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Diabetologia

, Volume 52, Issue 6, pp 1208–1211 | Cite as

IRS2 variants and syndromes of severe insulin resistance

  • W. E. Bottomley
  • M. A. Soos
  • C. Adams
  • T. Guran
  • T. A. Howlett
  • A. Mackie
  • J. Miell
  • J. P. Monson
  • R. Temple
  • Y. Tenenbaum-Rakover
  • J. Tymms
  • D. B. Savage
  • R. K. Semple
  • S. O’Rahilly
  • I. Barroso
Open Access
Research Letter

Keywords

Diabetes Genetics Insulin resistance IRS2 

To the Editor: Numerous lines of experimental evidence implicate IRS2 in insulin signal transduction in key insulin-responsive tissues, and the combination of insulin resistance and pancreatic beta cell failure of homozygous Irs2 knockout mice is highly reminiscent of both core abnormalities in human type 2 diabetes [1]. IRS2 is therefore a compelling candidate gene for involvement in prevalent forms of type 2 diabetes and insulin resistance in humans. Nevertheless, genetic studies to date in humans suggest that common polymorphisms found in the IRS2 coding or promoter regions are not associated with insulin resistance or type 2 diabetes [2, 3, 4]. Furthermore, these studies have revealed few rare non-synonymous variants, none of which appear to have a strong impact on insulin sensitivity [5, 6].

We have assembled a large cohort of patients with severe insulin resistance that is likely to be highly enriched for monogenic disorders of insulin action. All participants gave informed consent, and our investigations were carried out with the approval of the local research ethics committee in Cambridge, UK. This strategy has previously been validated, for example, by the finding of a convincing pathogenic mutation in AKT2 [7], despite a failure to find association between common genetic variation in AKT2 and metabolic traits [8]. We have now applied this robust approach to IRS2.

We sequenced the coding region of IRS2 in 161 predominantly Europid (n = 114) patients with severe insulin resistance. Severe insulin resistance was defined as (1) a fasting insulin level of >150 pmol/l or a peak insulin level on oral glucose tolerance testing of >1,500 pmol/l in those without diabetes and a BMI of <30 kg/m2, or (2) an exogenous insulin requirement of >3 U/kg/day in those with complete insulin deficiency and a BMI of <30 kg/m2. Those with partial beta cell decompensation or a BMI of >30 kg/m2 were included at the investigator’s discretion based on clinical and biochemical features of insulin resistance disproportionate to body weight, determined by reference to sex and BMI-specific 95th percentiles for plasma insulin from more than 500 non-diabetic volunteers.

Eight rare, novel, non-synonymous variants in the IRS2 gene were identified in eight patients from this cohort (designated as patients SIR1 to SIR8 in Table 1), four of whom are Europids. Wherever possible, family members of probands with non-synonymous variants were studied to look for co-segregation of the genetic variant with insulin resistance or diabetes.
Table 1

Non-synonymous variants found in the IRS2 gene of participants with severe insulin resistance (total SIR, n = 161; Europid SIR, n = 114) and Europid controls (n = 173)

Participant

Phenotype

SNP

PolyPhena/SIFTb

Genotype

Ethnicity

Fasting glucose (mmol/l)

Fasting insulin (pmol/l)

BMI (kg/m2)

SIR1

SIR, pseudoacromegaly

c.233G>A, p.S78N

Benign/Tolerated

GA

Asian

5.6

212

44.6

SIR2

SIR, pseudoacromegaly

c.1498C>G, p.L500V

Benign/Tolerated

CG

Asian

6.0

264

39.7

SIR3

SIR, short stature

c.1570A>G, p.I524V

Benign/Affect protein function

AG

Turkish

4.9

1,358

19

SIR4

SIR, pseudoacromegaly

c.2485C>T, p.P829S

Benign/Tolerated

CT

Ashkenazy

5.1

507

25.5

SIR5

SIR

c.2566G>A, p.A856T

Benign/Tolerated

GA

Europid

4.7

333

28

SIR6

SIR, FPLD1

c.2834C>T, p.S945F

Benign/Affect protein function

CT

Europid

5.5

155

Obese

SIR7

SIR, pseudoacromegaly

c.3424G>C, p.G1142R

Possibly damaging/Affect protein function

GC

Europid

8.5

880

47.3

SIR8

SIR, pseudoacromegaly

c.3983A>G, p.H1328R

Possibly damaging/Affect protein function

AG

Europid

6.8

246

37.3

Control 1

Healthy volunteer

c.1379C>T, p.P460L and c.3983A>G, p.H1328R

Benign/Possibly damaging

CT AG

Europid

5.5

47

24.6

Control 2

Healthy volunteer

c.3400A>G, p.K1134E

Benign/Affect protein function

AG

Europid

5.1

65

31.7

Control 3

Healthy volunteer

c.3788G>T, p.G1263V

Probably damaging/Affect protein function

GT

Europid

6.4

43

24.3

Control 4

Healthy volunteer

c.3983A>G, p.H1328R

Possibly damaging/Affect protein function

AG

Europid

4.9

45

23.4

aPolyPhen predicts the possible impact of an amino acid substitution on the structure and function of a protein based on physical and comparative considerations

bSIFT predicts whether an amino acid substitution affects protein function based on sequence homology and the physical properties of amino acids

FPLD1, familial partial lipodystrophy type 1, Online Mendelian Inheritance in Man (OMIM) database ID: 608600 (www.ncbi.nlm.nih.gov/omim, accessed 5 March 2009); IR, insulin resistance

In the case of SIR1 (c.233G>A, p.S78N), four of the five family members studied were found to be heterozygous for this mutation and had insulin levels commensurate with their degree of obesity [9] (fasting insulin levels 45–95 pmol/l, BMI 30–38.8 kg/m2); hence, there was no clear co-segregation of the mutation with severe insulin resistance in the family (Electronic supplementary material [ESM] Fig. 1), indicating that the S78N change in IRS2 within this family is either benign, as suggested by Polyphen (http://genetics.bwh.harvard.edu/pph, accessed 4 March 2008)/SIFT (http://blocks.fhcrc.org/sift/SIFT.html, accessed 22 December 2008) analysis, or is not the sole genetic determinant of insulin resistance within the kindred. The location of serine 78 within the Pleckstrin homology domain of the protein (ESM Fig. 2) and its strong evolutionary conservation support the view that a pathogenic role for this protein-altering variant should not be entirely discounted on the basis of these genetic data.

Eight members of the Turkish family of SIR3, with five affected members, were also studied, but only the unaffected father of the proband was found to carry the same heterozygous mutation (c.1570A>G, p.I524V). Thus, this non-synonymous change in IRS2 convincingly fails to co-segregate with insulin resistance in this family (ESM Fig. 3, ESM Table 1) and is thus most probably benign.

Of the four available relatives of Ashkenazy proband SIR4 (c.2485C>T, p.P829S), three were heterozygous for this variant, two of whom exhibited elevated fasting insulin levels with respect to 500 non-diabetic volunteers (ESM Fig. 4). However, incomplete clinical data precluded a definitive conclusion regarding co-segregation of the mutation with the phenotype in this family. It is of interest that this variant was absent in 185 Ashkenazy controls and that proline 829 is conserved in a diverse range of species, from Pan troglodytes (chimpanzee) down to Xenopus tropicalis (Western clawed frog) (ESM Fig. 5).

The non-synonymous variant found in SIR6 (c.2834C>T, p.S945F) was found to be absent in this patient’s mother, who presented with a similar but milder syndrome of partial lipodystrophy. Although the father of SIR6 was unavailable for study (ESM Fig. 6), this suggests that this variant does not underlie the observed phenotype.

In a study limited to the parents of SIR8 (ESM Fig. 7), there appeared to be co-segregation of the non-synonymous variant (c.3983A>G, p.H1328R) found in the proband with clinical phenotype, but because this change was also observed twice on sequencing additional controls, this variant was deemed to be benign.

Subsequent to our family studies, we also sequenced the IRS2 gene in 173 Europid controls (mean fasting insulin 30 pmol/l, mean BMI 27.3 kg/m2) to assess possible enrichment of non-synonymous variants in the patients with severe insulin resistance relative to insulin-sensitive controls. In the controls (Table 1), we detected four non-synonymous heterozygous variants, one of which (c.3983A>G, p.H1328R) was present in two controls (control 1 and 4), as well as in proband SIR8. These findings suggest that there is no significant difference in the frequency of non-synonymous variants in the IRS2 gene between the two groups studied, with four non-synonymous variants being found in each group of Europids (4/114 patients vs 4/173 controls, p = 0.54).

Our study has revealed several new non-synonymous variants in the IRS2 gene in both a cohort of predominately Europid individuals with severe insulin resistance and in insulin-sensitive Europid volunteers. Wherever possible, we have performed co-segregation studies on the available family members of severely insulin-resistant patients, but we have found no clear evidence that any of the non-synonymous variants studied has a fully penetrant pathogenic effect. Given the difficulty involved in performing detailed functional evaluation of each variant, we thought to describe these results in the hope that if others have similar data, aggregate data may provide the impetus required to carry out these additional analyses. However, we cannot rule out the possibility that some of these variants contribute to the phenotype of these patients in combination with environmental or other, unknown, genetic variants. Sequencing of IRS2 in insulin-sensitive controls demonstrated that there was no enrichment in the number of non-synonymous variants among the cases, but because this study only had 4.85% power (at p = 0.05 significance) to detect the modest differences in frequency observed here, much larger studies will be required to elucidate the role of rare IRS2 variants in disease. To obtain nominal levels of significance (p = 0.05), assuming the same proportions of mutation carriers and non-carriers as those we report here, the study would need to include at least tenfold more insulin-sensitive and insulin-resistant participants.

In conclusion, we have identified several novel mutations in the IRS2 gene, which, despite no clear segregation with insulin resistance in this study, merit further investigation in additional cohorts; some variants (e.g. P829S) may yet be shown to have an effect on insulin sensitivity in humans.

Notes

Acknowledgements

This study was supported by grants from the Wellcome Trust (R. K. Semple, Intermediate Clinical Fellowship 080952/Z/06/Z; S. O’Rahilly, Programme Grant 078986/Z/06/Z; I. Barroso, Wellcome Trust grant 077016/Z/05/Z), GlaxoSmithKline (D. B. Savage, GlaxoSmithKline Clinical Fellowship) and the UK National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. The authors would also like to thank A. Thompson and A. Daly from the Wellcome Trust Sanger Institute for technical and informatics support, respectively.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Supplementary material

125_2009_1345_MOESM1_ESM.pdf (126 kb)
ESM Fig. 1 Pedigree diagram demonstrating that the p.S78N variant in IRS2 does not segregate with severe insulin resistance in the relatives (R) of patient (P) SIR1. The numbers preceded by P or R are identification numbers. Circle, female; square, male; black fill, affected; white fill, unaffected (PDF 125 KB)
125_2009_1345_MOESM2_ESM.pdf (129 kb)
ESM Fig. 2 Domains within the Homo sapiens protein IRS2, with the positions of the changes found in patients with severe insulin resistance indicated. PH, pleckstrin homology domain; PTBI, phosphotyrosine-binding domain (IRS1-like) (PDF 129 KB)
125_2009_1345_MOESM3_ESM.pdf (44 kb)
ESM Fig. 3 Pedigree diagram demonstrating that the p.I524V variant in IRS2 does not segregate with severe insulin resistance in the relatives (R) of patient (P) SIR3. The numbers preceded by P or R are identification numbers. Circle, female; square, male; black fill, affected; white fill, unaffected; diagonal line, deceased (PDF 43.9 KB)
125_2009_1345_MOESM4_ESM.pdf (125 kb)
ESM Fig. 4 Pedigree diagram demonstrating that the p.P829S variant in IRS2 does not segregate with severe insulin resistance in the relatives (R) of patient (P) SIR4. The numbers preceded by P or R are identification numbers. Circle, female; square, male; black fill, affected; white fill, unaffected (PDF 125 KB)
125_2009_1345_MOESM5_ESM.pdf (187 kb)
ESM Fig. 5 ClustalW2 (www.ebi.ac.uk/Tools/clustalw2/, accessed 8 September 2008) multiple sequence alignment of IRS2 orthologues showing the evolutionary conservation of the amino acid residues, with the alternative residues found in SIR patients indicated (PDF 186 KB)
125_2009_1345_MOESM6_ESM.pdf (100 kb)
ESM Fig. 6 Pedigree diagram demonstrating that the p.S945F variant in IRS2 does not segregate with severe insulin resistance in the relatives (R) of patient (P) SIR6. The numbers preceded by P or R are identification numbers. Circle, female; square, male; black fill, affected; white fill, unaffected (PDF 99.8 KB)
125_2009_1345_MOESM7_ESM.pdf (94 kb)
ESM Fig. 7 Pedigree diagram demonstrating that the p.H1328R variant in IRS2 does not segregate with severe insulin resistance in the family of patient SIR8. The numbers preceded by P or R are identification numbers. Circle, female; square, male; black fill, affected; white fill, unaffected (PDF 93.9 KB)
125_2009_1345_MOESM8_ESM.pdf (10 kb)
ESM Table 1 Biochemical and clinical data for SIR3 and the studied relatives of this patient (PDF 10.2 KB)

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Copyright information

© The Author(s) 2009

Authors and Affiliations

  • W. E. Bottomley
    • 1
  • M. A. Soos
    • 2
  • C. Adams
    • 2
  • T. Guran
    • 3
  • T. A. Howlett
    • 4
  • A. Mackie
    • 5
  • J. Miell
    • 6
  • J. P. Monson
    • 7
  • R. Temple
    • 8
  • Y. Tenenbaum-Rakover
    • 9
  • J. Tymms
    • 10
  • D. B. Savage
    • 2
  • R. K. Semple
    • 2
  • S. O’Rahilly
    • 2
  • I. Barroso
    • 1
  1. 1.Wellcome Trust Sanger InstituteCambridgeUK
  2. 2.University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Treatment Centre, Addenbrooke’s HospitalCambridgeUK
  3. 3.Department of Pediatric Endocrinology and DiabetesMarmara UniversityIstanbulTurkey
  4. 4.Department of Diabetes and EndocrinologyLeicester Royal InfirmaryLeicesterUK
  5. 5.Ninewells HospitalDundeeUK
  6. 6.Department of EndocrinologyUniversity Hospital LewishamLondonUK
  7. 7.Centre for Endocrinology, William Harvey Research Institute, St Bartholomew’s and The Royal London Hospitals, QMULLondonUK
  8. 8.Elsie Bertram Diabetes Centre, Norfolk and Norwich University Hospital NHS TrustNorwichUK
  9. 9.Pediatric Endocrine UnitHa’Emek Medical CenterAfulaIsrael
  10. 10.Diabetes Centre, Royal Albert Edward InfirmaryWiganUK

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