Performance of mutation pathogenicity prediction tools on missense variants associated with 46,XY differences of sex development

Montenegro, Luciana R.; Lerário, Antônio M.; Nishi, Miriam Y.; Jorge, Alexander A.L.; Mendonca, Berenice B.

doi:10.6061/clinics/2021/e2052

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Sumário

ORIGINAL ARTICLE • Clinics 76 • 2021 • https://doi.org/10.6061/clinics/2021/e2052 copy

Performance of mutation pathogenicity prediction tools on missense variants associated with 46,XY differences of sex development

Authorship SCIMAGO INSTITUTIONS RANKINGS

OBJECTIVES:

Single nucleotide variants (SNVs) are the most common type of genetic variation among humans. High-throughput sequencing methods have recently characterized millions of SNVs in several thousand individuals from various populations, most of which are benign polymorphisms. Identifying rare disease-causing SNVs remains challenging, and often requires functional in vitro studies. Prioritizing the most likely pathogenic SNVs is of utmost importance, and several computational methods have been developed for this purpose. However, these methods are based on different assumptions, and often produce discordant results. The aim of the present study was to evaluate the performance of 11 widely used pathogenicity prediction tools, which are freely available for identifying known pathogenic SNVs: Fathmn, Mutation Assessor, Protein Analysis Through Evolutionary Relationships (Phanter), Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polymorphism Phenotyping v2 (Polyphen-2), Align Grantham Variation Grantham Deviation (Align-GVGD), CAAD, Provean, SNPs&GO, and MutPred.

METHODS:

We analyzed 40 functionally proven pathogenic SNVs in four different genes associated with differences in sex development (DSD): 17β-hydroxysteroid dehydrogenase 3 (HSD17B3), steroidogenic factor 1 (NR5A1), androgen receptor (AR), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF>0.01) benign SNVs found in the same four DSD genes. The quality of the predictions was analyzed using six parameters: accuracy, precision, negative predictive value (NPV), sensitivity, specificity, and Matthews correlation coefficient (MCC). Overall performance was assessed using a receiver operating characteristic (ROC) curve.

RESULTS:

Our study found that none of the tools were 100% precise in identifying pathogenic SNVs. The highest specificity, precision, and accuracy were observed for Mutation Assessor, MutPred, SNP, and GO. They also presented the best statistical results based on the ROC curve statistical analysis. Of the 11 tools evaluated, 6 (Mutation Assessor, Phanter, SIFT, Mutation Taster, Polyphen-2, and CAAD) exhibited sensitivity >0.90, but they exhibited lower specificity (0.42-0.67). Performance, based on MCC, ranged from poor (Fathmn=0.04) to reasonably good (MutPred=0.66).

CONCLUSION:

Computational algorithms are important tools for SNV analysis, but their correlation with functional studies not consistent. In the present analysis, the best performing tools (based on accuracy, precision, and specificity) were Mutation Assessor, MutPred, and SNPs&GO, which presented the best concordance with functional studies.

Disorders of Sex Development; Pathogenicity Prediction Tools; Genetics

Gene/Protein	Pathogenic allelic Variant	Benign allelic variant	Reference
HSD17B3 17β-hydroxysteroid dehydrogenase ENST00000375263 NP_000188	p.Ser65Leu	p.Val25Met	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Arg80Gln	p.Val31Leu	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Ala203Val	p.Gly289Arg	(⁹9. Dong C, Wei P, Jian X, Gibbs R, Boerwinkle E, Wang K, et al. Comparison and integration of deleteriousness prediction methods for nonsynonymous SNVs in whole exome sequencing studies. Hum Mol Genet. 2015;24(8):2125-37. https://doi.org/10.1093/hmg/ddu733. https://doi.org/10.1093/hmg/ddu733... )
	p.Val205Glu	p.Ile102Phe	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Phe208Ile	p.Glu114Lys	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Glu215Asp	p.Arg45Trp	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Ser232Leu	p.Arg45Gln	(¹⁰10. Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendonca BB, et al. Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3. Nat Genet. 1994;7(1):34-9. https://doi.org/10.1038/ng0594-34. https://doi.org/10.1038/ng0594-34... )
	p.Met235Val	p.Ser65Ala	(¹⁰10. Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendonca BB, et al. Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3. Nat Genet. 1994;7(1):34-9. https://doi.org/10.1038/ng0594-34. https://doi.org/10.1038/ng0594-34... )
	p.Pro282Leu	p.Ile223Val	(⁸8. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, et al. Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 1996;81(1):130-6. https://doi.org/10.1210/jcem.81.1.8550739. https://doi.org/10.1210/jcem.81.1.855073... )
	p.Cys268Tyr		(¹¹11. Lindqvist A, Hughes IA, Andersson S. Substitution mutation C268Y causes 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 2001;86(2):921-3. https://doi.org/10.1210/jcem.86.2.7172. https://doi.org/10.1210/jcem.86.2.7172... )
NR5A1 Steroidogenic factor 1 ENST00000373588.8 NP_004950	p.Val15Met	p.Glu11Asp	(¹¹11. Lindqvist A, Hughes IA, Andersson S. Substitution mutation C268Y causes 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 2001;86(2):921-3. https://doi.org/10.1210/jcem.86.2.7172. https://doi.org/10.1210/jcem.86.2.7172... )
	p.Val20Leu	p.Gly146Ala	(¹²12. Lin L, Philibert P, Ferraz-de-Souza B, Kelberman D, Homfray T, Albanese A, et al. Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol Metab. 2007;92(3):991-9. https://doi.org/10.1210/jc.2006-1672. https://doi.org/10.1210/jc.2006-1672... )
	p.His24Tyr	p.Val173Met	(¹²12. Lin L, Philibert P, Ferraz-de-Souza B, Kelberman D, Homfray T, Albanese A, et al. Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol Metab. 2007;92(3):991-9. https://doi.org/10.1210/jc.2006-1672. https://doi.org/10.1210/jc.2006-1672... )
	p.Arg39Pro	p.Gly178Arg	(¹³13. Camats N, Pandey AV, Fernández-Cancio M, Andaluz P, Janner M, Torán N, et al. Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals. J Clin Endocrinol Metab. 2012;97(7):E1294-306. https://doi.org/10.1210/jc.2011-3169. https://doi.org/10.1210/jc.2011-3169... )
	p.Met78Ile	p.Tyr211Cys	(¹¹11. Lindqvist A, Hughes IA, Andersson S. Substitution mutation C268Y causes 17 beta-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab. 2001;86(2):921-3. https://doi.org/10.1210/jcem.86.2.7172. https://doi.org/10.1210/jcem.86.2.7172... )
	p.Gly91Ser	p.Pro235Leu	(¹⁴14. Allali S, Muller JB, Brauner R, Lourenço D, Boudjenah R, Karageorgou V, et al. Mutation analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias. PLoS One. 2011;6(10):e24117. https://doi.org/10.1371/journal.pone.0024117 https://doi.org/10.1371/journal.pone.002... )
	p.Pro235Leu	p.Thr296Met	(¹⁵15. Lin L, Achermann JC. Steroidogenic factor-1 (SF-1, Ad4BP, NR5A1) and disorders of testis development. Sex Dev. 2008;2(4-5):200-9. https://doi.org/10.1159/000152036. https://doi.org/10.1159/000152036... )
	p.Trp279Arg	p.Val355Met	(¹³13. Camats N, Pandey AV, Fernández-Cancio M, Andaluz P, Janner M, Torán N, et al. Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals. J Clin Endocrinol Metab. 2012;97(7):E1294-306. https://doi.org/10.1210/jc.2011-3169. https://doi.org/10.1210/jc.2011-3169... )
	p.Arg313Cys		(¹³13. Camats N, Pandey AV, Fernández-Cancio M, Andaluz P, Janner M, Torán N, et al. Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals. J Clin Endocrinol Metab. 2012;97(7):E1294-306. https://doi.org/10.1210/jc.2011-3169. https://doi.org/10.1210/jc.2011-3169... )
	p.Leu437Gln		(¹⁴14. Allali S, Muller JB, Brauner R, Lourenço D, Boudjenah R, Karageorgou V, et al. Mutation analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias. PLoS One. 2011;6(10):e24117. https://doi.org/10.1371/journal.pone.0024117 https://doi.org/10.1371/journal.pone.002... )
AR Androgen receptor ENST00000374690.8 NP_000035	p.Cys579Phe	p.Ala45Gly	(¹⁶16. Imasaki K, Okabe T, Murakami H, Tanaka Y, Haji M, Takayanagi R, et al. Androgen insensitivity syndrome due to new mutations in the DNA-binding domain of the androgen receptor. Mol Cell Endocrinol. 1996;120(1):15-24. https://doi.org/10.1016/0303-7207(96)03812-9. https://doi.org/10.1016/0303-7207(96)038... )
	p.Phe582Tyr	p.Gln59Leu	(¹⁶16. Imasaki K, Okabe T, Murakami H, Tanaka Y, Haji M, Takayanagi R, et al. Androgen insensitivity syndrome due to new mutations in the DNA-binding domain of the androgen receptor. Mol Cell Endocrinol. 1996;120(1):15-24. https://doi.org/10.1016/0303-7207(96)03812-9. https://doi.org/10.1016/0303-7207(96)038... )
	p.Arg710Thr	p.Gln87His	(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
	p.Gly724Asp	p.Gln91Lys	(¹⁸18. Jääskeläinen J, Mongan NP, Harland S, Hughes IA. Five novel androgen receptor gene mutations associated with complete androgen insensitivity syndrome. Hum Mutat. 2006;27(3):291. https://doi.org/10.1002/humu.9405 https://doi.org/10.1002/humu.9405... )
	p.Gly750Asp	p.Gly216Arg	(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
	p.Ala765Thr	p.Leu272Phe	(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
	p.Arg774His	p.Leu341Met	(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
	p.Leu812Pro	p.Val731Met	(¹⁸18. Jääskeläinen J, Mongan NP, Harland S, Hughes IA. Five novel androgen receptor gene mutations associated with complete androgen insensitivity syndrome. Hum Mutat. 2006;27(3):291. https://doi.org/10.1002/humu.9405 https://doi.org/10.1002/humu.9405... )
	p.Arg855Cys	p.Arg856Leu	(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
	p.Asp864Gly		(¹⁷17. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, et al. Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 2000;85(2):658-65. https://doi.org/10.1210/jcem.85.2.6337. https://doi.org/10.1210/jcem.85.2.6337... )
LHCGR Luteinizing hormone/chorionic gonadotropin receptor ENST00000294954 NP_000224	p.Ile374Thr^* * Inactivating variants - phenotype: Leydig cell hypoplasia.	p.Ala57Thr	(¹⁹19. Pals-Rylaarsdam R, Liu G, Brickman W, Duranteau L, Monroe J, El-Awady MK, et al. A novel double mutation in the luteinizing hormone receptor in a kindred with familial Leydig cell hypoplasia and male pseudohermaphroditism. Endocr Res. 2005;31(4):307-23. https://doi.org/10.1080/07435800500430890. https://doi.org/10.1080/0743580050043089... )
	p.Thr392lle^* * Inactivating variants - phenotype: Leydig cell hypoplasia.	p.Ile103Lys	(¹⁹19. Pals-Rylaarsdam R, Liu G, Brickman W, Duranteau L, Monroe J, El-Awady MK, et al. A novel double mutation in the luteinizing hormone receptor in a kindred with familial Leydig cell hypoplasia and male pseudohermaphroditism. Endocr Res. 2005;31(4):307-23. https://doi.org/10.1080/07435800500430890. https://doi.org/10.1080/0743580050043089... )
	p.Phe194Val^* * Inactivating variants - phenotype: Leydig cell hypoplasia.	p.Tyr113His	(²⁰20. Themmen APN, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev. 2000;21(5):551-83. https://doi.org/10.1210/edrv.21.5.0409. https://doi.org/10.1210/edrv.21.5.0409... )
	p.Glu354Lys^* * Inactivating variants - phenotype: Leydig cell hypoplasia.	p.Ala118Glu	(²¹21. Stavrou SS, Zhu YS, Cai LQ, Katz MD, Herrera C, Defillo-Ricart M, et al. A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters. J Clin Endocrinol Metab. 1998;83(6):2091-8. https://doi.org/10.1210/jcem.83.6.4855. https://doi.org/10.1210/jcem.83.6.4855... )
	p.Leu502Pro^* * Inactivating variants - phenotype: Leydig cell hypoplasia.	p.Lys126Asn	(²²22. Leung MY, Al-Muslim O, Wu SM, Aziz A, Inam S, Awadh M, et al. A novel missense homozygous inactivating mutation in the fourth transmembrane helix of the luteinizing hormone receptor in leydig cell hypoplasia. Am J Med Genet A. 2004;130A(2):146-53. https://doi.org/10.1002/ajmg.a.20681. https://doi.org/10.1002/ajmg.a.20681... )
	p.Met398Thr^ Activating variants - phenotype: GIPP (gonadotropin-independent precocious puberty).	p.Lys137Asn	(²³23. Kraaij R, Post M, Kremer H, Milgrom E, Epping W, Brunner HG, et al. A missense mutation in the second transmembrane segment of the luteinizing hormone receptor causes familial male-limited precocious puberty. J Clin Endocrinol Metab. 1995;80(11):3168-72. https://doi.org/10.1210/jcem.80.11.7593421. https://doi.org/10.1210/jcem.80.11.75934... )
	p.Leu547Arg^ Activating variants - phenotype: GIPP (gonadotropin-independent precocious puberty).	p.Val144Leu	(²⁴24. Latronico AC, Abell AN, Arnhold IJ, Liu X, Lins TS, Brito VN, et al. A unique constitutively activating mutation in third transmembrane helix of luteinizing hormone receptor causes sporadic male gonadotropin-independent precocious puberty. J Clin Endocrinol Metab. 1998;83(7):2435-40. https://doi.org/10.1210/jcem.83.7.4968. https://doi.org/10.1210/jcem.83.7.4968... )
	p.Asp564Gly^ Activating variants - phenotype: GIPP (gonadotropin-independent precocious puberty).	p.Phe611Val	(²⁵25. Laue L, Chan WY, Hsueh AJ, Kudo M, Hsu SY, Wu SM, et al. Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty. Proc Natl Acad Sci U S A. 1995;92(6):1906-10. https://doi.org/10.1073/pnas.92.6.1906. https://doi.org/10.1073/pnas.92.6.1906... )
	p.Ala568Val^ Activating variants - phenotype: GIPP (gonadotropin-independent precocious puberty).	p.Cys543Tyr	(²⁶26. Latronico AC, Anasti J, Arnhold IJ, Mendonça BB, Domenice S, Albano MC, et al. A novel mutation of the luteinizing hormone receptor gene causing male gonadotropin-independent precocious puberty. J Clin Endocrinol Metab. 1995;80(8):2490-4. https://doi.org/10.1210/jcem.80.8.7629248. https://doi.org/10.1210/jcem.80.8.762924... )
	p.Ile575Leu^ Activating variants - phenotype: GIPP (gonadotropin-independent precocious puberty).	p.Gly504Ser	(²⁷27. Laue L, Wu SM, Kudo M, Hsueh AJ, Cutler GB Jr, Jelly DH, et al. Heterogeneity of activating mutations of the human luteinizing hormone receptor in male-limited precocious puberty. Biochem Mol Med. 1996;58(2):192-8. https://doi.org/10.1006/bmme.1996.0048. https://doi.org/10.1006/bmme.1996.0048... )

Program Name	URL and Key reference	Basis	Reference
Fathmn	http://fathmm.biocompute.org.uk/	Evolutionary conservation	(²⁸28. Shihab HA, Gough J, Cooper DN, Day IN, Gaunt TR. Predicting the functional consequences of cancer-associated amino acid substitutions. Bioinformatics. 2013;29(12):1504-10. https://doi.org/10.1093/bioinformatics/btt182. https://doi.org/10.1093/bioinformatics/b... ),(²⁹29. Shihab HA, Gough J, Cooper DN, Stenson PD, Barker GL, Edwards KJ, et al. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models. Hum Mutat. 2013;34(1):57-65. https://doi.org/10.1002/humu.22225. https://doi.org/10.1002/humu.22225... ),(³⁰30. Shihab HA, Gough J, Mort M, Cooper DN, Day IN, Gaunt TR. Ranking non-synonymous single nucleotide polymorphisms based on disease concepts. Hum Genomics. 2014;8(1):11. https://doi.org/10.1186/1479-7364-8-11 https://doi.org/10.1186/1479-7364-8-11... )
Mutation Assessor	http://mutationassessor.org/r3/	Evolutionary conservation	(³¹31. Reva B, Antipin Y, Sander C. Determinants of protein function revealed by combinatorial entropy optimization. Genome Biol. 2007;8(11):R232. https://doi.org/10.1186/gb-2007-8-11-r232 https://doi.org/10.1186/gb-2007-8-11-r23... )
Phanter	http://www.pantherdb.org/	Evolutionary conservation	(³²32. Tang H, Thomas PD. PANTHER-PSEP: predicting disease-causing genetic variants using position-specific evolutionary preservation. Bioinformatics. 2016;32(14):2230-2. https://doi.org/10.1093/bioinformatics/btw222. https://doi.org/10.1093/bioinformatics/b... )
SIFT	https://sift.bii.a-star.edu.sg/	Evolutionary conservation	(³³33. Sim NL, Kumar P, Hu J, Henikoff S, Schneider G, Ng PC. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 2012;40(Web Server issue):W452-7. https://doi.org/10.1093/nar/gks539. https://doi.org/10.1093/nar/gks539... )
Mutation Taster	http://www.mutationtaster.org/	Protein structure/function and Evolutionary conservation	(³⁴34. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 2014;11(4):361-2. https://doi.org/10.1038/nmeth.2890. https://doi.org/10.1038/nmeth.2890... )
Polyphen-2	http://genetics.bwh.harvard.edu/pph2/	Protein structure/function and Evolutionary conservation	(³⁵35. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248-9. https://doi.org/10.1038/nmeth0410-248. https://doi.org/10.1038/nmeth0410-248... )
Align-GVGD	http://agvgd.hci.utah.edu/	Protein structure/function and Evolutionary conservation	(³⁶36. Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB, et al. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet. 2006;43(4):295-305. https://doi.org/10.1136/jmg.2005.033878. https://doi.org/10.1136/jmg.2005.033878... )
MutPred	http://mutpred.mutdb.org/index.html	Protein structure/function and Evolutionary conservation	(³⁷37. Pejaver V, Urresti J, Lugo-Martinez J, Pagel KA, Lin GN, Nam HJ, et al. Inferring the molecular and phenotypic impact of amino acid variants with MutPred2. Nat Commun. 2020;11(1):5918. https://doi.org/10.1038/s41467-020-19669-x https://doi.org/10.1038/s41467-020-19669... )
CAAD	https://cadd.gs.washington.edu/	Protein structure/function and Evolutionary conservation	(³⁸38. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019;47(D1):D886-D894. https://doi.org/10.1093/nar/gky1016 https://doi.org/10.1093/nar/gky1016... )
Provean	http://provean.jcvi.org/index.php	Protein structure/function	(³⁹39. Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7(10):e46688. https://doi.org/10.1371/journal.pone.0046688 https://doi.org/10.1371/journal.pone.004... )
SNPs&GO	http://snps.biofold.org/snps-and-go//snps-and-go.html	Protein structure/function	(⁴⁰40. Calabrese R, Capriotti E, Fariselli P, Martelli PL, Casadio R. Functional annotations improve the predictive score of human disease-related mutations in proteins. Hum Mutat. 2009;30(8):1237-44. https://doi.org/10.1002/humu.21047. https://doi.org/10.1002/humu.21047... ),(⁴¹41. Capriotti E, Altman RB. Improving the prediction of disease-related variants using protein three-dimensional structure. BMC Bioinformatics. 2011;12 Suppl 4(Suppl 4):S3. https://doi.org/10.1186/1471-2105-12-S4-S3 https://doi.org/10.1186/1471-2105-12-S4-... ),(⁴²42. Capriotti E, Calabrese R, Fariselli P, Martelli PL, Altman RB, Casadio R. WS-SNPs&GO: a web server for predicting the deleterious effect of human protein variants using functional annotation. BMC Genomics. 2013;14 Suppl 3(Suppl 3):S6. https://doi.org/10.1186/1471-2164-14-S3-S6 https://doi.org/10.1186/1471-2164-14-S3-... )

Type of SNV	Classification by the sites	Fathmn	Mutation Assessor	Phanter	SIFT	Mutation Taster	Polyphen-2	Align-GVGD	MutPred	CAAD	Provean	SNPs&GO
Pathogenic (n=40)	Pathogenic	35	38	38	36	37	37	33	33	36	32	32
Pathogenic (n=40)	Benign	5	2	2	4	3	3	7	7	4	8	8
Benign (n=36)	Pathogenic	27	14	21	16	17	13	28	6	12	11	6
Benign (n=36)	Benign	9	22	15	18	19	13	8	30	24	25	30

Performance	Fathmn	Mutation Assessor	Phanter	SIFT	Mutation Taster	Polyphen-2	Align-GVGD	MutPred	CAAD	Provean	SNPs&GO
Accuracy	0.56	0.79	0.70	0.74	0.74	0.76	0.54	0.83	0.79	0.75	0.82
Precision	0.56	0.73	0.64	0.69	0.69	0.73	0.54	0.85	0.75	0.74	0.84
Specificity	0.16	0.61	0.42	0.53	0.53	0.53	0.22	0.83	0.67	0.69	0.83
Sensitivity	0.88	0.95	0.95	0.92	0.93	0.93	0.83	0.83	0.90	0.80	0.80
NPV	0.50	0.92	0.88	0.86	0.86	0.84	0.53	0.81	0.86	0.76	0.79
MMC	0.04	0.60	0.44	0.50	0.50	0.51	0.06	0.66	0.59	0.50	0.63

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E-mail: clinics@hc.fm.usp.br

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[1] *Corresponding Authors. E-mail: lucianam@usp.br