Neopterin is a pro-inflammatory molecule upregulated in several diseases; however, its role in pathophysiology is unclear and its genetic regulation is unexplored. We observed that neopterin levels increase during senescence (P-value = 1.88×10-13, beta = 0.96) and positively correlate with age-related neurodegeneration and inflammation markers. The heritability estimation of neopterin variation was 35%. We then conducted a genome-wide association study on 999 Sardinians, identifying two signals in the GTP cyclohydrolase (GCH1) gene that were suggestively associated with neopterin levels. The first signal, led by rs140884539-C (P-value = 7.05×10-08, beta = 0.59), was in strong linkage disequilibrium with variants associated with predisposition to rheumatoid arthritis, decrease in dopamine, increased levels of GCH1 transcript, dopamine metabolites, and galectin-3. The second signal, represented by rs12323905-T (P-value = 8.17×10-08, beta = 0.30), colocalised with GCH1 splicing and Parkinson’s disease signals. Transcriptome analysis of 605 Sardinians showed that the Parkinson’s disease-predisposing variant was significantly associated with an increase in a shorter and inactive form of GCH1, whose presence is predicted to reduce the GCH1 decamer stability. The GCH1 homo-decamer regulates neopterin and tetrahydrobiopterin production, a cofactor required for the synthesis of dopamine and serotonin. Our data motivate experimental work to test whether modulating GCH1 expression or isoform ratio alters dopaminergic function in Parkinson’s disease models.

BackgroundThe immunoglobulin level following vaccination against SARS-CoV-2 results from a multifaceted immunological process involving cells and molecules that determine its efficacy and protect us against severe infection outcomes. The observed heterogeneity in the immune response to vaccination is partly attributable to host genetic factors; however, this genetic contribution has only been partially explored.MethodsTo elucidate the mechanisms underlying the antibody response elicited by the Pfizer-BioNTech vaccine, we conducted a genome-wide association study on anti-spike immunoglobulin G levels measured in 1,968 Italian individuals who received two doses of the vaccine, selected from a larger cohort of 7,169 volunteers characterized for 8 million genetic variants. Sex, age, body mass index, smoking habit, and time elapsed between vaccine administration and blood draw were accounted as covariates in the linear regression model.ResultsWe identified a novel signal of association on chromosome 3 in the intronic region of the CMTM8 gene and confirmed one previously identified at the HLA locus close to the HLA-B gene. The lead SNP in the CMTM8 gene, rs7643677 (p-value = 2.095×10-8), is associated with anti-S IgG levels and with the expression level of CD66b on granulocytic/polymorphonuclear myeloid-derived suppressor cells.ConclusionsThese findings support a role for CMTM8 in regulating the suppressive activity of specific immune cells, and suggest a potential interplay among genetic, humoral, and cellular mechanisms underlying the immune response to SARS-CoV-2 vaccination.

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing-a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing and flow cytometry suggested FAS as the predominant colonic epithelial receptor for TNFSF13, which was confirmed by co-immunoprecipitation and binding assays. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in immunoglobulin-producing plasma cells compared to control colonoid cocultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells.

Genotype-phenotype associations can be context-dependent and dynamic in nature leading to heterogeneity of genetic effects across different parts of the phenotype distribution. Quantile regression, an alternative to linear regression for continuous phenotypes, is particularly well suited for detecting and characterizing heterogeneous genotype-phenotype associations. Here we propose a novel and computationally efficient whole-genome quantile regression technique, Regenie.QRS, for biobank-scale GWAS data with genetic structure. Our approach first estimates the polygenic effect, and then incorporates this effect as an offset in the non-mixed quantile regression model. Our simulations demonstrate robust control of type I error and higher power to detect heterogeneous associations relative to linear regression in GWAS, and improved power over the marginal quantile regression tests. We present applications using data from the UK Biobank and the ProgeNIA/SardiNIA project, where we show the advantages of Regenie.QRS in identifying and characterizing heterogeneous genetic effects. To cite just one interesting example, using quantile regression we are able to show that even though variants at the G6PC2 locus increase glucose levels, their effects are much stronger at lower quantiles of glucose level distribution than at higher quantiles, showing that G6PC2 serves as a guardian against low glucose levels without driving dangerous hyperglycemia, which may explain the lack of association with diabetes risk.

Supplementary Figure from Genetic Analysis in African American Children Supports Ancestry-Specific Neuroblastoma Susceptibility

Subtle population structure remains a significant concern in genome-wide association studies. Using human height as an example, we show how quantile regression, a natural extension of linear regression, can better correct for subtle population structure due to its inherent ability to adjust for quantile-specific effects of covariates such as principal components. We utilize data from the UK biobank and the SardiNIA/ProgeNIA project for demonstration.

Polygenic risk scores (PRS) are widely used in post-GWAS analyses to predict complex traits across humans, animals, and plants. While significant progress has been made in developing new PRS methods, much less attention has been given to quantifying the uncertainty associated with these predictions. In this work, we propose a method for individualized uncertainty quantification based on quantile regression. When paired with conformal prediction, this approach enables the construction of prediction intervals with guaranteed coverage, offering lower and upper bounds within which the phenotype is likely to fall with high probability. We apply this framework to data from the UK Biobank and the ProgeNIA/SardiNIA studies, showing that the resulting prediction intervals: (1) maintain valid coverage under minimal model assumptions, (2) provide more realistic individualized estimates of uncertainty by allowing for asymmetry and individual-specific interval lengths, and (3) exhibit reduced uncertainty compared to existing methods. Overall, we present a novel framework for individualized uncertainty quantification in PRS analyses and highlight the importance of incorporating uncertainty into predictive modeling. ### Competing Interest Statement The authors have declared no competing interest.

Gene co-expression network analysis enables identification of biologically meaningful clusters of co-regulated genes (modules) in an unsupervised manner. We present here the largest study conducted thus far of co-expression networks in white blood cells (WBC) based on RNA-seq data from 624 individuals. We identify 41 modules, 13 of them related to specific immune-related functions and cell types (e.g. neutrophils, B and T cells, NK cells, and plasmacytoid dendritic cells); we highlight biologically relevant lncRNAs for each annotated module of co-expressed genes. We further characterize with unprecedented resolution the modules in T cell sub-types, through the availability of 95 immune phenotypes obtained by flow cytometry in the same individuals. This study provides novel insights into the transcriptional architecture of human leukocytes, showing how network analysis can advance our understanding of coding and non-coding gene interactions in immune system cells.

Dysbiosis is associated with pediatric and adult-onset inflammatory bowel disease (IBD), but the role of dysbiosis and the microbiome in very early onset IBD (VEO-IBD) has not yet been described. Here, we aimed to demonstrate the impact of age and inflammation on microbial community structure using shotgun metagenomic sequencing in children with VEO-IBD, pediatric-onset IBD, and age-matched pediatric healthy controls (HC) observed longitudinally over the course of 8 weeks. We found disease-related differences in alpha and beta diversity between HC and children with IBD or VEO-IBD. Using a healthy microbial maturity index modeled from HC across the age range to characterize their gut microbiota, we found that children with pediatric-onset IBD and VEO-IBD had lower maturity than their age-matched HC groups, suggesting a disease effect on the microbial community. In addition, patients with pediatric IBD had significantly lower maturity than those with VEO-IBD, who had more heterogeneity at the youngest ages, highlighting differences in these two cohorts that were not captured in standard comparisons of alpha and beta diversity. These results demonstrate that young age and inflammation independently impact microbial community structure. However, the effect is not additive in the youngest patients, likely because of the heterogeneous and dynamic stool microbiome in this population.

IntroductionDisease-modifying therapies (DMTs) have been shown to improve disease outcomes in multiple sclerosis (MS) patients. They may also impair the immune response to vaccines, including the SARS-CoV-2 vaccine. However, available data on both the intrinsic immune effects of DMTs and their influence on cellular response to the SARS-CoV-2 vaccine are still incomplete.MethodsHere, we evaluated the immune cell effects of 3 DMTs on the response to mRNA SARS-CoV-2 vaccination by comparing MS patients treated with one specific therapy (fingolimod, dimethyl fumarate, or natalizumab) with both healthy controls and untreated patients. We profiled 23 B-cell traits, 57 T-cell traits, and 10 cytokines, both at basal level and after stimulation with a pool of SARS-CoV-2 spike peptides, in 79 MS patients, treated with DMTs or untreated, and 32 healthy controls. Measurements were made before vaccination and at three time points after immunization.Results and DiscussionMS patients treated with fingolimod showed the strongest immune cell dysregulation characterized by a reduction in all measured lymphocyte cell classes; the patients also had increased immune cell activation at baseline, accompanied by reduced specific immune cell response to the SARS-CoV-2 vaccine. Also, anti-spike specific B cells progressively increased over the three time points after vaccination, even when antibodies measured from the same samples instead showed a decline. Our findings demonstrate that repeated booster vaccinations in MS patients are crucial to overcoming the immune cell impairment caused by DMTs and achieving an immune response to the SARS-CoV-2 vaccine comparable to that of healthy controls.

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing- a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13 variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing, flow cytometry, and co-immunoprecipitation identified FAS as the predominant colonic epithelial receptor for TNFSF13. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in the production of IgA+ plasma cells compared to control colonoid co-cultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells.

Supplementary Table 1. Functional prioritization of SNPs in linkage disequilibrium (r2=0.6) with significant rs4673067 at SCG2 locus. Supplementary Table 2. SNPs in LD (r2>0.60) with the SNP rs118727. Supplementary Table 3. SNPs in LD (r2>0.60) with the SNP rs196830. Supplementary Table 4. SNPs in LD (r2>0.60) with the SNP rs169061. Supplementary Table 5. SNPs in LD (r2>0.60) with the SNP rs11994014. Supplementary Table 6. SNPs in LD (r2>0.60) with the SNP rs17830286. Supplementary Table 7. Functional prioritization of SNPs in 3'UTR region of NEFL and in linkage disequilibrium (r2=0.6) with significant typed SNPs rs118727, rs196830, rs17830286, rs11994014. Supplementary Table 8. Prediction of mircoRNAs whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by mrSNP web tool. Supplementary Table 9. Prediction of transcriptional factors whose binding is affected by the SNPs rs1059111, rs2979704, rs3761 by HaploReg V2. Supplementary Table 10. List of UTR motifs analyzed for the SNPs rs3761, rs2979704, and rs1059111. Supplementary Table 11. Imputation results of SNPs at SCG2 and NEFL loci (+/- 1Kb) by using 1000 Genomes data. Supplementary Table 12. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in European American cohort. Supplementary Table 13. Association of NEFL SNP genotypes with pathologic characteristics of neuroblastoma in Italian cohort. Supplementary Figure 1. Linkage disequilibrium (LD) plot of NEFL gene (chr8: positions 24,845,004 to 24,887,674) by Haploview 4.2 for HapMap CEU subjects. Supplementary Figure 2. REST gene silencing in neuroblastoma cells. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against REST (shREST) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (a-b). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. Induction of NEFL and REST gene expression levels after REST silencing in SH-SY5Y (c) and SK-N-BE2c (d). *P<0.05 Supplementary Figure 3. (a) Association between NEFL SNP genotype and gene expression in 16 neuroblastoma cell lines. The gene expression measure of the NB1 cell line was excluded from this analysis as affected the normal distribution of data (data not shown). The analysis was performed using the SNPs rs12545967 and rs2976427 in complete linkage disequilibrium (r2=1) with rs11994014 and rs1059111, respectively. Conventionally, we report the allele code of rs11994014 and rs1059111 in the plot. (b) Association between NEFL SNP genotype and gene expression in 198 LCLs. The analysis for rs1059111 (missing in the dataset) was performed using the SNP rs2979704 in complete linkage disequilibrium (r2=1). Supplementary Figure 4. NEFL gene silencing in neuroblastoma cells with rs1059111 TA genotype. Evaluation of cell growth (a) and invasion (b) after NEFL silencing in SH-SY5Y and SK-N-BE2c cell lines. The efficiency of gene silencing mediated by lentiviral delivery of hairpin RNA directed against NEFL (shNEFL) in SH-SY5Y and SK-N-BE2c cell lines was assessed by western blotting (c). The bar graphs show integral optical density (OD) value for each band, normalized respect to β-Actin expression. The same results were observed for mRNA measurements (data not shown). The results are shown as mean of three experiments and are represented as fold respect to shCTR cells which are infected by lentivirus-mediated delivery of non-silencing hairpin RNA. *P<0.05 Supplementary Figure 5. Association between NEFL SNP genotype and neuronal marker genes in neuroblastoma cell lines. Eight cell lines (IMR-32, SK-N-DZ, SK-N-AS, KP-N-SI9s with rs1059111 TT genotype and SIMA, SK-N-BE(2), SH-SY5Y, SK-N-FI with rs1059111 TA protective genotype) with the extremes of NEFL mRNA expression were selected based on the analysis shows in the Supplementary Figure 2a. The analysis was performed using the SNPs rs2976427 in complete linkage disequilibrium (r2=1) with and rs1059111. Conventionally, we report the allele code of rs1059111 in the plot.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3′UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

BACKGROUND:Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Treatment for patients who have a monogenic cause of their IBD, often the youngest children, known as very early onset IBD (VEO-IBD), can be different from standard treatment for polygenic cases. Yet, ascertainment of these patients is difficult. METHODS:We analyzed cases of VEO-IBD to understand the breadth of monogenic etiology and to identify clinical, laboratory, and flow cytometric correlates of this subpopulation. RESULTS:Genetic causes of very early onset inflammatory bowel disease are highly diverse ranging from pure epithelial defects to classic T cell defects. Flow cytometry, other than testing for chronic granulomatous disease, has a low sensitivity for monogenic etiologies. Poor growth was a clinical feature associated with monogenic causality. CONCLUSIONS:Genetic testing is, at this moment, the most robust method for the identification of monogenic cases of very early onset IBD.

BACKGROUND AND PURPOSE:Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2.METHODS:We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants.RESULTS:We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA.CONCLUSIONS:Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.

Monitoring immune responses to SARS-CoV-2 vaccination and its clinical efficacy over time in Multiple Sclerosis (MS) patients treated with disease-modifying therapies (DMTs) help to establish the optimal strategies to ensure adequate COVID-19 protection without compromising disease control offered by DMTs. Following our previous observations on the humoral response one month after two doses of BNT162b2 vaccine (T1) in MS patients differently treated, here we present a cross-sectional and longitudinal follow-up analysis six months following vaccination (T2, n=662) and one month following the first booster (T3, n=185). Consistent with results at T1, humoral responses were decreased in MS patients treated with fingolimod and anti-CD20 therapies compared with untreated patients also at the time points considered here (T2 and T3). Interestingly, a strong upregulation one month after the booster was observed in patients under every DMTs analyzed, including those treated with fingolimod and anti-CD20 therapies. Although patients taking these latter therapies had a higher rate of COVID-19 infection five months after the first booster, only mild symptoms that did not require hospitalization were reported for all the DMTs analyzed here. Based on these findings we anticipate that additional vaccine booster shots will likely further improve immune responses and COVID-19 protection in MS patients treated with any DMT.

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.