Recognition of the HLA-DQB1*03:96 allele in a Taiwanese individual.

One nucleotide substitution in codon 130 of HLA-DQB1*03:03:02:01 results in a novel allele HLA-DQB1*03:96.

Case report: Strong GAD antibody positivity and type 1 diabetes-HLA-susceptible haplotype-DRB1*04:05-DQB1*04:01 in a Japanese patient with immune checkpoint inhibitor-induced type 1 diabetes.

Immune checkpoint inhibitors (ICIs) are widely used in cancer treatment; however, they can lead to immune-related adverse events, including immune checkpoint inhibitor-induced type 1 diabetes mellitus (ICI-T1DM). While fulminant T1DM is common in East Asia, ICI-T1DM has predominantly been reported in Western countries. In this report, we present the case of a 66-year-old Japanese man with type 2 diabetes mellitus undergoing dialysis for diabetic nephropathy. The patient was diagnosed with left upper lobe lung cancer, and treatment with nivolumab and ipilimumab was initiated. After 48 days, the patient experienced impaired consciousness and difficulty moving. His blood glucose levels were 815 mg/dL, and metabolic acidosis was detected, leading to a diagnosis of diabetic ketoacidosis. The patient was subsequently treated with continuous intravenous insulin. However, his C-peptide levels rapidly depleted, and new-onset ICI-T1DM was diagnosed. Although most Japanese patients with ICI-T1DM test negative for glutamic acid decarboxylase (GAD) antibodies, this case exhibited a strong positivity. Thus, we reviewed the literature on 15 similar Japanese cases, revealing a mean HbA1c level at onset of 8.7% and a mean time from ICI administration to onset of 9.7 weeks, which was shorter than that in GAD-negative cases. Moreover, human leukocyte antigen typing revealed five cases of DRB1*04:05-DQB1*04:01, including the present case, and one case of DRB1*09:01-DQB1*03:03, both of which were susceptible to T1DM haplotypes. These findings suggest that GAD antibody positivity may be associated with acute onset and disease progression in some cases of Japanese patients with ICI-T1DM. Given that the prediction of new-onset ICI-T1DM is challenging, monitoring GAD antibody levels might be useful. However, further studies with large sample sizes and validation across different racial and ethnic populations are warranted.

Multiple polygenic risk scores can improve the prediction of systemic lupus erythematosus in Taiwan.

OBJECTIVE: To identify new genetic variants associated with SLE in Taiwan and establish polygenic risk score (PRS) models to improve the early diagnostic accuracy of SLE. METHODS: The study enrolled 2429 patients with SLE and 48 580 controls from China Medical University Hospital in Taiwan. A genome-wide association study (GWAS) and PRS analyses of SLE and other three SLE markers, namely ANA, anti-double-stranded DNA antibody (dsDNA) and anti-Smith antibody (Sm), were conducted. RESULTS: Genetic variants associated with SLE were identified through GWAS. Some novel genes, which have been previously reported, such as RCC1L and EGLN3, were revealed to be associated with SLE in Taiwan. Multiple PRS models were established, and optimal cut-off points for each PRS were determined using the Youden Index. Combining the PRSs for SLE, ANA, dsDNA and Sm yielded an area under the curve of 0.64 for the optimal cut-off points. An analysis of human leucocyte antigen (HLA) haplotypes in SLE indicated that individuals with HLA-DQA1*01:01 and HLA-DQB1*05:01 were at a higher risk of being classified into the SLE group. CONCLUSIONS: The use of PRSs to predict SLE enables the identification of high-risk patients before abnormal laboratory data were obtained or symptoms were manifested. Our findings underscore the potential of using PRSs and GWAS in identifying SLE markers, offering promise for early diagnosis and prediction of SLE.

Identification of the novel HLA-DQB1*06:466 allele by next-generation sequencing in a Chinese cord blood donor.

HLA-DQB1*06:466 differs from HLA-DQB1*06:01:01:01 by a single nucleotide substitution at position 571G→A.

Exploring the role of HLA variants in neuroblastoma susceptibility through whole exome sequencing.

Although a number of susceptibility loci for neuroblastoma (NB) have been identified by genome-wide association studies, it is still unclear whether variants in the HLA region contribute to NB susceptibility. In this study, we conducted a comprehensive genetic analysis of variants in the HLA region among 724 NB patients and 2863 matched controls from different cohorts. We exploited whole-exome sequencing data to accurately type HLA alleles with an ensemble approach on the results from three different typing tools, and carried out rigorous sample quality control to ensure a fine-scale ancestry matching. The frequencies of common HLA alleles were compared between cases and controls by logistic regression under additive and non-additive models. Population stratification was taken into account adjusting for ancestry-informative principal components. We detected significant HLA associations with NB. In particular, HLA-DQB1*05:02 (OR = 1.61; padj = 5.4 × 10-3) and HLA-DRB1*16:01 (OR = 1.60; padj = 2.3 × 10-2) alleles were associated to higher risk of developing NB. Conditional analysis highlighted the HLA-DQB1*05:02 allele and its residue Ser57 as key to this association. DQB1*05:02 allele was not associated to clinical features worse outcomes in the NB cohort. Nevertheless, a risk score derived from the allelic combinations of five HLA variants showed a substantial predictive value for patient survival (HR = 1.53; p = 0.032) that was independent from established NB prognostic factors. Our study leveraged powerful computational methods to explore WES data and HLA variants and to reveal complex genetic associations. Further studies are needed to validate the mechanisms of these interactions that contribute to the multifaceted pattern of factors underlying the disease initiation and progression.

Relationship between HLA genetic variations, COVID-19 vaccine antibody response, and risk of breakthrough outcomes.

The rapid global distribution of COVID-19 vaccines, with over a billion doses administered, has been unprecedented. However, in comparison to most identified clinical determinants, the implications of individual genetic factors on antibody responses post-COVID-19 vaccination for breakthrough outcomes remain elusive. Here, we conducted a population-based study including 357,806 vaccinated participants with high-resolution HLA genotyping data, and a subset of 175,000 with antibody serology test results. We confirmed prior findings that single nucleotide polymorphisms associated with antibody response are predominantly located in the Major Histocompatibility Complex region, with the expansive HLA-DQB1*06 gene alleles linked to improved antibody responses. However, our results did not support the claim that this mutation alone can significantly reduce COVID-19 risk in the general population. In addition, we discovered and validated six HLA alleles (A*03:01, C*16:01, DQA1*01:02, DQA1*01:01, DRB3*01:01, and DPB1*10:01) that independently influence antibody responses and demonstrated a combined effect across HLA genes on the risk of breakthrough COVID-19 outcomes. Lastly, we estimated that COVID-19 vaccine-induced antibody positivity provides approximately 20% protection against infection and 50% protection against severity. These findings have immediate implications for functional studies on HLA molecules and can inform future personalised vaccination strategies.

Exploring potential correlations between HLA class II and the risk of microvascular complications in Japanese patients with type 1 diabetes.

Managing complications in Type 1 diabetes (T1D) remains challenging. HLA genes, particularly DR and DQ, are linked to T1D susceptibility. We studied 48 Japanese T1D inpatients and revealed associations between DRB1*04:05-DQB1*04:01 and DRB1*09:01-DQB1*03:03 haplotypes and complications, offering a new perspective for future research.

HLA-DQB1 Allele Polymorphism Associated with Oral Submucous Fibrosis in Hunan, China.

Methods: 44 OSF patients and 44 healthy volunteers were included in this study. To detect the expression frequency of HLA-DQB1 alleles in the two groups and analyze significant allelic subtypes and their relative risk, polymerase chain reaction (PCR) sequence-specific primers were used. Subsequently, based on the identification of differential genes, we compare the gene expression levels of OSF patients and healthy volunteers expressing differential genes by real-time quantitative PCR. Results: The expression frequency of the HLA-DQB1 ∗05 : 02 allele in the OSF group (36.4%) was significantly higher than in the controls (13.6%), and exposure to the HLA-DQB1 ∗05 : 02 allele was strongly related to OSF (OR (95% CI) = 3.619 (1.257,10.421), Wald χ2 = 5.681, P=0.017). However, there were no significant differences in the allele expression frequencies of DQB1 ∗02 : 01, DQB1 ∗03 : 03, DQB1 ∗05 : 01, DQB1 ∗05 : 03, DQB1 ∗06 : 02, DQB1 ∗06 : 03, and DQB1 ∗06 : 04 in the OSF group compared with the controls (all P > 0.05). Furthermore, the relative expression level of the HLA-DQB1 ∗05 : 02 allele in the OSF group (3.98 ± 3.50) was significantly higher than in controls (0.70 ± 0.41). Conclusions: There are differences in the HLA-DQB1 allele polymorphisms between the healthy population and patients with oral submucosal fibrosis. Preliminarily, it is suggested that the HLA-DQB1 ∗05 : 02 allele, which has a strong correlation with OSF and great differential expression between patients with OSF and controls, might be a susceptibility gene for OSF in Hunan.

Two new HLA-DQB1*03:02:01 variants with substitutions in intron 2: HLA-DQB1*03:02:01:19 and -DQB1*03:02:01:21.

Two different single nucleotide substitutions in intron 2 give rise to novel HLA-DQB1*03:02:01 alleles.

Investigation of HLA susceptibility alleles and genotypes with hematological disease among Chinese Han population.

Several genes involved in the pathogenesis have been identified, with the human leukocyte antigen (HLA) system playing an essential role. However, the relationship between HLA and a cluster of hematological diseases has received little attention in China. Blood samples (n = 123913) from 43568 patients and 80345 individuals without known pathology were genotyped for HLA class I and II using sequencing-based typing. We discovered that HLA-A*11:01, B*40:01, C*01:02, DQB1*03:01, and DRB1*09:01 were prevalent in China. Furthermore, three high-frequency alleles (DQB1*03:01, DQB1*06:02, and DRB1*15:01) were found to be hazardous in malignant hematologic diseases when compared to controls. In addition, for benign hematologic disorders, 7 high-frequency risk alleles (A*01:01, B*46:01, C*01:02, DQB1*03:03, DQB1*05:02, DRB1*09:01, and DRB1*14:54) and 8 high-frequency susceptible genotypes (A*11:01-A*11:01, B*46:01-B*58:01, B*46:01-B*46:01, C*01:02-C*03:04, DQB1*03:01-DQB1*05:02, DQB1*03:03-DQB1*06:01, DRB1*09:01-DRB1*15:01, and DRB1*14:54-DRB1*15:01) were observed. To summarize, our findings indicate the association between HLA alleles/genotypes and a variety of hematological disorders, which is critical for disease surveillance.

Genome-Wide Association Study Identifies IFIH1 and HLA-DQB1*05:02 Loci Associated With Anti-NMDAR Encephalitis.

BACKGROUND AND OBJECTIVES: Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a rare autoimmune neurologic disorder, the genetic etiology of which remains poorly understood. Our study aims to investigate the genetic basis of this disease in the Chinese Han population. METHODS: We performed a genome-wide association study and fine-mapping study within the major histocompatibility complex (MHC) region of 413 Chinese patients with anti-NMDAR encephalitis recruited from 6 large tertiary hospitals and 7,127 healthy controls. RESULTS: Our genome-wide association analysis identified a strong association at the IFIH1 locus on chromosome 2q24.2 (rs3747517, p = 1.06 × 10-8, OR = 1.55, 95% CI, 1.34-1.80), outside of the human leukocyte antigen (HLA) region. Furthermore, through a fine-mapping study of the MHC region, we discovered associations for 3 specific HLA class I and II alleles. Notably, HLA-DQB1*05:02 (p = 1.43 × 10-12; OR, 2.10; 95% CI 1.70-2.59) demonstrates the strongest association among classical HLA alleles, closely followed by HLA-A*11:01 (p = 4.36 × 10-7; OR, 1.52; 95% CI 1.29-1.79) and HLA-A*02:07 (p = 1.28 × 10-8; OR, 1.87; 95% CI 1.50-2.31). In addition, we uncovered 2 main HLA amino acid variation associated with anti-NMDAR encephalitis including HLA-DQß1-126H (p = 1.43 × 10-12; OR, 2.10; 95% CI 1.70-2.59), exhibiting a predisposing effect, and HLA-B-97R (p = 3.40 × 10-8; OR, 0.63; 95% CI 0.53-0.74), conferring a protective effect. Computational docking analysis suggested a close relationship between the NR1 subunit of NMDAR and DQB1*05:02. DISCUSSION: Our findings indicate that genetic variation in IFIH1, involved in the type I interferon signaling pathway and innate immunity, along with variations in the HLA class I and class II genes, has substantial implications for the susceptibility to anti-NMDAR encephalitis in the Chinese Han population.

Characterization of the novel HLA-DQB1*02:01:01:21Q allele by sequencing-based typing.

HLA-DQB1*02:01:01:21Q differs from HLA-DQB1*02:01:01:01 by one nucleotide substitution in the splice site in the beginning of intron 3.

Next-generation sequencing identifies two novel HLA-DQB1 alleles, HLA-DQB1*03:519 and HLA-DQB1*06:01:35.

We identified two novel HLA-DQB1 alleles by NGS, HLA-DQB1*03:519 and HLA-DQB1*06:01:35.

Family-based association of HLA-DRB1 and DQB1 alleles and haplotypes in a group of Iranian Type 1 diabetes children.

This family-based study was conducted in a group of Iranians with Type 1 diabetes (T1D) to investigate the transmission from parents of risk and non-risk HLA alleles and haplotypes, and to estimate the genetic risk score for this disease within this population. A total of 240 T1D subjects including 111 parent-child trios (111 children with T1D, 133 siblings, and 222 parents) and 330 ethnically matched healthy individuals were recruited. High-resolution HLA typing for DRB1/DQB1 loci was performed for all study subjects (n = 925) using polymerase chain reaction-sequence-specific oligonucleotide probe method. The highest predisposing effect on developing T1D was conferred by the following haplotypes both in all subjects and in probands compared to controls: DRB1*04:05-DQB1*03:02 (Pc = 2.97e-06 and Pc = 6.04e-10, respectively), DRB1*04:02-DQB1*03:02 (Pc = 5.94e-17 and Pc = 3.86e-09, respectively), and DRB1*03:01-DQB1*02:01 (Pc = 8.26e-29 and Pc = 6.56e-16, respectively). Conversely, the major protective haplotypes included DRB1*13:01-DQB1*06:03 (Pc = 6.99e-08), DRB1*15:01-DQB1*06:02 (Pc = 2.97e-06) in the cases versus controls. Also, DRB1*03:01-DQB1*02:01/DRB1*04:02|05-DQB1*03:02 and DRB1*03:01-DQB1*02:01/DRB1*03:01-DQB1*02:01 diplotypes conferred the highest predisposing effect in the cases (Pc = 8.65e-17 and Pc = 6.26e-08, respectively) and in probands (Pc = 5.4e-15 and Pc = 0.001, respectively) compared to controls. Transmission disequilibrium test showed that the highest risk was conferred by DRB1*04:02-DQB1*03:02 (Pc = 3.26e-05) and DRB1*03:01-DQB1*02:01 (Pc = 1.78e-12) haplotypes and the highest protection by DRB1*14:01-DQB1*05:03 (Pc = 8.66e-05), DRB1*15:01-DQB1*06:02 (Pc = 0.002), and DRB1*11:01-DQB1*03:01 (Pc = 0.0003) haplotypes. Based on logistic regression analysis, carriage of risk haplotypes increased the risk of T1D development 24.5 times in the Iranian population (p = 5.61e-13). Also, receiver operating characteristic curve analysis revealed a high predictive power of those risk haplotypes in discrimination of susceptible from healthy individuals (area under curve: 0.88, p = 5.5e-32). Our study highlights the potential utility of genetic risk assessment based on HLA diplotypes for predicting T1D risk in individuals, particularly among family members of affected children in our population.

Determination of HLA class II risk alleles and prediction of self/non-self-epitopes contributing Hashimoto's thyroiditis in a group of Iranian patients.

One of the probable hypotheses for the onset of autoimmunity is molecular mimicry. This study aimed to determine the HLA-II risk alleles for developing Hashimoto's thyroiditis (HT) in order to analyze the molecular homology between candidate pathogen-derived epitopes and potentially self-antigens (thyroid peroxidase, TPO) based on the presence of HLA risk alleles. HLA-DRB1/-DQB1 genotyping was performed in 100 HT patients and 330 ethnically matched healthy controls to determine the predisposing/protective alleles for HT disease. Then, in silico analysis was conducted to examine the sequence homology between epitopes derived from autoantigens and four potentially relevant pathogens and their binding capacities to HLA risk alleles based on peptide docking analysis. We identified HLA-DRB1*03:01, *04:02, *04:05, and *11:04 as predisposing alleles and DRB1*13:01 as a potentially predictive allele for HT disease. Also, DRB1*11:04 ~ DQB1*03:01 (Pc = 0.002; OR, 3.97) and DRB1*03:01 ~ DQB1*02:01 (Pc = 0.004; OR, 2.24) haplotypes conferred a predisposing role for HT. Based on logistic regression analysis, carrying risk alleles increased the risk of HT development 4.5 times in our population (P = 7.09E-10). Also, ROC curve analysis revealed a high predictive power of those risk alleles for discrimination of the susceptible from healthy individuals (AUC, 0.70; P = 6.6E-10). Analysis of peptide sequence homology between epitopes of TPO and epitopes derived from four candidate microorganisms revealed a homology between envelop glycoprotein D of herpes virus and sequence 151-199 of TPO with remarkable binding capacity to HLA-DRB1*03:01 allele. Our findings indicate the increased risk of developing HT in those individuals carrying HLA risk alleles which can also be related to herpes virus infection.

Cretan HLA genetics supports its early Minoan culture as a link between North Africa and Europe.

HLA studies in Crete show that this population is related to North Africans and also Iberians. This may be a reflection of a common prehistoric first Europeans relationships with North Africans and drying Saharan emigration after 10,000 years BC; it may be specifically represented by a primitive and early cult to the bull in both Cretan (Minoan) and Iberian populations. In the present study, unrelated Cretans representing different Island parts have been studied for class II HLA-DRB1 and -DQB1 alleles. The most frequent ones were HLA-DRB1*11:01 and HLA-DRB1*07:01 and HLA-DQB1*03:01 and DQB1*05:01. Also, the Cretan HLA class II haplotype HLA-DBR1*11:01-DQB1*03:01 had the highest frequency and is also common to other Mediterraneans, including Iberians. In addition, DRB1*07:01-DQB1*02:01 and HLA-DRB1*04:02-DQB1*03:02 Cretan haplotypes are shared with North Africans (the latter with Algerians, Tunisians and Moroccans). In summary, Crete was one of the first European classic cultures (Minoan) which was probably an early link, like Iberia, between North Africa /Sahara and Europe,also supported by genetic results.

HLA-DRB1 and HLA-DQB1 genes in patients diagnosed with systemic lupus erythematosus in Guatemala.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that impacts connective tissue and can affect various organs and systems within the body. One important aspect of this disease is the role of the human leukocyte antigen (HLA) system, a protein complex that plays a role in the immune response. Specifically, the HLA-DRB1 and HLA-DQB1 genes have been implicated in the development of SLE. In order to better understand this relationship in the Guatemalan population, a study was conducted with the objective of characterizing the allelic and haplotype profiles of the HLA-DQB1 and HLA-DRB1 loci in 50 patients diagnosed with SLE who were receiving treatment at a hospital in Guatemala. Allele and haplotype frequencies were determined and compared to 127 healthy Guatemalan subjects as a control group. The results of the analysis showed a reduction in the frequencies of HLA-DQB1*03 and HLA-DRB1*14 in SLE patients, which could suggest a protective effect on the development of the disease. In contrast, a risk association was found between HLA-DRB1*07, HLA-DRB1*08, HLA-DQB1*02 and HLA-DQB1*06 in SLE patients. Finally, we observed an additional protective associated of haplotype HLA-DRB1*04∼DQB1*03 with SLE patients, while haplotypes HLA-DRB1*07∼DQB1*02 and DRB1*08-DQB1*06 showed a risk association.

Validation of tag SNPs for multiple sclerosis HLA risk alleles across the 1000 genomes panel.

Currently, the genetic variants strongly associated with risk for Multiple Sclerosis (MS) are located in the Major Histocompatibility Complex. This includes DRB1*15:01 and DRB1*15:03 alleles at the HLA-DRB1 locus, the latter restricted to African populations; the DQB1*06:02 allele at the HLA-DQB1 locus which is in high linkage disequilibrium (LD) with DRB1*15:01; and protective allele A*02:01 at the HLA-A locus. HLA allele identification is facilitated by co-inherited ('tag') single nucleotide polymorphisms (SNPs); however, SNP validation is not typically done outside of the discovery population. We examined 19 SNPs reported to be in high LD with these alleles in 2,502 healthy subjects included in the 1000 Genomes panel having typed HLA data. Examination of 3 indices (LD R2 values, sensitivity and specificity, minor allele frequency) revealed few SNPs with high tagging performance. All SNPs examined that tag DRB1*15:01 were in perfect LD in the British population; three showed high tagging performance in 4 of the 5 European, and 2 of the 4 American populations. For DQB1*06:02, with no previously validated tag SNPs, we show that rs3135388 has high tagging performance in one South Asian, one American, and one European population. We identify for the first time that rs2844821 has high tagging performance for A*02:01 in 5 of 7 African populations including African Americans, and 4 of the 5 European populations. These results provide a basis for selecting SNPs with high tagging performance to assess HLA alleles across diverse populations, for MS risk as well as for other diseases and conditions.

Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Longitudinal screening of HLA-risk and HLA-nonrisk children for celiac disease to age 15 years: CiPiS study.

OBJECTIVES: Autoantibodies against tissue transglutaminase (tTG) are serological markers of celiac disease. The aim was to study the applicability of human leukocyte antigen (HLA)-genotyping and tTG autoantibodies in the screening of celiac disease in a longitudinal birth cohort followed to age 15 years. METHODS: Included were 13,860 HLA-DQ-genotyped children at birth and previously invited to a screening at age 3 and 9 years, respectively. HLA-DQB1*02 and/or DQB1*03:02 (HLA-risk) children were compared with non-HLA-DQB1*02 and non-DQB1*03:02 (HLA-nonrisk) children. The present study reinvited 12,948/13,860 (93.4%) children at age 15 years of whom 1056/2374 (44.5%) participated in screening at both age 3 and 9 years. Both immunoglobulin A (IgA) and G (IgG) autoantibodies against tTG were analyzed separately in radiobinding assays. Persistently tTG autoantibody-positive children were examined with intestinal biopsy to confirm the diagnosis of celiac disease. RESULTS: At age 3 years, celiac disease was diagnosed in 56/1635 (3.4%) HLA-risk children compared with 0/1824 HLA-nonrisk children (p < 0.001). By age 9 years, celiac disease was diagnosed in 72/1910 (3.8%) HLA-risk children compared with 0/2167 HLA-nonrisk children (p < 0.001). Screening at age 15 years detected 14/1071 (1.3%) HLA-risk children positive for IgA-tTG and/or IgG-tTG of whom 12/1071 (1.1%) remained persistently positive. Among those, 10/1071 (0.9%, 95% confidence interval: 0.4%-1.7%) HLA-risk children were diagnosed with celiac disease compared with 0/1303 HLA-nonrisk children (p < 0.001) and 5/491 (1.0%) were negative in screenings at both 3 and 9 years of age. CONCLUSIONS: Screening for celiac disease needs to be performed at multiple timepoints to detect all cases but can be restricted to children at HLA-risk.