Supplemental Material - Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronis

Caroccia, Brasilina

Rossi, Gian Paolo

Research Data UNIPD

Supplemental Material Double CYP11B1/CYP11B2 Immunohistochemistry  and Detection of KCNJ5 Mutations in Primary Aldosteronism    Brasilina Caroccia*,1, Livia Lenzini*,1, Andrea Benetti2, Alessandra Giannella3, Hala Ajjour1,  Giulio Ceolotto1, Francesca Galuppini4, Teresa Maria Seccia1,  Celso Gomez-Sanchez5 and Gian Paolo Rossi1  1Specialized Center of Excellence for Hypertension of the European Society of Hypertension and Emergency Medicine Unit, Department of Medicine-DIMED, University of Padua, Italy 2First Medical Clinic, Department of Medicine-DIMED, University of Padua, Italy 3Division of Thrombotic and Hemorrhagic Diseases, Department of Medicine-DIMED, University of Padua, Italy 4Department of Medicine, Surgical Pathology Unit, University of Padua, Italy 5 G.V. (Sonny) Montgomery VA Medical Center and Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA    ________________________________________________________________________________ Corresponding author: Prof. Gian Paolo Rossi, MD. FACC, FAHA ORCID: :0000-0002-7963-0931 DIMED– Hypertension Unit University Hospital Via Giustiniani, 2 35126 Padova, Italy Phone: 39-049-821-2279 or 7821 Fax: 39-049-821-7873 E-mail: gianpaolo.rossi@unipd.it  * These Authors equally contributed to the work Disclosures  The authors have nothing to disclose.  La licenza scadrà tra 2 giorni.Logistic RegressionNotesOutput CreatedCommentsInput DataActive DatasetFilterWeightSplit FileN of Rows in Working Data FileMissing Value Handling Definition of MissingSyntaxResources Processor TimeElapsed Time28-FEB-2024 17:01:10/Users/utente/Desktop/NGS /FILE 2024/SPSS/BC_NGS_2024_2_26.savDataSet1<none><none><none>127User-defined missing values are treated as missingLOGISTIC REGRESSION VARIABLES Sanger1POS_2NEG  /METHOD=BSTEP(WALD) Tumor_Size Age_calc SBP_AVS DBP_AVS ALL_DRC PAC_ngdl_calc IHC  /CRITERIA=PIN(.05) POUT(.10) ITERATE(20) CUT(.5).00:00:00,0400:00:00,00[DataSet1] /Users/utente/Desktop/NGS /FILE 2024/SPSS/BC_NGS_2024_2_26.savCase Processing SummaryUnweighted Casesa N PercentSelected Cases Included in AnalysisMissing CasesTotalUnselected CasesTotal8 0 63.04 7 37.0127 100.00 .0127 100.0If weight is in effect, see classification table for the total number of cases.a. Dependent Variable EncodingOriginal Value Internal Value1.002.0001Page 1Block 0: Beginning BlockClassification Tablea,bObservedPredictedSanger1POS_2NEG Percentage Correct1.00 2.00Step 0 Sanger1POS_2NEG 1.002.00Overall Percentage0 4 0 .00 4 0 100.050.0Constant is included in the model.a. The cut value is .500b. Variables in the EquationB S.E. Wald d f Sig. Exp(B)Step 0 Constant .000 .224 .000 1 1.000 1.000Variables not in the EquationScore d f Sig.Step 0 Variables Tumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAAldo ng_dl Calc da pMIHCOverall Statistics3.501 1 .06113.290 1 <.0013.313 1 .0692.435 1 .1191.764 1 .184.188 1 .6651.000 1 .31724.739 7 <.001Block 1: Method = Backward Stepwise (Wald)Page 2Omnibus Tests of Model CoefficientsChi-square d f Sig.Step 1 StepBlockModelStep 2aStepBlockModelStep 3aStepBlockModelStep 4aStepBlockModelStep 5aStepBlockModel29.408 7 <.00129.408 7 <.00129.408 7 <.001- .030 1 .86329.378 6 <.00129.378 6 <.001- .366 1 .54529.012 5 <.00129.012 5 <.001- .806 1 .36928.206 4 <.00128.206 4 <.001-2 .438 1 .11825.768 3 <.00125.768 3 <.001A negative Chi-squares value indicates that the Chi-squares value has decreased from the previous step.a. Model SummaryStep-2 Log likelihoodCox & Snell R SquareNagelkerke R Square1234581.496a.308 .41081.525a.307 .41081.892a.304 .40682.698a.297 .39685.136b.275 .367Estimation terminated at iteration number 5 because parameter estimates changed by less than .001.a. Estimation terminated at iteration number 4 because parameter estimates changed by less than .001.b. Page 3Classification TableaObservedPredictedSanger1POS_2NEG Percentage Correct1.00 2.00Step 1 Sanger1POS_2NEG 1.002.00Overall PercentageStep 2 Sanger1POS_2NEG 1.002.00Overall PercentageStep 3 Sanger1POS_2NEG 1.002.00Overall PercentageStep 4 Sanger1POS_2NEG 1.002.00Overall PercentageStep 5 Sanger1POS_2NEG 1.002.00Overall Percentage2 6 1 4 65.01 2 2 8 70.067.52 6 1 4 65.01 2 2 8 70.067.52 6 1 4 65.01 2 2 8 70.067.52 4 1 6 60.01 1 2 9 72.566.32 6 1 4 65.01 1 2 9 72.568.8The cut value is .500a. Variables in the EquationB S.E. Wald d f Sig.Step 1aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAAldo ng_dl Calc da pMIHCConstantStep 2aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAIHCConstantStep 3aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAConstant.384 .172 4.999 1 .025.138 .037 14.170 1 <.001- .062 .024 6.846 1 .009.047 .032 2.187 1 .139.064 .098 .421 1 .516.002 .011 .029 1 .864.262 .444 .349 1 .555-3 .782 2.928 1.669 1 .196.388 .171 5.150 1 .023.139 .037 14.369 1 <.001- .063 .024 7.041 1 .008.047 .031 2.277 1 .131.063 .099 .411 1 .521.267 .442 .363 1 .547-3 .766 2.931 1.651 1 .199.383 .169 5.143 1 .023.140 .037 14.543 1 <.001- .063 .024 7.080 1 .008.049 .031 2.497 1 .114.078 .097 .646 1 .422-3 .516 2.895 1.475 1 .225.374 .167 4.984 1 .026 Page 4Variables in the EquationExp(B)Step 1aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAAldo ng_dl Calc da pMIHCConstantStep 2aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAIHCConstantStep 3aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineDRC_measured_DRCdaPRAConstant1.4681.148.9401.0481.0661.0021.300.0231.4741.149.9391.0491.0651.305.0231.4661.150.9391.0511.081.0301.453Variables in the EquationB S.E. Wald d f Sig.Step 4aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineConstantStep 5aTumor_Sizeage calc in yrs at studySBP baselineConstant.374 .167 4.984 1 .026.143 .037 15.191 1 <.001- .062 .023 6.974 1 .008.045 .030 2.284 1 .131-3 .146 2.762 1.297 1 .255.316 .158 4.021 1 .045.123 .033 13.964 1 <.001- .037 .016 5.455 1 .020-1 .484 2.539 .341 1 .559Page 5Variables in the EquationExp(B)Step 4aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineConstantStep 5aTumor_Sizeage calc in yrs at studySBP baselineConstant1.4531.153.9401.046.0431.3721.131.963.227Variable(s) entered on step 1: Tumor_Size, age calc in yrs at study, SBP baseline, DBP baseline, DRC_measured_DRCdaPRA, Aldo ng_dl Calc da pM, IHC.a. Variables not in the EquationScore d f Sig.Step 2aVariables Aldo ng_dl Calc da pMOverall StatisticsStep 3bVariables Aldo ng_dl Calc da pMIHCOverall StatisticsStep 4cVariables DRC_measured_DRCdaPRAAldo ng_dl Calc da pMIHCOverall StatisticsStep 5dVariables DBP baselineDRC_measured_DRCdaPRAAldo ng_dl Calc da pMIHCOverall Statistics.029 1 .864.029 1 .864.044 1 .834.365 1 .546.391 2 .822.681 1 .409.023 1 .879.682 1 .4091.099 3 .7772.407 1 .121.460 1 .497.136 1 .712.871 1 .3513.405 4 .492Variable(s) removed on step 2: Aldo ng_dl Calc da pM.a. Variable(s) removed on step 3: IHC.b. Variable(s) removed on step 4: DRC_measured_DRCdaPRA.c. Variable(s) removed on step 5: DBP baseline.d. Logistic RegressionPage 6NotesOutput CreatedCommentsInput DataActive DatasetFilterWeightSplit FileN of Rows in Working Data FileMissing Value Handling Definition of MissingSyntaxResources Processor TimeElapsed Time28-FEB-2024 17:01:10/Users/utente/Desktop/NGS /FILE 2024/SPSS/BC_NGS_2024_2_26.savDataSet1<none><none><none>127User-defined missing values are treated as missingLOGISTIC REGRESSION VARIABLES Sanger1POS_2NEG  /METHOD=BSTEP(WALD) Tumor_Size Age_calc SBP_AVS DBP_AVS FR_MUT_KCNJ5 sex1M2F IHC  /CONTRAST (sex1M2F)=Indicator  /CLASSPLOT  /PRINT=CORR ITER(1) SUMMARY CI(95)  /CRITERIA=PIN(0.05) POUT(0.10) ITERATE(20) CUT(0.5).00:00:00,0400:00:00,00Case Processing SummaryUnweighted Casesa N PercentSelected Cases Included in AnalysisMissing CasesTotalUnselected CasesTotal3 6 28.39 1 71.7127 100.00 .0127 100.0If weight is in effect, see classification table for the total number of cases.a. Dependent Variable EncodingOriginal Value Internal Value1.002.0001Page 7Categorical Variables CodingsFrequencyParameter coding(1)Sex_1male_2female 121 2 1.0002 4 .000Block 0: Beginning BlockIterat ion Historya,b,cIteration-2 Log likelihoodCoefficientsConstantStep 0 1234526.233 -1 .55625.142 -1 .99525.116 -2 .07725.116 -2 .07925.116 -2 .079Constant is included in the model.a. Initial -2 Log Likelihood: 25.116b. Estimation terminated at iteration number 5 because parameter estimates changed by less than .001.c. Classification Tablea,bObservedPredictedSanger1POS_2NEG Percentage Correct1.00 2.00Step 0 Sanger1POS_2NEG 1.002.00Overall Percentage3 2 0 100.04 0 .088.9Constant is included in the model.a. The cut value is .500b. Variables in the EquationB S.E. Wald d f Sig. Exp(B)Step 0 Constant -2 .079 .530 15.374 1 <.001 .125Page 8Variables not in the EquationScore d f Sig.Step 0 Variables Tumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCOverall Statistics1.386 1 .239.014 1 .907.355 1 .552.478 1 .4898.751 1 .003.562 1 .453.129 1 .72010.316 7 .171Block 1: Method = Backward Stepwise (Wald)Iterat ion Historya,b,c,d,eIteration-2 Log likelihoodCoefficientsConstant Tumor_Sizeage calc in yrs at study SBP baselineStep 1 12345678Step 7 12345620.714 -1 .539 .121 .022 - .00116.290 -2 .326 .263 .050 - .00214.794 -3 .155 .453 .086 - .00314.464 -4 .015 .609 .114 - .00514.438 -4 .477 .671 .127 - .00614.437 -4 .550 .679 .128 - .00614.437 -4 .551 .680 .129 - .00714.437 -4 .551 .680 .129 - .00721.565 .44018.041 1.18117.360 1.66517.304 1.84717.303 1.86817.303 1.868Page 9I terat ion Historya,b,c,d,eIterationCoefficientsDBP baseline FR_MUT_KCNJ5Sex_1male_2female(1) IHCStep 1 12345678Step 7 123456- .001 - .069 .159 .306- .014 - .113 .397 .767- .039 - .142 .756 1.396- .056 - .158 1.048 1.830- .061 - .163 1.166 1.961- .062 - .163 1.183 1.974- .062 - .163 1.183 1.974- .062 - .163 1.183 1.974- .072- .125- .160- .173- .175- .175Method: Backward Stepwise (Wald)a. Constant is included in the model.b. Initial -2 Log Likelihood: 25.116c. Estimation terminated at iteration number 8 because parameter estimates changed by less than .001.d. Estimation terminated at iteration number 6 because parameter estimates changed by less than .001.e. Omnibus Tests of Model CoefficientsChi-square d f Sig.Step 1 StepBlockModelStep 7aStepBlockModel10.678 7 .15310.678 7 .15310.678 7 .153- .745 1 .3887.813 1 .0057.813 1 .005A negative Chi-squares value indicates that the Chi-squares value has decreased from the previous step.a. Page 1 0Model SummaryStep-2 Log likelihoodCox & Snell R SquareNagelkerke R Square1714.437a.257 .51117.303b.195 .388Estimation terminated at iteration number 8 because parameter estimates changed by less than .001.a. Estimation terminated at iteration number 6 because parameter estimates changed by less than .001.b. Classification TableaObservedPredictedSanger1POS_2NEG Percentage Correct1.00 2.00Step 1 Sanger1POS_2NEG 1.002.00Overall PercentageStep 7 Sanger1POS_2NEG 1.002.00Overall Percentage3 1 1 96.91 3 75.094.43 2 0 100.02 2 50.094.4The cut value is .500a. Variables in the EquationB S.E. Wald d f Sig.Step 1aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCConstantStep 7aFR_MUT_KCNJ5Constant.680 .590 1.325 1 .250.129 .126 1.034 1 .309- .007 .048 .019 1 .892- .062 .076 .662 1 .416- .163 .085 3.643 1 .0561.183 1.779 .442 1 .5061.974 1.903 1.075 1 .300-4 .551 6.824 .445 1 .505- .175 .076 5.302 1 .0211.868 1.514 1.523 1 .217Page 1 1Variables in the EquationExp(B)95% C.I.for EXP(B)Lower UpperStep 1aTumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCConstantStep 7aFR_MUT_KCNJ5Constant1.973 .620 6.2761.137 .888 1.457.994 .905 1.091.940 .810 1.091.850 .719 1.0043.263 .100 106.7317.197 .173 300.050.011.839 .723 .9746.477Variable(s) entered on step 1: Tumor_Size, age calc in yrs at study, SBP baseline, DBP baseline, FR_MUT_KCNJ5, Sex_1male_2female, IHC.a. Correlation MatrixConstant Tumor_Sizeage calc in yrs at study SBP baselineStep 1 ConstantTumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCStep 7 ConstantFR_MUT_KCNJ51.000 - .595 - .630 - .054- .595 1.000 .594 - .192- .630 .594 1.000 - .464- .054 - .192 - .464 1.000.179 - .431 - .148 - .519- .133 .051 - .130 - .154- .518 .397 .438 - .056- .487 .585 .304 .0481.000- .911Correlation MatrixDBP baseline FR_MUT_KCNJ5Sex_1male_2female(1) IHCStep 1 ConstantTumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCStep 7 ConstantFR_MUT_KCNJ5.179 - .133 - .518 - .487- .431 .051 .397 .585- .148 - .130 .438 .304- .519 - .154 - .056 .0481.000 .112 - .372 - .631.112 1.000 .145 .062- .372 .145 1.000 .518- .631 .062 .518 1.000Page 1 2Correlation MatrixFR_MUT_KCNJ5Step 1 ConstantTumor_Sizeage calc in yrs at studySBP baselineDBP baselineFR_MUT_KCNJ5Sex_1male_2female(1)IHCStep 7 ConstantFR_MUT_KCNJ5- .9111.000Variables not in the EquationScore d f Sig.Step 7aVariables Tumor_Sizeage calc in yrs at studySBP baselineDBP baselineSex_1male_2female(1)IHCOverall Statistics.495 1 .482.767 1 .381.056 1 .813.022 1 .881.029 1 .865.020 1 .8882.218 6 .899Variable(s) removed on step 7: age calc in yrs at study.a.              Step number: 1             Observed Groups and Predicted Probabilities      16 +                                                                                                    +         I                                                                                                    I         I                                                                                                    IF        I                                                                                                    IR     12 +                                                                                                    +E        I1                                                                                                   IQ        I1                                                                                                   IU        I1                                                                                                   IE      8 +1                                                                                                   +N        I1                                                                                                   IPage 1 3C        I1 2                                                                                                 IY        I1 1                                                                                                 I       4 +111                                                                                                 +         I1111                                                                                                I         I1111       1                                                                                        I         I111111    111          1           1                12               2     2                        IPredicted ---------+---------+---------+---------+---------+---------+---------+---------+---------+----------  Prob:   0       .1        .2        .3        .4        .5        .6        .7        .8        .9         1  Group:  1111111111111111111111111111111111111111111111111122222222222222222222222222222222222222222222222222          Predicted Probability is of Membership for 2.00          The Cut Value is .50          Symbols: 1 - 1.00                   2 - 2.00          Each Symbol Represents 1 Case.Step Summarya,bStepImprovement ModelCorrect Class %Chi-square d f Sig. Chi-square d f Sig.234567- .019 1 .889 10.659 6 .100 94.4%- .463 1 .496 10.196 5 .070 91.7%- .797 1 .372 9.399 4 .052 91.7%- .306 1 .580 9.092 3 .028 94.4%- .534 1 .465 8.558 2 .014 94.4%- .745 1 .388 7.813 1 .005 94.4%Step Summarya,bStep Variable234567 OUT: SBP baseline OUT: Sex_1male_2female OUT: DBP baseline OUT: IHC OUT: Tumor_Size OUT: age calc in yrs at studyPage 1 4No more variables can be deleted from or added to the current model.a. End block: 1b.              Step number: 7             Observed Groups and Predicted Probabilities      16 +                                                                                                    +         I                                                                                                    I         I 2                                                                                                  IF        I 1                                                                                                  IR     12 + 1                                                                                                  +E        I 1                                                                                                  IQ        I 1                                                                                                  IU        I 1                                                                                                  IE      8 + 1                                                                                                  +N        I 1                                                                                                  IC        I 1                                                                                                  IY        I 11                                                                                                 I       4 + 11                                                                                                 +         I 11                                                                                                 I         I 1111                                                                                               I         I111111   11   1    1    1   1   1       1  2        2              2                                IPredicted ---------+---------+---------+---------+---------+---------+---------+---------+---------+----------  Prob:   0       .1        .2        .3        .4        .5        .6        .7        .8        .9         1  Group:  1111111111111111111111111111111111111111111111111122222222222222222222222222222222222222222222222222          Predicted Probability is of Membership for 2.00          The Cut Value is .50          Symbols: 1 - 1.00                   2 - 2.00          Each Symbol Represents 1 Case.Page 1 5

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism

https://researchdata.cab.unipd.it/1218/7/Supplemental_Material_NGS%202024_03_04_%282_file_uniti%29.pdf

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism

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