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. 2023 Jan 26;18(1):e0280872. doi: 10.1371/journal.pone.0280872

Low risk for diabetic complications in type 1 diabetes patients carrying a protective insulin gene variant

René van Tienhoven 1, Anh Nguyet Vu 1, John S Kaddis 1, Bart O Roep 2,*
Editor: Matthias G von Herrath3
PMCID: PMC9879388  PMID: 36701305

Abstract

Type 1 diabetes patients carrying a ‘protective’ insulin gene (INS) variant present a disease endotype with reduced insulin antibody titers, preserved beta cell function and improved glycemic control. We tested whether this protective INS variant associated with lowered risk for development of proliferative diabetic retinopathy (PDR) and diabetic kidney disease (DKD) as long-term diabetic complications. Insulin gene polymorphisms were evaluated in 1,363 type 1 diabetes patients participating in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study that compared intensive versus conventional insulin therapy in relation with development of PDR and DKD with a follow-up of over two decades. PDR and DKD were absent in type 1 diabetes patients carrying the protective INS variant and receiving intensive insulin therapy (the current standard of clinical care) 1–5 years from their diagnosis (n = 67; mean post-diagnosis follow up of 20.4 ± 1.6 years), versus 11 of 258 patients (4.3%) lacking this variant (20.4 ± 1.8 years follow up). In the secondary intervention group of the intensive therapy arm (1–15 years of disease), PDR was significantly less frequent in carriers of the protective INS variant than those without it (4 of 83 [4.8%] vs. 31 of 260 [11.9%]; p = 0.032; 26.1 ± 3.9 and 26.3 ± 4.1 years follow-up, respectively), whereas DKD frequencies were no different between those with or without this variant (5 of 83 [6.0%] vs. 11 of 260 [4.2%]). Carrying a copy of this protective INS variant further reduces the risk of diabetic complications achieved by intensive insulin therapy and marks a disease endotype with superior glycemic control, increased and extended beta cell function, and prevention of DKD and PDR.

Introduction

There is a growing insight that type 1 diabetes patients and their disease differ [1], even despite receiving the current standard of care to achieve glycemic control. Therefore, a great, and largely unmet, need exists to identity markers and correlates of type 1 diabetes patient subpopulations with differential disease progression, preservation in beta cell function, and glycemic control. This is essential for the design and allocation of personalized and precision medicine strategies, including future immune intervention therapy and the prognosis of disease progression [2].

Gene variation may be used to serve such a purpose, as genetic risk scores have been developed underscoring this notion [3]. Genetic variation in the insulin gene (INS) is the second largest contributor to genetic risk to type 1 diabetes (T1D) [4]. Differences have been identified in a variable number of tandem repeats (VNTR) in the INS promotor region from which the highest risk is associated with class I VNTR alleles, whereas class III VNTRs have been linked to a dominantly protective effect. These INS polymorphisms are believed to influence the expression of proinsulin, where increased thymic expression and decreased pancreatic expression associate with the protective class III VNTR haplotype; this contributes to improved central immune tolerance to proinsulin and protection from type 1 diabetes [5, 6]. This INS promoter polymorphism is in strong linkage disequilibrium with single nucleotide polymorphisms (SNPs) at the 3’ untranslated region (UTR) of the insulin gene [4], allowing to determine the INS-related risk by using tagging SNPs for the promoter polymorphisms and vice versa [7–9].

Yet, INS associated protection is not complete and around 20% of type 1 diabetes patients carries a copy of the ‘protective’ INS variant. Strikingly, pediatric patients developing type 1 diabetes, despite carrying this protective INS variant and receiving the current standard of clinical care, showed reduced insulin autoimmunity, preserved beta cell function, higher C-peptide levels, and improved glycemic control [9]. This points to the possibility that genetic variation may contribute to diagnosis of disease variants and patient stratification.

It has been well established that intensive insulin treatment compared to conventional therapy can prevent and delay diabetes complications [10]. We hypothesized that type 1 diabetes patients receiving this current standard of care and carrying a copy of the protective INS variant have a further reduced risk of developing diabetes complications compared to patients only carrying the susceptible INS variant.

Materials and methods

The association between INS variation and the probability of microvascular diabetes complications, proliferative diabetic retinopathy (PDR) and diabetic kidney disease (DKD) was investigated in participants of the Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study [10]. Following project approval, DCCT/EDIC clinical and SNP study data on 1,441 individuals was downloaded from the National Center for Biotechnology Information database of Genotypes and Phenotypes website, under accession number phs000086.v3.p1. The DCCT/EDIC trial contained two treatment arms (intensive insulin therapy vs conventional therapy), and two cohorts (primary prevention and secondary intervention) within each arm (four groups in total). The association between INS variation and complication probability was analyzed for each group separately. The patients in the primary prevention cohort had T1D for 1–5 years without evidence of retinopathy and albumin secretion rates of <40mg/24h. The patients in the secondary intervention cohort had T1D for 1–15 years and at least one microaneurysm in either eye and albumin secretion rates no greater than 200mg/24h. PDR was defined as any individual with an Early Treatment Diabetic Retinopathy Study score of 12 or more or receiving scatter laser treatment. DKD was defined as any individual with an estimated glomerular filtration rate less than 60 mL/min/1.73m2 on two or more consecutive visits, or an albumin excretion rate greater than 300 mg/24h on one visit. The INS tagging SNP was determined by genotyping rs3842752 (also known as +1127PstI) in the 3’ UTR of INS; the A allele represents the protective and G the susceptible variant. SNP data was available for 1,363 individuals. Since our a priori predictions were directional (i.e., protection reduces risk), statistical significance was determined by a one-sided log rank test with a significance level of p<0.05. Further details of the study cohort are provided in Table 1.

Table 1. DCCT/EDIC patient characteristics by insulin gene variant.

Characteristic DCCT/EDIC Patients by Insulin Variant
Protective Susceptible
n Value* n Value*
Age at T1D Onset (years)† 275 20.8 (±8.0) 1088 21.3 (±8.1)
Sex  Female 133 48% 509 47%
 Male 142 52% 579 53%
Race  White 266 97% 1047 96.4%
 Black 7 2% 22 2%
 Hispanic 2 1% 12 1%
 Asian/Pacific Islander 0 0% 6 0.5%
 American Indian/Alaska Native 0 0% 1 0.1%
BMI (kg/m2)  DCCT Start 275 23.2 (±2.7) 1088 23.5 (±2.8)
 DCCT End (Y9) 253 25.4 (±3.4) 1003 25.8 (±3.8)
 EDIC Start 254 25.6 (±3.5) 1002 25.8 (±3.7)
 EDIC Last Available (Y8) 242 27.6 (±4.1) 971 27.5 (±4.5)
HbA1c (%)†† Intensive therapy arm
 DCCT Start 150 8.9 (±1.7) 518 8.9 (±1.5)
 DCCT End (Y9) 149 7.5 (±1.1) 515 7.4 (±1.1)
Conventional treatment arm
 DCCT Start 125 8.9 (±1.8) 570 8.9 (±1.6)
 DCCT End (Y9) 124 9.2 (±1.6) 569 9.1 (±1.5)
Post disease diagnosis follow up period (years) Intensive arm
 Primary prevention group 67 20.4 (±1.6) 258 20.4 (±1.8)
 Secondary intervention group 83 26.1 (±3.9) 260 26.3 (±4.1)
Conventional treatment arm
 Primary prevention group 66 20.2 (±2.0) 292 19.8 (±2.7)
 Secondary intervention group 59 25.6 (±4.6) 278 25.1 (±4.5)
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* Mean ± SD reported for continuous variables; % reported for categorical variables

† Calculated as age minus T1D duration at baseline of DCCT

†† Listed in order from DCCT start to EDIC last available, mmol/mol values for those with the protective insulin variant are: A) 73.8, B) 67.2, C) 67.2, and D) 63.9. For those with the susceptible insulin variant, values were identical, i.e.: E) 73.8, F) 67.2, G) 67.2, and H) 63.9. Percentages were converted to mmol/mol using a DCCT to International Federation of Clinical Chemistry (IFCC) formula described elsewhere [11]. Reporting of standard deviation in mmol/mol is not possible because the formula only works for numbers between 4% and 13%.

Results

We report that patients carrying protective INS have a further reduced risk of developing diabetes complications compared to those lacking this gene variant. In the primary prevention group of the DCCT/EDIC study, none out of 67 intensively treated patients carrying the protective INS variant (homo- or heterozygous) developed PDR or DKD with follow-up of 20.4 ± 1.6 years after diagnosis. However, 11 out of 258 patients (4.3%) lacking the protective INS variant developed complications (PDR only n = 5, DKD only n = 3, or both PDR and DKD n = 3) in 20.4 ± 1.8 years of follow-up after diagnosis (Fig 1). In the secondary intervention group, intensively treated patients with the protective INS variant showed significantly less PDR (4 of 83 [4.8%]; 26.1 ± 3.9 years post-diagnosis follow up) compared to patients with only the susceptible INS variant (31 of 260 [11.9%]; 26.3 ± 4.1 years post-diagnosis follow up; p = 0.032). However, there were no differences in the DKD frequencies between those with (5 of 83 [6.0%]) and without (11 of 260 [4.2%]) the protective INS variant (Fig 2). None of the seven patients homozygous for the protective INS variant developed DKD 22 ± 1 years after diagnosis. In 695 conventionally treated patients, complication frequencies did not differ between those with or without the protective INS variant (primary prevention group: 3 of 66 developed PDR (5%) vs. 19 of 292 (7%), respectively, and 4 of 66 developed DKD (6%) vs. 14 of 292 (5%), respectively; secondary intervention group: 13 of 59 developed PDR (22%) vs. 72 of 278 (26%), respectively, and 11 of 59 developed DKD (19%) vs. 34 of 278 (12%), respectively. Interestingly, none of the eight patients homozygous for the protective INS variant developed DKD up to 25.2 ± 4.9 years of post-diagnosis follow up.

Fig 1. Association between INS variation and complication probability during intensive insulin treatment.

Fig 1

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The complication free probability of PDR (left) and DKD (right) is shown for type 1 diabetes patients carrying the protective (blue) and susceptible (red) INS variant in the primary prevention group of the DCCT/EDIC intensively treated study arm. None of the individuals carrying the protective INS variant developed PDR or DKD (0 of 67 and 0 of 67, respectively); however, PDR and DKD occurred in those carrying the susceptible INS variant (8 of 258 and 6 of 258, respectively). Homozygous and heterozygous protective patients were combined due to low numbers of protective homozygotes.

Fig 2. Association between INS variation and complication probability during secondary intervention.

Fig 2

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The complication free probability of PDR (left) and DKD (right) is shown for type 1 diabetes patients carrying the protective (blue) and susceptible (red) INS variant in the secondary intervention group of the DCCT/EDIC intensively treated study arm. Significantly fewer individuals carrying the protective INS variant developed PDR (4 of 83) compared to those with the susceptible INS variant only (31 of 260). The probability for developing DKD was not different between individuals carrying the protective INS variant (5 of 83) compared to those with the susceptible INS variant only (11 of 260). Homozygous and heterozygous protective patients were combined due to low numbers of protective homozygotes. Statistical significance was determined using a one-sided log rank test and indicated if p<0.05.

Discussion

We conclude that the protective INS variant further decreases the low risk for diabetes complications accomplished through intensive insulin treatment and glycemic control in patients with European ancestry. We propose that the combination of immune tolerance to insulin [5, 6] and preserved beta cell function [9] in patients carrying the protective INS variant contributes to superior glycemic control previously reported in carriers with recent onset pediatric T1D, thereby reducing the risk of development of diabetic complications. No complications were reported for patients carrying a protective INS variant in the primary prevention group. The lack of difference in complications frequencies between INS variants in the ‘conventionally’ treated group underscores that intensive treatment, which is the current standard of care, is necessary to reduce complications. The relationship between glycemic control and the association between INS variation and diabetic complication remains unclear. In the DCCT cohort, HbA1c levels were much better in the intensive treatment arm compared to the conventional treatment arm but did not differ between patients with and without the protective INS variant within the intensive treatment arm. Yet, in the Hvidøre cohort an association between superior glycemic control and the protective INS variant was observed in children in the first year after their T1D diagnosis [9]. We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset (‘metabolic memory’) and may change with improved glycemic control later in disease progression [12]. Future and independent validation of our findings in other large cohorts therefore remains warranted.

We concede that the proportion of type 1 diabetes patients receiving the current standard of care (‘intensive insulin therapy’ in DCCT nomenclature) and nonetheless developing diabetic complications is thankfully relatively small. Yet, we argue that ‘small numbers’ are inherent to diagnosis of type 1 diabetes disease heterogeneity and personalized medicine, and relevant to that minor subset of type 1 diabetes patients involved. Although the DCCT/EDIC cohort is the largest T1D complications cohort with the longest follow-up time, a limitation of this study is the relatively small sample size. Alternative T1D cohorts were considered to replicate our results but these cohorts had even smaller numbers of study subjects, diabetic complications were not (yet) recorded or defined differently or the relevant SNP typing was missing. Fortunately, the Hvidøre pediatric cohort showed proof in the same direction that carrying a copy of the protective INS variant has favorable outcome: in their case on glycemic control and preserved beta cell function [9], both known to significantly reduce or even prevent diabetic complications, and thus indirectly yet independently supporting our observations in the DCCT cohort.

Our new data add to the favorable clinical prognosis in those carrying a copy of the ‘protective’ INS variant thereby linking this ‘endotype’ with superior glycemic control and reduced risk for diabetic complication. This finding may contribute to better selection and stratification of type 1 diabetes patients participating in immune intervention trials, and ultimately personalizing medicine.

Acknowledgments

The authors thank Z. Chen and R. Natarajan for their advice in defining PDR and DKD.

Data Availability

The Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications Study dataset is available at the National Center for Biotechnology Information database of Genotypes and Phenotypes, under accession number phs000086.v3.p1.

Funding Statement

This study was supported by the Wanek Family Project for Type 1 Diabetes (Director: BOR).

References

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Decision Letter 0

Matthias G von Herrath

10 Nov 2022

PONE-D-22-27736Low risk for diabetic complications in type 1 diabetes patients carrying a protective insulin gene variantPLOS ONE

Dear Dr. Roep,

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Additional Editor Comments:

Dear investigators, Both reviewers raised foremost the point that the cohort size you are studying does not fully support your conclusions. this needs to be addressed, either by toning things down substantially or by expanding the studies with a validation cohort. Please let us know, what you decide to do. matthias von Herrath, MD

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Reviewer #1: Roep et al. state that they hypothesize that ‘type 1 diabetes patients receiving the current standard of care (intensive insulin therapy) and carrying a copy of the protective INS variant have a further reduced risk of developing diabetes complications compared to patients only carrying the susceptible INS variant’. However, the results are more focused on having the INS variant or not rather than intensive treatment with the variant vs. non-intensive therapy with the protective variant. This is a bit confusing.

The authors should also provided a brief summary of what is meant by primary & secondary prevention from DCCT/EDIC as not all readers may be familiar with the differences, including the differences in the mean time from diagnosis & the presence of mild/moderate retinopathy at baseline in the ‘secondary prevention’ cohort.

The numbers used in this current analyses are quite small for making strong interpretations of associations—will the authors further evaluate this in a separate cohort to assess the impact of the protective variant on progression to complications (or worsening of complications)? I appreciate the challenges with the expected sample limitations when discussing precision approaches to treating T1D or associated complications but the authors should provide more discussion regarding the limitations and how might the results be strengthened.

Reviewer #2: The authors Tienhoven and co-workers evaluated insulin gene polymorphism in 1,363 T1D patients. They then determined if there was an association between the protective INS variant and diabetic complications viz., proliferative diabetic retinopathy (PDR) and diabetic kidney disease (DKD). The authors concluded that the protective INS gene variant imparted some degree of protection against PDR but not DKD in intensively treated patient sub-population. In the conventionally treated arm, there was no difference in the rate of diabetic complication.

Comments:

1. There seems to be discrepancy in the number of patients (carrying the susceptible INS variant, in the primary prevention group) developing PDR or DKD in intensively treated arm i.e., in the text the authors write,”11/258 lacking the protective INS variant developed PDR (n=5), DKD (n=3), or both (n=3)”. However, in the figure legend (of Fig 1), the authors write,”however, PDR and DKD was observed in those carrying the susceptible INS variant (8/258 and 6/258 respectively)”.

2. In the secondary intervention group, intensive treatment in patients with protective INS variant has significantly less incidence of PDR while that of DKD did not change. Intensive insulin therapy in T2D is known to impact retinal vasculature. Is it plausible then that similar phenomenon might be occurring in T1D patients? Besides, is it possible there are other unknown epigenetic factors contributing to the observed effect of PDR.

3. In the conventionally treated patient group there was no difference in the frequency of developing diabetic complications in the primary prevention group regardless of the INS variant. This suggests that intensive treatment regimen is necessary for the suggested “protective” impact of INS variant on PDR.

Overall, while the authors put forth a potentially interesting hypothesis, the study subgroup is under-powered to enable such firm conclusion. It would immensely strengthen this interesting observation/hypothesis if the authors can replicate their findings in another cohort.

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Reviewer #2: No

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PLoS One. 2023 Jan 26;18(1):e0280872. doi: 10.1371/journal.pone.0280872.r002

Author response to Decision Letter 0

15 Dec 2022

Editor Comments:

Dear investigators,

Both reviewers raised foremost the point that the cohort size you are studying does not fully support your conclusions. this needs to be addressed, either by toning things down substantially or by expanding the studies with a validation cohort. Please let us know, what you decide to do. matthias von Herrath, MD

We are grateful for the thoughtful and constructive responses from the Reviewers and Associate Editor. We appreciate the value of validation cohorts and had thoroughly considered our options. Unfortunately, this proved to be challenging for several reasons:

1. The DCCT/EDIC cohort is the largest of its kind with the longest follow-up.

2. Other T1D cohorts do not (yet) feature long term diabetic complications (i.e., T1DGC, TrialNet, TRGIR, FR1DA, INNODIA and TEDDY). One cohort (GENIE) that does carry information on diabetic complications defined DKD differently (persistent proteinuria >05 g/24 hours), did not measure PDR, and did not include any data on the history of T1D treatment (see point 3 that follows). Thus, we were not able to directly compare our findings with data from that cohort.

3. A further complication involved the relationship with glycemic control that we report: it matters whether T1D patients receive intensive insulin therapy and guidance, which happened to be the incentive of the DCCT cohort.

As we are aware that obtaining corroborating results in other cohorts could strengthen our results and interpretation, we were delighted to note that a sufficiently large pediatric cohort (therefore lacking data on complications) showed proof in the very same direction that carrying a copy of the protective INS variant has favorable outcome (in their case on glycemic control, preserved beta-cell function and lack of insulin autoimmunity) that are known to significantly reduce or even prevent diabetic complication. This independent observation from Nielsen and colleagues gave us indirect support for our observations in DCCT. We added a sentence in our discussion pointing to the desire for future validation (page 9-10, lines 158-165), in appreciation of this notion:

‘Although the DCCT/EDIC cohort is the largest T1D complications cohort with the longest follow-up time, a limitation of this study is the relatively small sample size. Alternative T1D cohorts were considered to replicate our results but these cohorts had even smaller numbers of study subjects, diabetic complications were not (yet) recorded or defined differently, or the relevant SNP typing was missing. Fortunately, the Hvidøre pediatric cohort showed proof in the same direction that carrying a copy of the protective INS variant has favorable outcome: in their case on glycemic control and preserved beta cell function [Nielsen, Diabetologia, 2006], both known to significantly reduce or even prevent diabetic complications, and thus indirectly yet independently supporting our observations in the DCCT cohort.’

We further tone our report down by stating (Discussion, page 9, lines 144-153):

‘The relationship between glycemic control and the association between INS variation and diabetic complication remains unclear. In the DCCT cohort, HbA1c levels were much better in the intensive treatment arm compared to the conventional treatment arm but did not differ between patients with and without the protective INS variant within the intensive treatment arm. Yet, in the Hvidøre cohort an association between superior glycemic control and the protective INS variant was observed in children in the first year after their T1D diagnosis [Nielsen, Diabetologia, 2006]. We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset (‘metabolic memory’) and may change with improved glycemic control later in disease progression [Chen, Nat Metabolism, 2020]. Future and independent validation of our findings in other large cohorts therefore remains warranted.’

 

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Reviewer #1: Partly

Reviewer #2: No

________________________________________

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Roep et al. state that they hypothesize that ‘type 1 diabetes patients receiving the current standard of care (intensive insulin therapy) and carrying a copy of the protective INS variant have a further reduced risk of developing diabetes complications compared to patients only carrying the susceptible INS variant’. However, the results are more focused on having the INS variant or not rather than intensive treatment with the variant vs. non-intensive therapy with the protective variant. This is a bit confusing.

Thank you for this comment. We apologize for this confusion. The DCCT/EDIC trial contains two treatment arms: one where patients received intensive treatment and the other with patients receiving the treatment that was conventional at the time of the trial. We followed this stratification for our analysis and focused on the intensive treatment arm, since this is the standard of clinical care and therefore most relevant to patients nowadays. The INS variants were actually tested in both treatment arms to show that the rate of glycemic control (which by definition is better in the ‘intensive treatment’ arm) matters. In the intensive treatment arm DKD and PDR were completely absent in patients with the protective INS variant in the primary prevention group and PDR was significantly less frequent in patients with the protective INS variant in the secondary intervention group. In the conventional treatment arm, we report no difference in DKD or PDR occurrence, except that no DKD was observed in patients homozygous for the protective INS variant. For this reason, we stated that carrying a protective INS variant only reduces the risk of complications in patients that received the current standard of care (which is intensive insulin treatment). We hope this clears the confusion that our wording had caused and clarified the methods of our manuscript accordingly (page 4, lines 73-79):

‘The DCCT/EDIC trial contained two treatment arms (intensive insulin therapy vs conventional therapy), and two cohorts (primary prevention and secondary intervention) within each arm (four groups in total). The association between INS variation and complication probability was analyzed for each group separately. The patients in the primary prevention cohort had T1D for 1-5 years without evidence of retinopathy and albumin secretion rates of <40mg/24h. The patients in the secondary intervention cohort had T1D for 1-15 years and at least one microaneurysm in either eye and albumin secretion rates no greater than 200mg/24h.’

In addition, to further emphasize the difference between the intensive and conventional treatment arm, we revised the presentation of the HbA1c data in Table 1 to stratify the results by both therapy arms and insulin variant (not just INS variant alone). Please see the revised table below. We also clarified the relationship between glycemic control and the association between INS variation and diabetic complications in the discussion of our revised manuscript (page 9, lines 144-153):

‘The relationship between glycemic control and the association between INS variation and diabetic complication remains unclear. In the DCCT cohort, HbA1c levels were much better in the intensive treatment arm compared to the conventional treatment arm but did not differ between patients with and without the protective INS variant within the intensive treatment arm. Yet, in the Hvidøre cohort an association between superior glycemic control and the protective INS variant was observed in children in the first year after their T1D diagnosis [Nielsen, Diabetologia 2006]. We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset and may change with improved glycemic control later in disease progression [Chen, Nat Metabolism, 2020]. Future and independent validation of our findings in other large cohorts therefore remains warranted.’

 

Characteristic DCCT/EDIC Patients by Insulin Variant

Protective Susceptible

n Value* n Value*

Age at T1D Onset (years)† 275 20.8 (±8.0) 1088 21.3 (±8.1)

Sex

Female

Male

133

142

48%

52%

509

579

47%

53%

Race

White

Black

Hispanic

Asian/Pacific Islander

American Indian/Alaska Native

266

7

2

0

0

97%

2%

1%

0%

0%

1047

22

12

6

1

96.4%

2%

1%

0.5%

0.1%

BMI (kg/m2)

DCCT Start

DCCT End (Y9)

EDIC Start

EDIC Last Available (Y8)

275

253

254

242

23.2 (±2.7)

25.4 (±3.4)

25.6 (±3.5)

27.6 (±4.1)

1088

1003

1002

971

23.5 (±2.8)

25.8 (±3.8)

25.8 (±3.7)

27.5 (±4.5)

HbA1c (%)††

Intensive therapy arm

DCCT Start

DCCT End (Y9)

Conventional treatment arm

DCCT Start

DCCT End (Y9)

150

149

125

124

8.9 (±1.7)

7.5 (±1.1)

8.9 (±1.8)

9.2 (±1.6)

518

515

570

569

8.9 (±1.5)

7.4 (±1.1)

8.9 (±1.6)

9.1 (±1.5)

Post disease diagnosis follow up period (years)

Intensive arm

Primary prevention group

Secondary intervention group

Conventional treatment arm

Primary prevention group

Secondary intervention group

67

83

66

59

20.4 (±1.6)

26.1 (±3.9)

20.2 (±2.0)

25.6 (±4.6)

258

260

292

278

20.4 (±1.8)

26.3 (±4.1)

19.8 (±2.7)

25.1 (±4.5)

 

The authors should also provided a brief summary of what is meant by primary & secondary prevention from DCCT/EDIC as not all readers may be familiar with the differences, including the differences in the mean time from diagnosis & the presence of mild/moderate retinopathy at baseline in the ‘secondary prevention’ cohort.

Thank you for this comment. We added a brief statement regarding the differences between primary prevention and secondary intervention in the methods section of our revised manuscript (page 4, lines 73-79):

‘The DCCT/EDIC trial contained two treatment arms (intensive insulin therapy vs conventional therapy), and two cohorts (primary prevention and secondary intervention) within each arm (four groups in total). The association between INS variation and complication probability was analyzed for each group separately. The patients in the primary prevention cohort had T1D for 1-5 years without evidence of retinopathy and albumin secretion rates of <40mg/24h. The patients in the secondary intervention cohort had T1D for 1-15 years and at least one microaneurysm in either eye and albumin secretion rates no greater than 200mg/24h.’

The numbers used in this current analyses are quite small for making strong interpretations of associations—will the authors further evaluate this in a separate cohort to assess the impact of the protective variant on progression to complications (or worsening of complications)? I appreciate the challenges with the expected sample limitations when discussing precision approaches to treating T1D or associated complications but the authors should provide more discussion regarding the limitations and how might the results be strengthened.

Thank you for this comment. We appreciate the value of validation cohorts and had thoroughly considered our options. Unfortunately, this proved to be challenging for several reasons:

1. The DCCT/EDIC cohort is the largest of its kind with the longest follow-up.

2. Other T1D cohorts do not (yet) feature long term diabetic complications (i.e., T1DGC, TrialNet, TRGIR, FR1DA, INNODIA and TEDDY). One cohort (GENIE) that does carry information on diabetic complications defined DKD differently (persistent proteinuria >05 g/24 hours), did not measure PDR, and did not include any data on the history of T1D treatment (see point 3 that follows). Thus, we were not able to directly compare our findings with data from that cohort.

3. A further complication involved the relationship with glycemic control that we report: it matters whether T1D patients receive intensive insulin therapy and guidance, which happened to be the incentive of the DCCT cohort.

As we are equally aware that finding corroborating results in other cohorts could strengthen our results and interpretation, we were delighted to note that a sufficiently large pediatric cohort (therefore lacking data on complications) showed proof in the very same direction that carrying a copy of the protective INS variant has favorable outcome (in their case on glycemic control, preserved beta-cell function and lack of insulin autoimmunity) that are known to significantly reduce or even prevent diabetic complication. This independent observation from Nielsen and colleagues gave us indirect support for our observations in DCCT. We added a sentence in our discussion pointing to the desire for future validation (page 9-10, lines 158-165), in appreciation of this notion:

‘Although the DCCT/EDIC cohort is the largest T1D complications cohort with the longest follow-up time, a limitation of this study is the relatively small sample size. Alternative T1D cohorts were considered to replicate our results but these cohorts had even smaller numbers of study subjects, diabetic complications were not (yet) recorded or defined differently or the relevant SNP typing was missing. Fortunately, the Hvidøre pediatric cohort showed proof in the same direction that carrying a copy of the protective INS variant has favorable outcome: in their case on glycemic control and preserved beta cell function [Nielsen, Diabetologia, 2006], both known to significantly reduce or even prevent diabetic complications, and thus indirectly yet independently supporting our observations in the DCCT cohort.’

We further tone our report down by stating (Discussion, page 9, lines 144-153):

‘The relationship between glycemic control and the association between INS variation and diabetic complication remains unclear. In the DCCT cohort, HbA1c levels were much better in the intensive treatment arm compared to the conventional treatment arm but did not differ between patients with and without the protective INS variant within the intensive treatment arm. Yet, in the Hvidøre cohort an association between superior glycemic control and the protective INS variant was observed in children in the first year after their T1D diagnosis [Nielsen, Diabetologia, 2006]. We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset (‘metabolic memory’) and may change with improved glycemic control later in disease progression [Chen, Nat Metabolism, 2020]. Future and independent validation of our findings in other large cohorts therefore remains warranted.’

 

Reviewer #2: The authors Tienhoven and co-workers evaluated insulin gene polymorphism in 1,363 T1D patients. They then determined if there was an association between the protective INS variant and diabetic complications viz., proliferative diabetic retinopathy (PDR) and diabetic kidney disease (DKD). The authors concluded that the protective INS gene variant imparted some degree of protection against PDR but not DKD in intensively treated patient sub-population. In the conventionally treated arm, there was no difference in the rate of diabetic complication.

Comments:

1. There seems to be discrepancy in the number of patients (carrying the susceptible INS variant, in the primary prevention group) developing PDR or DKD in intensively treated arm i.e., in the text the authors write,”11/258 lacking the protective INS variant developed PDR (n=5), DKD (n=3), or both (n=3)”. However, in the figure legend (of Fig 1), the authors write,”however, PDR and DKD was observed in those carrying the susceptible INS variant (8/258 and 6/258 respectively)”.

Thank you for this comment. We appreciate that the reporting of these numbers was confusing, though correct. In the primary prevention group 11 patients developed complications, of which some developed both: 5 patients developed PDR only, 3 patients developed DKD only, and 3 patients developed both PDR and DKD. Therefore, the count of PDR occurrences was (5 alone + 3 both=)8 and the count of DKD occurrences was (3 alone + 3 both=)6. This has now been specified in our revised manuscript (page 7, lines 102-104):

‘However, 11 out of 258 patients (4.3%) lacking the protective INS variant developed complications (PDR only n=5, DKD only n=3, or both PDR and DKD n=3) in 20.4 ± 1.8 years of follow-up after diagnosis (Fig 1).’

and (page 8, lines 121-122):

‘however, PDR and DKD occurred in those carrying the susceptible INS variant (8 of 258 and 6 of 258, respectively).’

2. In the secondary intervention group, intensive treatment in patients with protective INS variant has significantly less incidence of PDR while that of DKD did not change. Intensive insulin therapy in T2D is known to impact retinal vasculature. Is it plausible then that similar phenomenon might be occurring in T1D patients? Besides, is it possible there are other unknown epigenetic factors contributing to the observed effect of PDR.

Thank you for raising these interesting thoughts. It is certainly plausible that similar features may apply in T2D. We assume that T2D patients carrying the INS variant protecting from T1D have better glycemic control than those carrying the susceptible INS variant, but this has not yet been investigated. While beyond the scope of our current report, epigenetic factors indeed contribute to (reduced) risk for PDR, as we recently reported that DNA methylation mediated development of HbA1c-associated complications in type 1 diabetes in the DCCT cohort (Nature Metab, 2020). It is not yet known whether these involve the INS region. We added a sentence speculating on this in the Discussion (page 9, lines 149-152):

‘We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset and may change with improved glycemic control later in disease progression [ref Chen, Nat Metabolism, 2020].’

3. In the conventionally treated patient group there was no difference in the frequency of developing diabetic complications in the primary prevention group regardless of the INS variant. This suggests that intensive treatment regimen is necessary for the suggested “protective” impact of INS variant on PDR.

Overall, while the authors put forth a potentially interesting hypothesis, the study subgroup is under-powered to enable such firm conclusion. It would immensely strengthen this interesting observation/hypothesis if the authors can replicate their findings in another cohort.

Thank you for this comment. We appreciate the value of validation cohorts and had thoroughly considered our options. Unfortunately, this proved to be challenging for several reasons:

1. The DCCT/EDIC cohort is the largest of its kind with the longest follow-up.

2. Other T1D cohorts do not (yet) feature long term diabetic complications (i.e., T1DGC, TrialNet, TRGIR, FR1DA, INNODIA and TEDDY). One cohort (GENIE) that does carry information on diabetic complications defined DKD differently (persistent proteinuria >05 g/24 hours), did not measure PDR, and did not include any data on the history of T1D treatment (see point 3 that follows). Thus, we were not able to directly compare our findings with data from that cohort.

3. A further complication involved the relationship with glycemic control that we report: it matters whether T1D patients receive intensive insulin therapy and guidance, which happened to be the incentive of the DCCT cohort.

As we are equally aware that finding corroborating results in other cohorts could strengthen our results and interpretation, we were delighted to note that a sufficiently large pediatric cohort (therefore lacking data on complications) showed proof in the very same direction that carrying a copy of the protective INS variant has favorable outcome (in their case on glycemic control, preserved beta-cell function and lack of insulin autoimmunity) that are known to significantly reduce or even prevent diabetic complication. This independent observation from Nielsen and colleagues gave us indirect support for our observations in DCCT. We added a sentence in our discussion pointing to the desire for future validation (page 9-10, lines 158-165), in appreciation of this notion:

‘Although the DCCT/EDIC cohort is the largest T1D complications cohort with the longest follow-up time, a limitation of this study is the relatively small sample size. Alternative T1D cohorts were considered to replicate our results but these cohorts had even smaller numbers of study subjects, diabetic complications were not (yet) recorded or defined differently or the relevant SNP typing was missing. Fortunately, the Hvidøre pediatric cohort showed proof in the same direction that carrying a copy of the protective INS variant has favorable outcome: in their case on glycemic control and preserved beta cell function [Nielsen, Diabetologia, 2006], both known to significantly reduce or even prevent diabetic complications, and thus indirectly yet independently supporting our observations in the DCCT cohort.’

We further tone our report down by stating (Discussion, page 9, lines 144-153):

‘The relationship between glycemic control and the association between INS variation and diabetic complication remains unclear. In the DCCT cohort, HbA1c levels were much better in the intensive treatment arm compared to the conventional treatment arm but did not differ between patients with and without the protective INS variant within the intensive treatment arm. Yet, in the Hvidøre cohort an association between superior glycemic control and the protective INS variant was observed in children in the first year after their T1D diagnosis [Nielsen, Diabetologia, 2006]. We speculate that the impact of the protective INS variant on complication probability involves epigenetic modifications early after disease onset (‘metabolic memory’) and may change with improved glycemic control later in disease progression [Chen, Nat Metabolism, 2020]. Future and independent validation of our findings in other large cohorts therefore remains warranted.’

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Decision Letter 1

Matthias G von Herrath

11 Jan 2023

Low risk for diabetic complications in type 1 diabetes patients carrying a protective insulin gene variant

PONE-D-22-27736R1

Dear Dr. Roep,

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Acceptance letter

Matthias G von Herrath

16 Jan 2023

PONE-D-22-27736R1

Low risk for diabetic complications in type 1 diabetes patients carrying a protective insulin gene variant

Dear Dr. Roep:

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    Data Availability Statement

    The Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications Study dataset is available at the National Center for Biotechnology Information database of Genotypes and Phenotypes, under accession number phs000086.v3.p1.

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