A quarter of patients with type 1 diabetes have co-existing non-islet autoimmunity: the findings of a UK population-based family study.

Individuals with type 1 diabetes (T1D) are at increased risk of coeliac disease (CD), autoimmune thyroiditis and autoimmune gastritis, but the absolute risks are unclear. The aim of this study was to investigate the prevalence of autoantibodies to tissue transglutaminase (TGA), thyroid peroxidase (TPOA) and gastric H+ /K+ -ATPase (ATPA) and their genetic associations in a well-characterized population-based cohort of individuals with T1D from the Bart's-Oxford family study for whom islet autoantibody prevalence data were already available. Autoantibodies in sera from 1072 patients (males/females 604/468; median age 11·8 years, median T1D duration 2·7 months) were measured by radioimmunoassays; HLA class II risk genotype was analysed in 973 (91%) using polymerase chain reaction with sequence specific primers (PCR-SSP). The prevalence of TGA (and/or history of CD), TPOA and ATPA in patients was 9·0, 9·6 and 8·2%, respectively; 3·1% had two or more autoantibodies. Females were at higher risk of multiple autoimmunity; TGA/CD were associated with younger age and TPOA with older age. ATPA were uncommon in patients under 5 years, and more common in older patients. Anti-glutamate decarboxylase autoantibodies were predictive of co-existing TPOA/ATPA. TGA/CD were associated with human leucocyte antigen (HLA) DR3-DQ2, with the DR3-DQ2/DR3-DQ2 genotype conferring the highest risk, followed by DR4-DQ8/DR4-DQ8. ATPA were associated with DR3-DQ2, DRB1*0404 (in males) and the DR3-DQ2/DR4-DQ8 genotype. TPOA were associated with the DR3-DQ2/DR3-DQ2 genotype. Almost one-quarter of patients diagnosed with T1D aged under 21 years have at least one other organ-specific autoantibody. HLA class II genetic profiling may be useful in identifying those at risk of multiple autoimmunity.

ATP4A autoimmunity and Helicobacter pylori infection in children with type 1 diabetes.

Persistent presence of ATP4A autoantibodies (ATP4AA) directed towards parietal cells is typical for atrophic body gastritis (ABG), an autoimmune disease associated with type 1 diabetes. We assessed whether Helicobacter pylori (Hp) infection might be associated with positivity for ATP4AA in children with type 1 diabetes. Sera were collected from 70 (38♀) type 1 diabetes children [aged 13·2 ± 4·5 years, age at diagnosis 8·8 ± 4·3 years, diabetes duration 4·5 ± 3·8 years, mean HbA1c 7·8 ± 1·6% (62 ± 17·5 mmol/mol)] seen at the regional diabetes clinic in Katowice, Poland. Patients were tested concurrently for Hp infection by means of a 13C urea breath test. ATP4AA were measured using a novel radioimmunoprecipitation assay developed at the Barbara Davies Center for Childhood Diabetes, University of Colorado. ATP4AA were present in 21 [30%, 95% confidence interval (CI) = 19-41%] and Hp infection was detected in 23 (33%, 95% CI = 22-44%) children. There was no statistically significant association between ATP4AA presence and Hp status. ATP4AA presence was not associated with current age, age at type 1 diabetes diagnosis, diabetes duration or current HbA1c. ATP4AA were more prevalent in females [42% (26-58%)] than males [16% (3-28%)], P = 0·016. ATP4A are found in nearly one-third of children with type 1 diabetes and more common among females. In this cross-sectional analysis, Hp infection was not associated with autoimmunity against parietal cells.

In antibody-positive first-degree relatives of patients with type 1 diabetes, HLA-A*24 and HLA-B*18, but not HLA-B*39, are predictors of impending diabetes with distinct HLA-DQ interactions.

AIMS/HYPOTHESIS: Secondary type 1 diabetes prevention trials require selection of participants with impending diabetes. HLA-A and -B alleles have been reported to promote disease progression. We investigated whether typing for HLA-B*18 and -B*39 may complement screening for HLA-DQ8, -DQ2 and -A*24 and autoantibodies (Abs) against islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8) for predicting rapid progression to hyperglycaemia. METHODS: A registry-based group of 288 persistently autoantibody-positive (Ab(+)) offspring/siblings (aged 0-39 years) of known patients (Ab(+) against insulin, GAD, IA-2 and/or ZnT8) were typed for HLA-DQ, -A and -B and monitored from the first Ab(+) sample for development of diabetes within 5 years. RESULTS: Unlike HLA-B*39, HLA-B*18 was associated with accelerated disease progression, but only in HLA-DQ2 carriers (p < 0.006). In contrast, HLA-A*24 promoted progression preferentially in the presence of HLA-DQ8 (p < 0.002). In HLA-DQ2- and/or HLA-DQ8-positive relatives (n = 246), HLA-B*18 predicted impending diabetes (p = 0.015) in addition to HLA-A*24, HLA-DQ2/DQ8 and positivity for IA-2A or ZnT8A (p ≤ 0.004). HLA-B*18 interacted significantly with HLA-DQ2/DQ8 and HLA-A*24 in the presence of IA-2 and/or ZnT8 autoantibodies (p ≤ 0.009). Additional testing for HLA-B*18 and -A*24 significantly improved screening sensitivity for rapid progressors, from 38% to 53%, among relatives at high Ab-inferred risk carrying at least one genetic risk factor. Screening for HLA-B*18 increased sensitivity for progressors, from 17% to 28%, among individuals carrying ≥ 3 risk markers conferring >85% 5 year risk. CONCLUSIONS/INTERPRETATION: These results reinforce the importance of HLA class I alleles in disease progression and quantify their added value for preparing prevention trials.

Screening for insulinoma antigen 2 and zinc transporter 8 autoantibodies: a cost-effective and age-independent strategy to identify rapid progressors to clinical onset among relatives of type 1 diabetic patients.

In first-degree relatives of type 1 diabetic patients, we investigated whether diabetes risk assessment solely based on insulinoma antigen 2 (IA-2) and zinc transporter 8 (ZnT8) antibody status (IA-2A, respectively, ZnT8A) is as effective as screening for three or four autoantibodies [antibodies against insulin (IAA), glutamate decarboxylase 65 kDa (GAD) glutamate decarboxylase autoantibodies (GADA) and IA-2A with or without ZnT8A] in identifying children, adolescents and adults who progress rapidly to diabetes (within 5 years). Antibodies were determined by radiobinding assays during follow-up of 6444 siblings and offspring aged 0-39 years at inclusion and recruited consecutively by the Belgian Diabetes Registry. We identified 394 persistently IAA(+) , GADA(+) , IA-2A(+) and/or ZnT8A(+) relatives (6·1%). After a median follow-up time of 52 months, 132 relatives developed type 1 diabetes. In each age category tested (0-9, 10-19 and 20-39 years) progression to diabetes was significantly quicker in the presence of IA-2A and/or ZnT8A than in their joint absence (P < 0·001). Progression rate was age-independent in IA-2A(+) and/or ZnT8A(+) relatives but decreased with age if only GADA and/or IAA were present (P = 0·008). In the age group mainly considered for immune interventions until now (10-39 years), screening for IA-2A and ZnT8A alone identified 78% of the rapid progressors (versus 75% if positive for ≥ 2 antibodies among IAA, GADA, IA-2A and ZnT8A or versus 62% without testing for ZnT8A). Screening for IA-2A and ZnT8A alone allows identification of the majority of rapidly progressing prediabetic siblings and offspring regardless of age and is more cost-effective to select participants for intervention trials than conventional screening.

An important minority of prediabetic first-degree relatives of type 1 diabetic patients derives from seroconversion to persistent autoantibody positivity after 10 years of age.

AIMS/HYPOTHESIS: The appearance of autoantibodies (Abs) before diabetes onset has mainly been studied in young children. However, most patients develop type 1 diabetes after the age of 15 years. In first-degree relatives aged under 40 years, we investigated the frequency of seroconversion to (persistent) Ab positivity, progression to diabetes and baseline characteristics of seroconverters according to age. METHODS: Abs against insulin (IAA), glutamate decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A) and zinc transporter 8 (ZnT8A) were measured during follow-up of 7,170 first-degree relatives. RESULTS: We identified 379 (5.3%) relatives with positivity for IAA, GADA, IA-2A and/or ZnT8A (Ab(+)) at first sampling and 224 (3.1%) at a later time point. Most seroconversions occurred after the age of 10 years (63%). During follow-up, Abs persisted more often in relatives initially Ab(+) (76%) than in seroconverters (53%; p < 0.001). In both groups diabetes developed at a similar pace and almost exclusively with Ab persistence (136 of 139 prediabetic individuals). For both groups, progression was more rapid if Abs appeared before the age of 10 years. Baseline characteristics at seroconversion did not vary significantly according to age. CONCLUSIONS/INTERPRETATION: Seroconversion to (persistent) Ab(+) occurs regardless of age. Although the progression rate to diabetes is higher under age 10 years, later seroconverters (up to age 40 years) have similar characteristics when compared with age-matched initially Ab(+) relatives and generate an important minority of prediabetic relatives, warranting their identification and, eventually, enrolment in prevention trials.

Genetic association of zinc transporter 8 (ZnT8) autoantibodies in type 1 diabetes cases.

AIMS/HYPOTHESIS: Autoantibodies to zinc transporter 8 (ZnT8A) are associated with risk of type 1 diabetes. Apart from the SLC30A8 gene itself, little is known about the genetic basis of ZnT8A. We hypothesise that other loci in addition to SLC30A8 are associated with ZnT8A. METHODS: The levels of ZnT8A were measured in 2,239 British type 1 diabetic individuals diagnosed before age 17 years, with a median duration of diabetes of 4 years. Cases were tested at over 775,000 loci genome wide (including 53 type 1 diabetes associated regions) for association with positivity for ZnT8A. ZnT8A were also measured in an independent dataset of 855 family members with type 1 diabetes. RESULTS: Only FCRL3 on chromosome 1q23.1 and the HLA class I region were associated with positivity for ZnT8A. rs7522061T>C was the most associated single nucleotide polymorphism (SNP) in the FCRL3 region (p = 1.13 × 10(-16)). The association was confirmed in the family dataset (p ≤ 9.20 × 10(-4)). rs9258750A>G was the most associated variant in the HLA region (p = 2.06 × 10(-9) and p = 0.0014 in family cases). The presence of ZnT8A was not associated with HLA-DRB1, HLA-DQB1, HLA-A, HLA-B or HLA-C (p > 0.05). Unexpectedly, the two loci associated with the presence of ZnT8A did not alter risk of having type 1 diabetes, and the 53 type 1 diabetes risk loci did not influence positivity for ZnT8A, despite them being disease specific. CONCLUSIONS/INTERPRETATION: ZnT8A are not primary pathogenic factors in type 1 diabetes. Nevertheless, ZnT8A testing in combination with other autoantibodies facilitates disease prediction, despite the biomarker not being under the same genetic control as the disease.

Development of a novel autoantibody assay for autoimmune gastritis in type 1 diabetic individuals.

BACKGROUND: Autoimmune atrophic body gastritis (ABG) and pernicious anaemia are prototypical, organ-specific autoimmune diseases whose prevalence in the general population is 2.0 vs 2 and 0.15-1%, respectively. The incidence of disease increases with age and is frequently associated with other autoimmune disorders such as type 1 diabetes mellitus (T1DM). Early diagnosis of ABG/pernicious anaemia is essential for the prevention and/or treatment before manifestations of chronic disease become irreversible. Parietal cell autoantibody detection via enzyme-linked immunosorbent assay is currently the most widely used biomarker of disease with diagnosis confirmed by subsequent immunohistochemistry via biopsy. METHODS: To improve the assay we designed a specific, molecularly defined radioimmunoprecipitation assay for early detection of ABG, targeting its major antigen, the gastric H+/K+ ATPase 4A subunit ATP4A. RESULTS: The major antigenic domain in ATP4A was tested against a panel of sera from new onset patients with T1DM which tested positive for the gold standard T1DM autoantibodies (IAA, IA2A, GAD65A, and ZnT8A). Significant immunoreactivity to ATP4A was measured (25%) while 6% of first-degree relatives of subjects with T1DM who were sero-negative for T1DM autoantigens were positive for ATP4A autoantibodies. ATP4A antibody prevalence increased with age of onset of T1DM, which is atypical of other T1DM autoantibodies. Immunoreactivity to ATP4A, unlike that of T1DM antigens, demonstrates a significant gender bias in newly diagnosed individuals with T1DM. CONCLUSION: Although the utility of the assay as a biomarker for T1DM is likely limited, it may serve as an improved indicator of ABG.

Predictive power of screening for antibodies against insulinoma-associated protein 2 beta (IA-2beta) and zinc transporter-8 to select first-degree relatives of type 1 diabetic patients with risk of rapid progression to clinical onset of the disease: implications for prevention trials.

AIMS/HYPOTHESIS: We investigated whether screening for insulinoma-associated protein (IA-2) beta (IA-2beta) autoantibodies (IA-2betaA) and zinc transporter-8 (ZnT8) autoantibodies (ZnT8A) improves identification of first-degree relatives of type 1 diabetic patients with a high 5-year disease risk, which to date has been based on assays for insulin autoantibodies (IAA), GAD autoantibodies (GADA) and IA-2 autoantibodies (IA-2A). METHODS: IA-2betaA and ZnT8A (using a ZnT8 carboxy-terminal hybrid construct, CW-CR, carrying 325Arg and 325Trp) were determined by radiobinding assay in 409 IAA(+), GADA(+) and/or IA-2A(+) siblings or offspring (<40 years) of type 1 diabetic patients consecutively recruited by the Belgian Diabetes Registry. The median (interquartile range) age of the first-degree relatives was 12 (6-19) years. RESULTS: Of the first-degree relatives, 24% were IA-2A(+) (n = 97), 14% (n = 59) IA-2betaA(+) and 20% (n = 80) ZnT8A(+). IA-2betaA and ZnT8A were significantly (p < 0.001) associated with IA-2A and prediabetes (n = 86); in IA-2A(-) first-degree relatives (n = 312) the presence of IA-2betaA and ZnT8A was associated with an increased progression rate to diabetes (p < 0.001). Positivity for IA-2A and/or ZnT8A emerged as the most sensitive combination of two markers to identify first-degree relatives with a 5-year progression rate to diabetes of 45% (survival analysis) and as strongest predictor of diabetes (Cox regression analysis). Omission of first-degree relatives protected by HLA-DQ genotypes or maternal diabetes reduced the group to be followed from n = 409 to n = 246 (40%) with minor loss in the number of prediabetic IA-2A(+) or ZnT8A(+) first-degree relatives identified (n = 3). CONCLUSIONS/INTERPRETATION: IA-2A(+) and/or ZnT8A(+) first-degree relatives may be the participants of choice in future secondary prevention trials with immunointervention in relatives of type 1 diabetic patients.

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

AIMS/HYPOTHESIS: We analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein. METHODS: Autoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268-369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype. RESULTS: Seventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed. CONCLUSIONS/INTERPRETATION: These results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Mobility of two optional G + C-rich clusters of the var1 gene of yeast mitochondrial DNA.

Yeast mtDNA contains two different kinds of mobile optional sequences, two group I introns and a short G + C-rich insertion to some var1 genes. Movement of each element in crosses has been called gene conversion though little is known about the mechanism of G + C cluster conversion. A new allele of the var1 gene found in mtDNA of Saccharomyces capensis is described that permitted a more detailed comparison between intron mobility and G + C cluster conversion. The S. capensis var1 gene lacks the cc+ element present in all S. cerevisiae var 1 genes and the previously described optional a+ element. In crosses with cc+ a- and cc+ a+ S. cerevisiae strains, both clusters were found to be mobile and, in the latter cross, appear to convert independently and only to homologous insertion sites. No evidence for flanking marker coconversion (a hallmark feature of intron conversion) was obtained despite the availability of nearby physical markers on both sides of cluster conversion sites. These data indicate that G + C cluster conversion has only a superficial resemblance to intron mobility; analogies to procaryotic transposition mechanisms are considered.

Kinetics of the post-onset decline in zinc transporter 8 autoantibodies in type 1 diabetic human subjects.

CONTEXT: Zinc transporter 8 (ZnT8) is a newly discovered islet autoantigen in human type 1A diabetes (T1D). OBJECTIVE: The objective was to document changes in ZnT8 autoantibody (ZnT8A) titer and prevalence after onset of disease in relationship to 65 kDa glutamate decarboxylase antibody (GADA) and islet cell antigen antibody (IA2A). DESIGN/PATIENTS: Autoantibody radioimmunoprecipitation assays were performed on sera from three groups: 21 individuals monitored every 3 months from diagnosis for 2.5 yr; 61 individuals monitored at six monthly intervals for 5-12 yr; and a cross-sectional study of 424 patients with T1D of 20-57 yr duration. Circulating C-peptide was determined as an index of residual ß-cell function. RESULTS: ZnT8A titers declined exponentially from clinical onset of T1D with a t(1/2) ranging from 26 to 530 wk, similar to C-peptide (23-300 wk). Life-table analysis of antibody prevalence to 12 yr indicated that ZnT8A measured with either Arg325 or Trp325 probes persisted for a shorter interval than IA2A. Although prevalence of ZnT8A, IA2A, and GADA were comparable at disease onset (70.4 vs. 73.4 vs. 64%), only 6.7% of individuals remained ZnT8A positive after 25 yr compared with 19.5% for IA2A and 25.9% for GADA. Titers of ZnT8A and IA2A in seropositive individuals decreased progressively, whereas GADA remained elevated consistent with periodic reactivation of GADA humoral autoimmunity. CONCLUSIONS: ZnT8 humoral autoreactivity declines rapidly in the first years after disease onset and is less persistent than IA2A or GADA in the longer term. ZnT8A determination may be a useful measure of therapeutic efficacy in the context of immune-based clinical interventions.

Conserved epitopes in the protein tyrosine phosphatase family of diabetes autoantigens.

IA2 and phogrin are important targets of humoral and cell-mediated autoimmunity in type 1 diabetes in man. They belong to a conserved subfamily of transmembrane protein tyrosine phosphatases (PTPs) associated with the regulatory pathway of secretion. To examine potential cross-reactivity between PTP family members we tested sera from T1D patients for reactivity to IA2, and the Drosophila (FLYDA) and C. elegans (IDA) orthologs using radioimmunoprecipitation assays of (35)S Met-labeled in vitro translated products of the cytosolic domains of these proteins. Approximately 80% of sera reacted with at least one probe. Of these, 82.5% showed reactivity to human IA2, 74.1% to FLYDA, and 33.7% to IDA. The majority of sera that bound FLYDA and/or IDA also recognized IA2. This raises the possibility that in some cases reactivity to IA2 may have arisen by molecular mimicry.

A latent intron-encoded maturase is also an endonuclease needed for intron mobility.

Some yeast mitochondrial introns encode proteins that promote either splicing (maturases) or intron propagation via gene conversion (the fit1 endonuclease). We surveyed introns in the coxl gene for their ability to engage in gene conversion and found that the group I intron, al4 alpha, was efficiently transmitted to genes lacking it. An endonucleolytic cleavage is detectable in recipient DNA molecules near the site of intron insertion in vivo and in vitro. Conversion is dependent on an intact al4 alpha open reading frame. This intron product is a latent maturase, but these data show that it is also a potent endonuclease involved in recombination. Dual function proteins that cleave DNA and facilitate RNA splicing may have played a pivotal role in the propagation and tolerance of introns.

cDNA cloning of the two subunits of human CAAX farnesyltransferase and chromosomal mapping of FNTA and FNTB loci and related sequences.

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in several cellular proteins. Substrates include the p21ras proteins, nuclear lamins, and several retinal proteins, all of which end with a "CAAXbox," where C is cysteine, A is an aliphatic amino acid, and X is methionine or serine. Full-length cDNAs for the alpha and beta subunits of the rat farnesyltransferase have been cloned, and both have been shown to be essential for catalytic activity. Here we have used the rat cDNAs to clone cDNAs for the human alpha and beta subunits. Comparison of the human and rat amino acid sequences revealed a remarkable degree of conservation (93% identity for the alpha subunit and 96% identity for the beta subunit). The functional genes for the alpha and beta subunits of human farnesyltransferase (gene symbols, FNTA and FNTB) were localized to human chromosome bands 8p22-q11 and 14q23-q24, respectively, by Southern blot hybridization and PCR analyses of panels of human x Chinese hamster somatic cell hybrid lines and by fluorescence chromosomal in situ hybridization. We also found several related farnesyltransferase genes. FNTAL1 was assigned to 11q13.4-q14.1, FNTAL2 to chromosome 13, and FNTBL1 to chromosome 9.