The R22X Mutation of the SDHD Gene in Hereditary Paraganglioma Abolishes the Enzymatic Activity of Complex II in the Mitochondrial Respiratory Chain and.

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The R22X Mutation of the SDHD Gene in Hereditary Paraganglioma Abolishes the Enzymatic Activity of Complex II in the Mitochondrial Respiratory Chain and Activates the Hypoxia Pathway Anne-Paule Gimenez-Roqueplo, Judith Favier, Pierre Rustin, Jean-Jacques Mourad, Pierre-François Plouin, Pierre Corvol, Agnès Rötig, Xavier Jeunemaitre The American Journal of Human Genetics Volume 69, Issue 6, Pages 1186-1197 (December 2001) DOI: 10.1086/324413 Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 1 Analysis of mutations in the SDHD gene. A, Schematic representation of the affected kindred. Paragangliomas are represented by a hatched box and pheochromocytoma by a blackened box. B, Sequence analysis of exon 2 of SDHD in DNA extracted from the peripheral blood of one affected subject (II:2) and from an unaffected relative (I:2). C, Amplification of exon 2 from the blood of all family members and its cleavage with BstBI. The BstBI restriction fragments, obtained after digestion at 65°C for 3 h, were separated by electrophoresis on a 3% agarose gel. The normal maternal allele gave two bands after enzymatic digestion (159 and 131 bp), whereas the paternal mutated allele gave a single undigested band (290 bp). A three-band heterozygous pattern (290, 159, and 131 bp) was observed for patients I:1, II:1, and II:2, whereas the unaffected subjects (I:2, III:1 and III:2) exhibit a two-band homozygous profile. ND = not digested. The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 2 Loss of maternal allele at the SDHD gene. A, Electrophoretograms corresponding to the forward (a) and reverse (b) sequences of exon 2 of SDHD from the pheochromocytoma of subject II:2. The small peak of C probably results from minor contamination from nontumoral DNA (blood vessel and/or leukocytes). B, Amplification of exon 2 and cleavage with BstBI: undigested control (lane a), digested profiles of maternal peripheral DNA (lane b), germline DNA (lane c), and pheochromocytoma DNA (lane d) of subject II:2. The normal maternal allele shows two bands (159 and 131 bp) after digestion and the undigested paternal mutated allele shows one band (290 bp). The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 3 Loss of maternal allele at chromosome 11q21-25. The LOH was typed by microsatellite analysis, using constitutive and tumor DNA from patient II:2. The positions of the markers tested are indicated on an ideogram of chromosome 11. The maternal allele lost is indicated by a line and homozygosity by NI. * = noninformative marker. The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 4 Mitochondrial enzyme function studies. A, Spectrophotometric assay of respiratory-chain enzyme activities in tumors with (first trace) and without (second trace) mutations in SDHD. The values given on the traces are nmol/min/mg protein. Mean ± 1 SD is indicated in brackets. LM = lauryl maltoside; DUQH2 = decylubiquinol. B, Enzyme activities in subject II:2 (inherited pheo) and controls (sporadic pheos). SCCR = malonate-sensitive succinate cytochrome c reductase activity (complexes II+III). SQDR = succinate quinone dichlorophenolindophenol reductase (complex II). SPDR = succinate phenazine methosulfate dichlorophenolindophenol reductase (succinate dehydrogenase). C, Schematic representation of the principal electron paths through the various subunits of complex II. The acceptor sites for quinones and phenazine methosulfate are indicated as well as the thenoyltrifluoroacetone inhibition site. Aa = antimycin; C = SDHC; CIII = complex III; CIV = complex IV; D = SDHD; DCPIP = dichlorophenolindophenol; DUQ = decylubiquinone; DUQH2 = decylubiquinol; FAD = flavine adenosine; Fp = flavoprotein; Ip = iron protein; KCN = potassium cyanide; PMS = phenazine methosulfate; Q = ubiquinone; Q▪− = semiubiquinol; QH2 = ubiquinol. Q, Q▪−, and QH2 are the various forms of the ubiquinone present in the mitochondrial membrane. The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 5 Gene-expression patterns in right carotid-body paraganglioma of patient I:1, as shown by in situ hybridization or immunostaining. EPAS1 mRNA is observed in both endothelial cells and tumor cells (A). The protein of the homolog transcription factor HIF1α is also present (B). Tyrosine hydroxylase (C), NSE (D and I) and VEGF (E and J) are present at a very high level in tumor cells within the Zellballen clusters, whereas VEGF receptors R1 (F and K) and R2 (G) transcripts are restricted to endothelial cells. Note the absence of VEGF-R2 in the wall of the central large vessel (*) and the almost undetectable signal with the Tie2 probe (H). High magnifications clearly reveal the expression of NSE (I) and VEGF (J) within the Zellballen structures (arrowheads), surrounded by a VEGF-R1 positive capillary network (K, arrows). The in situ hybridization signals are visualized either under dark (A and E–H; signal visible as white dots) or bright field illumination (J and K;labeling detected by black dots). Immunostaining (B, D, and I) is revealed by a brown coloration. Scale bars = 200 μm (A–H); scale bars = 100 μm (I–K). The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 6 EPAS1 mRNA expression; comparison between patient II:2 pheochromocytoma and a sporadic benign pheochromocytoma. The expression of EPAS1 within tumor cells is higher in the inherited pheochromocytoma (A and B) than in a sporadic pheochromocytoma (C and D). In contrast, the intensity of EPAS1 signal does not vary between the two samples in endothelial cells (black arrows). The in situ hybridization signals are observed under dark-field illumination for low magnifications (A and C [white dots]) and under bright field illumination for enlargements (B, D [black dots]). White arrows = blood vessels; +++ = very strong signal; + = weak signal. Scale bars = 100 μm. The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions

Figure 7 Real-time quantitative RT-PCR experiments. The level of EPAS1 and VEGF transcripts were compared between the SDHD mutated pheochromocytoma (inherited Pheo) with three sporadic pheochromocytomas (sporadic Pheo 1, 2, and 3). Comparison of quantitative DNA data obtained with EPAS1 as target and β-actin (A) or 18S ribosomal RNA (B) as endogenous control and of data obtained with VEGF as target and β-actin (C) or 18S ribosomal RNA (D) as endogenous controls. The circle represents the mean of 10 calculated measurements (ratio of target gene on reference gene for each of five log of concentration in duplicate). The superior bar shows the maximal value, and the inferior bar shows the minimal value. The American Journal of Human Genetics 2001 69, 1186-1197DOI: (10.1086/324413) Copyright © 2001 The American Society of Human Genetics Terms and Conditions