Raffaella Buzzetti is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: RB. Performed the experiments: MS SZ. Analyzed the data: GC. Contributed reagents/materials/analysis tools: LM MC. Wrote the paper: RB MS. Recruitment of patients: IMDIAB Study Group RB.
The incidence of type 1 diabetes has, progressively, increased worldwide over the last decades and also in Continental Italian population. Previous studies performed in northern European countries, showed, alongside a general increase in the disease incidence, a decreasing frequency of the highest risk HLA genotype in type 1 diabetes populations, thus emphasizing the role of environmental factors. The aim of the study was to evaluate whether a decreasing trend of high risk HLA, CTLA-4 and PTPN22 genotypes would be present in type 1 diabetes subjects of Continental Italy, a country considered at low incidence of the disease compared to northern European populations. N = 765 type 1 diabetes patients diagnosed from 1980 to 2012 in Lazio region were included. For HLA, CTLA4 and PTPN22 temporal trend evaluation, subjects were subdivided into groups of years according to age at diagnosis. All subjects were typed for HLA-DRB1 and DQB1 by a reverse line blot. The CT60 polymorphism of the CTLA4 and C1858T of the PTPN22 gene were genotyped using ABI PRISM 7900HT (n = 419 and n = 364 respectively). HLA genotypes were divided in high, moderate and low risk categories. The proportion of the HLA risk categories was not statistically different over the three decades in subjects with age of onset <15 years and ≥15 years. The genotype distribution of CT60 polymorphism of CTLA4 gene did not show any change in the frequencies during time. The analysis of the PTPN22 C1858T variant revealed, instead, that the frequency of CT+TT susceptibility genotypes decreased during time (23.9% vs 13.6%, p = 0.017). We can hypothesize that the pressure of the diabetogenic environment could be milder and therefore not sufficient to reduce the need of a strong genetic background (HLA) “to precipitate” diabetes; the increased pressure of the environment could have, instead, some effects on minor susceptibility genes in our population.
The incidence of type 1 diabetes has increased progressively over the last half century
Subjects for this study include n = 765 type 1 diabetes patients diagnosed from 1980 to 2012 recruited by participating centers of the IMDIAB group in Continental Italy (Lazio region). The diagnosis of type 1 diabetes was based on the American Diabetes Association (ADA) classification criteria
Our research involved human participants and has been approved by the “Sapienza” University ethical committee and a written informed consent was obtained from all participating subjects. Written inform consent has been obtained from parents or legal guardian for all the children participating to the study. All clinical investigation have been conducted according to the principles expressed in the Declaration of Helsinki.
All subjects were Caucasians with parents of Italian origin. The age at diagnosis ranged from 1 to 49 years (12.7±8.9). Subjects were sub-divided into two categories: <15 years of age at diagnosis and ≥15 of age at diagnosis. For HLA temporal trend evaluation, all subjects were grouped by three decades of diagnosis: 1980–1989, 1990–1999, 2000–2012; for PTPN22 and CTLA4 they were divided into two groups of years of diagnosis: 1980–1995, 1996–2012 due to the smaller number of subjects evaluated, n = 419 and n = 364 respectively.
The HLA genotypes were classified in three risk categories based on the absolute risk values (AR) previously estimated in Continental Italian population (Lazio region)
Blood samples were collected and were stored at −20°C until used for genomic extraction of DNA. Genomic DNA was extracted using QIAamp DNA Blood Kit (QIAGEN Genomics Inc., Bothell, WA). All subjects were typed for HLA-DRB1 and DQB1 loci by polymerase chain reaction (PCR) followed by a reverse line blot assay using an array of immobilized sequence–specific oligonucleotide probes, as previously described
Comparisons of HLA, PTPN22 and CTLA-4 genotype frequencies between the cohorts over time were assessed by the Chi square or by Fischer test. A probability value of 0.05 or less was considered to be statistically significant. Based on the genotype frequencies of HLA (DRB1 and DQB1), CTLA4 and PTPN22 in Italian type 1 diabetes population, we should be able to identify statistical differences between the groups analysed with a power of 96%, 93% and 90% respectively and a p value <0.05.
The mean age at diagnosis of type 1 diabetes was 12.7±8.9 years (mean ± SD), and did not differ across decades of diagnosis. The distribution of HLA genotype categories in subjects with age of onset <15 and ≥15 years, according to the three groups of decades is shown in
Age of onset <15 years | Year of diagnosis | |||
1980–1989 | 1990–1999 | 2000–2012 | p | |
n = 133 | n = 325 | n = 79 | ||
High risk HLA genotype |
31 (23.4) | 69 (21.2) | 21 (26.6) | ns |
Moderate risk HLA genotypes |
82 (61.6) | 204 (62.8) | 46 (58.2) | ns |
Low risk HLA genotypes |
20 (15) | 52 (16) | 12 (15.2) | ns |
n = 45 | n = 115 | n = 68 | ||
High risk HLA genotype |
4 (8.9) | 15 (13.0) | 9 (13.2) | ns |
Moderate risk HLA genotypes |
30 (66.7) | 68 (59.2) | 46 (67.7) | ns |
Low risk HLA genotypes |
11 (24.4) | 32 (27.8) | 13 (19.1) | ns |
Data are expressed as number (%).
High: DRB1*03-DQB1*0201/DRB1*04-DQB1*0302 genotype (DRB1*04 different from 0403, 06, 11).
Moderate: DRB1*04-DQB1*0302/DRB1*04-DQB1*0302,DRB1*03-DQB1*0201/DRB1*03-DQB1*0201, DRB1*04-DQB1*0302/X, and DRB1*03/X (X different from DRB1*03, DRB1*04-DQB1*0302, DRB1*04 not 0403, 06, 11, or DQB1*0602/03) genotypes.
Low: other genotypes.
CT60 SNP CTLA4 | Year of diagnosis | |
1980–1995 | 1996–2012 | |
n = 219 | n = 200 | |
AA | 46 (21.0) | 46 (23.0) |
AG+GG |
173 (79.0) | 154 (77.0) |
n = 180 | n = 184 | |
CC | 137 (76.1) | 159 (86.4) |
CT+TT |
43 (23.9) | 25 (13.6) |
Data are expressed as number (%).
AG+GG vs AA p = ns.
CT+TT vs CC p = 0.017.
In the present study we did not observe any significant temporal change in HLA class II genotype distributions, including the highest risk DRB1*03-DQB1*0201/DRB1*04-DQB1*0302, over the last decades, both in type 1 diabetes children <15 years and in adult subjects, alongside a general increase in the disease incidence. In Continental Italy (Lazio region) type 1 diabetes incidence doubled in the years 2004–2009 (15.68 new cases per 100,000 per year <15 years of age with a peak in 2004∶17.3 for 100,000) compared to 1989–1993 and to 1990–1999 periods of time (7.9 and 8.8 new cases per 100,000 per year <15 years of age respectively)
According to our results, a recent study showed no changes in HLA genotype frequencies in a large sample of type 1 diabetes subjects with age at onset ≤20 from Germany and Austria
This finding is, apparently, in contrast with a decreasing trend of the highest risk HLA genotype observed, over the last decades, in northern European countries
Authors | Country | High risk HLA genotype | Age at diagnosis | n | ≤1979 | 1980–1989 | 1990–1999 | ≥2000 | p | ||
(years) | % | % | % | % | |||||||
Spoletini M et al. | Italy | DRB1 |
<15 | 537 | 23.4 | 21.2 | 26.6 | ns | |||
Awa WL et al. |
Germany | DRB1 |
≤20 | 1445 | 24.8 | ns | |||||
Hermann R et al. |
Finland | DRB1 |
<15 | 736 | 25.3 | 18.2 | 0.007 | ||||
Lindehammer SA et al. |
Sweden | DQA1 |
<18 | 943 | 35.6 | 36.8 | 19.1 | <0.0001 | |||
Gillespie KM et al. |
UK | DRB1 |
≤15 | 776 | 47 | 35 | 0.003 | ||||
Fourlanos S et al. |
Australia | DRB1 |
<18 | 462 | 79 |
47 | 37 | 28 | <0.0001 | ||
Steck AK et al. |
USA | DRB1 |
≤18 | 4075 | 48 | 35 |
<0.0001 | ||||
Vehik K et al. |
USA | DRB1 |
≤17 | 364 | 39 | 28 | 0.05 |
1995–2006.
1950–1969.
Studies performed in northern European populations demonstrated the same decreasing trend of the highest HLA susceptible genotype in children <18 years, although this frequency decreased from 35.6% in the decade 1980–1989 to 19.1% in 2000- in Swedish population
Vehik K et al
The reason why the highest HLA susceptible genotype tends to be constant in some populations while decreasing in others is not clear.
The two groups of populations
The main difference between the two groups of populations regards the incidence of the disease, Italian
Then, in Italian and German populations, the DRB1*03-DQB1*0201/DRB1*04-DQB1*0302 frequency, in type 1 diabetes population, was around 25% in the 80′ compared to >35% of that reported in the northern European countries at that time, except Finland. This finding reflected the low frequency of DRB1*03-DQB1*0201/DRB1*04-DQB1*0302 in the general populations of Italy and German and could be, at least in part, the reason why the incidence of the disease has been low so far in these countries, assuming that the Odds Ratio (ORs) for every specific genotype tended, at that time, to be constant in all Caucasian populations.
Over the past few decades we assisted at a decreasing frequency of the highest HLA susceptible genotype in the diabetes population of countries with a high incidence of the disease, but not in those with a low incidence. Based on these evidences we can speculate that different environmental factors in various populations could differently influence the penetrance of HLA genes. Thus, populations with a lower incidence could be at an earlier stage of the natural evolution of diabetes history; the pressure of the diabetogenic environment could be milder and still “polarized” to the highest HLA genotype, as not sufficient to reduce the need of a strong genetic background (HLA) “to precipitate” diabetes. A logical consequence is that the HLA class II genotypes ORs appear now to be different in the different populations.
Viral infections
Environmental factors could either modify the penetrance of susceptibility genes, or, as triggering factors, could contribute directly to the incidence. It has been hypothesized that changes in penetrance might be linked to patterns of childhood immunization, but this has yet to be confirmed
Due to the limited sample size of subjects, we sub-divided the genotype distribution of CTLA4 and PTPN22 into two groups: 1980–1995 and 1996–2012 according to the year of diagnosis of type 1 diabetes. As well as for HLA genotypes, also the distribution of CT60 polymorphism of CTLA4 gene did not show any change in the frequencies during time, as previously demonstrated
Conversely, we observed that the frequency of CT+TT susceptibility genotypes of PTPN22 gene decreased during time. For our knowledge this is the first study to analyze the PTPN22 genotype frequency in type 1 diabetes during decades. The risk conferred by this genotype to the disease is quite small (OR = 2.48)
Our study has some limitations, the most important one is due to the small number of subjects who took part to this study in the last decade compared to the previous ones. Nevertheless our data contribute to clarify that the increase in type 1 diabetes over the last decades might be explained by a complex interactions between genes and environmental risk factors which may differ in the different populations. The epidemiology of type 1 diabetes suggests that varying gene–environment interactions are likely triggering and/or accelerating the autoimmune destruction of β-cells leading to complete insulin deficiency
We can hypothesize that also epigenetic regulation could be one way to explain the rapid increase in incidence and could be a central mechanism by which environmental factors can influence the development of type 1 diabetes