SUPPLEMENTARY INFORMATION. Rare independent mutations in renal salt handling genes contribute to blood pressure variation

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1 SUPPLEMENTARY INFORMATION Rare independent mutations in renal salt handling genes contribute to blood pressure variation Weizhen Ji, Jia Nee Foo, Brian J. O Roak, Hongyu Zhao, Martin G. Larson, David B. Simon, Christopher Newton-Cheh, Matthew W. State, Daniel Levy, Richard P. Lifton

2 Supplementary Figure 1. Mutation detection among Framingham cohort members. Examples demonstrating identification of DNA sequence variants in NCCT, ROMK and NKCC2. For each example, the results of temperature gradient capillary electrophoresis (left panel), demonstrating double peaks, and DNA sequencing (right panel), demonstrating heterozygous bases, are shown. The effect on inferred amino acid sequence is indicated above each variant.

3 Supplementary Figure 2. Sensitivity and specificity of different criteria for identifying functional mutations among sequence variants in NCCT identified in FHS determined by comparison to mutations identified in Gitelman syndrome subjects. Plot of percent sensitivity and false positive (1-specificity) was determined for each criterion as described in Methods and is shown. The labeled points correspond to a) disruptive variants + missense variants at sites conserved in all vertebrates and invertebrates, b) disruptive variants + missense variants at sites conserved in all vertebrates and majority of invertebrates, c) disruptive variants + all variants conserved in vertebrates and d) disruptive variants + all missense variants. Criteria b shows the best combination of sensitivity and specificity.

4 Supplementary Table 1. SNP frequencies in unrelated subjects of FHS, HapMap and Perlegen cohorts dbsnp Framingham HapMap Perlegen (%) (%) (%) rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs

5 Supplementary Table 2. Nonsynonymous and synonymous variants detected in NCCT, NKCC2 and ROMK in the offspring cohort of FHS. Putative functional mutations are shown in red. ++ indicates the mutation is predicted to be functional, + indicates a low confidence prediction of functional effects, while indicates the mutation is not predicted to be functional. NA is shown where predictions are not available. A. Variants in NCCT Nucleotide Amino Acid Carriers Allele Freq. Nucleotide Amino Acid Allele Freq. SIFT PANTHER PolyPhen Carriers Variation Substitution (n) (%) Variation Substitution (%) c37 G>C A13P NA - c27 G>C T9T c97 C>T P33S NA + c36 C>T D12D c187 G>A V63M NA - c366 A>G A122A 251 >4 c334 G>T E112X Disruptive c774 C>T N258N c334 G>C E112Q NA - c825 C>T S275S c363 G>C E121D NA - c1023 C>T F341F c361 G>A E121K NA - c1176 C>T T392T c379 G>A E127K NA - c1263 C>T C421C c463 C>T L155F NA + c1284 C>T T428T c563 C>T S188F NA ++ c1314 C>T Y438Y c644 T>G L215R NA + c1386 C>T F462F c694 G>A A232T NA + c1392 C>G A464A c766 C>T P256S NA + c1395 C>T T465T 729 >12 c781 C>T R261C NA ++ c1539 C>T Y513Y c791 G>C G264A NA ++ c1884 G>A S628S 549 >9 c874 G>A V292I NA - c2142 C>T A714A 553 >9 c928 G>A E310K NA - c2442 C>T G814G c1024 G>A G342R NA - c2625 C>T G875G 491 >8 c1196 G>T R399L NA ++ c2737 C>A R913R c1249 G>A E417K NA - c2937 T>A A979A c1315 G>A G439S NA ++ c3051 C>T I1017I c1477 G>A G493S c1485 C>A F495L c1486 G>T G496C c1549 G>A V517M c1679 C>A P560H c1696 A>T N566Y c1837 G>A G613S c1865 A>G N622S c2126 A>G Y709C c2182 G>A A728T c2221 G>A G741R c2336 G>A G779E c2367 C>G H789Q c2498 C>T S833L c2497 T>A S833T c2501 A>T E834V c2581 C>T R861C c2620 G>A V874I c2738 G>A R913Q 595 > c2782 C>T R928C c2783 G>A R928H c2830 C>T R944W c2891 G>A R964Q c2957 T>C L986S c2965 G>A G989R

6 B. Variants in NKCC2 Nucleotide Amino Acid Allele Freq. Nucleotide Amino Acid Allele Freq. Carriers SIFT PANTHER PolyPhen Carriers Variation Substitution (%) Variation Substitution (%) c119 C>G T40S NA - c354 C>T T118T c347 G>A R116H NA ++ c477 T>C P159P c497 A>G N166S NA - c651 C>T G217G c704 C>T T235M NA ++ c876 G>A S292S c721 G>A G241R NA ++ c708 T>C N236N c760 C>G P254A NA ++ c807 A>G A269A c904 del (C) R302-FS Disruptive c1548 C>T P516P c904 C>T R302W NA ++ c1633 C>T Y538Y 976 >15 c1043 C>T P348L NA ++ c1998 T>C N666N c1046 C>T S349F NA - c2028 C>T H676H c1099 G>A A367T c2067 G>A G689G c1196 A>G N399S NA ++ c2223 G>A K741K c1513 C>G L505V NA + c2253 G>A V751V c1706 C>A P569H c2385 C>T N785N c2107 G>A A703S c2532 T>A T844T c2255 C>T A752V c2787 C>T I929I c2342 A>G Y781C c2943 T>C I981I c2362 G>A A788T c3054 G>A P1018P c2545 G>A E849K c2570 T>A I857N c2573 G>T R858L c2613 G>C K871N c2920 C>G H974D c2996 G>A R999H c3053 C>T P1018L c3209 A>G Y1070C c3247 C>G P1083A

7 C. Variants in ROMK Nucleotide Amino Acid Allele Freq. Nucleotide Amino Acid Allele Freq. Carriers SIFT PANTHER PolyPhen Carriers Variation Substitution (%) Variation Substitution (%) c23 isoform c C>G S8C isoform c NA - c33 T>A T11T c56 isoform a G>A S19N isoform a NA - c45 G>A G15G c70 G>A V24I c252 G>T A84A c112 G>A E38K c330 C>T T110T c496 C>T P166S c750 C>T Y250Y c506 G>A R169H c798 C>T T266T c521 C>T T174M c837 T>C D279D c578 G>C R193P c601 C>T L201F c751 C>T H251Y c898 G>T V300L c del(aaag) T313-FS Disruptive c956 G>A R319Q c958 G>A V320M c1014 G>A M338I c1013 T>C M338T c1019 T>G L340R c1074 C>A N358K c1102 G>A D368N NA ++ 3

8 Supplementary Table 3. Mutations in NCCT, NKCC2 and ROMK in 148 Gitelman and 144 Bartter patients. ND indicates mutation not detected. A. NCCT Mutations in 148 Gitelman patients Kindred Nucleotide Amino Acid Nucleotide Amino Acid Variation 1 Substitution 1 Variation 2 Substitution c2576t>c L859P c2576t>c L859P 102 c1261t>c C421R c625c>t R209W 103 c851a>g K284R c1964g>a R655H 104 c1964g>t R655L c1763c>t A588V c2576t>c L859P 107 c1046c>t P349L c1046c>t P349L 108 c1889g>t G630V c del (CTT) Del S c1456g>a D486N c1899c>t R928C 110 c1486g>t G496C ND ND 111 c2891g>a R964Q c2891g>a R964Q 112 c2221g>a G741R c2929c>t R977X 113 c2581c>t R861C c2581c>t R861C 114 c2981g>a C994Y 115 c2221g>a G741R c2221g>a G741R c2221g>a G741R 118 c1315g>a G439S c205c>a H69N Splice gt>tt c2891g>a R964Q 120 c1315g>a G439S c1315g>a G439S 121 c2202g>c K734N c2891g>a R964Q 122 c bp del T697FS c2186g>t G729V 123 c1000a>t R334W ND ND 124 c1315g>a G439S c del(gg) G731FS 125 c2295 del(t) F765FS ND ND 126 c1000a>t R334W c1195c>t R399C 129 c2581c>t R861C ND ND 132 c2221g>a G741R c1925g>a R642H 133 1

9 134 c bp ins A401FS c1379g>a G460D 135 c1196g>t R399L 136 c961c>t R321W c2576t>c L859P 137 c626g>a R209Q c1489a>t K497X 140 del (1-7) exons c1924c>g R642G 141 c1028t>a M343K c1028t>a M343K 143 c2221g>a G741R c2221g>a G741R 146 c1742t>a M581K c3070g>a V1024M 150 c2221g>a G741R ND ND 153 c1456g>a D486N c2891g>a R964Q 155 c2221g>a G741R ND ND 158 c1928c>t P643L c2891g>a R964Q 163 c1196g>t R399L c2576t>c L859P 166 c1964g>a R655H Intron 5 5 Splice gt>at 168 c1928c>t P643L 170 c2965g>a G989R 172 c1858t>c S620P ND ND 173 c1456g>a D486N ND ND 175 c1899c>t R928C ND ND 177 c334g>t E112X c2186g>t G729V c2581c>t R861C c2687g>a R896Q 192 c2581c>t R861C ND ND 193 c bp del T697FS ND ND 194 c3052c>t R1018X c3052c>t R1018X 195 c bp del T697FS c2612g>c R871P c c L998FS ins(cgct) ins(cgct) L998FS 201 c1456g>a D486N ND ND 202 c1939g>a V647M c1939g>a V647M 203 c1664c>t S555L c2576t>c L859P c2576t>c L859P c3077c>t T1026I 2

10 213 c2221g>a G741R 216 c1925g>a R642H c3029t>g L1010R Splice 5 Splice gt>tt gt>tt 224 c1376c>a A459D c2965g>a G989R 228 c2842 del(t) W948FS 229 c961c>t R321W 230 c1456g>a D486N c1456g>a D486N 237 c2963t>c I988T 240 c2221g>a G741R ND ND 241 c bp ins A401FS c433c>t R145C 245 c1205c>a S402Y c1924c>g R642G 246 c bp ins A401FS c bp ins A401FS 248 c1315g>a G439S Intron 4 3 Splice ag>aa 249 c2344g>a V782M ND ND 250 c1945g>a T649M ND ND 253 c1664c>t S555L c1844c>t S615L 254 Intron 12 Intron 15 3 Splice ag>aa 5 Splice gt>at 255 c2573t>a L858H ND ND 260 c2710a>t I904F 261 c2221g>a G741R c bp del T697FS 262 c ins(cc) P79FS c2221g>a G741R c1390g>a A464T ND ND 271 c938c>t A313V ND ND 275 c ins(cc) P79FS ND ND 280 c671c>a A224D ND ND 281 c1899c>t R928C ND ND 284 c2581c>t R861C ND ND 285 c938c>t A313V c2186g>t G729V 288 c2186g>t G729V 289 c2221g>a G741R c2981g>a C994Y 3

11 297 c1067c>t A356V c1315g>a G439S 300 c2221g>a G741R c2221g>a G741R 301 ND ND 302 c bp ins A401FS c2221g>a G741R 305 c961c>t R321W 308 c812t>c L271P c2221g>a G741R 309 c1664c>t S555L Intron 25 5 Splice gt>at 314 c1195c>t R399C c1837g>a G613S C>T S178L c1928c>t P643L 317 c1489a>t K497X c1489a>t K497X 320 c2204c>t P735L c2576t>c L859P 322 c2221g>a G741R c2221g>a G741R 330 c3077c>t T1026I 336 c2686c>t R896X ND ND 337 c2221g>a G741R c2576t>c L859P 341 c460a>t I154F c2221g>a G741R 342 c2221g>a G741R ND ND 348 c2576t>c L859P c3052c>t R1018X 350 c1315g>a G439S c2730g>c Q910H 352 c575t>c I192S c2117a>c K706T 360 c1924c>g R642G ND ND 361 c1899c>t R928C c3053g>a R1018Q 362 c2221g>a G741R ND ND 363 c1195c>t R399C c2981g>a C994Y 364 c2221g>a G741R 365 c2221g>a G741R c2576t>c L859P 367 c bp del T697FS c bp del T697FS C>T S178L 533C>T S178L 369 c1844c>t S615L ND ND 378 c791g>c G264A c1928c>t P643L 385 c791g>c G264A ND ND 387 c2221g>a G741R ND ND 388 c815t>c L272P c2965g>a G989R 391 c2221g>a G741R 392 c2996a>g Y999C ND ND 4

12 398 ND ND 403 c1899c>t R928C c1899c>t R928C 407 c43-44 del (TT) L15FS c43-44 del (TT) L15FS 413 c1145c>t T382M c2221g>a G741R 416 c1145c>t T382M 417 c497c>t A166V c1315g>a G439S 419 c2221g>a G741R ND ND 427 c1315g>a G439S Intron c791g>c G264A ND ND 430 c2221g>a G741R ND ND 431 c1315g>a G439S ND ND 432 c736c>g L246V c736c>g L246V 433 ND ND 436 c1928c>t P643L c1928c>t P643L 438 c2221g>a G741R ND ND 445 c2965g>a G989R c ins(g) F994FS 447 c bp del M1000FS ND ND 454 c791g>c G264A ND ND 5

13 B. NKCC2 Mutations in 144 Bartter patients Kindred Nucleotide Variation 1 Amino Acid Substitution 1 Nucleotide Variation 2 Amino Acid Substitution c904 del(c) R302FS c904 del(c) R302FS 152 c Ins(A) M195FS c Ins(A) M195FS 156 c814 G>T V272F c2467 del(c) Q823FS 165 c1942 G>T D648N c1942 G>T D648N 179 c1519 T>C S507P c1519 T>C S507P 223 c2239 del(t) F747-FS c2239 del(t) F747FS 236 c769 G>A G257S c769 G>A G257S 258 c769 G>A G257S c769 G>A G257S 268 c1663 G>A A555T c1663 G>A A555T 270 c595 C>T R199C c1966 C>T Q656X 273 c del c del del N526 (AAC) (AAC) del N c1432 G>A G478R c1432 G>A G478R 303 c24-27 del(tgta) V9-FS ND ND 373 c1954 G>A G652S ND ND 383 c2495 A>C E832A c2495 A>C E832A 386 c606 G>C W202C ND ND 400 c1834 G>A G612R c1834 G>A G612R 437 c535 T>A W179R ND ND 472 c1966 C>T Q656X Intron 25 5'splice gt>at 6

14 C. ROMK Mutations in 144 Bartter patients Kindred Nucleotide Variation 1 Amino Acid Substitution 1 Nucleotide Variation 2 Amino Acid Substitution c180 C>G Y60X c180 C>G Y60X 161 c39-40 ins(t) F13FS c39-40 ins(t) F13FS 206 c del(aaag) T313FS c584 C>T A195V 208 c174g>a W58X c600 C>G S200R 231 c240 G>C W80C c1019 T>G L340R 307 c626 G>A G209E c1019 T>G L340R 424 c874 C>T R292W c874 C>T R292W 425 c242 A>T Y81F c242 A>T Y81F 426 c c T313FS del(aaag) del(aaag) T313FS 450 c955 C>T R319X c955 C>T R319X 7

15 Supplementary Table 4. Effects of variants on long term average SBP and DBP in FHS. # of carriers Mean effect on SBP (mmhg) p value Mean effect on DBP (mmhg) p value Carriers of rare variants (MAF <1%) Proven/inferred functional mutations Rare, not functionally significant missense variants Rare synonymous variants Carriers of common nonsynonymous variants (MAF >1%) ROMK M338T NCCT G264A NCCT R319Q NCCT A728T NCCT R928C Carriers of common synonymous variants (MAF >1%) NKCC2 Y538Y NCCT A122A NCCT F341F NCCT T465T NCCT S628S NCCT A741A NCCT G875G

16 Supplementary Table 5. PCR primers GENE EXON FORWARD PRIMER REVERSE PRIMER NCCT 1 AGTGCAGACGCAGCCTATAAA AGTGGCCAGTCTTCTGAGACA 2 CTCAGGCTCCTTCCTCTGTG GCCCTCGGGGTAGATCTTAT 3 GACTTGTCGTTTGGCCTTG GGGTCTGGGAAGAACAGGAT 4 GGGAAATGCCCTGCCTAAG ATTAGGAGCCCACGAGAGGA 5 CCAACCGACTCATCTGGTTT CGGAGGAGAGTGCAATGG 6 AATTCAGAGGGTGGCTTGC CTTCTCCACGTGACCACCTC 7 ATCTGACCTCTGGGGTCCTT CCTGGGTAGAGAGTCCCTGTC 8 CCTTACTCATCAGGCCTTGC GACACAGGAGGGGCAAGTA 9 AGGGGCTGCCTAATGTCCT GTCAGGGTTTTGGGACACTG 10 AGGACAGAGTAAGGAGGGAAGG TCTCCTAAGCCTAGGCCTCAAC 11 CACCGTGGAGTCCCTGAG CACCCCCTGTCATCTCGAC 12 ACTCCACCATTCAAGCTCTACC GGAGATGAGGAGACAGGAGAAG 13 AGCCAGTCCTTGGCAGAGT CTGGGTGCCTCCTCCTGA 14 GCAGCTCTGGCCTAGAAAGA ATGGCTACCCTGGGGGAGGA 15 CCACGTGTCTGGTTTCCTCT GGAAACTCCCAGGGTGGAG 16 CATGTAGGGTCATGCTGGTG CAGAGTGCTGGGTTTACAGG 17 ATCTCAGGCTGGAGGGTGAA CACCAAGCCGTAAGTCCTGTA 18 ACATCAGAAAGTTGGGGCTCTG CAATGGGCCCAAATTAACAGAC 19 TGGACCTCACCTCCTCTCTT TTCTAGAACTTTCTGGGAGTGG 20 GTGCCCTCAGACAAGGAGAG CACCTGTCCTCGACCAAGTT 21 CTGCTGGCTCTGCTCTGAC CAGGAGGGCTGATCCAAG 22 TTGAATTCAGTCAGCCATGTTC GACAATCTCAGTGCCCACCT 23 CCTGGGAGACAGAGCAAGAC GTCTCCAGGCACACAGTTGG 24 CACTTTGTAAATGGCAGTGTCC ACTGCCAGAGCTCACTCAAGAT 25 TGACCTCGATGATATGGGAAG ACAGACTGGTCCTCCCACCA 26 CTGAAAACAAACTGGGAGCTG TGTGGACAGGGATGTCAAAG ROMK 2 CCTTCACCCAAATGTCTTTCTT TGCGAAAGACCAACTCCTTT 4 CCACTGACTGTCGCCTACAA TGGCTTTCCAGAGAGGTGAT 5a GGAGTGTGATTGGCAGAAGG GGTCTTTCATGTGGGTCACCT 5b TGGTCTCCTGTGGTATGCAG CATTCCCAGCGTCAACTACA 5c GGAAAGCTTCTGAAGACCACA AGCACCTCCTCTGGGACATA 5d TGGTGTTTTTAGATGGCACAG TGACTGCTTCATAATGCTTCTAGG NKCC2 1 AACAACCACAAAGTAGATAGCTCAGT AAGGGAGGAGACTTGCTTGTG 2 & 3 TGGAACCCTTTGTTCATTGAC GCAAAATTATTTAGGAGGGGAAA 4a GCAGCAATAGGGAATCCAAG ACCCGTAGCAAGTGATGAGG 4b TGGTCCTTTGATGTTTACCC GCCTGTGTTGCCTTTACCTT 4f GGGAGGTGGATCTTTCTGTG AGCAATATGTTACTTTCACTTCCAAT 5 GCAGTTCCTCAATGTGAAGTATGT TATGCTGCTTGGCAGAAAAA 6 GCTGCAATAAGACTCACATGC CCTGACCAGCCACTGTTGAT 7 TCTGATTTGGTTTCCTTTTACCTT GAGGAGGGCAATGGAGAAGT 8 GGACTAGGGAAGCCAATGGT AGGACTGCAAAGCAGAGCAA 9 TGCTCTGTATTCTTCTACCTCCA GAACAACTGGACCCCTCGTA 10 GAAAACCGTAAGGGACCAGA AATAGCAGTGAACATTTTTGAATTT 11 TCCAAATCACAGAAAGTCTCCTT AGGGAGAAGCACAAGCTGTT 12 TGACTGTGCATAGCTATAAATGACAA CAAACTAAAAGGAAAGCCCTATGA 13 CCCCTGGTCTCATCACTCAT TGCTTAGGCATATTTTAGTTTGGA 14 TGGAAGTTTTCCTTCTGCAT TGGAAACGCTATTCCAGACA 15 TGCCAATTTCCTCCTTTATCC AACACCAGGATGCCTGAGAC 16 CCACTGGAATGGTTCTAAGGTT CCTCACCCAAAATAATCCAAGA 17 GGCATTGCTGGCTATTTTTG TGGAGCACTAATTGTCTTTTGC 18 CCCAGTACGGTAAGGATTGC CACGTCTTGAAAGCCATCAC 19 TCAAAATCCTAGAAGCAAGTGTAA CCATAACAATGTCAGGCACAA 20 TGAGTTAAGTAGGTGATTTTGTCTTC CGGACTCTTCATAGATGCTCAA 21 & 22 GCCCTCAAAAGCAAACAGAT GACCTAACATGTGAGTGGCAAA 23 TGCAATGATGAAGTATTTTCCTTT CACAAAAGAGAATGAGAGAGTACCA 24 GCCAGTCACACCTGGAGTATC TCAACTACTGTTTCCTTTCTCAGC 25 TGGTAGAACTGTACTCAACAAATCTGA CCTGAAGAGTCCCAAGCTTTT 26 CACTTTCATTTTTAAATTTTTCCTTCA GGTTTGCATATCCATAGATCAGA

17 Supplementary Note Human subjects and clinical data The Framingham Heart Study was initiated in 1948, with the ascertainment of 5209 subjects drawn from a sample of families and additional volunteers from a list of all residents in a local census of Framingham, Massachusetts 1. The original cohort comprised approximately half of the population aged at that time. The offspring cohort was then recruited in 1971, comprising 5124 adult offspring and spouses of offspring of the original cohort participants 1,2. Participants from both cohorts are broadly representative of a European American population. Offspring cohort subjects were evaluated by medical examination, laboratory testing, and lifestyle interview for up to 6 cycles at the time of the study, done 8, 12, 16, 20 and 24 years after their initial visit 2. Seated BP measurements were taken twice at each visit by an examining physician with a mercury column sphygmomanometer, with systolic and diastolic BPs determined by the first and fifth Korotkoff sounds respectively 2. The average of the two BP measures was recorded for each examination. Clinical data and DNA samples from 3125 participants (1477 males, 1648 females) of the offspring cohort were analyzed in this study, among which were 1985 unrelated subjects and 1140 of their first and second degree relatives. These constituted all the subjects from the offspring cohort for whom a blood or DNA sample was available at the time of the study. Separately, blood or DNA samples from 148 unrelated subjects with Gitelman syndrome and 144 subjects with Bartter syndrome were collected by referral from physicians. The diagnosis of Gitelman syndrome was established by the constellation of hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria, high plasma renin activity, high serum or 24-hour urinary aldosterone level and the absence of hypertension. The criteria for Bartter syndrome were the same except these patients all had hypercalciuria rather than hypocalciuria and hypomagnesemia was absent or mild 3. The former patients were virtually all diagnosed after adolescence while the latter were virtually all diagnosed from the neonatal period to age 3. The ancestry and country of origin of the Gitelman subjects is listed below: 1

18 Ancestry Country of Ascertainment Number of GS patients European Caucasians Australia 2 Austria 2 Belgium 4 Canada 10 Denmark 4 France 7 Germany 6 Italy 14 Netherlands 1 Portugal 1 Puerto Rico 1 Spain 4 Sweden 6 UK 17 Total = 128 USA 49 Asians Canada 3 Japan 2 Philippines 1 Taiwan 2 UK 1 Total =10 USA 1 Africans South Africa 1 Hispanics USA 3 Middle Easterns Saudi Arabia Turkey 1 4 Native Americans USA 1 References 1. Kannel, W.B., Feinleib, M., McNamara, P.M., Garrison, R.J. & Castelli, W.P. An investigation of coronary heart disease in families. The Framingham offspring study. Am J Epidemiol 110, (1979). 2. Levy, D. et al. Evidence for a gene influencing blood pressure on chromosome 17. Genome scan linkage results for longitudinal blood pressure phenotypes in subjects from the framingham heart study. Hypertension 36, (2000). 3. Bettinelli, A. et al. Use of calcium excretion values to distinguish two forms of primary renal tubular hypokalemic alkalosis: Bartter and Gitelman syndromes. J Pediatr 120, (1992). 2

Supplemental Table 1. The list of variants with their respective scores for each variant classifier Gene DNA Protein Align-GVGD Polyphen-2 CADD MAPP

Supplemental Table 1. The list of variants with their respective scores for each variant classifier Gene DNA Protein Align-GVGD Polyphen-2 CADD MAPP Frequency Domain Mammals a 3 S/P b Mammals a 3 S/P b