In the present study we did not observe any significant temporal change in HLA class II genotype distributions, including the highest risk DRB1*
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 
. They observed that the highest risk HLA genotype was associated with a lower age at onset, as previously described in our population 
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 
as well as in Australia, where a “predominantly Anglo-Celtic European population” was evaluated, and in United States 
Temporal trends of high risk HLA genotype distribution in type 1 diabetes subjects from different countries.
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 
, while decreased from 25.3% to 18.2%, in the same period of time, in the Finnish population 
. Gillespie et al. 
observed a significant decreasing frequency of the highest risk HLA genotype DR3-DQ2/DR4-DQ8 over decades in children ≤15 years of age, stressing the concept that a major environmental effect could have been accelerated the type 1 diabetes onset, thus diluting the concentration of high risk HLA susceptible genotypes in type 1 diabetes population. Fourlanos et al. showing a significant decreasing trend of the highest risk HLA genotype from 79% in 1950–1969 to 28% in 2000–2005 
, in presence of an increase in type 1 diabetes incidence, concluded that changing environmental conditions have increased the chance to get the disease.
Vehik K et al 
, showed that the frequency of the DRB1*03-DQB1*02/DRB1*04-DQB1*03 was higher (39%) in type 1 diabetes children both from Hispanic and non Hispanic origin diagnosed during the 1978–1988 period than in those diagnosed during 2002–2004 (28%). The Authors concluded that the increasing environmental exposure is now able to trigger type 1 diabetes in subjects who are less genetically susceptible. This result was confirmed by two larger data sets of mainly Caucasian subjects (86.4%) with a minority of Hispanic and African American, still from USA, where there appeared a significant decrease of the highest risk DR3/DR4-DQB1*0302, over time, together with an increased percent of other HLA genotypes without HLA-DR3 or DR4 
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 (Italian and German vs the others) mainly differ for a) type 1 diabetes incidence, b) “ab initio” (early 80′) frequency of the HLA class II genotypes in type 1 diabetes populations, both evidences being related, at least in part.
The main difference between the two groups of populations regards the incidence of the disease, Italian 
and German 
populations being characterized by a moderate/low incidence (approximately 15 new cases per 100,000 per year <15 years) compared to the other countries characterized by a high incidence of the disease (new cases per 100,000 per year <15 years: Finland 64.2, Sweden 39.6, UK 26.4, USA 26.4 and Australia 23.1 
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 
, physical inactivity, excess of food intake or excessive hygiene may contribute to the increase incidence of type 1 diabetes 
. It is conceivable that a decreased microbial load in early life may have a major impact on the programming of the immune system, particularly the gut-associated lymphoid tissue 
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 
. Environmental exposures to dietary antigens and microbes could be implicated in the increasing incidence of type 1 diabetes. However, no single pathogenic environmental agent has been identified that explains all cases 
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
compared to that conferred by HLA DRB1*03-DQB1*0201/DRB1*4-DQB1*0302 in our population OR
. The increased pressure of the environment could have some effects on minor susceptibility genes and still not on the major HLA susceptibility component in our population.
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 
. When the T susceptibility allele of PTPN22 gene is not present the environmental factors could have a predominant role in the type 1 diabetes pathogenesis.
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