mitochondrial succinate dehydrogenase complex II. Although prevalence, penetrance and clinical characteristics of patients carrying point mutations affecting the genes encoding succinate dehydrogenase have been well studied, little is known regarding these clinical features in patients with gross deletions. Recently, we found two unrelated Spanish families carrying the previously reported SDHB exon 1 deletion, and suggested that this chromosomal region could be a hotspot deletion area.

Methods: We present the molecular characterisation of this apparently prevalent mutation in three new families, and discuss whether this recurrent mutation is due either to the presence of a founder effect or to a hotspot.

Results: The breakpoint analysis showed that all Iberian Peninsular families described harbour the same exon 1 deletion, and that a different breakpoint junction segregates in an affected French pedigree.

Conclusions: After haplotyping the SDHB region, we concluded that the deletion detected in Iberian Peninsular people is probably due to a founder effect. Regarding the clinical characteristics of patients with this alteration, it seems that the presence of gross deletions rather than point mutations is more likely related to abdominal presentations and younger age at onset. Moreover, we found for the first time a patient with neuroblastoma and a germline SDHB deletion, but it seems that this paediatric neoplasia in a pheochromocytoma family is not a key component of this disease.

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Paragangliomas (PGLs) are tumours derived from parasympathetic (head and neck) and sympathetic (intra-abdominal and thoracic) nervous system paraganglia.1 In contrast, pheochromocytoma (PCC) is a PGL arising from chromaffin cells of the adrenal medulla.2 Three of the four genes encoding the subunits of the mitochondrial succinate dehydrogenase (SDH) complex (SDHB, SDHC, and SDHD) have been related to hereditary susceptibility to familial PCC and/or PGL,35 and have been suggested to be mitochondrial tumour suppressor genes.6

Since the first germline mutation in the SDHB gene was reported in 2001,5 various studies have tried to establish the clinical presentation and the penetrance of the disease, in relation to point mutations in the different SDH subunits.1 7 Thus, SDHB mutation carriers were shown to have almost twice as much risk of developing intra-abdominal PGLs as SDHD mutation carriers, whereas the prevalence of malignant disease is higher in SDHB-related patients.1 7 8 On the other hand, it seems that there is no genotype–phenotype correlation among patients with SDHB point mutations.9 In addition, little is known regarding clinical features of patients carrying gross SDHB deletions since only three studies have reported such alterations to date.1012 In this respect, prevalence, penetrance, and clinical characteristics related to gross deletions remain to be established in order to improve genetic counselling of patients affected with these mutations. In a previous report, we described two unrelated patients carrying a germline loss of exon 1.11 Since this alteration affected the same region as the only previously described family harbouring a gross SDHB deletion at that time,10 we hypothesised that this represented a putative hot spot for chromosomal rearrangements in this region.10 11 Recently, we found three more families, two Spanish and one of French origin, affected with this type of deletion. In the present study, we characterised the deletion breakpoints at the nucleotide level and discuss whether this recurrent mutation is due either to the presence of a founder effect or to a hotspot deletion region. In addition, we studied the clinical features related to the presence of gross SDHB deletions in order to improve the selection of patients at risk for carrying these alterations for molecular diagnosis.

PATIENTS AND METHODS

Patients

Three new PGL families tested positive for the presence of gross SDHB deletions (table 1).

Table 1 Clinical and molecular data of patients carrying the SDHB exon 1 deletion
  • Family 1: the proband was diagnosed at age 19 with retroperitoneal (para-renal) PGL, and multiple metastases in bone, hypophysis, retroperitoneum, and liver. In addition, her sister was diagnosed with an adrenal neuroblastoma with metastasis in five homolateral ganglia at 5 years of age.

  • Family 2: the proband was first diagnosed with abdominal PGL and renal oncocytoma at age 17; both were surgically resected. Ten years later she showed bone metastasis.

  • Family 3: the proband had malignant PCC at age 27 years and died as a result of the disease. He had one relative with benign PCC, diagnosed at age 30 years. In addition, we have reassessed the previously reported cases with deletions affecting SDHB exon 1, with available clinical data.10 11

Genomic DNA was extracted from the patients’ blood following a standard method.13 Informed consent was obtained from all patients. Initial multiplex-polymerase chain reaction (PCR) analysis was performed in 13 individuals, either patients with PGL (n = 8) or unaffected relatives (n = 5), aged 14 to 48 years, from the three new families plus the described pedigrees who tested positive for gross SDHB exon 1 deletions (table 1). The breakpoint characterisation was initially performed based on the two affected members of family 1.

Methods

Multiplex-PCR analysis

The gross deletion analysis was performed using multiplex-PCR primers and conditions previously described.11 In order to narrow down the deleted region in the patients carrying the SDHB exon 1 deletion, we performed a multiplex-PCR using pairs of primers from both SDHB intron 1 and the 5′ upstream SDHB region, separated on the average 2.2 kb from each other (table 2). We used control fragments from exon 6 of SDHB (SDHB6F: 5′-ATG CAC TGA CCC CAA AGG TA-3′; SDHB6R: 5′-CCC AGA TTT ACC GAA AGC AA-3′) and a control region on chromosome 3 as internal controls of the assay. Briefly, multiplex amplifications were performed in 25 μl of a mixture containing 1X multiplex-PCR master mix, 0.2 μM of each primer, and 100–200 ng of genomic DNA. The PCR program started with an initial step at 95°C for 15 min to activate HotStarTaq DNA polymerase. Amplification was for 20 cycles: 30 s at 94°C, 90 s at 60°C, and 90 s at 72°C, followed by 10 min of final extension at 72°C. PCR amplification products were used for fragment analysis on an ABI PRISM 310 capillary sequencer (Applied Biosystems, Perkin Elmer), and analysed using GeneScan v3.1 software (Applied Biosystems, Foster, California, USA). Normalisation was performed by overlapping sequencing runs of samples from patients with a control sample, determining the peak area of all fragments and calculating the normal peak fractions.14

Table 2 Primers used for multiplex-polymerase chain reaction (PCR) and quantitative PCR analysis.

Quantitative real-time PCR

Quantitative real-time PCR assays were carried out using the ABI Prism 7900 Sequence Detection System (Applied Biosystems), and the SYBR Green PCR master mix (Applied Biosystems). The DNA content of the samples was measured by picogreen, and 12.5 and 62.5 ng of DNA were used for independent amplifications in a total volume of 12.5 μl. The primers used for narrowing down the deleted region are described in table 2. SDHB exon 1 amplification (SDHB-1F: 5′-CTC CCA CTT GGT TGC TCG-3′; SDHB-1R: 5′-GGC TTT CCT GAC TTT TCC CT-3′) was quantified as control for deletion, and amplification of a control region located in chromosome 3 (C-F: 5′-CCT TGT ACT GAG ACC CTA GTC TGT CAC T-3′; C-R: 5′-CAA GAC TCA TCA GTA CCA TCA AAA GCT G-3′) was quantified for data normalisation. Primer pairs in SDHB exon 1 and exon 6 were used as controls for deletion and non-deletion, respectively. Data normalisation was performed with a non-deleted region in chromosome 3. The PCR reaction started with 10 min at 95°C, followed by 5 cycles of 20 s at 95°C, 30 s at 58°C, 45 s at 72°C and 45 cycles of 20 s at 95°C, 30 s at 56°C, and 45 s at 72°C. A standard curve, made using serial dilutions of DNA from a control sample without deletions in any of the selected genes, was used to determine the number of copies for each amplicon.15 The ratio of the copy number of SDHB-deleted region fragments to the copy number of the control gene normalises the amount and quality of genomic DNA. The ratio defining the level of amplification is termed N, where N is the copy number of the SDHB amplicon divided by the copy number of the control gene. A normal SDHB diploid sample is thus expected to yield a ratio of N = 1, compared to N = 0.5 for a haploid SDHB genotype. One sample carrying the SDHB exon 1 deletion and one control sample without deletions in the SDHB gene were used as controls. All samples were run in triplicate. The standard deviation of the results was calculated, and in all cases was <17% of the mean value.

Haplotype analysis

Haplotype distribution was determined for all mutation carriers through direct sequencing of regions containing five single nucleotide polymorphisms (SNPs) which define a linkage disequilibrium (LD) block within the SDHB gene locus (from intron 2 to intron 6): rs2235930, rs7550829, rs2746467, rs10887990 and rs4920653. These five “tag-SNPs” were selected using the Tagger tool of the Haploview software package.16 LD blocks and the seven observed haplotypes were previously inferred based on the HapMap Project information for the HapMap-CEU population.17 In addition, two microsatellite markers (D1S2697 and GATA29A05) were tested both in patients and in 100 healthy Spanish controls, in order to better determine the frequency of the haplotype containing the mutation in our population. We used PHASE software for haplotype reconstruction in the control population.18

RESULTS

Using a multiplex-PCR amplification test previously described,11 we found three additional and not previously reported PGL/PCC families, two Spanish and one French, carrying a specific deletion affecting exon 1 of the SDHB gene.

In order to establish the deletion breakpoints occurring in these mutation carriers, we first studied the genomic DNA of the proband and her sister affected with neuroblastoma of family 1 by means of a multiplex-PCR specifically designed for narrowing down the deleted region (fig 1A). Fragment separation and subsequent GeneScan analysis showed one-allele amplification for M1, M2, M4, M5 and M6 amplicons, with retention of the normal biallelic amplification of M3, M7, M8 and M9 amplicons. This result allowed us to limit the 5′ upstream and the intron 1 SDHB breakpoint regions to within −11.3 kb and +6.1 kb relative to the translation start codon, respectively. To further narrow down the regions containing the breakpoints, quantitative PCR analysis was performed using the same samples and a normal control DNA. One-allele amplification was found for Q1, Q2, Q6, Q7, Q8 and Q9, whereas Q3, Q4, Q5 and Q10 showed biallelic amplification (table 3). This allowed us to further narrow down the two regions harbouring the breakpoints to 800 pb each. PCR amplification using a pair of primers flanking the two regions allowed us to amplify a fragment of 2.3 kb that was subsequently sequenced to determine the breakpoints. The deleted region involved close to 16 kb (15.69 kb), and based on the sequence obtained, two new pairs of primers were used to efficiently amplify a fragment of 213 bp as a marker for the SDHB exon 1 deletion and the specific breakpoints identified. All the SDHB exon 1 mutation carriers studied in the present work (n = 12) tested positive for this marker, except for the French family proband. Three normal DNAs, and a DNA corresponding to a non-carrier relative, were used as negative controls.

Figure 1 Breakpoint characterisation of SDHB exon 1 deletion by multiplex-PCR amplification (primer pairs M1–M9) and quantitative PCR (primer pairs Q1–Q10) of: (A) Spanish families; (B) French patient.
Table 3 Results of the quantitative PCR analysis performed in family 1

The same procedure using multiplex and quantitative PCR was used to characterise the SDHB deletion breakpoints in the French family (fig 1B). In this case, all multiplex amplicons except for M3 showed loss, and only Q6, Q7, Q8 and Q9 amplicons were lost in the quantitative PCR analysis. After performing PCR with a pair of primers flanking the theoretical breakpoints we were able to amplify a 3 kb fragment, which was subsequently sequenced to determine the exact location of the breakpoint. A pair of primers was designed to specifically amplify a fragment of 187 pb in the allele carrying the deletion. The deletion was found to span a genomic region of 20.3 kb.

Haplotype analysis of five tag-SNPs covering the non-deleted SDHB region revealed a common haplotype (CCCCT) for all exon 1 positive families, which was also the most frequent one (frequency 58%) in the HapMap-CEU population. The microsatellite analysis also estimated that among all possible combinations in the Spanish population, the mutation was included within the most frequent haplotype (30%).

DISCUSSION

We recently described a procedure to detect gross deletions affecting the SDH genes based on quantification of DNA amplified by means of a multiplex assay.11 Here we present three new families carrying a deletion affecting SDHB exon 1. Molecular characterisation of the deletion revealed the same deletion breakpoints in all Spanish families and a different junction in the French pedigree. This may imply a cost effective mutational analysis by means of a single PCR without subsequent sequencing in Spanish patients.

The Spanish deletion, involving close to 16 kb, was found to harbour a described motif for recombination: the DNA polymerase α frameshift hotspot GGGGGA at position +2.19 On the other hand, the 20 kb deletion detected in the French family contained no evident motif of recombination/deletion at the breakpoint junction. Despite the high density of Alu repeats within the first intron of SDHB, and the presence of an Alu sequence that included the downstream 16 kb breakpoint, it seems that the mechanism of exon 1 deletion is different from the suggested Alu-mediated genomic recombination mechanism.11

We contacted the four Iberian Peninsular probands (including those previously described by our group)11 and although a known familial relationship among them could be discarded, it was striking that all of them originally came from a relatively small area restricted to the northwest of the Iberian Peninsula (fig 2). In addition, all patients from the Iberian Peninsula shared the same breakpoint, which was different from the French one. Haplotype analysis suggested a common origin for all these Peninsular cases and, therefore, most likely represents a founder mutation, although the shared haplotype was the most frequent one both in the Caucasian HapMap population (SNP analysis) and in the Spanish control sample (microsatellite analysis). Two new unrelated cases, with a presumed French origin, have been recently described as carriers of an SDHB deletion affecting exon 1.1 It seems likely that these deletions did involve a hot spot of recombination, but it would be interesting to check if a similar founder effect is responsible for this apparently high prevalence of gross SDHB exon 1 deletion in France.

Figure 2 Geographic distribution (grey circles) of the Iberian Peninsular families carrying the SDHB deletion.

Taking into account the clinical features of the eight PGL/PCC affected patients with deletion of SDHB exon 1 characterised in our laboratory (table 1), six of them were diagnosed with retroperitoneal PGL, and only the two cases from the French family, with a different breakpoint, developed PCC. The presence of retroperitoneal tumours in the affected carriers (75% of cases) is higher than the 50% described for SDHB point mutation carriers,7 but we cannot exclude that this may be due to the small size of our series of patients with gross deletions. In addition, two cases previously reported as carrying gross SDHB deletions also developed thoracic or head and neck PGL.10 11 The mean age of diagnosis in SDHB-exon 1 deletion patients was 24 years (from 8–48 years), which is somewhat lower than the described 31–33 years of age, although the latter studies are population based.7 20

Metastatic disease was found in four out of the eight patients (50%), which is consistent with the percentage described for patients with malignant extra-abdominal PGLs carrying SDHB point mutations causing either truncated proteins or amino acid changes.21 Recently, a shorter survival was reported for SDHB mutation carriers compared to non-related SDHB PGL cases,1 and therefore a timely genetic diagnosis of patients with malignant PGL might be able to help decide on management and treatment.

Key points

  • The deletion of SDHB exon 1 detected in Iberian Peninsular people is probably due to a founder effect.

  • It seems that the presence of gross deletions is related to abdominal presentation and young age at onset, though further studies in larger series are necessary to confirm this clinical association.

  • We report for the first time a patient with neuroblastoma and a germline SDHB deletion.

Two extra-paraganglial tumours were found among the five pedigrees: one renal oncocytoma occurring in the probandus of family 2, and one adrenal neuroblastoma diagnosed in one mutation carrier. The neuroblastoma case was diagnosed with a malignant neuroblastic tumour arising in the adrenal medulla at the age of 5 years, while her sister developed retroperitoneal PGL at 20 years of age. To our knowledge, this is the first reported case of an SDH mutation in a neuroblastoma patient. Because neuroblastoma and PCC have the same embryological origin in the neural crest cells, usually affecting the adrenal glands, and show frequent somatic deletions affecting 1p36 where SDHB maps to, a possible role for this gene in neuroblastoma development has been suggested. Nevertheless, no SDHB point mutations have been reported in neuroblastoma patients22 23; however, no gross deletion analysis has been performed to date. Both the PGL and the neuroblastoma tumour that arose in this family showed loss of 1p36 (data not shown), as expected in these tumours.24 25 Our patient has been closely followed since she first developed the neuroblastoma, and has not developed other adrenal or extra-adrenal pathology at 33 years of age. In addition, no other SDHB mutation carrier has been reported as developing a neuroblastoma at a young age. Although these tumours could be underdiagnosed because they do not show clinical signs,26 it seems that the apparition of this paediatric neoplasia in a PGL family is not a key feature of this disease.

Apart from the patient with neuroblastoma, there were two other unaffected carriers among the studied relatives belonging to two families. The first one was a 22-year-old man from a previously described family (Cascon 2006) that has been adequately followed-up to date, and the second one was the healthy mother of the two affected sisters from family 1. Family 1 is another interesting example of the incomplete penetrance of SDHB mutations since the 67-year-old mother has not developed any feature of the disease, and one of her daughters (aged 33 years) developed only neuroblastoma at 5 years of age. Since the mean age at diagnosis in our series of SDHB-exon 1 deletion patients is 24 years (from 8–48 years), and nothing is known regarding other genetic factors that can modulate the development of the disease, annual clinical examinations are strongly recommended in all carriers. This is even more important, taking into account the malignant outcome described for SDHB mutation carriers.

In summary, we have found that deletion of exon 1 is a common genetic alteration, suggesting the existence of a probable recombination hot spot region. In the Spanish population this alteration affected 25% (4/16) of all SDHB mutation probands analysed in our laboratory (data not shown) and is probably due to a founder effect; a similar founder effect may exist in the French population. Whereas these gross deletions seem to be related to a more frequent abdominal presentation and an earlier age at onset, further studies in larger series are necessary to confirm these clinical differences. Finally, we have shown for the first time a patient with neuroblastoma carrying an SDHB germline mutation. Although it is tempting to relate the development of this tumour to the SDHB deletions, the fact is that it is not a frequent manifestation of the disease.

Acknowledgments

We thank Guillermo Pita for helping us with the haplotype reconstruction.

REFERENCES

Footnotes

  • Funding: This work was supported in part by the Fondo de Investigaciones Sanitarias, projects PI061477 and PI042154, project ISCIII CIBER-ER from the Spanish Ministry of Health, the Deutsche Krebshilfe (70-3313-Ne 1 to HPHN), the Deutsche Forschungsgemeinschaft (NE 571/5-3 to HPHN), and the European Union (LSHC-CT-2005-518200 to HPHN). Alberto Cascon and Cristina Rodríguez-Antona have FIS and ‘Ramon y Cajal’ contracts, respectively, from the Spanish government. Charis Eng is a Doris Duke Distinguished Clinical Scientist.

  • Competing interests: None declared.

  • Patient consent: Informed consent was obtained for publication of the patients’ details in this report.

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