http://auburn.edu/academic/agriculture/fisheries/genomics/FISH7660%202006/DNA%20marker%20review%20paper-Aquaculture.pdf

DNA marker technologies and their applications in aquaculture genetics.

Background Wheat, rye, and barley proteins induce celiac disease, an autoimmune type of gastrointestinal disorder, in genetically susceptible persons. Because the disease may be underdiagnosed, we estimated the prevalence of the disease and tested the hypothesis that assays for serum autoantibodies can be used to detect untreated celiac disease and that positive findings correlate with specific HLA haplotypes. Methods Serum samples were collected from 3654 students (age range, 7 to 16 years) in 1994 and screened in 2001 for endomysial and tissue transglutaminase antibodies. HLA typing was also performed on stored blood samples. All antibody-positive subjects were asked to undergo small-bowel biopsy in 2001. Results Of the 3654 subjects, 56 (1.5 percent) had positive antibody tests, as determined in 2001. Results of the two antibody tests were highly concordant. As of 1994, none of the subjects had received a clinical diagnosis of celiac disease, but 10 who had positive tests for both antibodies in serum o...

https://www.nejm.org/doi/pdf/10.1056/NEJMoa021687

Prevalence of Celiac disease among children in Finland.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which destruction or damaging of the beta-cells in the islets of Langerhans results in insulin deficiency and hyperglycemia. We only know for sure that autoimmunity is the predominant effector mechanism of T1D, but may not be its primary cause. T1D precipitates in genetically susceptible individuals, very likely as a result of an environmental trigger. Current genetic data point towards the following genes as susceptibility genes: HLA, insulin, PTPN22, IL2Ra, and CTLA4. Epidemiological and other studies suggest a triggering role for enteroviruses, while other microorganisms might provide protection. Efficacious prevention of T1D will require detection of the earliest events in the process. So far, autoantibodies are most widely used as serum biomarker, but T-cell readouts and metabolome studies might strengthen and bring forward diagnosis. Current preventive clinical trials mostly focus on environmental triggers. Therapeutic trials test the efficacy of antigen-specific and antigen-nonspecific immune interventions, but also include restoration of the affected beta-cell mass by islet transplantation, neogenesis and regeneration, and combinations thereof. In this comprehensive review, we explain the genetic, environmental, and immunological data underlying the prevention and intervention strategies to constrain T1D.

/pdf/type-1-diabetes-etiology-immunology-and-therapeutic-55k7uqxdh1.pdf

Type 1 Diabetes: Etiology, Immunology, and Therapeutic Strategies

Type 1 diabetes mellitus (T1DM), also known as autoimmune diabetes, is a chronic disease characterized by insulin deficiency due to pancreatic β-cell loss and leads to hyperglycaemia. Although the age of symptomatic onset is usually during childhood or adolescence, symptoms can sometimes develop much later. Although the aetiology of T1DM is not completely understood, the pathogenesis of the disease is thought to involve T cell-mediated destruction of β-cells. Islet-targeting autoantibodies that target insulin, 65 kDa glutamic acid decarboxylase, insulinoma-associated protein 2 and zinc transporter 8 - all of which are proteins associated with secretory granules in β-cells - are biomarkers of T1DM-associated autoimmunity that are found months to years before symptom onset, and can be used to identify and study individuals who are at risk of developing T1DM. The type of autoantibody that appears first depends on the environmental trigger and on genetic factors. The pathogenesis of T1DM can be divided into three stages depending on the absence or presence of hyperglycaemia and hyperglycaemia-associated symptoms (such as polyuria and thirst). A cure is not available, and patients depend on lifelong insulin injections; novel approaches to insulin treatment, such as insulin pumps, continuous glucose monitoring and hybrid closed-loop systems, are in development. Although intensive glycaemic control has reduced the incidence of microvascular and macrovascular complications, the majority of patients with T1DM are still developing these complications. Major research efforts are needed to achieve early diagnosis, prevent β-cell loss and develop better treatment options to improve the quality of life and prognosis of those affected.

Type 1 Diabetes Mellitus

Several studies have shown that gut bacteria have a role in diabetes in murine models. Specific bacteria have been correlated with the onset of diabetes in a rat model. However, it is unknown whether human intestinal microbes have a role in the development of autoimmunity that often leads to type 1 diabetes (T1D), an autoimmune disorder in which insulin-secreting pancreatic islet cells are destroyed. High-throughput, culture-independent approaches identified bacteria that correlate with the development of T1D-associated autoimmunity in young children who are at high genetic risk for this disorder. The level of bacterial diversity diminishes overtime in these autoimmune subjects relative to that of age-matched, genotype-matched, nonautoimmune individuals. A single species, Bacteroides ovatus, comprised nearly 24% of the total increase in the phylum Bacteroidetes in cases compared with controls. Conversely, another species in controls, represented by the human firmicute strain CO19, represented nearly 20% of the increase in Firmicutes compared with cases overtime. Three lines of evidence are presented that support the notion that, as healthy infants approach the toddler stage, their microbiomes become healthier and more stable, whereas, children who are destined for autoimmunity develop a microbiome that is less diverse and stable. Hence, the autoimmune microbiome for T1D may be distinctly different from that found in healthy children. These data also suggest bacterial markers for the early diagnosis of T1D. In addition, bacteria that negatively correlated with the autoimmune state may prove to be useful in the prevention of autoimmunity development in high-risk children.

/pdf/toward-defining-the-autoimmune-microbiome-for-type-1-3zf6zprguh.pdf

Toward defining the autoimmune microbiome for type 1 diabetes

Aims/hypothesis. Population-wide genetic screening of susceptibility to multifactorial diseases will become relevant as knowledge of the pathogenesis of these diseases increases and preventive interventions are identified. Methods. Feasibility and acceptance of neonatal genetic screening for Type I (insulin-dependent) diabetes mellitus susceptibility and adherence of the at-risk children to frequent autoantibody follow-up were studied. Screening was offered to all families. The infants with HLA-DQB1 genotypes *02/*0302 and *0302/x (x?*02, *0301, *0602) were invited to autoantibody follow-up. The children who developed signs of β-cell autoimmunity were invited to a separate prevention trial. Results. The parents of 31 526 babies born between November 1994 and April 1999 (94.4 % of those eligible) agreed to genetic screening. We found that 4651 infants (14.8 %) had increased genetic risk (2.5 to 15 times that of the general population) for Type I (insulin-dependent) diabetes mellitus, and 80 % of them joined the autoantibody surveillance. At the age of 1, 2, 3 and 4 years, 74, 69, 68 and 76 % of the at-risk children, respectively, attended the follow-up. A total of 17 of the 22 children (77 %) who were born during the study period and have developed diabetes carry the risk genotypes we currently use for screening. Conclusions/interpretation. Population-based screening of genetic susceptibility for Type I diabetes, linked with a possibility to participate later in a prevention trial, is highly accepted in Finland and identifies about 75 % of those developing diabetes at an early age. Families adhere well to the frequent measurement of signs of β-cell autoimmunity in the children at-risk. [Diabetologia (2001) 44: 290–297]

/pdf/feasibility-of-genetic-and-immunological-prediction-of-type-1h5dk45l8y.pdf

Feasibility of genetic and immunological prediction of Type I diabetes in a population-based birth cohort

We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility ( * 0201, * 0302) alleles and two protective ( * 0301, * 0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1×10 7 , 3×10 8 and 3×10 8 molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

Summary
Aims To improve sensitivity and specificity of the diabetes risk assessment of the population-based genetic screening used in the Finnish Diabetes Prediction and Prevention (DIPP) trial.

Methods One thousand consecutive newborns enrolled in the DIPP were compared with 316 samples from children with Type 1 diabetes mellitus. A modification of the previously described technique based on hybridization of relevant PCR products with five lanthanide-labelled probes detected by time-resolved fluorometry (TRF) was used. A new probe was designed and allowed discrimination between DQB1*0602 and *0603 alleles, in addition to DQB1*02, *0301 or *0302, each of which required specific probes. A new, added screening strategy was developed for individuals carrying low-risk genotypes through specific typing of DQA1*05 and *0201 alleles in DQB1*02 positive, and DRB1 typing for DR4 subtypes in DQB1*0302 positive subjects, with a new specifically designed high-resolution TRF-based DR4 subtyping technique.

Results This two-step screening approach enhanced the sensitivity of the detection of genetic risk for Type 1 diabetes mellitus in this cohort up to 85.4%. In the general population cohort, 24.4% were identified for prospective follow-up, 2.6% of these are expected to develop Type 1 diabetes mellitus before the age of 15 years. Exclusive typing for HLA-DQB1 locus as an alternative screening strategy had sensitivities of 26.3–77.2% with general population cohorts of 2.3–23.1% identified for follow-up.

Conclusions The described strategy for genetic prediction of Type 1 diabetes mellitus relies on the convenient genotyping procedure and could be applied in large scale screening projects such as DIPP.

Population-based genetic screening for the estimation of Type 1 diabetes mellitus risk in Finland: selective genotyping of markers in the HLA-DQB1, HLA-DQA1 and HLA-DRB1 loci.

OBJECTIVE To study the effectiveness of MHC genotyping in the assessment of risk for IDDM based on the identification of alleles that are significantly associated with risk for IDDM (DQB1 *0302 and *0201) and protection from it (DQB1 \*0602/\*0603 and *0301).

RESEARCH DESIGN AND METHODS A long series of 649 index cases of IDDM, together with their healthy siblings and 756 healthy blood donors, was collected in Finland. The samples were analyzed using a large-scale assay procedure that was developed for rapid screening purposes. The method utilizes time-resolved fluorometry to detect the hybridization of lanthanide-labeled allele-specific oligonucleotide probes with amplified gene product.

RESULTS A total of 61.9% of IDDM index cases had high risk (DQB1 \*0201/\*0302) or moderate risk (DQB1 *0302/x [x meaning DQB1 *0302 or a nondefined allele]) genotypes compared with 14.3% of the reference population. In patients and control subjects, the frequencies of low risk genotypes were 28.0 and 22.1%, respectively, and those of decreased risk genotypes, 10.0 and 63.6%. The relative risk of a \*0201/\*0302 genotype was 53.5 (31.1–92.8) compared with the decreased risk genotypes (63.6% of controls). The graded risk estimation was equally efficient in assessing the risk of IDDM in siblings of child with IDDM.

CONCLUSION The near-automatic typing procedure developed is attractive for large-scale screening projects, such as diabetes prevention and intervention trials.

Rapid HLA-DQB1 Genotyping for Four Alleles in the Assessment of Risk for IDDM in the Finnish Population

The most important gene loci defining risk of type 1 diabetes mellitus (T1DM) are located within the HLA gene region. HLA-DQ molecules are of primary importance but HLA-DR gene products modify the risk conferred by HLA-DQ. The risk associated with an HLA genotype is defined by the particular combination of susceptible and protective alleles. The highest risk is associated with a combination of two different risk haplotypes (7% risk to develop T1DM in Finland) whereas protective genotypes covering 69% of population have a risk of less than 0.2%). The complicated analysis of HLA genotypes is simplified by strong linkage disequilibrium between HLA-DRB1, -DQA1 and -DQB1 loci. In many cases one can deduce the alleles of other loci based on determination of the alleles in one locus. Differences between various populations in the frequency of marker alleles and in the linkages between them has to be taken into account. We have developed PCR based typing methods that utilize blood spot samples, microtiter plate format and lanthanide labeled oligonucleotide probes to define HLA-DQ and -DR alleles relevant for T1DM risk. Typing is run stepwise so that after initial HLA-DQB1 typing only those samples will be further analyzed in which -DQA1 or -DRB1 typing is informative and expected to contribute to the risk estimation. This method has been used to screen more than 50,000 newborn infants in Finland over a time period of 6 years, and it has been able to identify most children who have developed T1D during the follow-up period. The efficiency of the procedure has also been tested in Finnish and Greek populations. © 2002 Wiley-Liss, Inc.

/pdf/estimation-of-genetic-risk-for-type-1-diabetes-2wp85a10qv.pdf

Minna Sjöroos

Papers

Feasibility of genetic and immunological prediction of Type I diabetes in a population-based birth cohort

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

Population-based genetic screening for the estimation of Type 1 diabetes mellitus risk in Finland: selective genotyping of markers in the HLA-DQB1, HLA-DQA1 and HLA-DRB1 loci.

Rapid HLA-DQB1 Genotyping for Four Alleles in the Assessment of Risk for IDDM in the Finnish Population

Estimation of genetic risk for type 1 diabetes.