MCP Papers in Press. Published on May 23, 2005 as Manuscript M500024-MCP200

Genetic Association, Post-translational Modification and Protein-protein Interactions in Type 2 Diabetes Mellitus. Amitabh Sharma£1, Sreenivas Chavali£1, Anubha Mahajan1, Rubina Tabassum1, Vijaya Banerjee1, Nikhil Tandon2, Dwaipayan Bharadwaj1∗

1

Functional Genomics Unit, Institute of Genomics and Integrative Biology, CSIR, Delhi, India

2

Department of Endocrinology, All India Institute of Medical Sciences, New Delhi, India

£

These authors contributed equally to this work.

∗

Corresponding author:

Dr. Dwaipayan Bharadwaj Functional Genomics Unit Institute of Genomics and Integrative Biology (CSIR) Mall Road, Delhi- 110 007 India

Tel

: +91 11 2766 6156/6157

Fax

: +91 112766 7471

E-mail : [email protected]

Running Title: Functional assessment of variations in Type 2 Diabetes Mellitus

1 Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Abbreviations Used: SNPs - Single Nucleotide Polymorphisms T2DM - Type 2 Diabetes Mellitus DCVs - Disease Causing Variations DAVs - Disease Associated nonsynonymous Variations PSIC - Position Specific Independent Count CNVs - Control Nonsynonymous Variations

2

Summary: Type 2 Diabetes Mellitus is a complex disorder with a strong genetic component. Inherited complex disease susceptibility in humans is most commonly associated with single nucleotide polymorphisms. The mechanisms by which this occurs are still poorly understood. Here, we focus on analyzing the effect of a set of disease causing missense variations of monogenetic form of Type 2 Diabetes Mellitus and a set of disease associated nonsynonymous variations in comparison with that of nonsynonymous variations without any experimental evidence for association with any disease. Analysis of different properties such as evolutionary conservation status, solvent accessibility, secondary structure, etc. suggests that disease causing variations are associated with extreme changes in the value of the parameters relating to evolutionary conservation and/or protein stability. Disease associated variations are rather moderately conserved and have milder effect on protein function and stability. Majority of the genes harboring these variations are clustered in or near insulin signaling network. Most of these variations are identified as potential sites for post-translational modifications; certain predictions have already reported experimental evidences. Overall, our results indicate that Type 2 Diabetes Mellitus may result from a large number of SNPs which impair modular domain function and post-translational modifications involved in signaling. Our emphasis is more on conserved corresponding residues than the variation alone. We believe that the approach of considering a stretch of peptide sequence involving a polymorphism would aid as a better method of defining its role in the manifestation of this disease. Since most of the variations associated with the disease are rare, we hypothesize that this disease is a ‘Mosaic model’ of interaction between a large number of rare alleles and a small number of common alleles along with the environment, which is little contrary to the existing Common Disease Common Variants model.

3

Introduction: Type 2 Diabetes Mellitus (T2DM) is a genetically heterogeneous, polygenic disease with complex inheritance pattern and is caused by genetic predisposition and environmental factors. The precise biochemical defects are unknown and almost certainly include impairments in insulin secretion and insulin action. T2DM is characterized by abnormal glucose homeostasis leading to hyperglycemia and is represented primarily by insulin resistance. The vast majority of insulin resistance in T2DM has been shown to arise due to defects at the post-receptor level [1]. T2DM is also heterogeneous in the associated pathological and physiological symptoms leading to a variety of complications such as coronary heart disease, neuropathy, retinopathy, etc. Genetic dissection of any complex trait is done based on two approaches, which include genome wide scan studies and association studies. The concept of association studies [2] is being widely applied as an experimental technique to identify Single Nucleotide Polymorphisms (SNPs) underlying complex phenotype, which represents the most common form (90%) of genetic variations in humans [3]. Association is defined as a statistical statement about the cooccurrence of alleles or phenotypes. Owing to the application of high-throughput SNP detection techniques, the number of identified SNPs is growing rapidly enabling detailed statistical studies. Over the past decade many laboratories have sought to clarify the etiology of T2DM by attempting to associate clear differences in metabolic phenotype with mutations or polymorphisms in the genes. As a result of this a large amount of data has accumulated, associating SNPs in a large number of candidate genes with the disease across different populations. Unlike fully penetrant mutations that cause Mendelian diseases, SNPs involved in complex human phenotypes are not a necessary and sufficient condition defining the phenotype

4

but their effect depends on many other genetic and environmental components. In other words SNPs are shown to comprise risk factors of having a specific phenotype more in a statistical sense. This raises the question as to whether the associated SNPs are only of statistical significance. If not then, what might be the reason for encountering differences in variation statistics across different populations as shown by Cargill et al. [4]. However, identifying SNPs responsible for specific phenotypes appears to be an enigma that is very difficult to solve. Several recent studies [5-10] have applied computational methods to predict the potential effects of the nonsynonymous coding SNPs in bringing about variations in humans. A focus on the individual factors that highlight their maximum potential effect (whether positive or deleterious) is often optimistic, as in practice they do not operate in isolation. Instead they work jointly to generate the disease gene architecture and hence a study to determine the contribution of these interactions towards the disease is essential. Ideally, the end point of disease gene identification should be functional analysis of the disease associated allele and an understanding of the molecular mechanism of causation of the disease phenotype. The functional characterization can be facilitated by the computational analysis. Vitkup et al. [9] have shown that the probability of a nonsynonymous mutation causing a genetic disease increases monotonically with an increase in the degree of evolutionary conservation of the mutation site and a decrease in the solvent accessibility of the site; opposite trends are observed for non disease polymorphisms. In the current study we have extensively analyzed the effect of nonsynonymous variations on the structure and function of proteins and have attempted to determine their possible role in the disease phenotype.

5

Experimental procedures: Data set extraction: The data set considered for the study includes a set of 29 mutations shown to cause monogenetic T2DM in families or Maturity Onset of Diabetes in Young (disease causing variations-DCVs); 113 polymorphisms, associated with the disease in various populations in a total of 76 different candidate genes and 92 random nonsynonymous variations in 32 genes that do not have any experimental evidence of association with any disease as a control dataset (Supplementary Table 1 online). The selection of these random variations would help to distinguish specific behavior patterns of the disease related variations from that of chance occurrence. Hence these random variations through out the sequence in those genes that have been implicated with the T2DM were selected. The disease associated polymorphisms fall into four major categories-nonsynonymous (45), regulatory (42), synonymous (11) and intronic SNPs (15). In this study we determine the effect of the disease associated nonsynonymous variations (referred here after as DAVs) in comparison to the control nonsynonymous variations (CNVs) on the phenotype. DCVs were obtained querying Medline for ‘Type 2 Diabetes, Mutations’ ; DAVs by querying for ‘Type 2 Diabetes, SNPs’, ‘Type 2 Diabetes, Polymorphisms’, and CNVs from the SWISSPROT database [11]. The extraction of protein sequences needed for the analysis of all these variations was done from SWISSPROT. Relationship between the genes harboring DAVs was determined using Pathway Assist [12]. Pathway Assist is a software application for navigation and analysis of biological pathways, gene-regulation networks and protein interaction maps. It comes with the built in natural language processing module MedScan and a comprehensive database.

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Evaluating evolutionary conservation status of the variations: The best method to evaluate the significance of a variation using evolutionary information is to consider the nature of the change with respect to the variability of the affected residue as estimated from the wild type sequences in different proteins of a protein family. Set of similar sequences can be characterized by a multiple sequence alignment within common sequence domains (in case of protein families) or just a small sequence region (motif). We have done systematic examination of positions of the variations in motif region of proteins, using Pfam database [13] of probablistic models of protein domains and families derived using the HMM method and eMATRIX database [14]. eMATRIX [15] is a minimum risk method for estimating the frequencies of amino acids at conserved position in a protein family. Minimum risk estimation, finds the optimal weighting between a set of observed amino acid counts and a set of pseudo frequencies. This provides the information regarding the position of the variations in specific domains and functional motifs respectively. The prediction of residues conservation amongst the homologous proteins was performed by Scorecons [16]. Scorecons algorithm scores each residue position with multiple sequence alignment in terms of conservation. Multiple sequence alignment of homologous protein was done by using ClustalW [17] algorithm and was formatted in ClustalX (1.81). The mutation matrix of Jones et al. [18] is used to determine the likelihood of particular residue being replaced by another and to calculate a score based on the variability of each position. Normalized Shannon entropy scores for each amino acid position were calculated using the general formulae [16]. Cent = - Σkapa log2 pa/log2 [min (N, K)] and pa = na/N

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na is the number of amino acid residue of type A, N is the number of residues in the sequence database and K is the number of residue type. The program Scorecons http://www.biochem.ucl.ac.uk/cgi-bin/valder/Scorecons_server.pl was used for all calculations. A score of zero indicates a lack of conservation at that position where as score of 1 indicates very high sequence conservation.

Determining the involvement in formation of specific patterns: Non-conserved residues adjacent to the conserved residues in the primary sequence are generally less substitutable than other non-conserved residues, reflecting their involvement in functionally important region [19]. Peptide sequence containing the variant along with ten neighboring residues on either side was selected from protein sequence and pattern search was done using PROSITE [20] database to determine the involvement of the variants in formation of specific patterns. PROSITE consists of biologically significant sites, patterns like phosphorylation, glycosylation, etc. and profiles that help to reliably identify specific motifs within a peptide sequence. Sequences involving variants showing potential phosphorylation sites were evaluated for the affect on phosphorylation using NetPhos 2.0. NetPhos 2.0 is an artificial neural-network method that predicts phosphorylation sites in independent sequences with sensitivity in the range from 69-96% [21].

Assessing the effect of variation on structural parameters of Proteins: It is apparent that amino acid allelic variants have an impact on the protein structure and function and this has been shown to be predicted by analysis of multiple sequence alignments

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and protein 3D structures [8]. To assess the effect of the variations on structure and function of proteins Polyphen [22] was used. Polyphen is a World Wide Web server to automate functional annotation of nonsynonymous SNPs, based on sequence-based characterization of the substitution site and structural parameters. This provides us with the PSIC score (Position Specific Independent Count) calculated from the overall similarity of the sequences that share the amino acid type at this position with the help of statistical concepts and predicts whether a nonsynonymous variation is damaging i.e. is supposed to affect the protein function, or benign i.e. most likely lacking a profound phenotypic effect. Large differences in PSIC values (difference range above 1.5) for specific genetic variants might indicate that the substitution of interest is rarely or never observed in protein family [23]. Variations in the protein core involving a change in the hydrophobic character of a buried residue may result in different degrees of protein destabilization [24]. The hydrophobic effect is measured by solvent accessible surface area of a protein that is part of a complex surface in direct contact with solvent. Solvent accessibility is predicted using RVPNET [25], which uses single residue information of neighbors and provides real predictions of accessible surface area. Hydrophobic interactions are considered to be the primary factor stabilizing β-sheets [26], therefore by Chou-Fasman predictions [27] identification of secondary structure elements was done. Statistical evaluation: To compare the DCVs and DAVs with CNVs during the assessment of their effect on the disease phenotype χ2 tests were performed and p-value was calculated.

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Results: Pathway Assist analysis establishes the products of the genes harboring DAVs to be potential interacting members of insulin signaling cascade (Fig.1). Nevertheless it is to be noted that Pathway Assist connects any two input proteins and some of the proteins identified by Pathway Assist during networking of the input proteins might not be involved in Type 2 Diabetes as is understood at this point of time. Functional segregation of the proteins harboring the DAVs categorized enzymes as the major class (31%) whereas, transcription regulators were the major class harboring DCVs (58%) (Fig.2). Pfam analyses showed that most of the DCVs (67%), 49% of DAVs and 63% of CNVs correspond to the functional domains of respective proteins (Supplementary Table 2 online). Therefore, of the total variations, an average of 56% lie in functional domains of proteins (p=0.02). Further, in the total sequence space of the identified proteins, 60% is occupied by functional domains. eMatrix analysis revealed that majority of DCVs (50%) and DAVs (62%) corresponded to functional signatures in comparison to only 27% of CNVs. This clearly indicates that the DCVs and DAVs correspond significantly more to the functional signatures in comparison to the randomly picked CNVs (p= 0.0002). Scorecons analysis (Fig. 3) reveals that DCVs are more of conservative changes (90% above the value of 0.5) whereas DAVs are radical (56% above 0.5) in comparison to CNVs (47% above 0.5) which are mostly changes in variable regions with low Scorecons value (p=0.0003). Most of the patterns obtained from PROSITE for DCVs (51.7%) and DAVs (51.1%) represented consensus post-translational modification motifs for phosphorylation, glycosylation and myristoylation (Supplementary Table 2 online) in contrast to only 37% of CNVs. Few peptides showed more than one post-translational motif. Phosphorylation changes predicted by NetPhos 2.0 for the patterns, indicated a probable decrease in the phosphorylation of DCV-

10

T608R of IRS1 ( Common variant Hollow > Rare variant

> Disease causing > No role in the disease

30

Supplementary Table 1:

List of Disease associated variations, Disease causing variations of Type 2 Diabetes Mellitus and Control Nonsynonymous variations

Regulatory disease associated variations

Gene

Position

Base Change

Susceptible allele

ALR2

Promoter (-12)

C>G

G

Li, Q. et al. Chin Med J . 115, 209-213 (2002)

ALR2

Promoter (-106)

C>T

T

Li, Q. et al. Chin Med J. 115, 209-213 (2002)

APM1

Promoter(-11426)

A>G

G

Harvest, F. Gu, et al. Diabetes. 53, S31-S35 (2004)

APM1

Promoter(-11377)

G>C

C

Harvest, F. Gu, et al. Diabetes. 53, S31-S35 (2004)

CCR 5

Promoter (59029)

G>A

A

Nakajima, K. et al . Diabetes. 51, 238-242 (2002)

COX 2

Promoter

G>C

C

Konheim, Y. L. et al . Hum.Genet. 113, 377-381(2003)

CRP

Promoter

T>C

T

Walford, J. K. et al . Mol Genet Metab. 78, 136-144(2003)

FOXC2

5'UTR (-512 )

C>T

C

Ridderstale, M. et al . Diabetes. 51,3554-3560 (2002)

Reference

GFPT2

3' UTR

C>T

T

Zhang, H. et al . J Clin Endocrinol Metab. 89, 748-55 (2004)

GLUT 2

Promoter (-269)

A>C

C

Cha, J. Y. et al . Ann Clin Lab Sci. 32, 114-122 (2002)

GLUT 2

Promoter (-44)

A>G

G

Cha, J. Y. et al. Ann Clin Lab Sci. 32, 114-122 (2002)

GLUT 2

Promoter (103)

A>G

G

Cha, J. Y. et al . Ann Clin Lab Sci. 32, 114-122 (2002)

Hepatic GCK

Promoter (-258)

G>A

A

Chiu, K. C. et al . BMC Genetics . 1(1):2(2000)

IL 6

Promoter (-634)

C>G

G

Kitamura, A. et al . Diabet Med. 19, 1000-1005 (2002)

IL 6

Promoter (-174)

C>G

C

Mohlig, M. et al . J Clin Endocrinol Metab . 89, 1885-1890 (2004)

IRS2

3'UTR (4064)

T>C

C

Zeng, W. M. et al . YI Chuan Xue Bao. 30, 785-789 (2003)

ISL1

Promoter (-47)

A>G

A

Barat-Hauari, M. et al . Diabetes. 51, 1640-1643 (2002)

LIPC

Promoter (-250)

G>A

G

Todorova, B. et al . J Clin Endocrinol Metab . 89, 2019-2023 (2004)

MCP-1

Promoter(-2518)

A>G

A

Simeoni, E. et al . Diabetologia. 47,1574-1580 (2004)

ORP150

Promoter (-429)

G>A

A

Kovacs, P. et al . Diabetes . 51, 1618-1621, 2002

Pancreatic GCK

Promoter (-30)

G>A

A

Marz, W. et al . Circulation. 109, 2844 (2004)

PC -1

3'UTR

(2897)

G>A

A

Frittitta, L. et al . Diabetes. 50, 1952-1955 (2001)

PC -1

3' UTR (2906)

G>C

C

Frittitta, L. et al . Diabetes. 50, 1952-1955 (2001)

PC -1

3'UTR

(2948)

C>T

T

Frittitta, L. et al . Diabetes. 50, 1952-1955 (2001)

PCK 1

Promoter (-232)

C>G

G

Cao, H. et al . J Clin Endocrinol Metab. 89, 898-903 (2004)

PKLR

3'UTR

C>T

T

Wang, H. et al . Diabetes. 51,2861-2865 (2002)

PON1

Promoter(-107)

T>C

T

James, R. W. et al . Diabetes. 49, 1390-1393 (2000)

PTEN

5'UTR (-9)

C>G

C

Ishihara, H. et al . FEBS Lett. 554, 450-454 (2003)

Resistin

Promoter (-420)

C>G

G

Osawa, H. et al . Am.J.Hum.Genet. 75, 678-86 (2004)

Resistin

3'UTR (+62)

G>A

G

Tan, M. S. et al . J Clin Endocrinol Metab. 88, 1258-1263 (2003)

TNFα

Promoter (-238)

G>A

A

Shiau, M. Y. et al .Tissue Antigens . 61 , 393-397 (2003)

TNFα

Promoter (-308)

G>A

A

Kabaszek, A. et al . Diabetes. 52, 1872-1876 (2003)

TSC-22(-396)

Promoter (-396)

A>G

A

Sugawara, F. et al . Diabetes Res Clin Pract. 60, 191-197 (2003)

UCP1

5'UTR

A>C

C

Mori, H. et al . Diabetologia. 44, 373-376 (2001)

UCP2

Promoter (-866)

G>A

A

D'Adamo, M. et al . Diabetes. 53,1905-1910 (2004)

UCP3

Promoter (-55)

C>T

C

Meirhaeghe, A. et al . Diabetologia. 43, 1424-1428 (2000)

VEGF

5'UTR (-634)

C>G

C

Awata, T. et al . Diabetes. 51,1635-1639 (2002)

Disease associated non-synonymous variations (DAVs)

Gene

Base change

Amino acid change

Susceptible allele

ADCYAP1R1

G>A

G54D

G

Gu, H. F. Hum Mutat. 19, 572-573 (2002)

ADRB2

C>G

R16G

R

Chang, T. J. et al . Clin Endocrinol (Oxf). 57, 685-690 (2002)

ADRB3

T>C

W64R

R

Oizumi, T. et al . Diabetes Care. 24:1579-1583, 2001

AGT

T>C

M235T

T

Chang, H. R. et al . J Chin Med Assoc. 66, 51-56 (2003)

AGT

C>T

T174M

M

Chang, H. R. et al . J Chin Med Assoc. 66, 51-56 (2003)

APM1

C>T

R112C

C

Kondo, H. et al . Diabetes. 51, 2325-2328 (2002)

Reference

CD38

C>T

R140W

W

Yagui, K. et al . Diabetologia 41, 1024-1028 (1998)

FABP2

G>A

A54T

T

Albala, C. et al . Obesity Research. 12, 340-345 (2004)

GCGR

G>A

G40S

S

Hager, J. et al . Nat Genet .9, 299-304 (1995)

GFPT2

A>G

I471V

V

Zhang, H. et al . J Clin Endocrinol Metab. 89, 748-55 (2004)

GYS1

A>G

M416V

V

Shimomura, H. et al . Diabetologia. 40, 947-952 (1997)

HFE

G>A

C282Y

Y

Moczulski, D. K. et al . Diabetes Care. 24, 1187-1191(2001)

HFE

C>G

H63D

D

Moczulski, D. K. et al. Diabetes Care. 24, 1187-1191(2001)

HNF1α

A>C

I27L

L

Chiu, K. C. et al . J Clin Endocrinol Metab. 85,2178-2183 (2000)

HNF4α

C>T

T130I

I

Zhu, Q. et al . Diabetologia .46,567-573 (2003)

ICAM1

A>G

E469K

K

Kamiuchi, K. et al . Diabet Med. 19, 371-376 (2002)

INSR

G>A

V985M

M

Hart, L. M. et al . Am J Hum Genet. 59,1119-1125 (1996)

IRS1

G>A

G972R

R

Jellema, A. et al . Diabetologia. 46, 990-995 (2003)

IRS2

G>A

G1057D

G

Mammarella, S. et al . Hum Mol Genet. 9, 2517-2521(2000)

KCNJ11

C>G

L270V

V

Nielsen, E. M. et al . Diabetes. 52, 573-577 (2003)

KCNJ11

G>A

E23K

K

Love-Gregory, L. et al. Diabetologia. 46, 136-137(2003)

LEPR

A>G

Q223R

R

Chiu, K C. et al . Eur J Endocrinol. 150, 725-729 (2004)

LMNA

C>T

P213S

P

Kamiuchi, K. et al . J Diabetes Complications. 16, 333-337 (2002)

NeuroD

G>A

A45T

T

Ye, L. et al . Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 19, 484-487 (2002)

NOS3

G>T

E298D

D

Monti, L. D. et al . Diabetes. 52, 1270-1275 (2003)

NPY

C>G

L7V

V

Niskanen, L. et al . Exp Clin Endocrinol Diabetes. 108,235-236 (2000)

NR3C1

A>G

N363S

S

Roussel, R. et al . Clin Endocrinol. 59, 237-241(2003)

NR3C1

G>A

R23K

R

van Rossum, E. F. et al . Diabetes. 51,3128-3134, 2002

PC-1

A>C

K121Q

Q

Kubaszek, A. et al . J Clin Endocrinol Metal. 89, 2044-2047 (2004)

PGC1

G>A

G482S

S

Hara, K. et al . Diabetologia. 45, 740-743 (2002)

PLA2G4A

C>G

F479L

L

Wolford, J. K. et al . Mol Genet Metab. 79, 61-66 (2003)

PON1

A>G

Q191R

R

Murata, M. et al . Diabet Med. 21 , 837-844 (2004)

PON2

T>A

C311S

C

Wang, X. Y. et al . Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 20, 215-219 (2003)

PON2

C>G

A148G

G

Hegele R. A. et al . J Clin Endocrinol Metal .82, 3373-3377 (1997)

PPARγ2

C>G

P12A

P

Mori, H. et al . Diabetes. 50, 886–890 (2001)

PPP1R3

G>T

D905Y

Y

Wang, G. et al . Chin Med J. 114, 1258-1262 (2001)

PTPN1

C>T

P387L

L

Soren, M. et al . Diabetes. 51,1-6 (2002)

RAGE

G>A

G82S

G

Kumaramnickavel, G. et al . J Diabetes Complications. 16, 391-394 (2002)

SOD2

C>T

V16A

V

Nomiyama, T. et al . J Hum Genet. 48, 138-141(2003)

TGFβ

T>C

L10P

P

Wong, T. Y. et al . Kidney Int. 63, 1831-1835 (2003)

UCP1

A>C

M229L

L

Mori, H. et al . Diabetologia. 44, 373-376 (2001)

UCP2

C>T

A55V

V

Wang, H. et al. Am J Physiol Endocrinol Metab. 286, E1-7(2004)

UTS2

G>A

S89N

N

Wenyi, Z. et al . Diabetologia. 46, 972-976 (2003)

WFS1

G>A

R456H

R

Minton, J. A. et al . Diabetes. 51,1287–1290 (2002)

WFS1

G>A

R611H

H

Minton, J. A. et al . Diabetes. 51,1287–1290 (2002)

Disease associated synonymous variations

Gene

Base Change

Amino acid change

Alpha 2 Integrin

C>T

F224F

Maeno, T, et al.Diabetes. 51:1523-1528 (2002).

Alpha 2 Integrin

G>A

T246T

Maeno, T, et al.Diabetes. 51:1523-1528 (2002).

Glucocorticoid receptor

G>A

E22E

PGC 1

A>G

T394T

Hara, K, et al .Diabetologia. 45:740-3 (2002).

PKLR

C>A

R569R

Wang, H, et al. Diabetes. 51:2861-2865 (2002).

PTP-1B

C>T

P303P

Mok, A ,et al . J Clin Endocrinol Metab. 87-724-727 (2002).

SREBP1

G>C

G952G

Eberle,D, et al. Diabetes. 53:2153-2157 (2004).

SUR 1

G>A

R1273R

Rissanen, J, et al.Diabetes care. 23:70-73 (2000).

SUR1

C>T

T759T

Reis, AF, et al.Diabetes Metab. 28:209-215 (2002).

SUR1

C>T

T759T

Reis AF, et al.Diabetes Metab. 28:209-215 (2002).

Syntaxin 1 A

T>C

D68D

Tsunoda, K, et al .Diabetologia. 44:2092-2097 (2001).

Reference

Van, Rossum Ef, et al . Diabetes. 51:3128-3134 (2002).

Disease associated Intronic variations

Gene

Base Change

Position

APM1

G>T

Intron 3

Hara,K, et al . Diabetes. 51:536-540 (2002).

APM1

G>T

Intron 1

Hara,K, et al. Diabetes. 51:536-540 (2003).

CAPN10

G>A

Intron 3

Michael, J Garant et al . Diabetes. 51, 231-237 (2002).

CAPN10

T>C

Intron 3

Weedon, MN, et al. Am.J.Hum.Genet. 73:1208 (2003).

GFPT-2

C>T

Intron 2

Zhang, H, et al . J Clin Endocrinol Metab. 89: 748-755 (2004).

GFPT-2

A>G

Intron 12

Zhang, H, et al . J Clin Endocrinol Metab. 89:748-755 (2004).

HNF 1α

C>G

Intron

NOS3

A>C

Intron 18

Monti, LD, et al. Diabetes. 52:1270-1275 (2003).

PKLR

C>T

Intron 3

Wang, H, et al. Diabetes. 51:2861-2865 (2002).

PKLR

C>T

Intron 5

Wang, H, et al. Diabetes. 51:2861-2865 (2002).

PPARγ

C>T

Intron

Tai, ES, et al . J Lipid Res. 45:674-685. (2004)

Reference

Gragnoli, C, et al . Diabetologia. 44:1326-1329 (2001).

SREBP-1C

C>T

Intron 18

Laudes, M,et al . Diabetes. 53:842-846 (2004).

SUR 1

T>C

Intron 24

Ji,L, et al. Zhonghua Yi Xue Za Zhi . 78:774-775 (1998).

Disease causing variations (DCVs)

Gene

Base Change

Amino acid change

Amylin

A>G

S20G

Seino,S, et al . Diabetologia. 44, 906-909 (2001)

APM1

T>C

I164T

Kondo,H, et al . Diabetes. 51, 2325-2328 (2002)

G3PD

T>C

F635S

Novialis, A, et al . Biochem Biophys Res Commun. 231, 570-572 (1997)

GCK

C>T

A456V

Henrik,B,T,et al . Diabetes. 51:1240–1246, 2002

GCK

G>C

G299R

Stoffel,M, et al . Nat Genet. 2, 153-1566 (1992)

GCK

G>A

A188T

Shimada,F, et al . Diabetologia. 36, 433-437(1993

GLUT2

G>A

V197I

Mueckler, M, et al . Diabetologia. 37, 420-427 (1994)

GLUT4

G>A

V383I

Choi, WH, et al . Diabetes. 40, 1712-1718 (1991)

Reference

HNF1α

G>A

G319S

Hegele, RA, et al . Diabetes Care. 22,524 (1999)

HNF1β

C>T

R177X

Tomura,H, et al . J Biol Chem. 274, 12975-12978 (1999)

HNF1β

C>G

S465R

Furuta, H, et al . J Clin Endocrinol Metab. 87, 3859-3863 (2002)

HNF1β

C>T

R276X

Furuta, H, et al . J Clin Endocrinol Metab. 87, 3859-3863 (2002)

HNF3β

G>A

A86T

Zhu, Q. et al . Diabetologia. 43, 1197-1200 (2000)

HNF4α

G>A

V393I

Hani, EH, et al . J Clin Invest. 101, 521-526 (1998)

HNF4α

C>T

R127W

Furuta,H, et al . Diabetes. 46, 1652-1657 (1997)

INSR

T>G

F382V

Accili,D, et al . EMBO J. 8, 2509-2517(1989)

IPF-1

T>C

C18R

Macfarlane, WM, et al . J Clin Invest. 104, R33-R39 (1999)

IPF-1

G>A

R197H

Macfarlane, WM, et al . J Clin Invest. 104, R33-R39 (1999)

IPF-1

G>A

D76N

Macfarlane, WM, et al . J Clin Invest. 104, R33-R39 (1999)

IRS1

C>G

T608R

Esposito, DL, et al . J Clin Endocrinol Metab. 88, 1468-1475 (2003)

IRS2

C>G

L647V

Almind,K, et al . Diabetologia. 42, 1244-1249 (1999)

ISL-1

C>T

Q310X

Shimomura,H, et al . Diabetes. 49, 1597–1600 (2000)

LPL

G>A

A71T

Yang,T, et al . Hum Mutat. 21, 453 (2003)

LPL

G>A

V181I

Yang,T, et al . Hum Mutat. 21, 453 (2003)

LPL

G>A

G188E

Yang,T, et al . Hum Mutat. 21, 453 (2003)

NeuroD

G>T

R111L

Malecki,MT, Nat Genet. 23, 323-328 (1999)

Pax4

C>T

R121W

Shimajiri,Y, et al . Diabetes. 50, 2864-2869 (2001)

PPARγ2

G>A

V318M

Barroso,I, et al . Nature .402:880-883 (1999)

PPARγ2

C>T

P467L

Barroso,I, et al . Nature.402:880-883 (1999)

Controls Nonsynonymous Variations(CNVs) extracted from Swiss-Prot Database

Gene

Amino acid change

ADRB2

I159F

ADRB2

I159L

ADRB2

K375R

ADRB2

V34M

ADRB3

T265M

ANGT

L392M

ANGT

L372V

APM1

H241P

APM1

R221S

APM1

V117M

GCGR

P114A

GLUT2

L478V

GLUT2

P68L

GLUT2

T110I

GLUT2

V101I

GLUT4

T78S

GLUT4

A358V

GYS1

E359G

GYS1

E619Q

GYS1

I108M

GYS1

K130E

GYS1

N283S

GYS1

P691A

GYS1

S706R

HFE

T217I

HFE

V53M

HFE

V59M

HNF1a

A98V

HNF1a

H514R

HNF1a

S487N

HNF1b

G492S

HNF4a

P436S

HXK4

A11T

HXK4

D4N

HXK4

M107T

ICAM1

K155N

ICAM1

K56M

ICAM1

P352L

ICAM1

R397Q

ICAM1

R478W

ICAM1

V315M

INSR

I1023F

INSR

K492Q

INSR

T448I

INSR

Y1361C

IRS1

A512P

IRS1

M209T

IRS1

P158R

IRS1

P679A

IRS1

S809F

IRS1

S892G

KCNJ11

R195H

KCNJ11

S385C

LEPR

K109R

LEPR

K204R

LEPR

K656N

LEPR

S675T

LEPR

T85A

LPL

A288T

LPL

A427T

LPL

D36N

LPL

T379A

LPL

V370M

NPY

L22M

NR3C1

D233N

NR3C1

F29L

NR3C1

F65V

NR3C1

L112F

PC1

K171Q

PC1

K173Q

PC1

T779P

PC1

Y268H

PGC1

T612M

PLA2G4

K651R

PLA2G4

V224I

PON1

M54L

PON2

V172L

PPARg

P40A

PPR3R1

R883S

RAGE

Q100R

SOD2

E66V

SOD2

I82T

SOD2

S10I

TGF1b

T263I

WFS1

A684V

WFS1

E737K

WFS1

G576S

WFS1

G674R

WFS1

I333V

WFS1

I720V

WFS1

R708C

WFS1

V871M

Supplementary Table 2 a) Characterization of functional properties of Disease Associated nonsynonymous Variations (DAVs) of Type 2 Diabetes Mellitus

Gene

Amino Residue acid change change

ADCYAP1 G54D

Nonpolar> Negative

ADRB2

ADRB3

RVP-NET PROSITE [1] patterns [2]

In Domain (PFAM) [3]

eMATRIX [4]

Hormone_2 83- No signature 110,132-159 motif

PSIC score (polyphen)

Poly Phen

ChouFasman [5]

0.137

Benign

Turn

1.26

Benign

Turn

2.051

Damaging

Helix

Exposed

Near Tyrosine sulfation site

R16G

Positive> Nonpolar

Exposed

Glycosylation site

7 tm 50-326

Beta-2 adrenergic receptor signature I

W64R

Aromatic> Positive

Buried

No pattern

7 tm 54-346

No signature motif

0.61

Benign

Sheet

1.416

Benign

Sheet

AGT

M235T

Polar> Polar

Exposed

No pattern

Selectin superfamily Serpin domain 99complement481 binding repeat signature I

AGT

T174M

Polar> Polar

Exposed

No pattern

Serpin domain 99-481

Angiotensinog en signature V

APM1

Positive> R112 C Polar

Exposed

No pattern

C1q 117-242

Fibrillar collagen Cterminal domain

No prediction

Damaging

Sheet

CD38

R140W

Positive> Aromatic

Exposed

No pattern

ADP-ribosyl cyclase56-296

ADP-ribosyl cyclase

2.296

Damaging

Helix

FABP2

A54T

Nonpolar> Polar

Exposed

Myristoylation site

Lipocolin 1-131

Cytosolic fattyacid binding protein

0.944

Benign

Helix

G40S

Nonpolar> Polar

Exposed

Glycosylation site

HRM 55-126

Glucagon family receptor signature II

0.35

Benign

Sheet

GCGR

GFPT

I471V

Nonpolar> Nonpolar

Buried

P_Phospho_ site

SIS (sugar No signature binding) 377-511 motif

0.913

Benign

Sheet

GYS1

M416V

Polar> Nonpolar

Buried

No pattern

Glycogen No signature synthase 31-663 motif

1.581

Damaging

Helix

3.143

Damaging

Sheet

1.37

Damaging

Helix

C282Y

Polar> Aromatic

HFE

HFE

Exposed

No pattern

H63D

Positive> Negative

Buried

PKC_phospho_ MHC 1 26-202 site

HNF1α

I27L

Nonpolar> Nonpolar

Buried

No pattern

HNF-1 N 1-176

No signature motif

0.45

Benign

Helix

HNF4α

T130I

Aromatic> Nonpolar

Exposed

No pattern

zf-c4 49-124

No signature motif

1.33

Benign

Sheet

ICAM1

E469K

Negative> Positive

Exposed

No pattern

Ig like 404-475

No signature motif

1.43

Benign

Sheet

INSR

V985M

Nonpolar> Polar

Buried

No pattern

No domain

No signature motif

1.512

Damaging

Sheet

IRS 1

G972R

Nonpolar> Positive

Exposed

Myristoylation site

PH 13-115

No signature motif

1.406

Benign

Near Turn

IRS 2

G1057D

Nonpolar> Negative

Exposed

Myristoylation site

PH 31-144

Synapsin

0.45

Benign

Near Turn

L270V

Nonpolar> Nonpolar

IRK 36-309

Kir6.2 inward rectifier K+ channel signature V

1.183

Benign

Helix

KCNJ11

Buried

No pattern

222-294

Immunoglobul in and major histocompatibi lity complex domain Major histocompatibi lity complex protein, Class I

No pattern

IRK 36-309

Kir6.2 inward rectifier K+ channel signature I

0.006

Benign

Helix

KCNJ11

E23K

Negative> Positive

LEPR

Q223R

Polar> Positive

Exposed

No pattern

Leptin receptor Iq No signature 331-422 motif

0.123

Benign

Helix

LECAM-1

P213S

Nonpolar> Polar

Exposed

Glycosylation site

Sushi

No signature motif

0.982

Benign

Helix

NeuroD

A45T

Nonpolar> Polar

Buried

PKC_phospho_ HLH 102-154 site

No signature motif

0.97

Benign

Helix

NOS3

E298D

Negative> Negative

Exposed

No pattern

NO synthase114- No signature 485 motif

1.301

Benign

Sheet

NPY

L7V

Nonpolar> Polar

Buried

No pattern

Signal peptide 1- No signature 26 motif

0.834

Benign

No prediction

NR3C1

N363S

Polar> Polar

Exposed

No pattern

gcr 26-401

No signature motif

0.078

Benign

No prediction

gcr 26-401

Glucocorticoid receptor (3C nuclear receptor) signature I

1.014

Benign

Helix

Exposed

NR3C1

R23K

Positive> Positive

Exposed

RGD Cell attachment sequence

PC-1

K121Q

Positive> Polar

Exposed

Glycosylation site

Somatomedin B Somatomedin 104-144, 145-189 B domain

0.093

Benign

Turn

PGC 1

G482S

Nonpolar> Polar

Exposed

ARG_RICH

PPAR interacting No signature domain 293-339 motif

0.9

Benign

Helix

PLA2G4A F479L

Aromatic> Nonpolar

Buried

CK2_Phospho_ No signature PLA2 B 190-675 site motif

1.775

Damaging

Sheet

PON 1

Q191R

Polar> Positive

Exposed

Myristoylation site

Arylaceterase 1354

Arylesterase

0.996

Benign

Sheet

PON 2

C311S

Polar> Polar

Buried

No pattern

Arylaceterase 1354

Arylesterase

0.453

Benign

Helix

PON2

G148A

Nonpolar> Nonpolar

Exposed

No pattern

Arylaceterase 1354

Arylesterase

0.932

Benign

Helix

PPAR γ2

P12A

Nonpolar> Nonpolar

Exposed

zf 137No signature CK2_phospho_ 211,hormone motif site receptor318-500

PPP1R3

D905Y

Negative> Aromatic

Exposed

No pattern

No signature motif

2.336

Damaging

No prediction

PTPN1

P387L

Nonpolar> Nonpolar

Exposed

PKC_phospho_ y phosphatase 40- No signature 276 motif site

1.603

Damaging

Helix

RAGE

G82S

Nonpolar> Polar

Exposed

Glycosylation site

1.581

Damaging

Turn

SOD2

A16V

Nonpolar> Nonpolar

Buried

Superoxide PKC_Phospho_ dismutase 25Site 106,110-217

0.379

Benign

Sheet

1.823

Damaging

Sheet

2

Benign

Helix

0.093

Benign

Helix

TGF β

L10P

Nonpolar> Nonpolar

Buried

UCP 1

M229L

Polar> Nonpolar

Buried

UCP2

A55V

Nonpolar> Nonpolar

Buried

No domain

VIg domain 31-101 domain(ligand interaction)

No signature motif

Transforming Phosphatidylino growth factor sitol-specific Signal peptide 1beta 2 phospholipase 24 precursor X-box domain signature I Mitochondrial energy CK2_phospho_ transfer mit carr 6-102 site proteins (carrier protein) Mitochondrial brown fat Myristoylation mit carr 12-102 uncoupling site protein signature II

1.8

Damaging Near Turn

UTS2

S89N

Polar> Polar

Exposed

No pattern

Urotensin 113124

RGD-Cell attachment sequence

1.163

Benign

Near Turn

WFS1

R456H

Positive> Positive

Exposed

Myristoylation site

429-451 transmembrane

No signature motif

1.125

Benign

Sheet

WFS1

R611H

Positive> Positive

Exposed

No pattern

588-610 transmembrane

No signature motif

1.125

Benign

Helix

PSIC score

Poly Phen

ChouFasman

1.504

Damaging

Turn

No No prediction prediction

Sheet

b) Characterization of functional properties of Disease Causing Variations (DCVs) of Type 2 Diabetes Mellitus

Gene

Amino acid change

Residue change

RVP-NET

Pattern (PROSITE)

IN Domain (PFAM)

eMATRIX

Exposed

Islet amyloid protein PKC_phospho_ calc_CGRP_IAP (amylin) signature I site P 32-73

I164T

Nonpolar> Polar

Buried

C1q domain signature

G3PD

F635S

Aromatic> Polar

Buried

CK2_phospho_ No signature site EF hand 627-655 motif

1.667

Damaging

Helix

GCK

A456V

Nonpolar> Nonpolar

Buried

No pattern

SIS 91-259

No signature motif

1.15

Benign

Helix

GCK

G299R

Nonpolar> Positive

Exposed

No pattern

Hexokinase Hexokinase_219- family 458 signature V

2.743

Damaging

Helix(from pdb)

GCK

A188T

Nonpolar> Polar

Buried

No pattern

Hexokinase_10217

2.082

Damaging

Helix(from pdb)

S20G

Polar> Nonpolar

APM1

Amylin

c1q 117-242

Complement C1Q domain signature II

Hexokinase family

Buried

Nmyristoylation site

V383I

Nonpolar> Nonpolar

Buried

Sugar Major facilitator Sug tr domain26- transporter superfamily 483 signature IV

HNF 1α

G319S

Nonpolar> Polar

Buried

No pattern

HNF--1b_C280541

HNF 3β

A 86 T

Nonpolar> Polar

Buried

No pattern

HNF1β

R177X

Positive> Termination Termination No pattern

HNF1β

S465R

Polar> Positive

HNF1β

R276X

HNF4α

V393I

V197I

Nonpolar> Nonpolar

GLUT4

GLUT2

Glu tr 13-499 No signature (transmembrane) motif

3

Benign

Sheet

0.09

Benign

Helix

No signature motif

1.555

Damaging

Sheet

Fork head domain 159-254

No signature motif

1.427

Benign

No prediction

HNF 1 N 1-182

No signature motif

HNF 1b C 312551

No signature motif

Positive> Termination Termination No pattern

HOX 231-314

No signature motif

Nonpolar> Nonpolar

No pattern

Hormone No signature receptor 183-364 motif

0.693

Benign

Helix

Buried

No pattern

Zinc finger, C4 type 49-124

Retinoid X receptor (2B nuclear receptor) signature I

2.675

Damaging

No prediction

Buried

No pattern

Recep_L_domain No signature 359-474 motif

1.912

Damaging Near Turn

HNF4α

Positive> R127W Aromatic

INSR

F382V

Aromatic> Nonpolar

Buried

Buried

Nmyristoylation site

Termination Damaging

1.779

Damaging

Termination Damaging

No prediction

Helix

No prediction

IPF-1

R197H

Positive> Positive

Exposed

No pattern

Homeobox 147203

Lambda and other repressor Helix-TurnHelix signature II

IPF-1

C18R

Polar> Positive

Buried

Proline-rich region

Homeobox 147203

No signature motif

3.168

Damaging

IPF-1

D76N

Negative> Polar

Exposed

CK2_phospho_ Homeobox 147site 203

No signature motif

1.088

Damaging Near Turn

IRS1

T608R

Polar> Positive

Exposed

ASPPPH 13-115, IRS phosphoserine 160-262

No signature motif

1.297

Damaging

Sheet

IRS2

L647V

Nonpolar> Nonpolar

Buried

Tyrosine sulfation site

PH 31-144,IRS 194-296

No signature motif

1.135

Benign

Near Turn

ISL-1

Q310X

Polar> Termination Termination No pattern

Homeobox 182238

No signature motif

LPL

A71T

Nonpolar> Polar

Buried

Nmyristoylation site

Lipase 12-338

Triacylglycerol lipase family signature II

1.656

Damaging

Helix

LPL

V181I

Nonpolar> Nonpolar

Buried

No pattern

Lipase 12-338

No signature motif

1.114

Benign

Helix

G188E

Nonpolar> Negative

Lipase 12-338

Lipoprotein lipase signature V

0.19

Benign

Helix

HLH 102-154

Helix-LoopHelix dimerization domain

2.565

Damaging

Helix

LPL

NeuroD

R111L

Positive> Nonpolar

Buried

No pattern

Exposed

Bipartite nuclear targeting sequence

1.582

Damaging

Helix

No prediction

Termination Damaging

No prediction

Pax4

PPARγ2

PPARγ2

Positive> R121W Aromatic

V318M

P467L

Nonpolar> Polar

Nonpolar> Nonpolar

Exposed

Nmyristoylation site

Pax 5-129

Paired box domain

3.143

Damaging

Helix

Buried

Peroxisome proliferatoractivated zf 137receptor Ligand-binding 211,hormone gamma domain receptor318-500 signature IV

1.479

Damaging

Helix

Exposed

Peroxisome proliferatoractivated receptor (1C zf 137nuclear Ligand-binding 211,hormone receptor) domain receptor318-500 signature VII

2.956

Damaging

Sheet

PSIC score

Poly Phen

ChouFasman

b) Characterization of functional properties of Control Non-Synonymous variations(CNVs)

Gene

Amino acid change

ADRB2

I -> F

Nonpolar> Aromatic

Buried

ADRB2

I -> L

Nonpolar> Nonpolar

ADRB2

K -> R

ADRB2

Pattern (PROSITE)

IN Domain (PFAM)

eMATRIX

No pattern

7tm_1(50-326)

GPCR superfamily signature IV

1.5

Benign

Helix

Buried

No pattern

7tm_1(50-326)

GPCR superfamily signature IV

0.6

Benign

Helix

Positive> Positive

Exposed

No pattern

No domain

No signature

0.9

Benign

Helix

V -> M

Nonpolar> Nonpolar

Buried

Nmyristoylation site(37-42)

No domain

No signature

0.26

Benign

Helix

ADRB3

T -> M

Polar> Nonpolar

Exposed

No pattern

7tm_1(54-346)

No signature

0.175

Benign

No prediction

ANGT

L -> M

Nonpolar> Nonpolar

Buried

No pattern

Serpin(99-481)

No signature

0.7

Benign

Helix

Buried

cAMP- and cGMPdependent Serpin(99-481) protein kinase phosphorylation site(370-373)

No signature

1

Benign

No prediction

ANGT

L->V

Residue change

Nonpolar> Nonpolar

RVP-NET

APM1

H -> P

Positive> Nonpolar

Exposed

Nglycosylation site(230-233)

C1q(114-239)

No signature

Benign

Sheet

APM1

R -> S

Positive> Polar

Exposed

Tyrosine sulfation site(218-232)

C1q(114-239)

No signature

Benign

Loop

Benign

Sheet

1.026

Benign

No prediction

APM1

V -> M

Nonpolar> Nonpolar

Buried

No pattern

C1q(114-239)

C-terminal tandem repeated domain in type 4 procollagen

GCGR

P -> A

Nonpolar> Nonpolar

Buried

No pattern

HRM(55-126)

Glucagon receptor signature IV

GLUT2

L -> V

Nonpolar> Nonpolar

Buried

Nmyristoylation site(471-476)

Sugar_tr(13-499) No signature

0.4

Benign

Sheet

GLUT2

P -> L

Nonpolar> Nonpolar

Exposed

N-glycosylation Sugar_tr(13-499) No signature site (62-65)

1.2

Benign

Sheet

GLUT2

T -> I

Polar> Nonpolar

Buried

Nmyristoylation site(107-112)

Sugar_tr(13-499) No signature

1.9

Damaging

Sheet

GLUT2

V -> I

Nonpolar> Nonpolar

Buried

No pattern

Sugar_tr(13-499) No signature

1.4

Benign

Sheet

GLUT4

Polar> T -> S Nonpolar

Buried

No pattern

Glucose transporter Sugar_tr(26-483) type 4 (GLUT4)

0.5

Benign

Sheet

GLUT4

A -> V

Nonpolar> Nonpolar

Exposed

Nmyristoylation site(359-364)

Sugar_tr(26-483) No signature

0.3

Benign

No prediction

GYS1

E -> G

Negative> Nonpolar

Exposed

N-glycosylation Glycogen_syn(31No signature 663) site(356-359)

1.26

Benign

Sheet

GYS1

E -> Q

Negative> Polar

Exposed

Tyrosine sulfation site(616-629)

Glycogen_syn(31No signature 663)

1

Benign

Helix

GYS1

I->M

Nonpolar> Nonpolar

Buried

No pattern

Glycogen_syn(31No signature 663)

1.4

Benign

Sheet

GYS1

K -> E

Positive> Negative

Exposed

No pattern

Glycogen_syn(31No signature 663)

1.62

Damaging

Helix

GYS1

N -> S

Exposed

No pattern

Glycogen_syn(31No signature 663)

1.8

Damaging

Helix

Polar>Polar

GYS1

P -> A

Nonpolar> Nonpolar

Exposed

cAMP- and cGMPdependent No domain protein kinase phosphorylation site(695-698)

GYS1

S->R

Nonpolar> Positive

Exposed

No pattern

HFE

HFE

T -> I

V -> M

Polar> Nonpolar

Nonpolar> Nonpolar

No signature

2.01

Damaging

Helix

No domain

No signature

1.1

Benign

No prediction

C1-set(211-294)

Major histocompatibi lity complex protein, Class I

0.54

Benign

No prediction

Exposed

No pattern

Buried

Casein kinase II MHC_I(26-202) phosphorylation site (45-48)

A Major histocompatibi lity complex protein, Class I

1.3

Benign

Sheet

A Major histocompatibi lity complex protein, Class I

0.56

Benign

Sheet

HFE

V -> M

Nonpolar> Nonpolar

Buried

Protein kinase C MHC_I(26-202) phosphorylation site(65-67)

HNF1a

A -> V

Nonpolar> Nonpolar

Buried

No pattern

HNF-1_N(1-176) No signature

1.6

Damaging

Helix

HNF1a

H -> R

Positive> Positive

Exposed

No pattern

HNF-1B_C(282541)

No signature

2.2

Damaging

No prediction

HNF1a

S -> N

Nonpolar> Polar

Buried

No pattern

HNF-1B_C(282541)

No signature

0.201

Benign

No prediction

HNF1b

G -> S

Nonpolar> Nonpolar

Exposed

Nmyristoylation site(464-469)

HNF-1B_C(314551)

No signature

No prediction

No prediction

HNF4a

P -> S

Nonpolar> Nonpolar

Exposed

No pattern

No domain

No signature

0.16

Benign

No prediction

HXK4

A -> T

Nonpolar> Polar

Exposed

No pattern

Hexokinase_1 (10-217)

No signature

0.193

Benign

Helix

HXK4

D -> N

Negative> Polar

Buried

No pattern

No domain

No signature

0.13

Benign

Helix

HXK4

Nonpolar> M -> T Polar

No signature

1.1

Benign

Sheet

ICAM1

Positive> K -> N Polar

Intercellular adhesion molecule/vasc ular cell

0.9

Benign

No prediction

Buried

Exposed

Casein kinase II Hexokinase_1 phosphorylation (10-217) site (437-440) Nmyristoylation No domain site(140-145)

Exposed

No pattern

ICAM_N(5-115)

Intercellular adhesion molecule/vasc ular cell

Exposed

No pattern

No domain

No signature

2.1

Damaging

Sheet

Positive>Po Exposed lar

N-glycosylation No domain site(385-388)

No signature

0.7

Benign

No prediction

R -> W

Positive> Aromatic

Exposed

No pattern

No domain

No signature

1.65

Damaging

Sheet

ICAM1

V -> M

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

0.7

Benign

Sheet

INSR

I -> F

Nonpolar> Aromatic

Buried

Protein kinase domain(10231298)

Furin-like(179340)

No signature

2.3

Damaging

Helix

INSR

K -> Q

Positive> Polar

Exposed

No pattern

No domain

No signature

0.767

Benign

Sheet

INSR

T -> I

Polar> Nonpolar

Exposed

No pattern

No domain

No signature

1.295

Benign

No prediction

INSR

Y -> C

Aromatic> Polar

Buried

Tyrosine kinase phosphorylation No domain site(1353-1361)

No signature

2.8

Damaging

No prediction

IRS1

A -> P

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

1.042

Benign

No prediction

IRS(160-262)

Insulin receptor substrate-1 PTB domain signature II

2.3

Damaging

Loop

1.7

Damaging

Loop

ICAM1

Positive> K -> M Nonpolar

ICAM1

P -> L

Nonpolar> Nonpolar

ICAM1

R -> Q

ICAM1

IRS1

M -> T

Nonpolar> Polar

Buried

No pattern

0.9

Benign

Loop

IRS1

P -> R

Nonpolar> Positive

Exposed

No pattern

No domain

Insulin receptor substrate-1 PTB domain signature I

IRS1

P-A

Nonpolar> Nonpolar

Exposed

No pattern

No domain

No signature

1.4

Benign

No prediction

IRS1

S -> F

Nonpolar> Aromatic

Buried

Nmyristoylation site(677-682)

No domain

No signature

1.3

Benign

No prediction

IRS1

S -> G

Nonpolar> Nonpolar

Exposed

No pattern

No domain

No signature

0.4

Benign

No prediction

KCNJ11

R -> H

Positive> Positive

Exposed

No pattern

No domain

No signature

KCNJ11

S -> C

Nonpolar> Polar

Buried

No pattern

No domain

No signature

LEPR

K -> R

Positive> Positive

Exposed

N-glycosylation No domain site(101-104)

No signature

LEPR

K -> R

Positive> Positive

Exposed

No pattern

No domain

No signature

LEPR

Positive> K -> N Polar

LEPR

Nonpolar> S -> T Polar

Exposed

No pattern

0.18

Benign

Sheet

No prediction

No prediction

0.123

Benign

No prediction

0.141

Benign

No prediction

No domain

Long hematopoietin receptor, gp130 family

1.4

Benign

Helix

No domain

Long hematopoietin receptor, gp130 family

1.3

Benign

Sheet

Buried

No pattern

Exposed

N-glycosylation No domain site(81-84)

No signature

0.5

Benign

Sheet

LPL

Nonpolar> A -> T Polar

Buried

Casein kinase II Lipase(12-338) phosphorylation site(292-295)

Triacylglycerol lipase family signature VI

1.6

Damaging

Turn

LPL

A -> T

Nonpolar> Polar

Buried

No pattern

PLAT(341-427)

Lipoprotein lipase signature VII

0.455

Benign

Sheet

LPL

D -> N

Negative> Polar

Exposed

No pattern

Lipase(12-338)

No signature

1.3

Benign

Helix

LPL

T -> A

Polar> Nonpolar

Buried

No pattern

No domain

No signature

1.66

Damaging

No prediction

LPL

Nonpolar> V -> M Nonpolar

Buried

Casein kinase II PLAT(341-427) phosphorylation site(385-387)

No signature

0.98

Benign

No prediction

NPY

L -> M

Nonpolar> Nonpolar

Buried

Nmyristoylation site(11-16)

No signature

0.7

Benign

Helix

Exposed

Casein kinase II GCR(26-401) phosphorylation site(241-244)

No signature

1.4

Benign

No prediction

LEPR

NR3C1

T-A

Polar> Nonpolar

Negative> D -> N Polar

No domain

Glucocorticoid receptor (3C nuclear receptor) signature I

GCR(26-401)

No signature

Glucocorticoid receptor (3C nuclear receptor) signature III

NR3C1

F -> L

Aromatic> NonPolar

Buried

Protein kinase C GCR(26-401) phosphorylation site(32-34)

NR3C1

F -> V

Aromatic> NonPolar

Buried

Nmyristoylation site(68-73)

0.793

Benign

Helix

2

Damaging

No prediction

1.2

Benign

Sheet

NR3C1

L -> F

NonPolar> Aromatic

Buried

No pattern

GCR(26-401)

PC1

K -> Q

Positive> Polar

Exposed

No pattern

Somatomedin_B Somatomedin (147-189) B domain

0.169

Benign

No prediction

PC1

K -> Q

Positive> Polar

Exposed

No pattern

Somatomedin_B Somatomedin (147-189) B domain

0.093

Benign

No prediction

PC1

T -> P

Polar> Nonpolar

Exposed

No pattern

No domain

DNA/RNA nonspecific endonuclease

0.29

Benign

Helix

PC1

Y -> H

Aromatic> Positive

Buried

No pattern

No domain

No signature

0.17

Damaging

Sheet

PGC1

T -> M

Polar> Nonpolar

Buried

No pattern

No domain

No signature

No prediction

No prediction

Exposed

Bipartite nuclear PLA2_B(190targeting 675) sequence(651667)

No signature

0.141

Benign

Sheet

Casein kinase PLA2_B(190II phosphorylation 675) site(215-218)

Lysophospholi pase catalytic domain

0.089

Benign

Sheet

K -> R

Positive> Positive

PLA2G4

V -> I

Nonpolar> Nonpolar

Buried

PON1

M -> L

Nonpolar> Nonpolar

Buried

PON2

V -> L

Nonpolar> Nonpolar

Buried

PLA2G4

PPARg

P -> Polar> A Nonpolar

Exposed

Nmyristoylation site(46-51) Casein kinase II phosphorylation site(165-168)

No pattern

Arylesterase(1354)

No signature

0.8

Benign

Sheet

Arylesterase(2354)

No signature

0.283

Benign

Sheet

No domain

Peroxisome proliferatoractivated receptor gamma signature I

1.7

Damaging

No prediction

PPR3R1

R -> S

Positive> Nonpolar

Exposed

No pattern

No domain

No signature

0.2

Benign

No prediction

RAGE

Q -> R

Polar> Positive

Exposed

Nmyristoylation site(95-100)

No domain

No signature

0.192

Benign

Sheet

SOD2

Negative> E -> V Nonpolar

Exposed

No pattern

Sod_Fe_N(25106)

Manganese and iron superoxide dismutase

0.3

Benign

Helix

SOD2

I -> T

Nonpolar> Polar

Buried

No pattern

Sod_Fe_N(25106)

No signature

0.9

Benign

Helix

SOD2

S -> I

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

1.4

Benign

Helix

0.3

Benign

No prediction

Buried

No pattern

No domain

Transforming growth factor beta 1 precursor signature VI

Buried

Casein kinase II No domain phosphorylation site(691-694)

No signature

0.9

Benign

Helix

Negative> Positive

Exposed

No pattern

No domain

No signature

0.9

Benign

No prediction

G -> S

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

1.3

Benign

Helix

WFS1

G -> R

Nonpolar> Positive

Buried

No pattern

no domain

No signature

1.8

Damaging

Helix

WFS1

I -> V

Nonpolar> Nonpolar

Buried

no pattern

No domain

No signature

0.225

Benign

Sheet

WFS1

I -> V

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

0.22

Benign

Helix

WFS1

R -> C

Positive> Polar

Exposed

No pattern

No domain

No signature

1.8

Damaging

Sheet

WFS1

V -> M

Nonpolar> Nonpolar

Buried

No pattern

No domain

No signature

0.6

Benign

Sheet

Polar> Nonpolar

TGF1b

T -> I

WFS1

Nonpolar> A -> V Nonpolar

WFS1

E -> K

WFS1

References: 1. Ahmad, S., Gromiha, M. M. & Sarai, A. Bioinformatics. 19, 1849-1851 (2003). 2. Falquet, L. et al. Nucleic Acids Res. 30, 235-238 (2002). 3. Bateman, A. et al . Nucleic Acids Res. 30, 276-280 (2002). 4. Bennett, S. P., Lu, L. & Brutlag, D. L. Nucleic Acids Res. 31, 3328-3332 (2003). 5. Chou, P. Y. & Fasman, G. D. Biochemistry. 13, 211- 222 (1974).