A total of 2,822 Mexican dyslipidemic cases and controls were recruited at INCMNSZ in

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1 Supplemental Material The N342S MYLIP polymorphism is associated with high total cholesterol and increased LDL-receptor degradation in humans by Daphna Weissglas-Volkov et al. Supplementary Methods Mexican dyslipidemic cases and controls A total of 2,822 Mexican dyslipidemic cases and controls were recruited at INCMNSZ in Mexico City, as described in detail previously (1,2). Briefly, the inclusion criteria were fasting serum TGs > 2.3 mmol/l (2 mg/dl) for the cases and < 1.7 mmol/l (15 mg/dl) for the controls (3). Exclusion criteria were type 2 diabetes mellitus or morbid obesity (body mass index > 4 kg/m 2 ), and the use of lipid lowering drugs for the controls. For the association analysis with the binary TC trait, the Third Report of The National Cholesterol Education Program (NCEP) cut point of high TC was employed (3). Accordingly, the subjects were classified as high-tc if their serum TC levels were 6.2 mmol/l (24 mg/dl) (n=492) and normal TC if their serum TC levels were < 6.2 mmol/l in the absence of lipid lowering medication (n=2,233). The subjects were also classified as combined hyperlipidemia if their serum TC and TG levels were 6.2 mmol/l and 2.3 mmol/l, respectively (n=35), and as controls if TC and TG levels were < 6.2 mmol/l and 1.7 mmol/l, respectively, in the absence of lipid lowering medication (n= 1,356). Individual Ancestry (IA) Analyses In order to adjust for the admixed Mexican ancestry (4,5) individual ancestry (IA) estimates were used as a covariate in the regression analyses. IA was estimated using 82 ancestry informative markers (AIMs), i.e. SNPs that discriminate between the parental subpopulations that were evenly distributed along the genome (6). These AIMs were selected based on a published list of European/Amerindian AIMs (7) and were used to calculate IA estimates using the STRUCTURE 2.2 software (8), as we described previously (6). The AIMs were not available for the 512 additional dyslipidemic study samples that were further genotyped for rs Therefore, their IA estimates were imputed based on TC levels using the following model: AI i

2 =α+β TC Y i,, where the intercept α and the slope β were estimated by regressing the AI estimates from the sample with AIMs (n=2,31) against the rank of TC residuals adjusted for age, sex, and hypertriglyceridemia affection status.

3 Supplementary References 1. Weissglas-Volkov D, Aguilar-Salinas CA, Sinsheimer JS, Riba L, Huertas-Vazquez A, Ordoñez-Sánchez ML, et al. Investigation of variants identified in caucasian genome-wide association studies for plasma high-density lipoprotein cholesterol and triglycerides levels in mexican dyslipidemic study samples. Circ Cardiovasc Genet. 21 Feb 1;3(1): Weissglas-Volkov D, Plaisier CL, Huertas-Vazquez A, Cruz-Bautista I, Riaño-Barros D, Herrera-Hernandez M, et al. Identification of two common variants contributing to serum apolipoprotein B levels in Mexicans. Arterioscler. Thromb. Vasc. Biol. 21 Feb;3(2): Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). Jama. 21 May 16;285(19): Price AL, Patterson N, Yu F, Cox DR, Waliszewska A, McDonald GJ, et al. A genomewide admixture map for Latino populations. Am J Hum Genet. 27 Jun;8(6): Salari K, Burchard EG. Latino populations: a unique opportunity for epidemiological research of asthma. Paediatr Perinat Epidemiol. 27 Nov;21 Suppl 3: Weissglas-Volkov D, Plaisier CL, Huertas-Vazquez A, Cruz-Bautista I, Riaño-Barros D, Herrera-Hernandez M, et al. Identification of Two Common Variants Contributing to Serum Apolipoprotein B Levels in Mexicans. Arterioscler. Thromb. Vasc. Biol [Internet]. 29 Dec 3;Available from: 7. Tian C, Hinds DA, Shigeta R, Adler SG, Lee A, Pahl MV, et al. A genomewide singlenucleotide-polymorphism panel for Mexican American admixture mapping. Am J Hum Genet. 27 Jun;8(6): Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2 Jun;155(2):

4 Figure legends Supplementary Figure S1. Trait distributions in the Mexican subjects that were sequenced for the MYLIP gene and in the Mexican dyslipidemic cases and controls. Frequency distributions of LDL-C values in the low-ldl resequencing sample (A) and in the high-ldl resequencing sample (B). Frequency distributions of standardized TC (C) and non-hdl-c (D) residuals corrected for age, sex and hypertriglyceridemia affection status in the dyslipidemic study sample. Supplementary Figure S2. LD across the MYLIP gene (±5 kb) in the Mexican and European ancestries. LD was calculated in r 2 using the 6 Mexican-American founders (Left panel) and 6 CEU founders with European ancestry (Right panel) of the HapMap project. The red points represent the MYLIP gene. The Caucasian genome-wide significant variants are indicated in black and the most significantly associated SNP in Mexicans in blue. Supplementary Figure S3. Analysis of the effect of MYLIP point mutations on LDLR degradation. (A) Immunoblot of HEK293T whole cell lysate co-transfected with LDLR and MYLIP expression plasmids with either isoleucine (I22) or leucine (L22) at residue 22. The ratio of MYLIP:LDLR expression vector was altered while maintaining a constant amount of total DNA transfected. (B) Immunoblot of HEK293T whole cell lysate co-transfected with LDLR and MYLIP expression plasmids with either valine (V339) or isoleucine (I339) at residue 339. The ratio of MYLIP:LDLR expression vector was altered while maintaining a constant amount of total DNA transfected. (C) Quantification of LDLR expression in HEK293T whole cell lysate following co-transfection with LDLR and either N342 or S342 MYLIP expression plasmids. The ratio of MYLIP:LDLR expression vector was altered while maintaining a constant amount of total DNA transfected. Data are expressed as mean±sem (n=4/group). (D) Immunoblot of HEK293T whole cell lysate co-transfected with LDLR and FLAG-tagged WT (N342/V339/I22), C387A ring domain mutant (RING MUT), I339 or L22. The thin white line indicates that the lanes were run on the same gel but were noncontiguous. (E) Amino-acid sequence alignment of MYLIP homologs at position 342; N stands for asparagine and S for serine. (F) Immunoblot of HEK293T whole cell lysate following co-transfection with LDLR and

5 mouse MYLIP S342, N342 or RING MUT expression constructs as indicated. (G) Immunoblot of HEK293T cell surface protein isolated by biotinylation following transfection with LDLR and MYLIP N342 (WT), S342, I339 or RING MUT expression constructs as indicated.

6 Supplementary Table S1. Clinical characteristics of the Mexican subjects that were sequenced for the MYLIP gene and of the Mexican dyslipidemic case/control study sample. Trait Study Sample A Low-LDL High-LDL B Hypertriglyceridemic Normotriglyceridemic N (% Females) 56 (7%) 66 (83%) 1,29 (49%) 1,532 (54%) Age, years 41.2± ± ± ±.28 LDL-C, mmol/l 1.83± 5.6± ± ±.2 TC, mmol/l 3.52±.7 7.7± ± 4.87± TG,mmol/L.97± ± 3.21± 1.11± apob, g/l 62.84± ± ± ±.62 BMI, kg/m ± ± ± ±.11 Amerindian.49±.46±.5±.52± ancestry estimates Trait values represent the marginal means evaluated at the average age and sex ± SEM. A The coding sequence of the MYLIP gene was sequenced in the Mexican subjects with low LDL-C and high LDL-C, and the GWA associated region of the MYLIP gene was fine mapped in the Mexican dyslipidemic case/control study sample. B Marginal means ± SEM were calculated in a linear mixed effects model using the kinship library in R to adjust for family relations.

7 Supplementary Table S2. Sequence variants identified in the coding and exon-intron boundaries of MYLIP. Variant a Position b Location Major, Low-LDL High-LDL (Type) minor Genotype Genotype MAF e allele c count d MAF count d rs 'UTR G/T /2/48.2 /1/59.8 I22L Exon 4 (Nonsynonymous) A/C /2/54 8 /2/62 9 rs Intron4 (-34bp) A/G 4/19/3.25 7/21/ rs Exon 5 G/A /1/52 /3/61.28 (Synonymous) L338L Exon 6 C/T /1/54.9 /1/63.9 (Synonymous) V339I Exon 6 (Nonsynonymous) G/A /2/53.9 //64. rs Exon 6 (Nonsynonymous) G/A 4/26/ /23/ rs Exon 6 C/T /6/49.56 /1/63.9 (Synonymous) rs 'UTR T/C /7/46.67 /5/61 5 C.*871T>C 'UTR T/C //53. /1/6 rs 'UTR G/T 4/29/ /28/ rs 'UTR G/A /2/53 9 //66. All variants had at least a 9% genotype call rate and were in Hardy-Weinberg equilibrium (P >.5). a The unknown coding variants are called according to the amino-acid position and/or substitution, and the unknown 3 UTR variants are called according to the HGVS nomenclature guidelines ( b Basepair position is indicated according to the human reference sequence NCBI36/hg18. c Alleles are shown in + strand relative to the human reference sequence. d Genotype count of the homozygote rare/heterozygote/homozygote common alleles. e Minor allele frequency in unrelated founder individuals.

8 Supplementary Table S3 Sequencing variants investigated in the Mexican normotriglyceridemic study samples Variant LDL-C mmol/l a ALL Low-LDL High-LDL C/C R/X Genotype count b MAF Genotype count b MAF Genotype count b MAF V339I 3.15± ±1.9 /3/173.1 /1/265.2 /2/379.3 rs ± 2.99±.13 1/32/415 7 /13/12 6 1/1/127 3 rs ± 2.82±.23 /13/423 4 /5/131 8 /4/126 5 a C/C represent the mean ± SEM in homozygotes of the common allele and R/X the mean in carriers of the rare allele. b Genotype count of the homozygote rare/heterozygote/homozygote common alleles.

9 Supplementary Table S4. Association results for the MYLIP region with TC and non-hdl-c in the Mexican dyslipidemic study sample. Position type Minor (SE) P P.ADJ P.CON (SE) P P.ADJ P.CON SNP BP SNP Major/ MAF High TC Status TC residuals Non HDL-C residuals Best GWA tag F TC TC (SE) C P P.ADJ D P.CON E SNP r 2 A B High Normal Effect D E Effect D E Effect rs g G/A (.12) 7.21E E E-1 () 5.48E E E-1 () 8.13E E E-1 rs i C/T (.12) 7.21E E E-1 () 5.48E E E-1 () 8.12E-1 9.2E E-1 rs g G/A (.9) 9.95E-1 8.9E-1 9.7E-1 () 6.94E E E-1 () 9.52E E E-1 rs g G/T (.9) 7.38E-1 7.1E E-2 () 5.1E E-1 1.6E-1 () 9.93E E-1 4.4E-1 rs i T/C (.9) 9.79E E E-1 () 6.86E E E-1 () 9.69E E E-1 rs i T/C (.9) 9.79E E E-1 () 6.86E E E-1 () 9.69E E E-1 rs g A/G (.12) 7.21E E E-1 () 5.48E E E-1 () 8.13E E E-1 rs i T/C (.8) 7.57E E E-1 () 3.94E E E-1 () 8.6E E-1 7.6E-1 rs i G/A (.8) 7.61E E E-1 () 3.97E E E-1 () 8.63E E E-1 1 rs i T/A (.9) 9.85E E E-1 () 6.94E-1 8.6E E-1 () 9.61E E E-1 rs i A/G (.11) 1.3E E E-1.6 () 1.5E E-1 3.6E-1 () 4.84E E E rs g C/A (.9) 4.48E-2 2.1E E-1 () 3.35E E E-2 () 9.25E E-2 1.4E-1 rs rs g T/G (.9) 1.71E E E-1 () 2.13E E E-1 () 1.87E E E-1 rs rs i G/T (.9) 3.98E E E-1 () 4.22E-3 2.5E-2 1.6E-1 () 1.11E E E-1 rs rs i G/C (.47) 3.89E-1 4.3E E-1 (.15) 3.27E E E-1 (.15) 4.75E E-1 4.2E-1 rs i C/T (.9) 7.64E E E-1 () 2.6E E E-1 () 4.12E E E-1 rs g G/A (.8) 1.13E E E-1.5 () 8.51E E E-1.6 () 6.77E E E-1 rs rs g T/C (.12) 2.2E E E-1 (.5) 4.25E E E-1 (.5) 7.82E E E-1 rs g A/G E E E E E-2 7.1E-2 7.9E-3 2.3E E-2 rs

10 (.9) () () rs i T/A (.8) 2.57E E E-1 () 2.85E-1 4.7E E-1 () 1.99E E E-1 rs i T/C (.8) 2.55E E E-1 () 2.89E E E-1 () 2.8E E-1 9.7E rs i T/C (.8) 2.44E-1 4.7E E-1 () 2.87E-1 4.8E E-1 () 2.7E E-1 9.7E-1 rs g C/A 1.19 (.41) 6.74E E E-1 - (.15) 8.15E-1 7.1E E-1 (.15) 6.53E E E-1 rs g C/T (.8) 2.53E E E-1 () 2.92E-1 4.6E E-1 () 2.12E E E rs i G/A.4.37 (.8) 1.49E E E-1 () 1.52E E E-1 () 1.18E E-1 6.7E-1 rs g A/G (.8) 1.66E E-2 9.1E-1.6 () 2.76E E-2 5.7E-1.7 () 1.98E E E-1 rs rs i A/G 1.44 (.51) 4.49E E E-1.5 (.15) 7.51E-1 8.6E-1 6.6E-1 - (.15) 9.29E E E-1 rs g C/T (.15) 4.11E E-1 2.2E-1 - (.5) 5.27E E-1 1.E+ (.5) 6.54E E E-1 rs i C/T (.9) 2.24E E E () 3.77E E E-1 () 2.37E E E-1 rs rs g C/A (.9) 1.26E E E-1.8 () 2.3E-2 6.9E E-1.8 () 2.8E E E-1 rs rs i T/C (.9) 2.E E-2 5.6E-1.8 () 2.18E E E-1.7 () 2.3E E E-1 rs rs g G/A (.9) 2.62E-2 9.5E E () 6.56E E E () 3.2E E E-2 rs rs g T/C (.8) 2.57E-4 3.8E E-1.9 () 7.E-3 3.4E E-1.8 () 1.26E E E-1 rs rs g A/G (.9) 1.14E E-1 9.5E-1 () 6.15E E E-1 () 5.45E-1 8.1E E-1 rs rs g T/G (.9) 1.15E E E-1 () 6.55E E-1 4.4E-1 () 5.24E E E-1 rs rs g G/A.9.9 (.14) 6.15E E-1 3.2E-1 (.5) 9.93E-1 6.4E E-1 (.5) 7.8E-1 5.E E rs i C/T (.8) 4.97E-6 5.4E E-1 () 3.43E-4 8.5E E-1 () 2.8E E-2 5.1E-1 rs rs g C/T (.9) 9.62E E-2 5.7E-1 () 5.36E E E-1 () 1.39E E-2 2.5E-1 rs rs g A/G (.9) 1.27E E E-1 - () 2.66E E E-1 - () 3.97E E E-1 rs rs i A/G E E E-1-4.4E-1 9.1E E E E E-1 rs

11 (.12) () () rs i C/T (.9) 1.98E-2 1.3E E-1 - () 2.6E E-1 8.2E-1 - () 3.44E-1 6.1E E-1 rs rs i C/T (.12) 2.86E-2 2.2E E-1 - () 4.52E E E-1 () 5.74E E E-1 rs rs i A/G.59 (.67) 3.79E E E-1.13 (.17) 4.8E-1 4.5E E-1.15 (.17) 4.29E-1 4.8E-1 4.9E-1 rs i A/G (.8) 1.86E E E-1 () 1.24E E E-1 - () 2.55E E E-1 rs rs g T/G (.9) 3.57E E E-1 - () 3.8E-1 5.5E E-1 - () 6.4E E E-1 rs rs i A/G (.9) 2.8E E E-1 () 9.94E E E-1 - () 1.57E E E-1 rs rs g T/C (.8) 1.39E-1 2.1E-1 3.6E-1 () 8.71E E-1 9.2E-1 - () 1.73E-1 2.3E E-1 rs rs g G/A (.9) 1.51E E-5 NA.12 () 2.26E E-3 NA.11 () 1.81E E-3 NA rs rs i C/T.5.78 (.21) 2.24E E E (.7) 1.19E E E (.7) 7.57E E E-1 rs g T/C (.18) 4.41E E E-1 (.6) 2.75E E E-1 (.6) 1.87E E E-1 rs i G/T (.9) 1.36E E-1 3.2E-1 () 1.8E E E-1 () 4.51E E E-1 rs rs i G/T.6.9 (.19) 4.95E E-2 6.6E-2 (.5) 4.27E E E-1 (.5) 6.27E E-1 9.1E-1.97 rs g G/T (.8) 7.5E E E-2 () 5.46E E E-1 () 9.14E E E-1 rs rs g G/A (.9) 3.55E E E-1 () 3.95E E E-1 () 8.87E-1 8.6E-1 2.2E-1 rs rs g T/C.3.37 (.9) 1.7E-4 1.2E E-2 () 7.36E E E-1 () 3.48E E E-2 rs rs i C/G (.1) 9.4E-2 4.4E E-1 () 1.22E E-1 9.2E-1 () 3.96E-1 7.6E E-1 rs rs i C/T.3.37 (.9) 8.16E E E-2 () 8.23E-3 3.3E E-1 () 3.99E E-2 9.8E-2 rs rs g G/A (.9) 7.94E-5 7.8E-4 2.E-2 () 6.87E E E () 3.38E E E-2 rs rs i A/G (.9) 6.6E E E-2 () 8.28E E E () 4.39E E E-2 rs rs g A/C (.11) 5.38E E E-2 () 5.65E E E-1 () 8.61E E E-1 rs rs i G/C E-3 1.1E E E E E E-1 2.9E E-1 rs

12 (.12) () () rs g G/A (.9) 8.95E E E-1.11 () 8.37E E E-1.9 () 7.26E E E-1 rs rs i A/G (.12) 3.36E E E-2 () 6.59E E-1 3.4E-1 () 1.19E-1 2.4E E-1 rs rs g G/A (.12) 4.3E E E-2 () 5.6E E E-1 () 9.36E E E-1 rs rs i C/G (.27) 7.52E E E-1 (.8) 7.33E E-1 9.8E-1 (.8) 5.91E E E-1 rs i C/A (.12) 3.71E E E-2 () 6.55E E E () 1.17E-1 2.3E E-1 rs rs i A/G (.55) 3.97E E E-1 (.15) 3.81E E E-1 (.15) 1.73E E-1 2.8E rs i A/C (.1) 6.69E E-1 5.6E-1 () 5.48E E E-1 () 2.38E E E-1 rs rs i G/T (.9) 1.9E E E-1 () 1.37E E E-1 () 8.17E E E-1 rs rs g T/C (.9) 1.23E-2 3.8E-2 4.1E-1 () 1.33E E E-1 () 7.75E E E-1 rs rs i G/A (.9) 1.11E E E-1 () 1.38E E E-1 () 8.28E E E-1 rs rs g G/A (.9) 1.14E E E-1 () 1.42E E E-1 () 8.1E-2 1.2E E-1 rs SNPs surpassing the multiple-testing correction threshold (P<7.2x1-4) are designated in red and results at P.5 are in boldface. AThe genotyped SNPs are indicated by g and imputed SNPs by i'. BMajor/minor alleles are in + strand relative to the human reference sequence (Build 36.1). CThe effect size represents the odds-ratio of each copy of the risk allele for the TC affection status and the proportion of 1 SD change in standardized residual values for each copy of the risk allele for the continuous TC and non- HDL-C levels. DP-value adjusted for admixture ancestry using the individual ancestry estimates as a covariate in the regression analysis. EP-value for conditional analysis including rs937867genotypes as a covariate in the regression analysis. FThe GWA signal (rs , rs937867, or rs ) that captures best this allele with r2 threshold.3.

13 Supplementary Figure S1.

14 Supplementary Figure S2.

15 Supplementary Figure S3