Cross-decoration of dendritic cells by non-inherited maternal antigen-containing extracellular vesicles: Potential mechanism for PD-L1-based tolerance in cord blood and organ transplantation.

Exposure to non-inherited maternal antigens (NIMA) during the fetal period induces lifelong split tolerance to grafts expressing these allo-antigens. In adult mice, the production of extracellular vesicles (EVs) from maternal microchimeric cells causes cross-decoration (XD) of offspring dendritic cells (DC) with NIMA and upregulation of PD-L1, contributing to NIMA tolerance. To see how this may apply to humans, we tested NIMA acquisition by fetal DCS in human cord blood. The average percentage of NIMA-XD among total DCs was 2.6% for myeloid and 4.5% for Plasmacytoid DC. These cells showed higher PD-L1 expression than their non-XD counterparts (mDC: p = .0016; pDC: p = .024). We detected CD9+ EVs bearing NIMA and PD-L1 in cord blood. To determine if this immune regulatory mechanism persists beyond the pregnancy, we analyzed NIMA-expressing kidney and liver transplant recipients. We found donor antigen XD DCs in peripheral blood and graft-infiltrating DCs. As in cord blood, the pattern of donor antigen expression was punctate, and PD-L1 expression was upregulated, likely due to both protein and miRNA acquired from EV. Our findings support a mechanism for split tolerance to NIMAs that develops during pregnancy and is recapitulated in adult transplant recipients.

Immunogenicity of a rheumatoid arthritis protective sequence when acquired through microchimerism.

HLA class II genes provide the strongest genetic contribution to rheumatoid arthritis (RA). HLA-DRB1 alleles encoding the sequence DERAA are RA-protective. Paradoxically, RA risk is increased in women with DERAA+ children born prior to onset. We developed a sensitive qPCR assay specific for DERAA, and found 53% of DERAA-/- women with RA had microchimerism (Mc; pregnancy-derived allogeneic cells) carrying DERAA (DERAA-Mc) vs. 6% of healthy women. DERAA-Mc quantities correlated with an RA-risk genetic background including DERAA-binding HLA-DQ alleles, early RA onset, and aspects of RA severity. CD4+ T cells showed stronger response against DERAA+ vs. DERAA- allogeneic cell lines in vitro, in line with an immunogenic role of allogeneic DERAA. Results indicate a model where DERAA-Mc activates DERAA-directed T cells that are naturally present in DERAA-/- individuals and can have cross-reactivity against joint antigens. Moreover, we provide an explanation for the enigmatic observation that the same HLA sequence differentially affects RA risk through Mendelian inheritance vs. microchimeric cell acquisition.

Mitochondrial N-formyl methionine peptides associate with disease activity as well as contribute to neutrophil activation in patients with rheumatoid arthritis.

OBJECTIVES: Literature suggests that neutrophils of patients with rheumatoid arthritis (RA) are primed to respond to N-formyl methionine group (formylated peptides). Animal models indicate that formylated peptides contribute to joint damage via neutrophil recruitment and inflammation in joints. Non-steroidal anti-inflammatory drugs are also known to inhibit formyl peptide-induced neutrophil activation. The predominant source of formylated peptides in sterile inflammatory conditions like RA is mitochondria, organelles with prokaryotic molecular signatures. However, there is no direct evidence of mitochondrial formyl peptides (mtNFPs) in the circulation of patients with RA and their potential role in neutrophil-mediated inflammation in RA, including their clinical significance. METHODS: Levels of mtNFPs (total fMet, MT-ND6) were analyzed using ELISA in plasma and serum obtained from patients in 3 cross-sectional RA cohorts (n = 275), a longitudinal inception cohort (n = 192) followed for a median of 8 years, and age/gender-matched healthy controls (total n = 134). Neutrophil activation assays were done in the absence or presence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H. RESULTS: Elevated levels of total fMet were observed in the circulation of patients with RA as compared to healthy controls (p < 0.0001) associating with disease activity and could distinguish patients with the active disease from patients with inactive disease or patients in remission. Baseline levels of total fMet correlated with current and future joint involvement, respectively and predicted the development of rheumatoid nodules (OR = 1.2, p = 0.04). Further, total fMet levels improved the prognostic ability of ACPA in predicting erosive disease (OR of 7.9, p = 0.001). Total fMet levels correlated with markers of inflammation and neutrophil activation. Circulating mtNFPs induced neutrophil activation in vitro through FPR1-dependent mechanisms. CONCLUSIONS: Circulating mtNFPs could be novel biomarkers of disease monitoring and prognosis for RA and in investigating neutrophil-mediated inflammation in RA. We propose, FPR1 as a novel therapeutic target for RA.

Predictors of maternal-origin microchimerism in young women in the Philippines.

OBJECTIVES: Microchimerism is the presence of a small quantity of cells or DNA from a genetically distinct individual. This phenomenon occurs with bidirectional maternal-fetal exchange during pregnancy. Microchimerism can persist for decades after delivery and have long-term health implications. However, little is known about why microchimerism is detectable at varying levels in different individuals. We examine the variability and the following potential determinants of maternal-origin microchimerism (MMc) in young women in the Philippines: gestational duration (in utero exposure to MMc), history of being breastfed (postpartum exposure to MMc), maternal telomere length (maternal cells' ability to replicate and persist), and participant's pregnancies in young adulthood (effect of adding fetal-origin microchimerism to preexisting MMc). MATERIALS AND METHODS: Data are from the Cebu Longitudinal Health and Nutrition Survey, a population-based study of infant feeding practices and long-term health outcomes. We quantified MMc using quantitative PCR (qPCR) in 89 female participants, ages 20-22, and analyzed these data using negative binomial regression. RESULTS: In a multivariate model including all predictors, being breastfed substantially predicted decreased MMc (detection rate ratio = 0.15, p = 0.007), and there was a trend of decreasing MMc in participants who had experienced more pregnancies (detection rate ratio = 0.55, p = 0.057). DISCUSSION: These results might be explained by breastfeeding having lasting impact on immune regulatory networks, thus reducing MMc persistence. MMc may also decrease in response to the introduction of fetal-origin microchimerism with pregnancies experienced in adulthood.

Preeclampsia and scleroderma: a prospective nationwide analysis.

INTRODUCTION: In a preliminary case-control study, women with scleroderma more frequently reported having had hypertensive complications during pregnancy compared with healthy women. MATERIAL AND METHODS: To prospectively investigate this possible association, we conducted a nation-wide cohort analysis of a major hypertensive complication during pregnancy, namely preeclampsia, and later scleroderma. Analyses were based on Danish register-based birth and hospital contact data on preeclampsia and scleroderma. We followed 778,758 women from time of giving birth between 1978 and 2010 to end of follow-up, emigration, death, or scleroderma diagnosis, whichever occurred first. The association was evaluated by incidence rate ratios, obtained in Poisson regression models. RESULTS: We report that preeclampsia is associated with a 69% significantly increased risk of later developing scleroderma. CONCLUSIONS: Though these findings do not impact clinical care directly, the association of preeclampsia with scleroderma underscores the significant relation of preeclampsia and other adverse pregnancy outcomes with later disease in women and should be included in patient counseling and education.

Maternal Microchimerism Predicts Increased Infection but Decreased Disease due to Plasmodium falciparum During Early Childhood.

Background: A mother's infection with placental malaria (PM) can affect her child's susceptibility to malaria, although the mechanism remains unclear. The fetus acquires a small amount of maternal cells and DNA known as maternal microchimerism (MMc), and we hypothesized that PM increases MMc and that MMc alters risk of Plasmodium falciparum malaria during infancy. Methods: In a nested cohort from Muheza, Tanzania, we evaluated the presence and level of cord blood MMc in offspring of women with and without PM. A maternal-specific polymorphism was identified for each maternal-infant pair, and MMc was assayed by quantitative polymerase chain reaction. The ability of MMc to predict malaria outcomes during early childhood was evaluated in longitudinal models. Results: Inflammatory PM increased the detection rate of MMc among offspring of primigravidae and secundigravidae, and both noninflammatory and inflammatory PM increased the level of MMc. Detectable MMc predicted increased risk of positive blood smear but, interestingly, decreased risk of symptomatic malaria and malaria hospitalization. Conclusions: The acquisition of MMc may result in increased malaria infection but protection from malaria disease. Future studies should be directed at the cellular component of MMc, with attention to its ability to directly or indirectly coordinate anti-malarial immune responses in the offspring.

Statistical Methods for Unusual Count Data: Examples From Studies of Microchimerism.

Natural acquisition of small amounts of foreign cells or DNA, referred to as microchimerism, occurs primarily through maternal-fetal exchange during pregnancy. Microchimerism can persist long-term and has been associated with both beneficial and adverse human health outcomes. Quantitative microchimerism data present challenges for statistical analysis, including a skewed distribution, excess zero values, and occasional large values. Methods for comparing microchimerism levels across groups while controlling for covariates are not well established. We compared statistical models for quantitative microchimerism values, applied to simulated data sets and 2 observed data sets, to make recommendations for analytic practice. Modeling the level of quantitative microchimerism as a rate via Poisson or negative binomial model with the rate of detection defined as a count of microchimerism genome equivalents per total cell equivalents tested utilizes all available data and facilitates a comparison of rates between groups. We found that both the marginalized zero-inflated Poisson model and the negative binomial model can provide unbiased and consistent estimates of the overall association of exposure or study group with microchimerism detection rates. The negative binomial model remains the more accessible of these 2 approaches; thus, we conclude that the negative binomial model may be most appropriate for analyzing quantitative microchimerism data.

Incidence, risk factors, and outcomes of sclerosis in patients with chronic graft-versus-host disease.

Sclerotic chronic graft-versus-host disease (GVHD) can result in disability after allogeneic hematopoietic cell transplantation. We assessed the incidence and risk factors of sclerosis and its association with transplant outcomes among 977 consecutive patients treated with systemic immunosuppression for chronic GVHD. Sclerosis was defined when cutaneous sclerosis, fasciitis, or joint contracture was first documented in the medical record. Seventy (7%) patients presented with sclerosis at the time of initial systemic treatment for chronic GVHD, and the cumulative incidence of sclerosis increased to 20% at 3 years. Factors associated with an increased risk of sclerosis included the use of a mobilized blood cell graft and a conditioning regimen with > 450 cGy total body irradiation. Factors associated with a decreased risk of sclerosis included the use of an HLA-mismatched donor and a major ABO-mismatched donor. Development of sclerosis was associated with longer time to withdrawal of immunosuppressive treatment but not with risks of overall mortality, nonrelapse mortality, or recurrent malignancy. We found a substantial incidence of sclerosis in patients with chronic GVHD. Development of sclerosis can cause disability but does not affect mortality or recurrent malignancy in patients with chronic GVHD.

The otherness of self: microchimerism in health and disease.

Microchimerism (Mc) refers to the harboring of a small number of cells (or DNA) that originated in a different individual. Naturally acquired Mc derives primarily from maternal cells in her progeny, or cells of fetal origin in women. Both maternal and fetal Mc are detected in hematopoietic cells including T and B cells, monocyte/macrophages, natural killer (NK) cells and granulocytes. Mc appears also to generate cells such as myocytes, hepatocytes, islet ß cells and neurons. Here, the detrimental and beneficial potential of Mc is examined. The prevalence, diversity and durability of naturally acquired Mc, including in healthy individuals, indicates that a shift is needed from the conventional paradigm of 'self versus other' to a view of the normal 'self' as constitutively chimeric.

Intrauterine environment and multiple sclerosis: a population- based case-control study.

BACKGROUND: Associations of several autoimmune disorders with intrauterine and early life exposures have been reported. OBJECTIVE: We used population-based linked hospital discharge-birth records data to explore maternal, prenatal, and infant characteristics in relation to MS-related hospitalization among Washington State residents. METHODS: 272 cases hospitalized for MS during 1988-2008 and 2720 birth record controls were identified from linked hospital discharge-birth certificate data. Exposure information from their birth records were compared in a population-based case-control study to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95%CIs) for associations with MS hospitalization. RESULTS: Most factors examined were not associated with MS. Having a mother with 3+ prior live births (OR 0.54, 95%CI 0.31-0.95) or having 3+ older siblings (OR 0.49, 95%CI 0.28-0.85) were negatively associated. Maternal prenatal smoking (OR 3.09, 95%CI 1.22-7.84) was positively associated. CONCLUSION: Transplacental exposure to smoke constituents including chemicals affecting myelin may help explain any association with maternal prenatal smoking; however, we were unable to assess childhood or adult smoke exposures which may also account at least partly for this effect. The negative associations observed with greater maternal parity and number of siblings are consistent with some other studies. Reasons for these associations may involve various pathways.

Microchimerism in the rheumatoid nodules of patients with rheumatoid arthritis.

OBJECTIVE: The rheumatoid nodule is a lesion commonly found on extraarticular areas prone to mechanic trauma. When present with inflammatory symmetric polyarthritis, it is pathognomonic of rheumatoid arthritis (RA), an autoimmune disease in which naturally acquired microchimerism has previously been described and can sometimes contribute to RA risk. Since RA patients harbor microchimerism in the blood, we hypothesized that microchimerism is also present in rheumatoid nodules and could play a role in rheumatoid nodule formation. This study was undertaken to investigate rheumatoid nodules for microchimerism. METHODS: Rheumatoid nodules were tested for microchimerism by real-time quantitative polymerase chain reaction (qPCR). The rheumatoid nodules of 29 female patients were tested for a Y chromosome-specific sequence. After HLA genotyping of patients and family members, rheumatoid nodules from 1 man and 14 women were tested by HLA-specific qPCR, targeting a nonshared HLA allele of the potential microchimerism source. Results were expressed as genome equivalents of microchimeric cells per 10(5) patient genome equivalents (GE/10(5)). RESULTS: Rheumatoid nodules from 21% of the female patients contained male DNA (range <0.5, 10.3 GE/10(5)). By HLA-specific qPCR, 60% of patients were microchimeric (range 0, 18.5 GE/10(5)). Combined microchimerism prevalence was 47%. A fetal or maternal source was identified in all patients who tested positive by HLA-specific qPCR. Unexpectedly, a few rheumatoid nodules also contained microchimerism without evidence of a fetal or maternal source, suggesting alternative sources. CONCLUSION: Our findings indicate that microchimerism is frequently present in the rheumatoid nodules of RA patients. Since microchimerism is genetically disparate, whether microchimerism in rheumatoid nodules serves as an allogeneic stimulus or allogeneic target warrants further investigation.

Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation.

Increasing mixed chimerism (reemerging recipient cells) after allogeneic hematopoietic cell transplant (allo-HCT) can indicate relapse, the leading factor determining mortality in blood malignancies. Most clinical chimerism tests have limited sensitivity and are primarily designed to monitor engraftment. We developed a panel of quantitative polymerase chain reaction assays using TaqMan chemistry capable of quantifying chimerism in the order of 1 in a million. At such analytic sensitivity, we hypothesized that it could inform on relapse risk. As a proof-of-concept, we applied our panel to a retrospective cohort of patients with acute leukemia who underwent allo-HCT with known outcomes. Recipient cells in bone marrow aspirates (BMAs) remained detectable in 97.8% of tested samples. Absolute recipient chimerism proportions and rates at which these proportions increased in BMAs in the first 540 days after allo-HCT were associated with relapse. Detectable measurable residual disease (MRD) via flow cytometry in BMAs after allo-HCT showed limited correlation with relapse. This correlation noticeably strengthened when combined with increased recipient chimerism in BMAs, demonstrating the ability of our ultrasensitive chimerism assay to augment MRD data. Our technology reveals an underappreciated usefulness of clinical chimerism. Used side by side with MRD assays, it promises to improve identification of patients with the highest risk of disease reoccurrence for a chance of early intervention.

Prediction of Erosive Disease Development by Antimitochondrial Antibodies in Rheumatoid Arthritis.

OBJECTIVE: Mitochondria are found in the extracellular space in rheumatoid arthritis (RA). However, whether mitochondria are a source of autoantigens in RA has not been carefully addressed. Thus, we undertook this study to investigate the presence and significance of antimitochondrial antibodies (AMAs) in patients with RA. METHODS: AMAs were measured in serum samples from 3 cross-sectional cohorts of RA patients (n = 95, n = 192, and n = 117) and healthy individuals (n = 38, n = 72, and n = 50) using a flow cytometry-based assay. Further, AMAs were detected using an anti-mitofusin-1 (anti-MFN-1) IgG enzyme-linked immunosorbent assay and Western blot analysis. A longitudinal inception cohort, followed up for a median of 8 years, was used to study disease progression. RESULTS: AMA levels were elevated in RA patients from all 3 cohorts as compared to healthy individuals (P < 0.001, P < 0.05, and P < 0.01), with a range of 14-26% positivity. Levels of anti-MFN-1 antibodies correlated with AMA levels (r = 0.31, P = 0.006) and were elevated in RA patients as compared to healthy individuals (P < 0.001). The presence of AMAs was associated with erosive disease (P < 0.05) and interstitial lung disease (P < 0.01). Further, AMA levels were found to predict erosive disease (odds ratio [OR] 4.59, P = 0.006) and joint space narrowing (OR 3.08, P = 0.02) independent of anti-citrullinated protein antibodies. Finally, anti-MFN-1 antibodies identified seronegative patients developing erosive disease (OR 9.33; P = 0.02). CONCLUSION: Our findings demonstrate the presence of novel autoantibodies targeting mitochondria in the setting of RA. AMAs were used to stratify patients based on disease phenotype and to predict development of erosive disease, including in patients with seronegative disease. Our results highlight the essential role of mitochondria in the pathogenesis of RA and suggest a possible benefit of therapies targeting mitochondrial-mediated inflammation and clearance in these patients.

Increased fetal microchimerism in immune and stem cell subsets in preeclampsia.

PROBLEM: Preeclampsia (PE) is associated with an increased risk of maternal cardiovascular disease (CVD), however, it is unclear whether this is due to shared underlying physiology or changes which occur during the disease process. Fetal microchimerism (FMc) within the maternal circulation can durably persist decades after pregnancy, is known to occur at greater frequency in PE, and can potentially affect local and systemic immune programming, thus changes in cellular FMc may provide a mechanism for long-term health outcomes associated with PE. METHOD OF STUDY: We investigated whether PE is associated with alterations in FMc immune and stem cell populations. We analyzed maternal peripheral blood mononuclear cells (PBMC) from PE cases (n = 16) and matched controls from normal pregnancies (n = 16), from which immune and stem cell subsets were isolated by flow cytometry. Genomic DNA was extracted from total PMBC and individual cell subsets, and FMc frequency was quantified by quantitative polymerase chain reaction assays targeting a fetal-specific non-shared polymorphism identified from family genotyping. RESULTS: There was a significant increase in FMc concentration in immune cell subsets in PE cases compared to controls, predominantly in B cell, and NK cell lymphocyte populations. There was no significant difference in FMc frequency or concentration within the stem cell population between PE and controls. CONCLUSIONS: The altered concentrations of immune cells within FMc in the maternal blood provides a potential mechanism for the inflammation which occurs during PE to induce long-lasting changes to the maternal immune system and may potentially promote chronic maternal disease.

Pregnancy-associated systemic gene expression compared to a pre-pregnancy baseline, among healthy women with term pregnancies.

Background: Pregnancy is known to induce extensive biological changes in the healthy mother. Little is known, however, about what these changes are at the molecular level. We have examined systemic expression changes in protein-coding genes and long non-coding (lnc) RNAs during and after pregnancy, compared to before pregnancy, among healthy women with term pregnancies. Methods: Blood samples were collected from 14 healthy women enrolled in our prospective pregnancy cohort at 7 time-points (before, during and after pregnancy). Total RNA from frozen whole blood was used for RNA sequencing. Following raw read alignment and assembly, gene-level counts were obtained for protein-coding genes and long non-coding RNAs. At each time-point, cell type proportions were estimated using deconvolution. To examine associations between pregnancy status and gene expression over time, Generalized Estimating Equation (GEE) models were fitted, adjusting for age at conception, and with and without adjusting for changes in cell type proportions. Fold-changes in expression at each trimester were examined relative to the pre-pregnancy baseline. Results: Numerous immune-related genes demonstrated pregnancy-associated expression, in a time-dependent manner. The genes that demonstrated the largest changes in expression included several that were neutrophil-related (over-expressed) and numerous immunoglobulin genes (under-expressed). Estimated cell proportions revealed a marked increase in neutrophils, and less so of activated CD4 memory T cells, during pregnancy, while most other cell type proportions decreased or remained unchanged. Adjusting for cell type proportions in our model revealed that although most of the expression changes were due to changes in cell type proportions in the bloodstream, transcriptional regulation was also involved, especially in down-regulating expression of type I interferon inducible genes. Conclusion: Compared to a pre-pregnancy baseline, there were extensive systemic changes in cell type proportions, gene expression and biological pathways associated with different stages of pregnancy and postpartum among healthy women. Some were due to changes in cell type proportions and some due to gene regulation. In addition to providing insight into term pregnancy among healthy women, these findings also provide a "normal" reference for abnormal pregnancies and for autoimmune diseases that improve or worsen during pregnancy, to assess deviations from normal.

Pre-pregnancy gene expression signatures are associated with subsequent improvement/worsening of rheumatoid arthritis during pregnancy.

BACKGROUND: While many women with rheumatoid arthritis (RA) improve during pregnancy and others worsen, there are no biomarkers to predict this improvement or worsening. In our unique RA pregnancy cohort that includes a pre-pregnancy baseline, we have examined pre-pregnancy gene co-expression networks to identify differences between women with RA who subsequently improve during pregnancy and those who worsen. METHODS: Blood samples were collected before pregnancy (T0) from 19 women with RA and 13 healthy women enrolled in our prospective pregnancy cohort. RA improvement/worsening between T0 and 3rd trimester was assessed by changes in the Clinical Disease Activity Index (CDAI). Pre-pregnancy expression profiles were examined by RNA sequencing and differential gene expression analysis. Weighted gene co-expression network analysis (WGCNA) was used to identify co-expression modules correlated with the improvement/worsening of RA during pregnancy and to assess their functional relevance. RESULTS: Of the 19 women with RA, 14 improved during pregnancy (RAimproved) while 5 worsened (RAworsened). At the T0 baseline, however, the mean CDAI was similar between the two groups. WGCNA identified one co-expression module related to B cell function that was significantly correlated with the worsening of RA during pregnancy and was significantly enriched in genes differentially expressed between the RAimproved and RAworsened groups. A neutrophil-related expression signature was also identified in the RAimproved group at the T0 baseline. CONCLUSION: The pre-pregnancy gene expression signatures identified represent potential biomarkers to predict the subsequent improvement/worsening of RA during pregnancy, which has important implications for the personalized treatment of RA during pregnancy.

Effect of parity on fetal and maternal microchimerism: interaction of grafts within a host?

Small amounts of genetically foreign cells (microchimerism, Mc) traffic between a mother and fetus during pregnancy. Commonly, these grafts durably persist. For women, multiple naturally acquired Mc grafts can accrue, as they harbor Mc from their own mothers (maternal Mc, MMc) and subsequently acquire fetal Mc (FMc) through pregnancy. The nature of interactions between these naturally acquired grafts may inform, and be informed by, observations in transplantation, including the effect of noninherited maternal HLA antigens (NIMA) and double-unit cord blood transplantation (CBT). We asked whether FMc and MMc are impacted by the addition of new grafts as evaluated by increasing parity. Mc was identified by quantitative PCR for a nonshared polymorphism unique to the Mc source. Despite increasing sources of Mc, FMc did not increase with increasing parity. MMc concentration was significantly lower with increasing parity. The odds ratio for detection of MMc for 2 or more births compared with 1 birth was .11 (95% CI 0.03-0.42, P = .001). These observations suggest that interactions occur among naturally acquired grafts and are of interest in light of recent observations of graft-graft interaction resulting in predominance of 1 unit in double-unit CBT and the correlation of MMc with the NIMA effect.

Acquisition of the rheumatoid arthritis HLA shared epitope through microchimerism.

OBJECTIVE: HLA-DRB1 alleles associated with risk of rheumatoid arthritis (RA) encode similar HLA-DRB1 sequences, called the shared epitope (SE). The most common SE sequences are QKRAA and QRRAA. Nevertheless, a substantial number of RA patients lack the SE. Bidirectional fetal-maternal trafficking results in long-term persistence of fetal cells in the mother and maternal cells in her offspring, a process known as microchimerism. This study was undertaken to discover whether RA patients who lack the SE can acquire it through microchimerism. METHODS: We studied a total of 86 female subjects who were genotypically negative for the SE, comprising 52 patients with RA and 34 healthy controls. We developed specific real-time quantitative polymerase chain reaction assays for the SE-encoded sequences QKRAA and QRRAA, and used them to test DNA extracted from peripheral blood mononuclear cells. RESULTS: Microchimerism with the SE was found significantly more often in RA patients than controls (odds ratio 4.1 [95% confidence interval 1.6-10.0], P = 0.003). Concentrations of SE microchimerism were also significantly higher among RA patients than controls (P = 0.002). In separate analyses for SE type, the prevalence of QKRAA microchimerism in RA patients versus healthy controls was 17% versus 3% (9 of 52 versus 1 of 34; P = 0.03) and the prevalence of QRRAA microchimerism was 40% versus 18% (21 of 52 versus 6 of 34; P = 0.04), respectively. Microchimerism concentrations were also higher in RA patients than healthy subjects for QKRAA (P = 0.03) and QRRAA (P = 0.03). CONCLUSION: These results indicate that RA patients who genotypically lack the SE can acquire the SE as persistent microchimerism from fetal-maternal cell exchange, suggesting that SE-encoding microchimerism could be a risk factor for RA.

Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms.

Genomic chimerism represents co-existing cells with different genotypes and has diagnostic significance in transplant engraftment monitoring, residual cancer detection, and other contexts. We previously described an approach to chimerism detection by interrogating variably present or absent genomic loci using single-molecule molecular inversion probes (smMIPs) and next-generation sequencing, which provided ultrasensitive limits of detection (<1 in 10,000 cells) but was not reliably quantitative. Herein, smMIP testing was modified to accurately quantitate chimeric cells by incorporating copy number neutral control loci for data normalization and computationally modeling cell mixtures from individual-specific genotypes. Data demonstrate precision and accuracy over three orders of magnitude (0.01% to 50% chimerism). Seventy hematopoietic stem cell transplant specimens from single (n = 42) or double (n = 28) donors were evaluated, benchmarking smMIP against conventional variable number tandem repeat (VNTR) analysis and an unrelated, ultrasensitive polymorphism-specific quantitative PCR (PS-qPCR) assay. Quantitative concordance of all three assays was high (P < 0.0005, Pearson correlation coefficient), although smMIP correlated better with VNTR testing than PS-qPCR. smMIP and PS-qPCR collectively identified low-level chimerism in all specimens testing negative by VNTR (n = 41 and n = 45 of 48 specimens, respectively). This work demonstrates the feasibility of smMIP-based chimerism testing for quantitative and ultrasensitive measurement of genomic chimerism at practical levels approaching one in one million cells, and cross-validates the approach.