Effective Detection of Human Leukocyte Antigen Risk Alleles in Celiac Disease Using Tag Single Nucleotide Polymorphisms

Background: The HLA genes, located in the MHC region on chromosome 6p21.3, play an important role in many autoimmune disorders, such as celiac disease (CD), type 1 diabetes (T1D), rheumatoid arthritis, multiple sclerosis, psoriasis and others. Known HLA variants that confer risk to CD, for example, include DQA1*05/DQB1*02 (DQ2.5) and DQA1*03/DQB1*0302 (DQ8). To diagnose the majority of CD patients and to study disease susceptibility and progression, typing these strongly associated HLA risk factors is of utmost importance. However, current genotyping methods for HLA risk factors involve many reactions, and are complicated and expensive. We sought a simple experimental approach using tagging SNPs that predict the CD-associated HLA risk factors. Methodology: Our tagging approach exploits linkage disequilibrium between single nucleotide polymorphism (SNPs) and the CD-associated HLA risk factors DQ2.5 and DQ8 that indicate direct risk, and DQA1*0201/DQB1*0202 (DQ2.2) and DQA1*0505/DQB1*0301 (DQ7) that attribute to the risk of DQ2.5 to CD. To evaluate the predictive power of this approach, we performed an empirical comparison of the predicted DQ types, based on these six tag SNPs, with those executed with current validated laboratory typing methods of the HLA-DQA1 and -DQB1 genes in three large cohorts. The results were validated in three European celiac populations. Conclusion: Using this method, only six SNPs were needed to predict the risk types carried by >95% of CD patients. We determined that for this tagging approach the sensitivity was >0.991, specificity >0.996 and the predictive value >0.948. Our results show that this tag SNP method is very accurate and provides an excellent basis for population screening for CD. This method is broadly applicable in European populations

The SPINK gene family and celiac disease susceptibility

The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n = 15) and diet-treated patients (n = 31) and controls (n = 16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population

Contribution of SARS-CoV-2 infection preceding COVID-19 mRNA vaccination to generation of cellular and humoral immune responses in children

Primary COVID-19 vaccination for children, 5-17 years of age, was offered in the Netherlands at a time when a substantial part of this population had already experienced a SARS-CoV-2 infection. While vaccination has been shown effective, underlying immune responses have not been extensively studied. We studied immune responsiveness to one and/or two doses of primary BNT162b2 mRNA vaccination and compared the humoral and cellular immune response in children with and without a preceding infection. Antibodies targeting the original SARS-CoV-2 Spike or Omicron Spike were measured by multiplex immunoassay. B-cell and T-cell responses were investigated using enzyme-linked immunosorbent spot (ELISpot) assays. The activation of CD4+ and CD8+ T cells was studied by flowcytometry. Primary vaccination induced both a humoral and cellular adaptive response in naive children. These responses were stronger in those with a history of infection prior to vaccination. A second vaccine dose did not further boost antibody levels in those who previously experienced an infection. Infection-induced responsiveness prior to vaccination was mainly detected in CD8+ T cells, while vaccine-induced T-cell responses were mostly by CD4+ T cells. Thus, SARS-CoV-2 infection prior to vaccination enhances adaptive cellular and humoral immune responses to primary COVID-19 vaccination in children. As most children are now expected to contract infection before the age of five, the impact of infection-induced immunity in children is of high relevance. Therefore, considering natural infection as a priming immunogen that enhances subsequent vaccine-responsiveness may help decision-making on the number and timing of vaccine doses

A spitting image: molecular diagnostics applied to saliva enhance detection of Streptococcus pneumoniae and pneumococcal serotype carriage

BackgroundDespite strong historical records on the accuracy of saliva testing, oral fluids are considered poorly suited for pneumococcal carriage detection. We evaluated an approach for carriage surveillance and vaccine studies that increases the sensitivity and specificity of pneumococcus and pneumococcal serotype detection in saliva samples.MethodsQuantitative PCR (qPCR)-based methods were applied to detect pneumococcus and pneumococcal serotypes in 971 saliva samples collected from 653 toddlers and 318 adults. Results were compared with culture-based and qPCR-based detection in nasopharyngeal samples collected from children and in nasopharyngeal and oropharyngeal samples collected from adults. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic curve analysis and accuracy of different approaches was assessed using a composite reference for pneumococcal and for serotype carriage based on isolation of live pneumococcus from the person or positivity of saliva samples determined with qPCR. To evaluate the inter-laboratory reproducibility of the method, 229 culture-enriched samples were tested independently in the second center.ResultsIn total, 51.5% of saliva samples from children and 31.8% of saliva samples from adults were positive for pneumococcus. Detection of pneumococcus by qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to diagnostic culture of nasopharyngeal samples in children (Cohen’s κ: 0.69–0.79 vs. 0.61–0.73) and in adults (κ: 0.84–0.95 vs. 0.04–0.33) and culture of oropharyngeal samples in adults (κ: 0.84–0.95 vs. −0.12–0.19). Similarly, detection of serotypes with qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to nasopharyngeal culture in children (κ: 0.73–0.82 vs. 0.61–0.73) and adults (κ: 0.90–0.96 vs. 0.00–0.30) and oropharyngeal culture in adults (κ: 0.90–0.96 vs. −0.13 to 0.30). However, results of qPCRs targeting serotype 4, 5, and 17F and serogroups 9, 12, and 35 were excluded due to assays’ lack of specificity. We observed excellent quantitative agreement for qPCR-based detection of pneumococcus between laboratories. After exclusion of serotype/serogroup-specific assays with insufficient specificity, moderate agreement (κ 0.68, 95% CI 0.58–0.77) was observed.ConclusionMolecular testing of culture-enriched saliva samples improves the sensitivity of overall surveillance of pneumococcal carriage in children and adults, but limitations of qPCR-based approaches for pneumococcal serotypes carriage detection should be considered

Long-Term Effects of Pneumococcal Conjugate Vaccine on Nasopharyngeal Carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis

BACKGROUND: Shifts in pneumococcal serotypes following introduction of 7-valent pneumococcal conjugate vaccine (PCV-7) may alter the presence of other bacterial pathogens co-inhabiting the same nasopharyngeal niche. METHODOLOGY/PRINCIPAL FINDINGS: Nasopharyngeal prevalence rates of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis were investigated before, 3 and 4.5 years after introduction of PCV-7 in the national immunisation program in children at 11 and 24 months of age, and parents of 24-month-old children (n≈330/group) using conventional culture methods. Despite a virtual disappearance of PCV-7 serotypes over time, similar overall pneumococcal rates were observed in all age groups, except for a significant reduction in the 11-month-old group (adjusted Odds Ratio after 4.5 years 0.48, 95% Confidence Interval 0.34-0.67). Before, 3 and 4.5 years after PCV-7 implementation, prevalence rates of S. aureus were 5%, 9% and 14% at 11 months of age (3.59, 1.90-6.79) and 20%, 32% and 34% in parents (1.96, 1.36-2.83), but remained similar at 24 months of age, respectively. Prevalence rates of H. influenzae were 46%, 65% and 65% at 11 months (2.22, 1.58-3.13), 52%, 73% and 76% at 24 months of age (2.68, 1.88-3.82) and 23%, 30% and 40% in parents (2.26, 1.58-3.33), respectively. No consistent changes in M. catarrhalis carriage rates were observed over time. CONCLUSIONS/SIGNIFICANCE: In addition to large shifts in pneumococcal serotypes, persistently higher nasopharyngeal prevalence rates of S. aureus and H. influenzae were observed among young children and their parents after PCV-7 implementation. These findings may have implications for disease incidence and antibiotic treatment in the post-PCV era

Is MYO9B the missing link between schizophrenia and celiac disease?

There has long been discussion on the correlation between schizophrenia and autoimmune diseases (especially celiac disease), which makes the recently discovered celiac disease risk factor, MYO9B, an attractive functional and positional candidate gene for schizophrenia. To test this hypothesis we compared allele frequencies of three MYO9B tag SNPs in 315 schizophrenia cases and 1,624 healthy controls in a genetic association study. Highly significant differences in allele frequencies between schizophrenia cases and healthy controls were observed for SNP rs2305767 in intron 14 of MYO9B (P = 1.16 x 10(-4); OR 1.41,95% CI 1.18-1.67). We demonstrate significant association of allelic variants in MYO9B with schizophrenia. To our knowledge, this is the first molecular genetic evidence for a correlation between autoimmune diseases and the risk of developing schizophrenia. (C) 2007 Wiley-Liss, Inc

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants

Antibody Levels at 3-Years Follow-Up of a Third Dose of Measles-Mumps-Rubella Vaccine in Young Adults.

Mumps outbreaks and breakthrough infections of measles and rubella have raised concerns about waning of vaccine-induced immunity after two doses of measles-mumps-rubella (MMR) vaccination. In the present follow-up study, serum IgG antibodies against mumps, measles and rubella, as well as the functional neutralizing antibodies against both the mumps vaccine strain and mumps outbreak strains were measured longitudinally in young adults that received a third MMR (MMR3) dose. The mumps-specific IgG and virus neutralizing antibody levels at 3 years after vaccination were still elevated compared to pre-vaccination antibody levels, although the differences were smaller than at earlier timepoints. Interestingly, subjects with low antibody levels to mumps before vaccination benefited the most as they showed the strongest antibody increase after an MMR3 dose. Three years after an MMR3 dose, all subjects had antibody levels to measles and rubella above the internationally agreed antibody cutoff levels for clinical protection. Our data support the recommendation that an MMR3 dose may provide additional protection for those that have become susceptible to mumps virus infection during outbreaks. MMR3 also resulted in an increase in anti-measles and rubella antibody levels that lasted longer than might have been expected

The MY09B gene is a strong risk factor for developing refractory Celiac disease

Background & Aims: Celiac disease (CD) is associated with HLA-DQ2 and HLA-DQ8 and has been linked to genetic variants in the MY09B gene on chromosome 19. HLA-DQ2 homozygosity is associated with complications of CD such as refractory celiac disease type II (RCD II) and enteropathy-associated T-cell lymphoma (EATL). We investigated whether MY09B also predisposes to RCD II and EATL. Methods: Genotyping of ATY09B and molecular HLA-DQ2 typing were performed on 62 RCD II and EATL patients, 421 uncomplicated CD patients, and 1624 controls. Results: One single nucleotide polymorphism in MY09B showed a significantly different allele distribution in RCD II and EATL patients compared with controls (P = .00002). The rs7259292 T allele was significantly more frequent in RCD II and EATL patients compared with CD patients (P = .0003; odds ratio [OR], 3.61; 95% confidence interval [CI], 1.78-7.31). The frequency of the haplotype carrying the T allele of this single nucleotide polymorphism was significantly increased in RCD II and EATL patients (11%), compared with controls (2%) and CD patients (3%) (OR, 6.76; 95% CI, 3.40-13.46; P = 2.27E-09 and OR, 4.22; 95% CI, 1.95-9.11; P = .0001, respectively). Both MY09B rs7259292 and HLA-DQ2 homozygosiry increase the risk for RCD II and EATL to a similar extent when compared with uncomplicated CD patients (OR, 4.3; 95% CI, 1.9-9.8 and OR, 5.4; 95% Cl, 3.0-9.6, respectively), but there was no evidence for any interaction between these 2 risk factors. Conclusions: We show that both MY09B and HLA-DQ2 homozygosity might be involved in the prognosis of CD and the chance of developing RCD II and EATL