Clinical benefits and adverse effects of genetically ...

[Pages:16]Clinical benefits and adverse effects of genetically-elevated free testosterone levels: a Mendelian randomization analysis

Pedrum Mohammadi-Shemirani1,2,3, Michael Chong MSc1,2,4, Marie Pigeyre MD PhD1,6, Robert W. Morton PhD5, Hertzel C. Gerstein MD MSc1,6, Guillaume Par? MD MSc1,2,7,8

Supplementary Methods.

Supplementary Text.

Supplementary Figure 1. Rationale for Mendelian randomization analysis. Supplementary Figure 2. Distribution of natural log-transformed sex hormone-binding globulin levels in males from the UK Biobank cohort Supplementary Figure 3. Distribution of free testosterone levels calculated using the Vermeulen equation in males from the UK Biobank cohort. Supplementary Figure 4. Manhattan plot showing distribution of p-values from genome-wide association study of calculated free testosterone according to chromosomal location of each genetic variant. Supplementary Figure 5. Association of genetically-predicted calculated free testosterone with significant outcomes using one-sample Mendelian randomization analysis stratified by age in males from the UK Biobank. Supplementary Figure 6. Quantile-quantile plot showing observed test statistics in genomewide association study of calculated free testosterone levels relative to expected test statistics under a null model. Supplementary Figure 7. Screenshot of options shown to male UK Biobank participants for selection of hair/baldness pattern.

Supplementary Table 1. Definitions for 31 health outcomes from the UK Biobank study. Supplementary Table 2. Detectable odds ratios for MR analyses at 80% power for dichotomous outcomes in males from the UK Biobank. Supplementary Table 3. Publicly available sources of summary statistics of genome-wide association studies used for two-sample MR analyses. Supplementary Table 4. Characteristics at recruitment for study population of males from UK Biobank cohort study. Supplementary Table 5. Independent genetic variants associated with calculated free testosterone levels and not associated with sex hormone-binding globulin in males from the UK Biobank. Supplementary Table 6. Effect of CFT on 10 case-control outcomes from independent genomewide association studies using two-sample Mendelian randomization analysis.

eReferences.

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eMethods

UK Biobank Genotyping Individual-level genetic data was available for 488,317 participants that consented to blood collection and genotyping. Genotyping was performed with the Applied Biosystems UK Biobank Lung Exome Variant Evaluation (UK BiLEVE) and UK Biobank Axiom arrays (Affymetrix Research Services Laboratory, Santa Clara, California, USA). Description of quality control has been previously described in detail1. In brief, UK Biobank centrally excluded poor quality markers or samples based on standard metrics, such as batch effects, plate effects, HardyWeinberg equilibrium, sex effects, array effects, missing rate, and heterozygosity. This data is available via application directly to the UK Biobank (). For our analyses, data was derived from the second release of the UK Biobank genetic data (June 2017) 1.

Assessment of Testosterone and Sex Hormone-Binding Globulin in UK Biobank In the UK Biobank, total testosterone and sex hormone-binding globulin (SHBG) were measured on a Beckman Coulter Unicel DXI 800 using a one-step competitive analysis and two-step sandwich immunoassay, respectively. Testosterone and SHBG measurements were flagged if they fell outside the manufacturer's observed reportable range, or samples reported high levels of bilirubin, haemoglobin or lipids/turbidity that might interfere with the assay. Testosterone measurements were flagged if levels of total protein (85 g/L) or triglycerides (>20 mmol/L) could interfere with the assay measurements. To monitor assay consistency, all samples were run with internal quality control samples between batches and operations used external quality assurance schemes against the ISO 17025:2005 standard.

UK Biobank Outcome Definitions ? Dichotomous Outcomes Erectile dysfunction, fracture, arterial embolism and thrombosis, benign prostatic hyperplasia, heart failure, prostate cancer, testicular cancer, type 2 diabetes, and venous thromboembolism cases were derived based on ICD-10 codes from hospital inpatient episode, death registry, and cancer registry records linked to each participant. Myocardial infarction, stroke, and dementia outcomes were defined using data derived from algorithmically-defined outcomes as recommended by the UK Biobank adjudication committee, which included hospital inpatient episode, death registry, and cancer registry records as well as self-reported medical conditions, medications and operations2. Depression was coded using a "broad" definition as previously described, which included self-reported depressive symptoms with associated impairment, or having sought help for "nerves, anxiety, tensions or depression"3. Androgenic alopecia was defined based on participants' responses to the question, "Which of the following best describes your hair/balding pattern?" (field ID 2395). Available options were four pictures of hair patterns (Supplementary Figure 7). Individuals with pattern 3 or 4 were cases, pattern 1 and 2 were controls, and "do not know" or "prefer not to answer" responses were excluded (Supplementary Figure 1).

UK Biobank Outcome Definitions ? Continuous Outcomes Physical activity was assessed using the overall acceleration average from wrist-worn accelerometer devices over the course of approximately 7 days. Following UK Biobank recommendations, individuals were excluded from the analysis based on poorly calibrated data (field ID: 90016) or having worn the device for insufficient time to get a stable measure of physical activity (field ID: 90015)4. Blood pressure measures were coded as the average of two automated measurements of blood pressure taken a few moments apart by a registered nurse using an Omron 705 IT electronic blood pressure monitor. Body fat percentage and whole body fat-free mass were estimated based on impedance measurements from a Tanita BC418MA body composition analyser. Heel BMD was estimated as a Tscore based on quantitative ultrasound index through the calcaneus relative to that expected in someone of the same sex. Handgrip strength was calculated as the average of right and left hands measured using a Jamar J00105 hydraulic hand dynamometer. Haemoglobin A1C was measured using high performance liquid chromatography analysis on a Bio-Rad VARIANT II Turbo. Glucose was measured using hexokinase analysis on a Beckman Coulter AU5800. Liver fat was measured using magnetic resonance imaging and defined as previously described 5. Haematocrit percentage was measured using a Coulter LH750 and calculated as the relative volume of packed erythrocytes to whole blood, computed by the formula: !"# %&''# ("&&) +",- ('!./)(/&,! 0'&/+".

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Genome-wide Association Study ? Quality Control To investigate potential confounding by population stratification, genomic inflation (l) was estimated by calculating the ratio of the median test statistic from the GWAS relative to the expected median test statistic under a null model.

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To distinguish between an inflated l due to population stratification or polygenic inheritance of the trait, the intercept of an LD score regression line was determined. LD score regression was performed and intercept was calculated with LDSC software 6 using 1000 Genomes Europeans phase 3 data as the LD reference panel7.

Supplementary Text

Quality Control of Genome-wide Association Study Test statistics appeared inflated relative to estimates expected under a null distribution (l=1.112) (Supplementary Figure 6). However, the intercept of the LD score regression (1.025) suggested the observed inflation could be attributed to polygenicity rather than uncontrolled population stratification.

Supplementary Figure 1. Rationale for Mendelian randomization analysis.

Supplementary Figure 2 Legend. Comparison of randomized controlled trial (RCT) and Mendelian randomization (MR) study designs demonstrating the common foundation behind interpretation of a causal effect of testosterone on cardiovascular disease (CVD). In accordance with Mendel's second law, random and independent inheritance of alleles can be thought of akin to random allocation of treatment vs. placebo in RCT. Therefore, by the same reasoning, if MR finds genetic variants affecting testosterone are associated with a difference in CVD risk, it provides evidence that testosterone causally affects CVD.

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Supplementary Figure 2. Distribution of natural log-transformed sex hormone-binding globulin levels in males from the UK Biobank cohort.

Supplementary Figure 3. Distribution of free testosterone levels calculated using the Vermeulen equation in males from the UK Biobank cohort.

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Supplementary Figure 4. Manhattan plot showing distribution of p-values from genome-wide association study of calculated free testosterone according to chromosomal location of each genetic variant.

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Supplementary Figure 5. Association of genetically-predicted calculated free testosterone with significant outcomes using one-sample Mendelian randomization analysis stratified by age in males from the UK Biobank.

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Supplementary Figure 6. Quantile-quantile plot showing observed test statistics in genome-wide association study of calculated free testosterone levels relative to expected test statistics under a null model.

Supplementary Figure 7. Screenshot of options shown to male UK Biobank participants for selection of hair/baldness pattern.

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Supplementary Table 1. Definitions for 31 health outcomes from the UK Biobank

study.

Outcome

UK Biobank field ID

ICD-10 codes for cases (if applicable)

Ncases/Ncontrols Ntotal

Outcomes with Expected Clinical Benefits

S02-, S12-, S22-, S32-,

All Fracture

41270, 40001, 40002

S42-, S52-, S62-, S72-,

9,133/148,098

S82-, S92-, T02-

Body Fat Percentage

23099

??

154,095

Dementia

42018

??

1,003/156,228

Alzheimer's Dementia

42020

??

349/156,882

Depression

2090, 2100, 41270

F32-, F33-, F34-, F38-, F39-

4,725/152,506

Erectile Dysfunction

41270, 40001, 40002

N48.4, F52.2

221/157,010

Handgrip Strength

46 and 47

??

156,403

Heel Bone Mineral Density

3148

??

90,597

Accelerometer-based Physical Activity

90012

??

30,439

Whole Body Fat-Free Mass

23101

??

154,262

Outcomes with Potential Adverse Effects

All-cause Stroke

42006

??

4,569/152,662

Ischaemic Stroke

42008

??

2,122/155,109

Intracerebral Haemorrhage

42010

??

411/156,820

Subarachnoid Haemorrhage

42012

??

348/156,883

Androgenic Alopecia

2395

??

70,283/85,757

Arterial Embolism and Thrombosis

41270, 40001, 40002

I74-

629/156,602

Benign Prostatic Hyperplasia

41270, 40001, 40002

N40-

10,894/146,337

Diastolic Blood Pressure

4079

??

145,156

Glucose

30740

??

138,308

Hematocrit Percentage

30030

??

152,893

Hemoglobin A1c

30750

??

149,829

Heart Failure

41270, 40001, 40002

I50-

4,288/152,943

Liver Fat

22402

??

1,595

Prostate Cancer

41270, 40001, 40002, 40006

C61-

7,586/149,645

Myocardial Infarction

42000

??

9,398/147,833

Systolic Blood Pressure

4080

??

145,155

Testicular Cancer

41270, 40001, 40002, 40006

C62-

410/156,821

Type 2 Diabetes

41270, 40001, 40002

E11-

11,079/146,152

Venous Thromboembolism

41270, 40001, 40002

I26-, I80-, I81-, I82-

4,127/153,104

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