Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

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1 Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension Felix Beuschlein, Sheerazed Boulkroun, Andrea Osswald, Thomas Wieland, Hang N. Nielsen, Urs D. Lichtenauer, David Penton, Vivien R. Schack, Laurence Amar, Evelyn Fischer, Anett Walther, Philipp Tauber, Thomas Schwarzmayr, Susanne Diener, Elisabet Graf, Bruno Allolio, Benoit Samson-Couterie, Arndt Benecke, Marcus Quinkler, Francesco Fallo, Pierre-Francois Plouin, Franco Mantero, Thomas Meitinger, Paolo Mulatero, Xavier Jeunemaitre, Richard Warth, Bente Vilsen, Maria-Christina Zennaro, Tim M. Strom, and Martin Reincke Supplementary information: - Supplementary Figure Supplementary Tables 1-5 1

2 Supplementary Figure 1: Mutations in ATPases in aldosterone producing adenomas. (A) Examples of somatic mutations found in ATP1A1 (ATP1A1c.311T>G, ATP1A1c.995T>G, and ATP1A1c299_313del) resulting in Leu104Arg and a Val332Gly substitution and Phe100_Leu104 deletion, respectively. Both positions display a high grade of homology among species. (B) Structure model of Na +,K + -ATPase highlights close proximity of mutated residues (shown in yellow and red) (C) Examples for somatic deletions within ATP2B3 (ATP2B3 c.1272_1277delgctggt) implementing an in-frame deletion at position 425/426. Both amino acids (Leu425 and Val426) are highly conserved among species. 2

3 Supplementary Figure 2: ATP1A1 and ATP2B3 expression in control adrenal cortex and in APAs. (A) Immunohistochemistry of ATP1A1 in control adrenal, non-mutated APA (KCNJ5- /ATPase-) and APAs carrying an ATP1A1p.Leu104Arg mutation. (B) Immunohistochemistry of ATP2B3 in control adrenal and non-mutated APA. (C) Quantification of ATP1A1 immunolabelling for membrane (grey) or membrane+cytoplasmic (white) localization in APA. (D) mrna expression levels of ATP1A1 and ATP2B3 retrieved from a transcriptome analysis performed on 91 APA samples and 11 control adrenals. (E) mrna expression levels of ATP1A1 and ATP2B3 in APAs as a function of the mutation status. Data were obtained as in C. Black dash line, boundary between ZG and ZF. Magnification x200. 3

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5 Supplementary Table 1: Clinical characteristics of initial patient cohort included in exome sequencing patient # age at diagnosis (years) gender ARR tumor diameter (mm) hypertension lowest potassium (mmol/l) ATPase mutation 1 60 male yes 2.6 ATP1A1 c.311t>g 2 55 male yes 2.2 ATP2B3 c.1273_1278delctggtc 3 55 male yes 1.97 ATP1A1c.311T>G 4 51 male yes 3.1 wt 5 62 male 94 n.a. yes 2.8 wt 6 71 male 24.2 n.a. yes 3.7 wt 7 65 male yes 3 wt 8 40 male yes 2 ATP1A1 c.995t>g 9 54 male yes 2.5 ATP2B3 c.1272_1277delgctggt Mean SD

6 1 Supplementary Table 2: Overview on mutations found in individual tumor samples upon exome sequencing Pat.# Type Genomic position Transcript cdna level Protein level Gene symbol Effect on protein # of read from tumor Nonref. all % of Ref. allele allele reads # of reads from blood Nonref. Ref. allele allele #1 missense chr1:g t>g NM_ c.311t>g p.leu104arg ATP1A1 Leu104Arg ,3% missense chr10:g c>g NM_ c.3796g>c p.asp1266his TTC40 Asp1266His ,3% 21 0 missense chr11:g g>t NM_ c.555c>a p.phe185leu RAPSN Phe185Leu ,5% missense chr15:g t>c NM_ c.878a>g p.asn293ser GPR176 Asn293Ser ,0% 58 0 missense chr17:g a>c NM_ c.1700a>c p.his567pro KPNB1 His567Pro ,2% missense chr2:g t>c NM_ c.2685a>g p.ile895met PLA2R1 Ile895Met ,5% missense chr20:g c>t NM_ c.287c>t p.ala96val RTEL1 Ala96Val ,4% 43 0 RNF123,AMI missense chr3:g g>c NM_ c.207c>g p.ser69arg GO3 Ser69Arg ,1% 95 0 missense chr4:g g>c NM_ c.34g>c p.gly12arg MRPS18C Gly12Arg ,5% missense chr5:g t>a NM_ c.2347a>t p.asn783tyr RICTOR Asn783Tyr ,9% TRPA1,LOC10 missense chr8:g c>g NM_ c.3141g>c p.gln1047his Gln1047His ,7% indel chrx:g _ delGAGACGGGG GCCGCCGCGGCTGC GGCGGCCACC NM_ c.421_453del p.thr141_glu1 51del LONRF3 Thr141_Glu1 51del ,6% 8 0 missense chrx:g g>c NM_ c.653g>c p.ser218thr TKTL1 Ser218Thr ,1% 95 0 #2 missense chr17:g g>c NM_ c.566c>g p.ala189gly DUS1L Ala189Gly ,7% nonsense chr18:g c>t NM_ c.1735c>t p.arg579* GREB1L Arg579Stop ,8% missense chr5:g g>a NM_ c.991g>a p.gly331arg PHAX Gly331Arg ,5% 62 0 indel chrx:g _ delCTGGTC NM_ c.1273_1278 del p.leu425_val4 26del ATP2B3 Leu425_Val4 26del ,5% 38 0 #3 missense chr1:g t>g NM_ c.311t>g p.leu104arg ATP1A1 Leu104Arg ,9% missense chr15:g c>t NM_ c.2860g>a p.val954met FMN1 Val954Met ,8% missense chr16:g c>t NM_ c.1408g>a p.ala470thr COG8 Ala470Thr ,8% 46 0 chr2:g _25469 c.1157_1163 p.val386alafs Val386Alafs frameshift 611delTGGCACA NM_ del *19 DNMT3A * ,3% missense chr2:g t>c NM_ c.4874t>c p.ile1625thr ZNF638 Ile1625Thr ,0% 63 0 missense chr2:g t>g NM_ c.2062t>g p.phe688val EIF5B Phe688Val ,3%

7 1 missense chr5:g a>g NM_ c.1289a>g p.gln430arg PPWD1 Gln430Arg ,0% 86 0 missense chr5:g t>g NM_ c.1293t>g p.asn431lys PPWD1 Asn431Lys ,2% 86 0 missense chr6:g a>g NM_ c.727a>g p.lys243glu TTBK1 Lys243Glu ,1% 61 0 missense chr19:g c>t NM_ c.683c>t p.thr228ile MIDN Thr228Ile ,0% 6 0 #4 ENST GOPC, missense chr6:g g>c 8.5 c.655g>c p.asp219his DCBLD1 Asp219His ,6% 5 0 #7 missense chr17:g a>c NM_ c.41a>c p.asn14thr DNAH9 Asn14Thr ,4% 10 0 #8 missense chr1:g t>g NM_ c.995t>g p.val332gly ATP1A1 Val332Gly ,2% 86 0 missense chr8:g a>t NM_ c.1211t>a p.met404lys GSDMC Met404Lys ,3% 50 1 missense chr9:g c>a NM_ c.94c>a p.arg32ser C9orf7 Arg32Ser ,0% 4 0 missense chr20:g c>t NM_ c.82g>a p.val28met CYP24A1 Val28Met ,9% 15 1 #9 AK097500, nonsense chr17:g g>a NM_ c.4276c>t p.gln1426* MYH4 Gln1426Stop ,0% frameshift chr3:g _ insT NM_ c.845dup p.ser283glufs* 5 RFC4 Ser283Glufs * ,3% 90 0 missense chr6:g t>c NM_ c.92a>g p.asp31gly SCAND3 Asp31Gly ,1% indel chrx:g _ delGTGCTG NM_ c.1272_1277 del p.leu425_val4 26del ATP2B3 Leu425_Val4 26del ,6%

8 1 Supplementary Table 3: Genomic variants of ATP1A1 and ATP2B3 in tumor tissues Table 2. ATP1A1 and ATP2B3 variants Genomic change cdna change Protein change dbsnp ref/ref + ref/alt + alt/alt (hem) + ATP1A1 (NM_ ) non-synonymous *chr1:g _ deltctcaatgtt ACTGT 8 Control adrenal Control lymph. c.299_313deltctcaatgttactgt p.phe100_leu104del /1 2/1 *chr1:g t>g c.311t>g p.leu104arg rs /3 10/2 *chr1:g t>g c.995t>g p.val332gly /1 1/0 chr1:g a>g c.1738a>g p.ile580val rs /3 4/4 6/3 synonymous chr1:g g>a c.546g>a p.ala182ala rs /1 chr1:g c>a c.741c>a p.thr247thr rs /1 1/0 chr1:g g>a c.2541g>a p.glu847glu rs /1 0/1 chr1:g c>t c.2739c>t p.ile913ile rs /1 1/0 chr1:g g>a c.3057g>a p.lys1019lys rs /1 1/0 ATP2B3 (NM_ ) non-synonymous *chrx:g _ delgctggt c.1272_1277delgctggt p.leu425_val426del 306 male 3 0 0/2 3/0 *chrx:g _ delctggtc c.1273_1278delctggtc p.leu425_val426del 308 male 1 0 0/1 1/0 *chrx:g _ deltcgtgg c.1277_1282deltcgtgg p.val426_val427del 308 female 1 0 0/1 0/1 chrx:g a>c c.694a>c p.ile232leu rs male 0 1 1/1 1/0 synonymous chrx:g g>c c.387g>c p.pro129pro rs male 0 1 1/1 1/0 chrx:g g>c c.2592g>c p.val864val rs male/ female male /2 23/0 female /3 0/39 chrx:g t>c c.3276t>c p.his1092his rs male 0 1 1/1 1/0 The table contains all ATP1A1 and ATP2B23 variants that were indentified by exome sequencing in 9 adenomas, Sanger sequencing of the entire coding sequence in additional 100 adenomas, and targeted sequencing of the identified somatic mutations in 199 adenomas. Somatic mutations are indicated by an asterisk. + ref = reference allele, alt= alternative allele, hem=hemizygous alternative allele. 2 3

9 1 Supplementary Table 4: Overview on distribution of ATPases mutations in relation to gender, centers and KCNJ5 mutational status 2 Center 9 No. with successful sequencing ATP mutations total ATP mutations (%) KCNJ5 mutations total KCNJ mutations (%) ATPase mutations in KCNJ5 wt patients (%) ATPase mutation/all (%) - males ATPase mutation/all (%) - females KCNJ5 mutation/all (%) - males KCNJ5 mutation/all (%) - females males/females ATPase mutation (%) males/females KCNJ5 mutation (%) 3 München % % 23.5% 25.6% 6.1% 12.8% 48.5% 83.3% 23.8% Paris % % 7.5% 8.2% 1.4% 26.2% 52.1% 83.3% 29.6% Torino % % 6.3% 0.0% 6.7% 21.4% 66.7% 0.0% 23.1% Berlin % % 8.3% 11.1% 0.0% 0.0% 78.6% 100% 0.0% Würzburg % % 0.0% 0.0% 0.0% 25.0% 60.0% NA 25.0% Padova I+II % % 3.8% 5.3% 0.0% 26.3% 45.5% 100% 33.3% TOTAL % % 11.1% 11.6% 2.5% 20.5% 54.3% 81.0% 25.4% 4 5

10 1 Supplementary Table 5: Clinical characteristics of Munich and Paris patient cohort in relation to KCNJ5 and ATPase mutational status KCNJ5-/ATPase- KCNJ5 ATPase all KCNJ5 vs. KCNJ5- /ATPase- ATPase vs. KCNJ5- /ATPase- KCNJ5 vs. ATPase n median IQR n median IQR n median IQR p p p p age at diagnosis (years) ns adenoma size (mm) ns ns lowest recorded serum potassium (mmol/l) ns ns serum creatinine (mg/dl) ns ns ns albuminuria* (mg/dl) ns ns urinary aldosterone* (mmol/l) ns ns ns plasma aldosterone (ng/l) ns ns plasma renin concentration (mu/l) ns aldosterone to renin ratio (ng/mu) ns systolic blood pressure (mmhg) ns ns diastolic blood pressure (mmhg) ns no. of antihypertensive agents ns N indicates the number of subjects for each group; IQR, inter quartile range; ns indicates not significant. *Values based on 24h urine samples. Data between groups were compared using Kruskal-Wallis test followed by 2-sided test ( ) for pairwise comparison of two groups. 2 10