Somatic Loss of Maternal Chromosome 11 Causes Parent-Of-Origin-Dependent Inheritance in SDHD-Linked Paraganglioma and Phaeochromocytoma Families

Somatic loss of maternal chromosome 11 causes parent-of-origin-dependent inheritance in SDHD-linked paraganglioma and phaeochromocytoma families

Erik F Hensen1,2, Ekaterina S Jordanova2, Ivonne JHM van Minderhout3, Pancras CW Hogendoorn2, Peter EM Taschner3, AndelGL van der Mey 1, Peter Devilee2,3 and Cees J Cornelisse*,2

1Department of Otolaryngology and Head and Neck Surgery, Leiden University Medical Centre, Albinusdreef 2, H4Q, 2333 ZA, Leiden, The Netherlands; 2Department of Pathology, Leiden University Medical Center, Albinusdreef 2, L1Q, 2333 ZA, Leiden, The Netherlands; 3Department of Human Genetics, Wassenaarseweg 72, 2333 AL, Leiden University Medical Center, Leiden, The Netherlands

Germline mutations in succinate dehydrogenase subunits Introduction B, C and D (SDHB, SDHC and SDHD), genes encoding subunits of mitochondrial complex II, cause hereditary Paragangliomas (PGL) of the head and neck are paragangliomas and phaeochromocytomas. In SDHB neuroendocrine tumours arising in branchiomeric and (1p36)- and SDHC (1q21)-linked families, disease in- intravagalparaganglia.They are rare, highlyvascular, heritance is autosomal dominant. In SDHD (11q23)- mostly benign tumours usually characterized by an linked families, the disease phenotype is expressed only indolent growth pattern. Paragangliomas, like normal upon paternal transmission of the mutation, consistent paraganglia, consist of two cell types: the type I or chief with maternal imprinting. However, SDHD shows bialle- cells, which represent the neoplastic population in lic expression in brain, kidney and lymphoid tissues paragangliomas (van Schothorst et al., 1998a), and the (Baysal et al., 2000). Moreover, consistent loss of the type II or sustentacular cells. The most common site is wild-type (wt) maternal allele in SDHD-linked tumours the carotid body, a chemoreceptive organ in the suggests expression of the maternal SDHD allele in bifurcation of the carotid artery that senses oxygen normal paraganglia. Here we demonstrate exclusive loss levels in peripheral blood in a way that is not yet fully of the entire maternal chromosome 11 in SDHD-linked understood. Most paragangliomas appear to be spora- paragangliomas and phaeochromocytomas, suggesting dic, but a significant minority of the cases (10–50%) has that combined loss of the wt SDHD allele and maternal been shown to be familial. Recently, several genes have 11p region is essential for tumorigenesis. We hypothesize been implicated in these familial forms of the disease. that this is driven by selective loss of one or more Analysis of families carrying the PGL1 gene revealed imprinted genes in the 11p15 region. In paternally, but not germline mutations in the succinate dehydrogenase in maternally derived SDHD mutation carriers, this can complex-subunit D (SDHD) gene on 11q23 (Baysal be achieved by a single event, that is, non-disjunctional et al., 2000). This gene encodes a mitochondrialprotein, loss of the maternal chromosome 11. Thus, the exclusive an anchoring subunit of the mitochondrialrespiratory paternal transmission of the disease can be explained by a chain complex II. Subsequently, mutations in other somatic genetic mechanism targeting both the SDHD subunits of the same mitochondrial complex II were also gene on 11q23 and a paternally imprinted gene on found to be associated with hereditary paraganglioma. 11p15.5, rather than imprinting of SDHD. The SDHB gene (1p36.1–p35) encodes a catalytic Oncogene (2004) 23, 4076–4083. doi:10.1038/sj.onc.1207591 subunit of mitochondrialcomplexII and has been Published online 5 April 2004 implicated in familial paraganglioma of the head and neck as well as in familial paraganglioma of the adrenal Keywords: paraganglioma; phaeochromocytoma; im- medulla, better known as pheochromocytoma (Astuti printing; SDHD; inheritance; monosomy 11 et al., 2001). Both SDHD and SDHB appear to act as tumour-suppressor genes in hereditary paraganglioma. The SDHC gene (1q21) encodes the second anchoring subunit of the mitochondrialcomplexII and mutations in this gene have recently been shown to cause hereditary paraganglioma as well (Niemann and Muller, 2000). Furthermore, a hereditary paraganglioma family *Correspondence: CJ Cornelisse; E-mail: [email protected] with linkage to a region on 11q13.1, the PGL2 locus, has Received 22 October 2003; revised 23 January 2004; accepted 30 January been described (Mariman et al., 1993). However, no 2004; Published online 5 April 2004 mitochondrial complex II genes are known to be located Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4077 in this region. Interestingly, strikingly different inheritance patterns have been found for paragangliomas of different genetic background. Whereas SDHB-and SDHC-linked pedigrees show autosomal dominant inheritance, SDHD- and PGL2-linked pedigrees exhibit a clear parent-of-origin effect: inheritance of paraganglioma occurs in an autosomal dominant way only when paternally transmitted, while no phenotype develops after maternaltransmission. This pattern is consistent in all SDHD- linked pedigrees, and suggests sex-specific epigenetic modification of the maternal SDHD allele, consistent with genomic imprinting (van der Mey et al., 1989). However, no evidence of a physicalimprint, for example, methylation of the 11q22.1–23 region, has been found. Furthermore, the SDHD gene is biallelicly expressed in human brain, kidney and lymphoid tissue (Baysal et al., 2000). It has been suggested that the imprinting of SDHD is restricted to the paraganglia cells, but loss of the maternal SDHD allele is frequently observed in paraganglioma from SDHD-mutation carriers, an event that is unlikely to promote tumour growth when the maternal allele is already silenced by an imprint (Baysal et al., 1997, 2000; Dannenberg et al., 2001). We hypothesized that somatic, selective loss of the whole maternal chromosome 11 could explain the exclusive paternal inheritance of the disease, mimicking maternalimprinting of the SDHD gene. We performed fluorescent in situ hybridisation (FISH) studies on 23 SDHD-linked tumours using different probe sets in order to test for loss of chromosome 11, and loss of heterozygosity (LOH) analysis using several microsatel- lite markers to determine the parental origin of the lost chromosome. Complete loss of a chromosome 11 copy was found in all tumours, and LOH analysis on a subset of seven tumours from patients for whom parental DNA samples were available revealed the exclusive Figure 1 Results obtained from interphase FISH analysis of maternalorigin of the lostchromosome. We propose paraffin-embedded sections of five SDHD-linked paragangliomas that the selective loss of the maternal chromosome 11 (P1–5). (a) Frequency distribution of signals obtained with the centromere 1 (PUC1.77) probe (upper panel) and the centromere copy is driven by the allelic phasing of the SDHD 11 (pLC11A) (lower panel). Compared to chromosome 1, there is a germline mutation and a paternally imprinted tumour- clear loss of chromosome 11 centromere signals. More than two suppressor gene on 11p15. chromosome 1 signals are observed in 9–17% of the nuclei, indicating aneuploidy or tetraploidy. (b) Loss of centromere 11 relative to centromere 1 signals (red and orange) is observed in 46– 65% of the nuclei. Loss of centromere 1 signals relative to centromere 11 (‘other combinations’) is 2–11% Results

We started with FISH experiments on tissue sections from ﬁve paragangliomas from D92Y carriers. The signals for chromosome 11, whereas of the nuclei with rationale for initially choosing sections rather than cell only one signal for chromosome 1, 0–7% had no signals suspensions was the expectation that this would facil- for chromosome 11. itate the visual selection of nuclei of the type I (chief) To exclude the possibility that loss of signals due to cells. The sections were hybridized with centromere tissue sectioning could have interfered with the results, probes for chromosomes 11 and 1, the latter chromo- we next hybridized isolated whole nuclei of 10 para- some serving as a ploidy reference. Loss of centromere gangliomas, three of which were also studied in the ﬁrst 11 relative to centromere 1 was found in all tumours, in study. Whereas the use of suspensions precluded the 45–65% of nuclei (Figure 1). Of the nuclei with three selection of type I cells, evaluation of an unselected signals for chromosome 1, 13–54% had two signals for sample of 200 nuclei still demonstrated the relative loss chromosome 11, 5–54% had one signalfor chromosome of centromere 11 in all samples in 35–63% of nuclei 11 and 5–32% had no signals for chromosome 11. Of (Figure 2). To discriminate between loss of the entire the nuclei with two signals for chromosome 1, 44–66% chromosome and subchromosomal loss due to complex had one signalfor chromosome 11 and 8–31% had no rearrangements, we next analysed isolated whole nuclei

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4078

Figure 2 Interphase FISH results from isolated whole nuclei of 10 SDHD-linked paragangliomas (P1–10). (a) Frequency distribution of signals obtained with the centromere 1 (PUC1.77) probe (upper panel) and the centromere 11 (pLC11A) probe (lower panel). Compared to chromosome 1, there is a clear loss of chromosome 11 centromere signals. More than two chromosome 1 signals are observed in 12–40% of the nuclei, indicating aneuploidy or tetraploidy. (b) Loss of centromere 11 relative to centromere 1 signals (red and orange) is observed in 35–63% of the nuclei. Loss of centromere 1 signals relative to centromere 11 (‘other combinations’) is negligible (0–1%)

of nine paragangliomas and two pheochromocytomas of paraganglioma and 31–62% of pheochromocytoma from D92Y mutation carriers that were not used in the nuclei (Figure 4a). previous studies, using a triple colour FISH technique. Next, we used BAC probes for the subtelomeric This allows simultaneous detection of two probes on regions of 11p and 11q, with the centromere 1 probe as a chromosome 11 and one probe on centromere 1 reference (Figure 3b and d). Concomitant loss of both (Figure 3). First, we studied the centromere 1 and 11 probes located on chromosome 11 relative to centro- probes in combination with a BAC probe that covers the mere 1 was found in 26–70% of paraganglioma and SDHD gene on 11q23 (Figure 3a and c). Concomitant 23–54% of pheochromocytoma nuclei (Figure 4b). In loss of both probes located on chromosome 11 relative both triple-colour experiments, loss of one of the two to centromere 1 was observed in all samples, in 24–65% probes located on chromosome 11 relative to the other

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4079 chromosome arms. For two paragangliomas, the analysis was informative for only one marker, either on 11p or 11q. In aneuploid- or G2,M-cell populations, all evaluable LOH experiments showed loss of maternal alleles. As expected, retention of heterozygosity was not observed (Figure 5). In the diploid cell populations and patient PBL DNA samples, no LOH was found (data not shown).

Discussion

The results obtained in this study demonstrate the loss of an entire copy of chromosome 11 in all investigated SDHD-linked paragangliomas. By LOH analysis, we were able to unequivocally demonstrate the maternal origin of the lost chromosome copy in a subset of seven paraganglioma and two phaeochromocytoma cases from which parental blood DNA samples were available. However, even without this direct proof, Knud- son’s two-hit modelpredicts that in case of paternal transmission of the germline mutation, loss of the wild- Figure 3 Triple colour FISH on whole nuclei isolated from type maternal allele should have occurred in the tumour. paraffin-embedded tissue. Probe/colour combinations are centro- Although loss of a centromere 11 already indicates mere 11 (pLC11A, green), centromere 1 (PUC1.77, blue) and 11q23 loss of the entire chromosome 11, we obtained (RP11-3F7, red) (a, c), and subtelomere 11p (RP11-645I8, green), subtelomere 11q (RP11-469N6, red) and centromere 1 (blue) (b, d). additional evidence by the triple colour FISH experi- Each panel is a composite of individually captured nuclei. (a) ments with telomeric probes and the 3F7 probe Paraganglioma cell nuclei. Top left: diploid nucleus with two containing the SDHD gene. Since it was not possible signals for each probe, top right: monosomy for chromosome 11, to accurately discriminate type I cells in the FISH bottom: tetraploidy for centromere 1 and diploidy for each chromosome 11 probe. (b) Paraganglioma cell nuclei. Top left: experiments on isolated nuclei, the evaluation of an diploid nucleus, top right: chromosome 11 monosomy and relative unselected sample of 200 nuclei unavoidably included chromosome 11 loss in a tetraploid nucleus (bottom). (c) non-neoplastic cells as well. This explains most of the Phaeochromocytoma cell nuclei. Top left: diploid nucleus, top variation in loss of chromosome 11 between the right: chromosome 11 monosomy, bottom left: relative chromo- different cases and the concordance of the results some 11 loss in a tetraploid nucleus, bottom right: tetraploid nucleus without relative chromosome 11 loss. (d) Phaeochromocy- obtained with different probe sets for the individual toma cell nuclei. Top left: diploid nucleus, top right: chromosome tumours (Figure 4). FISH on tissue sections, while 11 monosomy, bottom left: relative chromosome 11 loss in a permitting selection of type I cell nuclei, did not yield tetraploid nucleus, bottom right: a tetraploid nucleus without significantly higher percentages of nuclei with relative relative chromosome 11 loss chromosome 11 loss because of loss of signals from sliced nuclei. The latter problem would have seriously was observed in only a small minority of nuclei (1–7% complicated, if not precluded, the interpretation of triple and 2–4%, respectively), demonstrating that the ob- colour FISH experiments on tissue sections and thus served relative loss of chromosome 11 involves the entire nuclear suspensions were used in all further experiments. copy. Thus, relative loss of chromosome 11 signals was The selective loss of the entire maternal chromosome observed in all 23 tumours, ranging from 23 to 70% 11 explains why SDHD-linked tumours appear to arise (mean ¼ 40%). only upon paternal transmission of the mutation, even To determine the parentalorigin of the lostchromo- though the SDHD gene itself is not imprinted. The latter some 11, we performed LOH analysis on seven is supported by the observed biallelic expression of paragangliomas and two pheochromocytomas that were SDHD in severalhuman tissues (Baysal et al., 2000). also analysed by triple-colour FISH. For these cases, Although it is not uncommon for the somatic ’second patient- as well as parental PBL-derived DNA samples hit’ in the Knudson modelof tumorigenesis to involve a were available. LOH analysis was performed after gross chromosomalmechanism such as non-disjunc- tumour cell populations were enriched by fluorescence- tionalchromosome loss,it is intriguing that in SDHD- activated cell sorting (FACS) of the aneuploid G0,1 linked paragangliomas this appears to be the preferred fraction, or the often increased G2,M fraction of diploid mechanism for the second hit. We hypothesize that a tumours, with the diploid G0,1 fraction as a reference second target gene on chromosome 11, which is subject (van der Mey et al., 1991; van Schothorst et al., 1998a). to genomic imprinting, is involved in tumour formation. We used three markers on 11p and five on 11q. In five A growth advantage is gained when the wild-type paragangliomas and two pheochromocytomas, LOH maternal SDHD allele on 11q23 and the active maternal analysis was informative for at least one marker on both copy of this second, paternally imprinted gene are lost

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4080

Figure 4 Counts of whole nuclei isolated from parafﬁn-embedded material of paragangliomas (P6–14) and phaeochromocytomas (Ph 1–2), analysed by triple colour interphase FISH. (a) Results for centromere 11 (pLC11A), centromere 1 (PUC1.77) and 11q23 (RP11- 3F7) probes. Simultaneous loss of both chromosome 11 probes relative to centromere 1 (red and orange) was observed in 24–65% of paragangliomas and 31–62% of phaeochromocytomas. (b) Results for centromere 1, subtelomeric 11p (RP11-645I8) and 11q (RP11- 469N6) probes. Simultaneous loss of both chromosome 11 probes relative to centromere 1 (red and orange) was observed in 26–70% of paragangliomas and 23–54% of phaeochromocytomas. For each tumour, distributions are very similar in (a) and (b) indicating high reproducibility of the technique. Note that in both (a) and (b) nonsimultaneous loss of chromosome 11 probes or loss of centromere 1 signals relative to chromosome 11 signals (white) is infrequent (3–8 and 2–7%, respectively)

Within that model, the most parsimonious mechanism would be a single event, viz. the loss of the entire maternalchromosome 11 copy in case of a maternalwt SDHD allele and paternal inheritance of the SDHD mutation (Figure 6a). Loss of the maternalwt SDHD allele only, for example, by loss of a part of 11q, would not target the second tumour-suppressor gene on 11p15 and therefore not lead to tumour formation (Figure 6b). In case of maternalinheritance of the SDHD mutation, loss of paternal alleles would not lead to tumour formation for the same reason (Figure 6c and d). At least two events caused by different chromosomal mechanisms will be required to inactivate both SDHD and the imprinted gene on 11p15 when SDHD is maternally transmitted. These are successive loss of the paternalwild-type SDHD allele by, for example, mitotic recombination, followed by loss of the recombined paternalchromosome containing the paternal11q23 region and the maternal11p15 region (Figure 6e). Given the evidence for complex LOH mechanisms in solid Figure 5 LOH analysis of sorted aneuploid G0,1 or diploid G2,M fractions of isolated nuclei of parafﬁn-embedded paragangliomas tumours (Devilee et al., 2001; Balmain et al., 2003), it is (P12–19) and phaeochromocytomas (Ph1–2). LOH involved the somewhat surprising that the probability of this maternal allele in all cases in which the parental origin of the lost occurring in paraganglioma formation appears to be allele could be assessed (black). Retention of heterozygosity was very low or zero, since no cases of maternal transmission not found for any of the informative markers have been reported to date. One explanation might be that the number of cell divisions in normal paraganglia simultaneously. As the only region known to harbour an is simply too low since in most head and neck imprinted gene cluster on chromosome 11 is 11p15, we paragangliomas the growth fraction is lower than 1% further hypothesize that this second gene is located here. (Douwes Dekker et al., 2003). Selective loss of the whole

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4081

Figure 6 Modelfor the imprinted transmission of SDHD-linked paraganglioma. Maternal (white) and paternal (grey) chromosomes are depicted. (a) Both the maternal11q region, containing the wt SDHD allele, and the maternal 11p region, containing the active tumour suppressor allele, are targeted. In case of an event targeting only the wt maternal SDHD allele on 11q (b), the active maternal tumour suppressor allele on 11p15 is not affected and tumour development is inhibited. In case of maternal inheritance of the SDHD mutation, a second hit targeting the wt paternal allele by, for example, a deletion of the paternal 11q region (c) or even the whole paternalchromosome 11 ( d) will leave the maternal 11p15 region intact and tumour formation is not initiated. When the SDHD mutation is maternally transmitted, at least two events caused by different chromosomal mechanisms will be required to inactivate both the wt SDHD allele and the active maternal allele of the imprinted tumour-suppressor gene on 11p15, namely loss of the paternal wild-type SDHD allele by, for example, mitotic recombination, followed by loss of the recombined paternal chromosome containing the paternal11q23 region and the maternal11p15 region ( e). Apparently, this sequence of events is very unlikely in vivo maternal chromosome 11 would explain the exclusive LOH of maternal11p15, often with duplicationof paternaltransmission of disease in paraganglioma paternal11p15, occurs frequentlyin human paediatric linked to the PGL2 locus as well, because it is also tumours including Wilm’s tumours, embryonal rhabdo- located on 11q (Mariman et al., 1993). It would also myosarcomas, hepatoblastoma and adrenocortical car- explain the absence of generation skipping of tumour cinomas (Gicquel et al., 1997; Tycko, 2000). There is susceptibility in SDHB (1p36–p35)- and SDHC (1q21)- convincing evidence that LOH of 11p15 leads to linked families. In the latter two, loss of the maternal disruption of the regulation of expression of oppositely 11p15 region is probably also essential for tumour imprinted genes, in particular H19 and IGF2,ina development, since SDHB, SDHC and SDHD encode variety of tumours (Gicquel et al., 1997; Tycko, 2000). subunits from the same mitochondrialcomplex. Dan- The IGF2 gene product is a survivalfactor and strong nenberg et al. (2001) detected loss of 11p in two out of mitogen that is overexpressed in a variety of human nine sporadic paragangliomas by comparative genomic tumours including hereditary paragangliomas and hybridization, but the mutation status of SDHB, SDHC pheochromocytomas (Stewart and Rotwein, 1996). or SDHD was not investigated. Furthermore, loss of H19 codes for an untranslated RNA that acts a negative 11p has been reported in 45% of 11 sporadic abdominal trans regulator of IGF2 expression (Li et al., 1998; paragangliomas (Edstrom et al., 2000). Since data on the Runge et al., 2000). parental origin of the 11p losses are lacking, a major role Disruption of imprinted expression of 11p15 has also for loss of maternal 11p in sporadic paragangliomas, been implicated in the Beckwith–Wiedemann syndrome although likely, still remains to be proven. and focal hyperplasia of Langerhans islets causing Interestingly, a high percentage (86%) of loss of congenitalhyperinsulinism(FoCHI) (Waziri et al., chromosome 11 was also found in 31/36 (86%) of Von 1983; Weksberg and Squire, 1996; Fournet et al., Hippel-Lindau (VHL)-related pheochromocytomas, of 2001). There is an interesting parallel between our which 25/31 had loss of both 11p and 11q whereas six ﬁndings of maternalchromosome 11 lossin hereditary had only 11p loss (Lui et al., 2002). The investigators paraganglioma and loss of maternal 11p15 in FoCHI suggested that this observation might indicate the (Fournet et al., 2001). This disease is caused by a involvement of a different but essential and comple- paternally inherited, recessive mutation of the ABCC8- mentary genetic pathway in VHL-linked pheochromo- or KCNJ11-gene, which is located on 11p15.4, that is, cytoma tumorigenesis. The results of our study outside the imprinted region. The lesions show a emphasize a role for loss of 11p, and in particular the strongly decreased expression of H19 and increased maternalcopy, in SDHD-linked pheochromocytoma expression of IGF2. Thus, like in paraganglioma, a formation as well. single somatic event targets the wild-type allele of a

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4082 non-imprinted susceptibility gene on the maternal Mutation detection chromosome 11 as well as the maternally imprinted The D92Y mutation in the SDHD gene was detected by direct 11p15 region, and in both types of diseases this results in sequencing of PCR products obtained from peripheralblood exclusive paternal transmission. Although the develop- lymphocyte (PBL) DNA as described previously (Baysal et al., ment of solid tumours in general is a multi-step genetic 2000). evolution process, it is unclear why tumour development in SDHD mutation carriers specifically requires loss of a Interphase FISH on paraffin-embedded tissue sections putative maternally expressed tumour-suppressor gene, We performed interphase FISH on paraffin-embedded sections SDHD in addition to loss of wt . It has been speculated as previously described (Haralambieva et al., 2002). The that the tumorigenic effects of SDHD inactivation pLC11A probe and the PUC1.77 probe for the centromeric might be explained by either mitogenic effects of alphoid repeat DNA of chromosomes 11 and 1, respectively, elevated levels of reactive oxygen species or blocking were kindly provided by Dr J Wiegant (Deptartment of of apoptosis due to mitochondrialdysfunction (Astrom Molecular Cell Biology, LUMC, Leiden, The Netherlands) et al., 2003; Eng et al., 2003). On the other hand, (Cooke and Hindley, 1979; Waye et al., 1987). We have chosen oxidative stress may trigger pro-apoptotic signalling and the PUC1.77 probe as a reference because of our extensive create a selection pressure for mutational activation of experience with the interpretation of the signals given by this antiapoptotic pathways. Since the IGF pathway has probe and a previous LOH study did not indicate involvement found to be involved in antiapoptotic signalling, loss of of chromosome 1 in PGL1/SDHD-linked paragangliomas (Devilee et al., 1994). The probes were labelled by standard H19 the maternally expressed gene, a known suppressor nick translation with biotin-16-aUTP or digoxigenin-11-dUTP of IGF2, might be an essentialstep in paraganglioma (Roche, Basel, Switzerland). A total of 200 nuclei were development (Datta et al., 1997; Burns and Hassan, analysed for each sample by two independent investigators 2001). (EFH and ESJ). SDHD-linked paraganglioma is a striking, and to our knowledge, first example of the effect of allelic phasing Triple colour interphase FISH on nuclei isolated from on the penetrance of a hereditary tumour syndrome in paraffin-embedded tissue man. Recently, allelic phasing of mouse chromosome 11 deficiency was found to influence p53 tumorigenicity Isolation of intact nuclei, hybridization and immunodetection were performed as previously described (Jordanova et al., (Biggs et al., 2003). The deletion on chromosome 11 2002), with slight modifications. The hybridization mix elevated the tumour susceptibility and modified the contained 50% formamide, 3 ng/mlof each of the three probes tumour spectrum when in trans with the p53 mutation. (either PUC1.77, pLC11A and 3F7 or PUC1.77, 371C18 and Many genes display differential expression of parental 469N6) and a 50-fold excess of human Cot-1 DNA (Invitrogen alleles, due to genomic imprinting or genetic regulation Life tech., Paisley, UK). A volume of 5 mlof the mix was (Schadt et al., 2003). Conceivably, a certain dosage ratio applied directly onto the slides and covered with an of cancer-related alleles, which are coincidentally 18 Â 18 mm2 coverslip. After a denaturation step of 8 min at located on the same chromosome in cis-configuration, 801C, the slides were incubated overnight at 371C in a moisture may provide a selective growth advantage. The tumori- chamber. The BAC probes 371C18 (telomere 11p), 469N6 genic potentialof acquired chromosome aneuploidy,a (telomere 11q) and 3F7 (11q23, containing the SDHD gene) were obtained from the Children’s Hospital Oakland Research hallmark of many solid tumours, would then be Institute (Peter de Jong BAC library RP11). All probes were dependent on the allelic phasing or imprinting status labelled by standard nick translation with biotin-16-aUTP, of these genes. Our study provides a clear-cut example digoxigenin-11-dUTP or fluorescein-12-dUTP (Roche). A of this mechanism, which might also apply to an total of 200 nuclei were analysed for each sample and probe individual’s overall susceptibility to more common combination by two independent investigators (EFH and forms of cancer. ESJ).

Flow cytometry analysis and flow sorting Cell preparation and staining procedures were performed as described elsewhere (Beerman et al., 1990). Pepsin digestion Materials and methods was used to isolate whole nuclei from 45 mm thick paraffin sections. Nuclei were subsequently stained with propidium Patients and families iodide. DNA content was determined with a FACscan Diagnosis of paraganglioma was based on medical history, flow cytometer (Becton & Dickson, Immunocytometry physical and otolaryngological examination, radiological Systems, San Jose, CA, USA). On average, 100.000 nuclei imaging and histopathology of the excised tumour. After were measured in each sample. If the DNA histogram obtaining informed consent, peripheralbloodwas obtained showed a single G0,1 and G2 peak both populations were from patients and their parents for genomic DNA isolation. subsequently sorted on a FACsorter (FACSVantage SE, Routinely processed archival paraffin-embedded carotid Becton & Dickson, Immunocytometry Systems, San Jose, body paraganglioma or phaeochromocytoma tissue from CA, USA). Owing to the G2 arrest often detected in patients with the D92Y Dutch founder mutation in the paraganglioma cells, the G2,M population was considered SDHD gene were obtained from the archives of the enriched for tumour cells (Douwes Dekker et al., 2003). If the Department of Pathology of the Leiden University DNA histogram showed G0,1 peaks, the left peak was MedicalCenter (van Schothorst et al., 1998b; Baysal et al., considered to represent the diploid and the right peak the 2000). aneuploid population. Cells were sorted directly into 1.5 ml

Oncogene Somatic loss of chromosome 11 mimics imprinting of SDHD EF Hensen et al 4083 microfuge tubes and DNA was subsequently isolated as alleles could be unambiguously derived. Subsequently, in previously described (Abeln et al., 1994). informative cases both diploid and aneuploid or diploid and the G2,M fractions were tested. Loh analysis LOH analysis was performed as previously described (van Schothorst et al., 1998a). Genotypes of patients and their Acknowledgements parents were established for the markers D11S1984 and We thank CJ Haven for help with the FISH experiments and D11S2362 (11p15), D11S4183 (11p11), D11S1335, D11S1765 NJ Kuipers-Dijkshoorn for technicalassistance. We acknowl- and D11S4075 (11q13) and D11S1647, D11S3178 and edge T van Wezeland M Lombaerts for helpfulcommentson pDJ159Ogt1R (11q23). Markers were informative if they were the manuscript. This research was supported by a grant from heterozygous in the patient, and the parentalorigin of the the JANIVO foundation.

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Oncogene