Epigenetic analysis of cell-free DNA by fragmentomic profiling.

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Nuclease deficiencies alter plasma cell-free DNA methylation profiles.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

Genetics of Neonatal Lupus Erythematosus Risk and Specific Manifestations.

OBJECTIVE: Neonatal lupus erythematosus (NLE) is a passively acquired autoimmune disease of infants born to anti-Ro and/or La autoantibody positive mothers. Genetics may impact NLE risk. We analyzed the genetics of infants and anti-Ro antibody positive mothers, with NLE and NLE specific manifestations. METHODS: Infants and mothers from a tertiary care clinic underwent genotyping on the Global Screening Array. We created additive non-HLA and HLA polygenic risk scores (PRSs) for systemic lupus erythematosus (SLE), from one of the largest genome wide association studies. Outcomes were any NLE manifestations, cardiac NLE, and cutaneous NLE. We tested the association between SLE-PRSs in the infant, mother, and the PRS difference between the mother and infant with NLE outcomes, in logistic regression and generalized linear mixed models (Bonferroni P<0.02). We also performed HLA-wide analyses for the outcomes (P<5.00x10-8). RESULTS: The study included 332 infants, 270 anti-Ro antibody positive mothers, and 253 mother-infant pairs. A large proportion of mothers (40.3%) and infants (41.3%) were European, and 50.0% of infants were female. More than half of the infants had NLE (53.0%), including 7.2% with cardiac NLE and 11.7% with cutaneous NLE. We did not identify significant associations between infant, maternal, or maternal-infant PRSs and any NLE outcomes. HLA-wide analyses did not identify NLE risk alleles. CONCLUSION: In a multiethnic cohort of infants and anti-Ro antibody positive mothers, we did not identify a significant association between SLE genetics and risk of NLE outcomes.

Transmission disequilibrium analysis of whole genome data in childhood-onset systemic lupus erythematosus.

Childhood-onset systemic lupus erythematosus (cSLE) patients are unique, with hallmarks of Mendelian disorders (early-onset and severe disease) and thus are an ideal population for genetic investigation of SLE. In this study, we use the transmission disequilibrium test (TDT), a family-based genetic association analysis that employs robust methodology, to analyze whole genome sequencing data. We aim to identify novel genetic associations in an ancestrally diverse, international cSLE cohort. Forty-two cSLE patients and 84 unaffected parents from 3 countries underwent whole genome sequencing. First, we performed TDT with single nucleotide variant (SNV)-based (common variants) using PLINK 1.9, and gene-based (rare variants) analyses using Efficient and Parallelizable Association Container Toolbox (EPACTS) and rare variant TDT (rvTDT), which applies multiple gene-based burden tests adapted for TDT, including the burden of rare variants test. Applying the GWAS standard threshold (5.0 × 10-8) to common variants, our SNV-based analysis did not return any genome-wide significant SNVs. The rare variant gene-based TDT analysis identified many novel genes significantly enriched in cSLE patients, including HNRNPUL2, a DNA repair protein, and DNAH11, a ciliary movement protein, among others. Our approach identifies several novel SLE susceptibility genes in an ancestrally diverse childhood-onset lupus cohort.

Plasma DNA Profile Associated with DNASE1L3 Gene Mutations: Clinical Observations, Relationships to Nuclease Substrate Preference, and In Vivo Correction.

Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

Genetics of osteonecrosis in children and adults with systemic lupus erythematosus.

OBJECTIVES: Genetics plays an important role in SLE risk, as well as osteonecrosis (ON), a significant and often debilitating complication of SLE. We aimed to identify genetic risk loci for ON in people with childhood-onset (cSLE) and adult-onset (aSLE) SLE. METHODS: We enrolled participants from two tertiary care centres who met classification criteria for SLE. Participants had prospectively collected clinical data and were genotyped on a multiethnic array. Un-genotyped single nucleotide polymorphisms (SNPs) were imputed, and ancestry was inferred using principal components (PCs). Our outcome was symptomatic ON confirmed by imaging. We completed time-to-ON and logistic regression of ON genome-wide association studies (GWASs) with covariates for sex, age of SLE diagnosis, five PCs for ancestry, corticosteroid use and selected SLE manifestations. We conducted separate analyses for cSLE and aSLE and meta-analysed results using inverse-variance weighting. Genome-wide significance was P < 5 × 10-8. RESULTS: The study included 940 participants with SLE, 87% female and 56% with cSLE. ON was present in 7.6% (n = 71). Median age of SLE diagnosis was 16.9 years (interquartile range [IQR]: 13.5, 29.3), with median follow-up of 8.0 years (IQR: 4.2, 15.7). Meta-GWAS of cSLE and aSLE time-to-ON of 4 431 911 SNPs identified a significant Chr.2 SNP, rs34118383 (minor allele frequency = 0.18), intronic to WIPF1 (hazard ratio = 3.2 [95% CI: 2.2, 4.8]; P = 1.0 × 10-8). CONCLUSION: We identified an intronic WIPF1 variant associated with a 3.2 times increased hazard for ON (95% CI: 2.2, 4.8; P = 1.0 × 10-8) during SLE follow-up, independent of corticosteroid exposure. The effect of the SNP on time-to-ON was similar in cSLE and aSLE. This novel discovery represents a potential ON risk locus. Our results warrant replication.

Genetics of longitudinal kidney function in children and adults with systemic lupus erythematosus.

OBJECTIVES: Genome-wide association studies (GWAS) have identified loci associated with estimated glomerular filtration rate (eGFR). Few LN risk loci have been identified to date. We tested the association of SLE and eGFR polygenic risk scores (PRS) with repeated eGFR measures from children and adults with SLE. METHODS: Patients from two tertiary care lupus clinics that met ≥4 ACR and/or SLICC criteria for SLE were genotyped on the Illumina MEGA or Omni1-Quad arrays. PRSs were calculated for SLE and eGFR, using published weighted GWA-significant alleles. eGFR was calculated using the CKD-EPI and Schwartz equations. We tested the effect of eGFR- and SLE-PRSs on eGFR mean and variance, adjusting for age at diagnosis, sex, ancestry, follow-up time, and clinical event flags. RESULTS: We included 1158 SLE patients (37% biopsy-confirmed LN) with 36 733 eGFR measures over a median of 7.6 years (IQR: 3.9-15.3). LN was associated with lower within-person mean eGFR [LN: 93.8 (s.d. 26.4) vs non-LN: 101.6 (s.d. 17.7) mL/min per 1.73 m2; P < 0.0001] and higher variance [LN median: 157.0 (IQR: 89.5, 268.9) vs non-LN median: 84.9 (IQR: 46.9, 138.2) (mL/min per 1.73 m2)2; P < 0.0001]. Increasing SLE-PRSs were associated with lower mean eGFR and greater variance, while increasing eGFR-PRS was associated with increased eGFR mean and variance. CONCLUSION: We observed significant associations between SLE and eGFR PRSs and repeated eGFR measurements, in a large cohort of children and adults with SLE. Longitudinal eGFR may serve as a powerful alternative outcome to LN categories for discovery of LN risk loci.

Jagged Ends on Multinucleosomal Cell-Free DNA Serve as a Biomarker for Nuclease Activity and Systemic Lupus Erythematosus.

BACKGROUND: Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. METHODS: Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. RESULTS: Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290 bp, 330-380 bp, and 420-470 bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. CONCLUSIONS: Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.

Association of systemic lupus erythematosus (SLE) genetic susceptibility loci with lupus nephritis in childhood-onset and adult-onset SLE.

OBJECTIVE: LN is one of the most common and severe manifestations of SLE. Our aim was to test the association of SLE risk loci with LN risk in childhood-onset SLE (cSLE) and adult-onset SLE (aSLE). METHODS: Two Toronto-based tertiary care SLE cohorts included cSLE (diagnosed <18 years) and aSLE patients (diagnosed ⩾18 years). Patients met ACR and/or SLICC SLE criteria and were genotyped on the Illumina Multi-Ethnic Global Array or Omni1-Quad arrays. We identified those with and without biopsy-confirmed LN. HLA and non-HLA additive SLE risk-weighted genetic risk scores (GRSs) were tested for association with LN risk in logistic models, stratified by cSLE/aSLE and ancestry. Stratified effect estimates were meta-analysed. RESULTS: Of 1237 participants, 572 had cSLE (41% with LN) and 665 had aSLE (30% with LN). Increasing non-HLA GRS was significantly associated with increased LN risk [odds ratio (OR) = 1.26; 95% CI 1.09, 1.46; P = 0.0006], as was increasing HLA GRS in Europeans (OR = 1.55; 95% CI 1.07, 2.25; P = 0.03). There was a trend for stronger associations between both GRSs and LN risk in Europeans with cSLE compared with aSLE. When restricting cases to proliferative LN, the magnitude of these associations increased for both the non-HLA (OR = 1.30; 95% CI 1.10, 1.52; P = 0.002) and HLA GRS (OR = 1.99; 95% CI 1.29, 3.08; P = 0.002). CONCLUSION: We observed an association between known SLE risk loci and LN risk in children and adults with SLE, with the strongest effect observed among Europeans with cSLE. Future studies will include SLE-risk single nucleotide polymorphisms specific to non-European ancestral groups and validate findings in an independent cohort.

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

The Genetic Landscape of Renal Complications in Type 1 Diabetes.

Diabetes is the leading cause of ESRD. Despite evidence for a substantial heritability of diabetic kidney disease, efforts to identify genetic susceptibility variants have had limited success. We extended previous efforts in three dimensions, examining a more comprehensive set of genetic variants in larger numbers of subjects with type 1 diabetes characterized for a wider range of cross-sectional diabetic kidney disease phenotypes. In 2843 subjects, we estimated that the heritability of diabetic kidney disease was 35% (P=6.4×10-3). Genome-wide association analysis and replication in 12,540 individuals identified no single variants reaching stringent levels of significance and, despite excellent power, provided little independent confirmation of previously published associated variants. Whole-exome sequencing in 997 subjects failed to identify any large-effect coding alleles of lower frequency influencing the risk of diabetic kidney disease. However, sets of alleles increasing body mass index (P=2.2×10-5) and the risk of type 2 diabetes (P=6.1×10-4) associated with the risk of diabetic kidney disease. We also found genome-wide genetic correlation between diabetic kidney disease and failure at smoking cessation (P=1.1×10-4). Pathway analysis implicated ascorbate and aldarate metabolism (P=9.0×10-6), and pentose and glucuronate interconversions (P=3.0×10-6) in pathogenesis of diabetic kidney disease. These data provide further evidence for the role of genetic factors influencing diabetic kidney disease in those with type 1 diabetes and highlight some key pathways that may be responsible. Altogether these results reveal important biology behind the major cause of kidney disease.

Improved performance of epidemiologic and genetic risk models for rheumatoid arthritis serologic phenotypes using family history.

OBJECTIVE: To develop and validate rheumatoid arthritis (RA) risk models based on family history, epidemiologic factors and known genetic risk factors. METHODS: We developed and validated models for RA based on known RA risk factors, among women in two cohorts: the Nurses' Health Study (NHS, 381 RA cases and 410 controls) and the Epidemiological Investigation of RA (EIRA, 1244 RA cases and 971 controls). Model discrimination was evaluated using the area under the receiver operating characteristic curve (AUC) in logistic regression models for the study population and for those with positive family history. The joint effect of family history with genetics, smoking and body mass index (BMI) was evaluated using logistic regression models to estimate ORs for RA. RESULTS: The complete model including family history, epidemiologic risk factors and genetics demonstrated AUCs of 0.74 for seropositive RA in NHS and 0.77 for anti-citrullinated protein antibody (ACPA)-positive RA in EIRA. Among women with positive family history, discrimination was excellent for complete models for seropositive RA in NHS (AUC 0.82) and ACPA-positive RA in EIRA (AUC 0.83). Positive family history, high genetic susceptibility, smoking and increased BMI had an OR of 21.73 for ACPA-positive RA. CONCLUSIONS: We developed models for seropositive and seronegative RA phenotypes based on family history, epidemiological and genetic factors. Among those with positive family history, models using epidemiologic and genetic factors were highly discriminatory for seropositive and seronegative RA. Assessing epidemiological and genetic factors among those with positive family history may identify individuals suitable for RA prevention strategies.

Exploring the genetic architecture of circulating 25-hydroxyvitamin D.

The primary circulating form of vitamin D is 25-hydroxy vitamin D (25(OH)D), a modifiable trait linked with a growing number of chronic diseases. In addition to environmental determinants of 25(OH)D, including dietary sources and skin ultraviolet B (UVB) exposure, twin- and family-based studies suggest that genetics contribute substantially to vitamin D variability with heritability estimates ranging from 43% to 80%. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) located in four gene regions associated with 25(OH)D. These SNPs collectively explain only a fraction of the heritability in 25(OH)D estimated by twin- and family-based studies. Using 25(OH)D concentrations and GWAS data on 5,575 subjects drawn from five cohorts, we hypothesized that genome-wide data, in the form of (1) a polygenic score comprised of hundreds or thousands of SNPs that do not individually reach GWAS significance, or (2) a linear mixed model for genome-wide complex trait analysis, would explain variance in measured circulating 25(OH)D beyond that explained by known genome-wide significant 25(OH)D-associated SNPs. GWAS identified SNPs explained 5.2% of the variation in circulating 25(OH)D in these samples and there was little evidence additional markers significantly improved predictive ability. On average, a polygenic score comprised of GWAS-identified SNPs explained a larger proportion of variation in circulating 25(OH)D than scores comprised of thousands of SNPs that were on average, nonsignificant. Employing a linear mixed model for genome-wide complex trait analysis explained little additional variability (range 0-22%). The absence of a significant polygenic effect in this relatively large sample suggests an oligogenetic architecture for 25(OH)D.

Being overweight or obese and risk of developing rheumatoid arthritis among women: a prospective cohort study.

OBJECTIVES: To examine the relationship between being overweight or obese and developing rheumatoid arthritis (RA) in two large prospective cohorts, the Nurses' Health Study (NHS) and Nurses' Health Study II (NHSII). METHODS: We followed 109 896 women enrolled in NHS and 108 727 in NHSII who provided lifestyle, environmental exposure and anthropometric information through biennial questionnaires. We assessed the association between time-varying and cumulative Body Mass Index (BMI) in WHO categories of normal, overweight and obese (18.5-<25, 25.0-<30, ≥30.0 kg/m(2)) and incident RA meeting the 1987 American College of Rheumatology (ACR) criteria. We estimated HRs for overall RA and serologic subtypes with Cox regression models adjusted for potential confounders. We repeated analyses restricted to RA diagnosed at age 55 years or younger. RESULTS: During 2 765 195 person-years of follow-up (1976-2008) in NHS and 1 934 518 person-years (1989-2009) in NHSII, we validated 1181 incident cases of RA (826 in NHS, 355 in NHSII). There was a trend toward increased risk of all RA among overweight and obese women (HR (95% CI) 1.37 (0.95 to 1.98) and 1.37 (0.91, 2.09), p for trend=0.068). Among RA cases diagnosed at age 55 years or younger, this association appeared stronger (HR 1.45 (1.03 to 2.03) for overweight and 1.65 (1.34 to 2.05) for obese women (p trend <0.001)). Ten cumulative years of being obese, conferred a 37% increased risk of RA at younger ages (HR 1.37 (1.11 to 1.69)). CONCLUSIONS: Risks of seropositive and seronegative RA were elevated among overweight and obese women, particularly among women diagnosed with RA at earlier ages.

Circulating 25-hydroxyvitamin D level and risk of developing rheumatoid arthritis.

OBJECTIVE: The aim of this study was to examine the relationship between preclinical circulating 25-hydroxyvitamin D [25(OH)D] and RA in two nested case-control studies within the prospective cohort Nurses' Health Study (NHS) and NHS II (NHSII). METHODS: We included 166 women with RA and blood specimens collected 3 months to 16 years prior to the first RA symptom and 490 matched controls (3:1, matched on age, date of blood draw, hormonal factors). We calculated the odds ratio (OR) and 95% CI for incident RA using conditional logistic regression multivariable adjusted models, including additional covariates for smoking status, parity and breastfeeding, alcohol consumption, BMI, median income and region of residence in the USA. We repeated analyses stratified by time from blood draw to RA diagnosis (3 months to <4 years or ≥4 years) and meta-analysed estimates from the two cohorts using fixed effects models. RESULTS: Incident RA was confirmed in 120 NHS [mean age 63.8 years (s.d. 8.2)] and 46 NHSII participants [mean age 48.5 years (s.d. 4.7)]. Mean time from blood draw to RA diagnosis was 7.8 years (s.d. 4.2) for NHS and 4.2 years (s.d. 2.0) for NHSII participants. Meta-analysis of crude and multivariable-adjusted conditional logistic models did not show significant associations between circulating 25(OH)D and RA. However, among NHSII women with blood drawn between 3 months and <4 years prior to RA diagnosis, there was a 20% decreased risk of RA associated with each 1 ng/ml increase in 25(OH)D [OR 0.80 (95% CI 0.64, 0.99)]. CONCLUSION: We did not observe a significant association between circulating 25(OH)D levels and RA, except for among a small subset of NHSII women with levels measured closest to RA diagnosis.

Epidemiology and sociodemographics of systemic lupus erythematosus and lupus nephritis among US adults with Medicaid coverage, 2000-2004.

OBJECTIVE: Systemic lupus erythematosus (SLE) and lupus nephritis (LN) disproportionately affect individuals who are members of racial/ethnic minority groups and individuals of lower socioeconomic status (SES). This study was undertaken to investigate the epidemiology and sociodemographics of SLE and LN in the low-income US Medicaid population. METHODS: We utilized Medicaid Analytic eXtract data, with billing claims from 47 states and Washington, DC, for 23.9 million individuals ages 18-65 years who were enrolled in Medicaid for >3 months in 2000-2004. Individuals with SLE (≥3 visits >30 days apart with an International Classification of Diseases, Ninth Revision [ICD-9] code of 710.0) and with LN (≥2 visits with an ICD-9 code for glomerulonephritis, proteinuria, or renal failure) were identified. We calculated SLE and LN prevalence and incidence, stratified by sociodemographic category, and adjusted for number of American College of Rheumatology (ACR) member rheumatologists in the state and SES using a validated composite of US Census variables. RESULTS: We identified 34,339 individuals with SLE (prevalence 143.7 per 100,000) and 7,388 (21.5%) with LN (prevalence 30.9 per 100,000). SLE prevalence was 6 times higher among women, nearly double in African American compared to white women, and highest in the US South. LN prevalence was higher among all racial/ethnic minority groups compared to whites. The areas with lowest SES had the highest prevalence; areas with the fewest ACR rheumatologists had the lowest prevalence. SLE incidence was 23.2 per 100,000 person-years and LN incidence was 6.9 per 100,000 person-years, with similar sociodemographic trends. CONCLUSION: In this nationwide Medicaid population, there was sociodemographic variation in SLE and LN prevalence and incidence. Understanding the increased burden of SLE and its complications in this low-income population has implications for resource allocation and access to subspecialty care.

Adolescent Health Care Needs and Relationship to Disease in Patients With Childhood-Onset Systemic Lupus Erythematosus.

OBJECTIVE: Our objective was to characterize adolescent health and psychosocial issues in patients with childhood-onset systemic lupus erythematosus (cSLE) and evaluate demographic and disease characteristics associated with adolescent health. METHODS: We retrospectively examined adolescents aged 12 to 18 years with cSLE seen at the Hospital for Sick Children meeting the American College of Rheumatology/Systemic Lupus International Collaborating Clinics classification criteria, assessed by adolescent medicine in the cSLE clinic between 2018 and 2020. Adolescent health issues were characterized using the Home, Education/Employment, Activities, Diet/Drugs, Sexuality, Suicide/mood (HEADDSS) framework. Issues were classified as presenting and/or identified; adolescent health burden was tabulated as the number of distinct adolescent issues per patient. Multiple Poisson regression models examined associations between patient and disease characteristics (age, sex, material deprivation, disease activity, disease damage, and high-dose glucocorticoid exposure) and adolescent health issues. RESULTS: A total of 108 (60%) of 181 adolescents with cSLE were seen by adolescent medicine, with a median of 2 (interquartile range [IQR] 1-3) visits and a median of 2 (IQR 1-5) adolescent health issues during the study period. Common issues were mood (presenting in 21% vs identified in 50%), sleep (27% vs 2%), school and education (26% vs 1%), and nonadherence (23% vs 8%). Psychoeducation was provided by adolescent medicine to 54% of patients. High-dose glucocorticoids (risk ratio [RR] 1.82, 95% confidence interval [CI] 1.41-2.35, P < 0.001), material deprivation (RR 1.17, 95% CI 1.04-1.30, P = 0.007), and lower SLE Disease Activity Index scores (RR 0.95, 95% CI 0.92-0.98, P = 0.004) were associated with higher adolescent health burden. CONCLUSION: Adolescents with cSLE experience many adolescent issues, especially low mood. High-dose glucocorticoids and social marginalization are associated with greater adolescent health burden. This study highlights the importance of addressing adolescent health needs as part of routine care.

Causal relationship between obesity and vitamin D status: bi-directional Mendelian randomization analysis of multiple cohorts.

BACKGROUND: Obesity is associated with vitamin D deficiency, and both are areas of active public health concern. We explored the causality and direction of the relationship between body mass index (BMI) and 25-hydroxyvitamin D [25(OH)D] using genetic markers as instrumental variables (IVs) in bi-directional Mendelian randomization (MR) analysis. METHODS AND FINDINGS: We used information from 21 adult cohorts (up to 42,024 participants) with 12 BMI-related SNPs (combined in an allelic score) to produce an instrument for BMI and four SNPs associated with 25(OH)D (combined in two allelic scores, separately for genes encoding its synthesis or metabolism) as an instrument for vitamin D. Regression estimates for the IVs (allele scores) were generated within-study and pooled by meta-analysis to generate summary effects. Associations between vitamin D scores and BMI were confirmed in the Genetic Investigation of Anthropometric Traits (GIANT) consortium (n = 123,864). Each 1 kg/m(2) higher BMI was associated with 1.15% lower 25(OH)D (p = 6.52×10⁻²7). The BMI allele score was associated both with BMI (p = 6.30×10⁻6²) and 25(OH)D (-0.06% [95% CI -0.10 to -0.02], p = 0.004) in the cohorts that underwent meta-analysis. The two vitamin D allele scores were strongly associated with 25(OH)D (p≤8.07×10⁻57 for both scores) but not with BMI (synthesis score, p = 0.88; metabolism score, p = 0.08) in the meta-analysis. A 10% higher genetically instrumented BMI was associated with 4.2% lower 25(OH)D concentrations (IV ratio: -4.2 [95% CI -7.1 to -1.3], p = 0.005). No association was seen for genetically instrumented 25(OH)D with BMI, a finding that was confirmed using data from the GIANT consortium (p≥0.57 for both vitamin D scores). CONCLUSIONS: On the basis of a bi-directional genetic approach that limits confounding, our study suggests that a higher BMI leads to lower 25(OH)D, while any effects of lower 25(OH)D increasing BMI are likely to be small. Population level interventions to reduce BMI are expected to decrease the prevalence of vitamin D deficiency.

Prevalence, incidence, and demographics of systemic lupus erythematosus and lupus nephritis from 2000 to 2004 among children in the US Medicaid beneficiary population.

OBJECTIVE: To investigate the nationwide prevalence, incidence, and sociodemographics of systemic lupus erythematosus (SLE) and lupus nephritis among children in the US Medicaid beneficiary population. METHODS: Children ages 3 years to <18 years with a diagnosis of SLE (defined as ≥3 claims with an International Classification of Diseases, Ninth Revision [ICD-9] code of 710.0 for SLE, each >30 days apart) were identified from the US Medicaid Analytic eXtract database from 2000 to 2004. This database contains all inpatient and outpatient Medicaid claims for 47 US states and the District of Columbia. Lupus nephritis was identified from ≥2 ICD-9 billing codes for glomerulonephritis, proteinuria, or renal failure, each recorded >30 days apart. The prevalence and incidence of SLE and lupus nephritis were calculated among Medicaid-enrolled children overall and within sociodemographic groups. RESULTS: Of the 30,420,597 Medicaid-enrolled children during these years, 2,959 were identified as having SLE. The prevalence of SLE was 9.73 (95% confidence interval [95% CI] 9.38-10.08) per 100,000 Medicaid-enrolled children. Among the children with SLE, 84% were female, 40% were African American, 25% were Hispanic, 21% were White, and 42% resided in the South region of the US. Moreover, of the children with SLE, 1,106 (37%) had lupus nephritis, representing a prevalence of 3.64 (95% CI 3.43-3.86) per 100,000 children. The average annual incidence of SLE was 2.22 cases (95% CI 2.05-2.40) and that of lupus nephritis was 0.72 cases (95% CI 0.63-0.83) per 100,000 Medicaid enrollees per year. The prevalence and incidence rates of SLE and lupus nephritis increased with age, were higher in girls than in boys, and were higher in all non-White racial/ethnic groups. CONCLUSION: In the current study, the prevalence and incidence rates of SLE among Medicaid-enrolled children in the US are high compared to studies in other populations. In addition, these data represent the first population-based estimates of the prevalence and incidence of lupus nephritis in the US to date.

Maternal Anti-Ro Antibody Titers Obtained With Commercially Available Immunoassays Are Strongly Associated With Immune-Mediated Fetal Heart Disease.

OBJECTIVE: Anti-Ro antibody-positive mothers are frequently referred for serial echocardiography due to the fetal risk of developing heart block and endocardial fibroelastosis. Little is known why only some and not all offspring develop these cardiac manifestations of neonatal lupus (CNL). This prospective study examined associations between anti-Ro antibody titers and fetal CNL. METHODS: Antibody-positive mothers referred since 2018 for fetal echocardiography at risk of CNL (group 1; n = 240) or with CNL (group 2; n = 18) were included. Maternal antibody titers were measured with a chemiluminescent immunoassay (CIA). Additional testing on diluted serum samples was used to quantify anti-Ro 60 antibody titers above the analytical measuring range (AMR) of the standard CIA (≥1,375 chemiluminescent units [CU]). RESULTS: Among 27 total mothers with a fetal diagnosis of CNL, all displayed anti-Ro 60 antibody titers that exceeded the AMR of the CIA at least 10-fold. Of 122 mothers in group 1 who underwent additional anti-Ro 60 antibody testing, event rates of CNL (n = 9) were 0% (0 of 45) among mothers with anti-Ro 60 antibody titers from 1,375-10,000 CU, 5% (3 of 56) among mothers with titers from 10,000-50,000 CU, but 29% (6 of 21) among mothers with titers >50,000 CU (odds ratio 13.1, P = 0.0008). Of mothers in group 2 with a primary diagnosis of CNL, 0% (0 of 18 mothers) had anti-Ro 60 antibody titers <10,000 CU, 44% (8 of 18 mothers) had titers from 10,000-50,000 CU, and 56% (10 of 18 mothers) had titers >50,000 CU. CONCLUSION: CNL is associated with substantially higher anti-Ro antibody titers than are obtained using a standard CIA. Enhancing the assay measuring range allows an improved specificity of identifying pregnancies at risk of CNL.