Supplementary Table I. List of mutations presented by the Portuguese pediatric cohort with FH

Transcription

1 Supplementary Table I. List of mutations presented by the Portuguese pediatric cohort with FH Index patients (n=89) FH children Cascade screening (n=82) cdna Mutation Protein 3 0 c.10580g>a p.arg3527gln APOB exon 26 Gene Location Allele classification Functional studies & product activity 32% of protein activity (Innerarity TL et al Proc Natl Acad Sci 84(19): ) Amino acid Segregation analysis conservation Normolipidemic panel (C/A;C/NA) Reported in other populations Moderately conserved 21/21;0/10 (9 families) Innerarity TL et al Proc Natl Acad Sci 84(19): ; Miserez et al J Lipid Res. 34: ; Myant et al Genomics 45: 78-87; Rabés et al Hum Mutat. 10: ; Wenham et al Atherosclerosis. 129(2):185-92; Borén et al J Biol Chem. 276(12): c.10679a>g p.tyr3560cys APOB exon 26 Moderately conserved 4/4;0/0 Liyanage et al Ann Clin Biochem 45(Pt 2): c.-135c>g LDLR Promotor 5-15% LDLR activity in homozygote (Hobbs et al Hum Mutat 1(6):445-66); RT-PCR show no transcription from this allele (Bourbon et al J Med Genet 46(5):352-7) 25/26;0/8 (10 families) Hobbs et al Hum Mutat. 1(6):445-66; Koivisto et al PNAS 91 : ; Jensen et al Hum Mutat. 7(1):82-4; Mozas et al 2004 Hum Mutat c.[-30-?_190+?del;1061-?_1845+?del] LDLR Promotor FH Siracusa (deletion promotor to exon 2): 45% LDLR activity in heterozygous fibroblasts (Garuti et al Atherosclerosis 121(1):105-17) 39/39;0/14 (13 families) Garuti et al Atherosclerosis 121(1): (20kb deletion of promotor to exon 2 reported as FH Siracusa); Chaves et al Eur J Clin Invest 31(4): (18kb deletion of promoter to exon 2 reported as FH Valencia-1) 1 1 c.1a>c p.met1leu LDLR exon 1 Note: luciferase assays showed that an alteration in the same amoniacid position but for a different nucleotide change (c.1a>t; p.met1leu) produces no detectable protein (Langenhoven et al Atherosclerosis. 125(1):111-9) Moderately conserved 5/5;0/0 Chaves et al J Clin Endocrinol Metab 86(10): ; Fouchier et al Hum Mutat 26(6):550-6; Tosi et al Atherosclerosis 194(1): c.58g>a p.gly20arg LDLR exon 1 Moderately conserved 1/2;0/0 Amsellem et al Hum Genet 111(6):501-10; Widhalm et al J Inherit Metab Dis 30(2):239-47; Chmara et al J Appl Genet 51(1): c.68-?_313+?del LDLR exon 2 40% of LDLR activity in homozygote with FH Tomani-2 (Mabuchi et al Ann. NY Acad. Sci. 598: ) 5/5;0/0 Hobbs et al Hum Mutat 1(6): (10kb deletion of exons 2 to 3 reported as FH Tonami-2); Ma et al Clin Genet 36(4): (5kb deletion of exons 2 to 3 reported as FH French Canadian-5); Yamakawa et al Hum Genet 82(4): (5kb deletion of exons 2 to 3 reported as FH Tsukuba-1) 0 1 c.190+4instg LDLR intron 2 Retention of 2 nt of intron 2 generating premature stop codon at aa 206 (Medeiros et al Atherosclerosis 212(2):553-8) 1 1 c.235inst p.val79cysfs*51 LDLR exon 3 4/5;0/1 4/4;0/2 0 1 c.241c>t p.arg81cys LDLR exon 3 Highly conserved 1/1;1/3 Nissen et al Clin Genet 53(6):433-9; Loubser et al Clin Genet 55(5):340-5; Fouchier et al Hum Genet 109(6): c.325t>c p.cys109arg LDLR exon % LDLR activity in heterozygote (Hobbs et al Hum Mut 1(6): ) Hobbs et al Hum Mutat. 1(6):445-66; Robles-Osorio et al Arch Med Res. 37(1):102-8; Fouchier et al Hum Mutat. 26(6): c.326g>t p.cys109phe LDLR exon 4 4/4;0/1 1 0 c.369_370deltc p.arg124alafs*5 LDLR exon c.369_393del p.arg124glyfs*74 LDLR exon 4 1/1;0/0 2/2;0/2 1 1 c.427t>c p.cys143arg LDLR exon 4 Nauck et al Hum Mutat 18(2):165-6; Rukavina et al Clin Chem Lab Med 39(6): c.502g>c p.asp168his LDLR exon 4 <2% LDLR activity in homozygote (Leitersdorf et al Hum Genet 91(2):141-7) 2/2;0/5 Leitersdorf et al Hum Genet Hum Genet. 91(2):141-7; Fouchier et al Hum Mutat. 26(6): c.530c>t p.ser177leu LDLR exon 4 <2% LDLR activity in homozygote (Hobbs et al J Clin Invest 84(2): ) 31/33;0/8 (9 families) Hobbs et al J Clin Invest. 84(2):656-64; Schuster et al Clin Investig. 71(2):172-5; Hobbs et al Hum Mutat. 1(6):445-66; Cenarro et al Clin Genet. 49(4):180-5; Gorski et al Hum Genet. 102(5):562-5; Chmara et al J Appl Genet. 51(1):95-106

2 2 0 c.551g>a p.cys184tyr LDLR exon 4 8/10;0/0 Lee et al J Med Genet 35(7):573-8; Graham et al Atherosclerosis 147(2):309-16; Wang et al Hum Mutat 18(4):359; Fouchier et al Hum Genet 109(6): c.590g>t p.cys197phe LDLR exon 4 <2% LDLR activity in heterozygote (Hobbs et al Hum Mut 1(6):445-66) 3/3;0/2 Hobbs et al Hum Mutat. 1(6): c.618_638del p.gly207_ser213del LDLR exon 4 14/15;0/7 (5 families) 0 1 c.[631c>g;1816g>t] p.[his211asp;ala606ser] LDLR exon 4 & exon 12 Both amino acids 4/4;1/0 1 1 c.661g>t p.asp221tyr LDLR exon 4 5/5;0/2 Koivisto et al Am J Hum Genet 57(4):789-97; Giesel et al Hum Genet 96(3):301-4; Kotze et al Clin Genet 51(6):394-8; Cenarro et al Hum Mutat 11(5): c.662a>g p.asp221gly LDLR exon % LDLR activity in heterozygote (Hobbs et al Hum Mut 1(6):445-66) Hobbs et al Hum Mutat. 1(6):445-66; Ekstrom et al Eur J Clin Invest. 28(9):740-7; Kotze et al S Afr Med J. 76(8): ; Fouchier et al Hum Genet. 109(6):602-15; Chmara et al J Appl Genet. 51(1): c.670g>a p.asp224asn LDLR exon 4 <2% LDLR activity in homozygote (Hobbs et al Hum Mut 1(6): ) 41/44;0/12 (14 families) Hobbs et al Hum Mutat. 1(6):445-66; Wang et al J Lipid Res. 46(2): c.682g>a p.glu228lys LDLR exon 4 <2% LDLR activity in homozygote (Leitersdorf et al J Clin Invest.85(4): ) Leitersdorf et al J Clin Invest. 85(4): ; Hobbs et al Annu Rev Genet. 24:133-70; Pimstone et al Arterioscler Thromb Vasc Biol. 17(5):826-33; Pimstone et al Arterioscler Thromb Vasc Biol. 18(2):309-15; Bertolini et al Arterioscler Thromb Vasc Biol. 20(9):E41-52; Huijgen et al Hum Mutat. 31(6): c.818-2a>g LDLR intron 5 Retention of 10 nt of intron 5 generating a premature stop codon at aa 303 (Medeiros et al Atherosclerosis 212(2):553-8) 9/9;0/5 1 0 c.862g>a p.glu288lys LDLR exon 6 Varret et al Nucleic Acids Res 1;26(1):248-52; Fouchier et al Hum Genet 109(6):602-15; Bunn et al Hum Mutat 19(3): c.1016_1017insg p.cys340valfs*18 LDLR exon 7 4/4;0/0 1 0 c.1027g>a p.gly343ser LDLR exon % LDLR activity in heterozygote (Hobbs et al Hum Mut 1(6):445-66) 2/2;0/5 Hobbs et al Hum Mutat. 1(6):445-66; Reshef et al.1996 Hum Genet. 98(5):581-6; Thiart et al Mol Cell Probes. 14(5): ; Fouchier et al Hum Genet. 109(6):602-15; Mozas et al Hum Mutat. 24(2): c.1033c>t p.gln345* LDLR exon 7 3/3;0/2 0 3 c g>a LDLR exon 7 Skipping of exon 7 generating a premature stop codon at aa 779 (Medeiros et al Atherosclerosis 212(2):553-8) 7/9;0/2 1 0 c.1061-?_1845+?del LDLR exon 8 Note: 2.1 kb deletion of exons 8 12 reported in Bourbon et al Atherosclerosis 196(2): /6;0/1 1 0 c.1085dela p.asp362alafs*8 LDLR exon c.1216c>t p.arg406trp LDLR exon 9 2/2;0/1 27/29;5/7 (13 families) Reshef et al Hum Genet 98(5):581-6; Chiou et al Am J Cardiol 105(12): c.1222g>a p.glu408lys LDLR exon 9 <2% LDLR activity in homozygote (Hobbs et al Hum Mut 1(6):445-66)); 5-10% LDLR activity in homozygote (very low or virtually undetectable LDLreceptor activity) (Webb et al J Lipid Res 37(2):368-81; Amsellem et al Hum Genet 111(6):501-10) 8 9 c.1291g>a p.ala431thr LDLR exon 9 20% LDLR activity in COS-7 cells (Chang et al J Lipid Res 44(10):1850-8) 103/112;0/23 (33 families) Hobbs et al Annu Rev Genet. 24:133-70; Hattori et al Hum Mutat. 14(1):87; Fouchier et al Hum Genet. 109(6):602-15; Chang et al J Lipid Res. 44(10):1850-8; Dedoussis et al Eur J Clin Invest. 34(6):402-9; Chmara et al J Appl Genet. 51(1):95-106; Chiou KR, Charng MJ Am J Cardiol. 105(12):1752-8

3 2 0 c.1322t>c p.ile441thr LDLR exon 9 9/10;0/2 (4 families) Fouchier et al Hum Genet 109(6):602-15; Chiu et al Metabolism 54(8):1082-6; Chiou et al Am J Cardiol 105(12): c c>g LDLR intron 9 Retention of intron 9 (Bourbon et al J Med Genet 46(5):352-7) 4/5;2/2 1 1 c.1432g>a p.gly478arg LDLR exon % LDLR activity in heterozygote (Hobbs et al Hum Mut 1(6): ) 6/6;0/4 Hobbs et al Hum Mutat. 1(6):445-66; Koivisto et el Am J Hum Genet. 57(4): ; Mak et al Arterioscler Thromb Vasc Biol. 18(10):1600-5; Fouchier et al Hum Genet. 109(6):602-15; Ward et al Hum Mutat. 6(3): c.1455c>g p.his485gln LDLR exon 10 1/1;2/3 1 1 c.1468t>c p.trp490arg LDLR exon 10 5% LDLR activity in COS-7 cells (Silva S. et al Atherosclerosis. 225(1):128-34) 4/4;0/2 1 0 c.1587-?_1845+?del LDLR exon 11 4kb deletion of exons 11 to 12 reported as FH Genoa-1 produces a sequence of 48 novel amino acids preceding a stop codon (Bertolini et al Arterioscler Thromb Vasc Biol 15(1):81-8) 1/1;0/1 1 0 c.1599g>a p.trp533* LDLR exon c.1618_1620delgcc p.ala540del LDLR exon c.1633g>t p.gly545trp LDLR exon c.1756delt p.ser586glnfs*79 LDLR exon 12 2/3;0/0 23/24;1/9 (7 families) 3 1 c.1775g>a p.gly592glu LDLR exon % LDLR activity when compound heterozygote (Hobbs et al Hum Mut 1(6): ) 17/18;0/3 (7 families) Hobbs et al Hum Mut 1(6): ; Gorski et al Hum Genet. 102(5):562-5; Zakharova et al Bioorg Khim. 27(5):393-6; Bochmann et al Hum Mutat. 17(1):76-7; Mozas et al Hum Mutat. 24(2):187; Chmara et al J Appl Genet. 51(1):95-106; Huijgen et al Hum Mutat 31(6): c.1802a>t p.asp601val LDLR exon c.1816g>t p.ala606ser LDLR exon 12 4/5;0/4 1 0 c.1830_1839delcttggccgt p.ala612argfs*50 LDLR exon 12 7/7;0/2 (5 families) 1/1;0/0 Sun et al Arterioscler Thromb Vasc Biol 17(11): ; Dedoussis et al Hum Mutat 23(3): c g>a LDLR intron 12 <2% LDLR activity in homozygote (Hobbs et al Hum Mutat 1(6):445-66); Mutant allele reduced to ~1/3 of the normal allele in heterozygous (Jensen et al Clin Genet 49: 175-9); RT-PCR of exon 10 to 15 revealed 3 transcripts: exon 12 joined to partially deleted exon 13; exon 11 joined to the partially deleted exon 13; exon 12 joined to exon 15 with complete skipping of exons 13 and 14 (Bertolini et al Arterioscler Thromb Vasc Biol 19(2):408-18) 1/1;0/0 0 1 c.1846-?_2311+?dup LDLR exon 13 25% of reduction in LDLR binding/internalization ( Lelli et al Hum Genet. 86(4):359-62) 4/4;0/0 Lelli et al Hum Genet. 86(4): (7kb duplication of exons 13 to 15 reported as FH Bologna-2) 2 0 c.1876g>a p.glu626lys LDLR exon 13 8/8;0/3 Fouchier et al Hum Mutat 26(6): c.1936delc p.leu646tyrfs*19 LDLR exon 13 2/2;0/1 0 1 c.1966c>a p.his656asn LDLR exon 13 2/3;3/6 Mozas et al Hum Mutat 24(2):187; 180(1): c.2054c>t p.pro685leu LDLR exon % LDLR activity in homozygote (Soutar et al Proc Natl Acad Sci 86(11): ) 9/11;0/4 Soutar et al Proc Natl Acad Sci 86(11): ; King-Underwood et al Clin Genet. 40(1):17-28; Maruyama et al Arterioscler Thromb Vasc Biol. 15(10):1713-8; Defesche et al Clin Genet. 42(6):273-80; Mak et al Arterioscler Thromb Vasc Biol. 18(10):1600-5

4 0 1 c.2056c>t p.gln686* LDLR exon 14 4/5;0/1 Thiart et al Mol Cell Probes 11(6):457-8; Gorski et al Hum Genet 102(5):562-5; Chmara et al J Appl Genet 51(1): c.2077_2078delaa p.lys693valfs*23 LDLR exon 14 4/4;0/2 Fouchier et al Hum Mutat 26(6): c.2093g>t p.cys698phe LDLR exon 14 ~50% of reduction in LDL binding/internalization in heterozygote (Alves et al in prep ) 4/5;0/0 Humphries et al J Mol Med (Berl) 84(3): c g>a LDLR intron 14 50% LDLR activity in heterozygote (Takada et al J Hum Genet 47(12):656-64) Takada et al J Hum Genet 47(12):656-64; Mozas et al Hum Mutat. 24(2): c.2389g>a p.val797met LDLR exon 16 Donor site splicing error (Mak et al Arterioscler Thromb Vasc Biol 18(10): ) Weakly conserved 5/5;1/2 Mak et al Arterioscler Thromb Vasc Biol. 18(10):1600-5; Fouchier et al Hum Genet. 109(6):602-15; Amsellem et al Hum Genet. 111(6):501-10; Robles-Osorio et al Arch Med Res. 37(1):102-8; Chmara et al J Appl Genet. 51(1): c.2312-?_*220+?del LDLR exon 16 Internalization- phenotype (Lehrman et al Science 227(4683):140-6) Rodningen et al Clin Genet 42(6): (9.6kb deletion of exon 16 to 18 reported as FH Helsinki); Lehrman et al J Biol Chem 262(7): (7.8kb deletion of exons 16 to 18 reported as FH Osaka-1); Lehrman et al Science 227(4683):140-6 (5.5kb deletion of exons 16 to 18 reported as FH Rochester) 0 2 c.2389g>t p.val797leu LDLR exon 16 Skiping of exon 16 (Bourbon et al J Med Genet 46(5):352-7) Weakly conserved Lombardi et al Clin Genet. 57(2): c.2397_2412del p.val800glyfs*124 LDLR exon c g>a LDLR intron 17 Skipping of exon 17 generating a premature stop codon at aa 805 (Medeiros et al Atherosclerosis 212(2):553-8) 6/6;0/2 Amino acid conservation was analysed considering 11 species; 95 Portuguese normolipidemic individuals; Segregation analysis is presented as mutation carriers/affected individuals; mutation carriers/non-affected individuals (C/A;C/NA), considering all patients recruited to the PFHS Carriers of mutations shaded in grey were not included in ROC curves and in clinical criteria analysis due to non existence of pre-treatment values

5 Supplementary Table II. Comparison of lipid profile in children identified with FH as index patients versus relatives identified in cascade screening FH children Index patients (n=89) Cascade screening (n=82) Age (years) 10.2± ±4.0 Mae gender 53.9% 50.0% Basic lipid profile (mg/dl) a n=89 n=68 TC 311.1±58.8* 273.0±59.0* LDL-C 233.3±59.7* 204.3±52.0* HDL-C 54.0± ±12.0 TG 92.0± ±44.7 non-hdl-c/hdl-c ratio 5.2± ±1.6 Advanced lipid profile (mg/dl) b n=50 n=50 Lp(a) 50.3± ±49.0 apo A ±25.1* 120.7±20.7* apo B 118.6± ±40.3 apo B/apo A1 ratio 0.9± ±0.6 Specific lipid profile (mg/dl) c n=25 n=13 apo A2 27.8±5.5* 23.5±5.5* apo C2 3.2± ±1.8 apo C3 7.6± ±2.0 apo E 3.4± ±0.8 sdldl 40.1±15.7* 28.1±14.4* Data are expressed as mean±standard deviation (SD) unless otherwise noted. FH: familial hypercholesterolemia; TC: total cholesterol; non-hdl-c: non-high-density lipoprotein cholesterol; Lp(a): lipoprotein(a); sdldl: small, dense LDL cholesterol; a: highest values reported without treatment; b: advanced lipid profile performed at INSA recorded only for children without treatment; c: specific lipid profile performed at INSA (only available after 2010) recorded only for children without treatment. *P<0.05 index patients vs. relatives.

6 Supplementary Table III. Lipid profile of children identified with FH according to the type of mutation Null mutation (n=47) LDLR gene Defective mutation (n=105) Splicing mutation (n=14) APOB gene (n=5) Age (years) 10.23± ± ± ±2.78 Male gender 51.0% 54.2% 50.0% 60.0% Basic lipid profile (mg/dl) a n=46 n=92 n=12 n=5 TC 313.7±60.3* 282.6±60.5* 314.2± ±37.4 LDL-C 240.3±63.7* 205.7±52.9* 238.3± ±38.2 HDL-C 53.3± ± ± ±9.6 TG 102.1± ± ± ±23.4 non-hdl-c/hdl-c ratio 5.1± ± ± ±0.4 Advanced lipid profile (mg/dl) b n=30 n=58 n=9 n=3 Lp(a) 51.1± ± ± ±72.4 apo A ± ± ± ±15.8 apo B 125.6± ± ± ±4.9 apo B/apo A1 ratio 0.99± ± ± ±0.1 Specific lipid profile (mg/dl) c n=10 n=25 n=1 n=2 apo A2 27.0± ±6.5 n.d. 24.7±1.0 apo C2 3.6± ±1.5 n.d. 3.8±0.7 apo C3 7.7± ±1.8 n.d. 6.4±0.5 apo E 3.6± ±0.9 n.d. 2.6±0.3 sdldl 42.3± ±17.1 n.d. 32.8±20.5 Data are expressed as mean±standard deviation (SD) unless otherwise noted. n.d. not determined. FH: familial hypercholesterolemia; TC: total cholesterol; non-hdl-c: non-high-density lipoprotein cholesterol; Lp(a): lipoprotein(a); sdldl: small, dense LDL cholesterol; a: highest values reported without treatment; b: advanced lipid profile performed at INSA recorded only for children without treatment; c: specific lipid profile performed at INSA (only available after 2010) recorded only for children without treatment. *P<0.05 mutation vs. mutation.

7 Supplementary Table IV. Simon Broome criteria and different criteria established for clinical diagnosis of FH. Results obtained for sensitivity, specificity, number of true and false positives, true and false negatives are also presented. Sensitivity Specificity True positives True negatives False positives False negatives A. Index cases*: 50 FH vs. 105 non-fh (n=155) Criteria # 1 (Simon Broome Criteria) 76.0% 68.6% Criteria #2 70.0% 76.2% Criteria #3 88.0% 55.2% Criteria #4 82.9% 66.0% Criteria #5 86.0% 68.6% B. Relatives*: 50 FH vs. 56 non-fh (n=106) Criteria # 1 (Simon Broome Criteria) 36.0% 100.0% Criteria #2 82.0% 75.0% Criteria #3 86.0% 75.0% Criteria #4 28.0% 100.0% Criteria #5 84.0% 75.0% C. Index cases + Relatives*: 100 FH vs. 161 non-fh (n=261) Criteria # 1 (Simon Broome Criteria) 56.0% 79.5% Criteria #2 76.0% 75.8% Criteria #3 87.0% 62.1% Criteria #4 47.0% 88.8% Criteria #5 85.0% 70.8% *Criteria were determined using pretreatment values (n=155). Bold indicates the best criteria representing optimal balance between sensitivity and specificity. Criteria #1 (Simon Broome Criteria): (TC 260 mg/dl OR LDL-C 155 mg/dl) AND (family history of pcvd OR family history of hypercholesterolemia); Criteria #2: apob/apoa1 ratio 0.68; Criteria #3: [(TC 260 mg/dl OR LDL-C 155 mg/dl) AND (family history of pcvd OR family history of hypercholesterolemia)] OR apob/apoa1 ratio 0.68; Criteria #4: (TC 260 mg/dl OR LDL-C 190 mg/dl) AND (family history of pcvd OR family history of hypercholesterolemia); Criteria #5: [(TC 260 mg/dl OR LDL-C 190 mg/dl) AND (family history of pcvd OR family history of hypercholesterolemia)] OR apob/apoa1 ratio (pcvd: premature cardiovascular disease; TC: total cholesterol).