Nancy A. Shadick

Principal components analysis corrects for stratification in genome-wide association studies

Population stratification—allele frequency differences between cases and controls due to systematic ancestry differences—can cause spurious associations in disease studies. We describe a method that enables explicit detection and correction of population stratification on a genome-wide scale. Our method uses principal components analysis to explicitly model ancestry differences between cases and controls. The resulting correction is specific to a candidate markers variation in frequency across ancestral populations, minimizing spurious associations while maximizing power to detect true associations. Our simple, efficient approach can easily be applied to disease studies with hundreds of thousands of markers.

Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci

To identify new genetic risk factors for rheumatoid arthritis, we conducted a genome-wide association study meta-analysis of 5,539 autoantibody-positive individuals with rheumatoid arthritis (cases) and 20,169 controls of European descent, followed by replication in an independent set of 6,768 rheumatoid arthritis cases and 8,806 controls. Of 34 SNPs selected for replication, 7 new rheumatoid arthritis risk alleles were identified at genome-wide significance (P < 5 × 10−8) in an analysis of all 41,282 samples. The associated SNPs are near genes of known immune function, including IL6ST, SPRED2, RBPJ, CCR6, IRF5 and PXK. We also refined associations at two established rheumatoid arthritis risk loci (IL2RA and CCL21) and confirmed the association at AFF3. These new associations bring the total number of confirmed rheumatoid arthritis risk loci to 31 among individuals of European ancestry. An additional 11 SNPs replicated at P < 0.05, many of which are validated autoimmune risk alleles, suggesting that most represent genuine rheumatoid arthritis risk alleles.

Two independent alleles at 6q23 associated with risk of rheumatoid arthritis

To identify susceptibility alleles associated with rheumatoid arthritis, we genotyped 397 individuals with rheumatoid arthritis for 116,204 SNPs and carried out an association analysis in comparison to publicly available genotype data for 1,211 related individuals from the Framingham Heart Study. After evaluating and adjusting for technical and population biases, we identified a SNP at 6q23 (rs10499194, ∼150 kb from TNFAIP3 and OLIG3) that was reproducibly associated with rheumatoid arthritis both in the genome-wide association (GWA) scan and in 5,541 additional case-control samples (P = 10−3, GWA scan; P < 10−6, replication; P = 10−9, combined). In a concurrent study, the Wellcome Trust Case Control Consortium (WTCCC) has reported strong association of rheumatoid arthritis susceptibility to a different SNP located 3.8 kb from rs10499194 (rs6920220; P = 5 × 10−6 in WTCCC). We show that these two SNP associations are statistically independent, are each reproducible in the comparison of our data and WTCCC data, and define risk and protective haplotypes for rheumatoid arthritis at 6q23.

Common variants at CD40 and other loci confer risk of rheumatoid arthritis

To identify rheumatoid arthritis risk loci in European populations, we conducted a meta-analysis of two published genome-wide association (GWA) studies totaling 3,393 cases and 12,462 controls. We genotyped 31 top-ranked SNPs not previously associated with rheumatoid arthritis in an independent replication of 3,929 autoantibody-positive rheumatoid arthritis cases and 5,807 matched controls from eight separate collections. We identified a common variant at the CD40 gene locus (rs4810485, P = 0.0032 replication, P = 8.2 × 10−9 overall, OR = 0.87). Along with other associations near TRAF1 (refs. 2,3) and TNFAIP3 (refs. 4,5), this implies a central role for the CD40 signaling pathway in rheumatoid arthritis pathogenesis. We also identified association at the CCL21 gene locus (rs2812378, P = 0.00097 replication, P = 2.8 × 10−7 overall), a gene involved in lymphocyte trafficking. Finally, we identified evidence of association at four additional gene loci: MMEL1-TNFRSF14 (rs3890745, P = 0.0035 replication, P = 1.1 × 10−7 overall), CDK6 (rs42041, P = 0.010 replication, P = 4.0 × 10−6 overall), PRKCQ (rs4750316, P = 0.0078 replication, P = 4.4 × 10−6 overall), and KIF5A-PIP4K2C (rs1678542, P = 0.0026 replication, P = 8.8 × 10−8 overall).

HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software

We present a robust, fully automatable technology platform that includes computer software for the detailed analysis of low femtomoles of N-linked sugars released from glycoproteins. Features include (i) sample immobilization in 96-well plates, glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence, linkage, and arm-specific information for charged and neutral glycans; (iii) automatic structural assignment of peaks from HPLC profiles via web-based software that accesses our database (GlycoBase) of more than 350 N-glycan structures, including 117 present in the human serum glycome; and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions to produce a refined list of final structures. The N-glycans from a plate of 96 samples can be released and purified in 2 or 3 days and profiled in 2 days. This strategy can be used for (i) identification and screening of disease biomarkers and (ii) monitoring the production of therapeutic glycoproteins, allowing optimization of production conditions. This technology is also suitable for preparing released glycans for other analytical techniques. Here we demonstrate its application to rheumatoid arthritis using 5 microl of patient serum.

De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot

Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. Through a genome-wide survey of 114 subjects with TOF and their unaffected parents, we identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. We then examined a second, independent TOF cohort (n = 398) for additional CNVs at these loci. We identified CNVs at chromosome 1q21.1 in 1% (5/512, P = 0.0002, OR = 22.3) of nonsyndromic sporadic TOF cases. We also identified recurrent CNVs at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single subject with TOF occurred at six loci, two that encode known (NOTCH1, JAG1) disease-associated genes. Our findings predict that at least 10% (4.5–15.5%, 95% confidence interval) of sporadic nonsyndromic TOF cases result from de novo CNVs and suggest that mutations within these loci might be etiologic in other cases of TOF.

Genetic variants at CD28, PRDM1, and CD2/CD58 are associated with rheumatoid arthritis risk

To discover new rheumatoid arthritis (RA) risk loci, we systematically examined 370 SNPs from 179 independent loci with P < 0.001 in a published meta-analysis of RA genome-wide association studies (GWAS) of 3,393 cases and 12,462 controls. We used Gene Relationships Across Implicated Loci (GRAIL), a computational method that applies statistical text mining to PubMed abstracts, to score these 179 loci for functional relationships to genes in 16 established RA disease loci. We identified 22 loci with a significant degree of functional connectivity. We genotyped 22 representative SNPs in an independent set of 7,957 cases and 11,958 matched controls. Three were convincingly validated: CD2-CD58 (rs11586238, P = 1 × 10−6 replication, P = 1 × 10−9 overall), CD28 (rs1980422, P = 5 × 10−6 replication, P = 1 × 10−9 overall) and PRDM1 (rs548234, P = 1 × 10−5 replication, P = 2 × 10−8 overall). An additional four were replicated (P < 0.0023): TAGAP (rs394581, P = 0.0002 replication, P = 4 × 10−7 overall), PTPRC (rs10919563, P = 0.0003 replication, P = 7 × 10−7 overall), TRAF6-RAG1 (rs540386, P = 0.0008 replication, P = 4 × 10−6 overall) and FCGR2A (rs12746613, P = 0.0022 replication, P = 2 × 10−5 overall). Many of these loci are also associated to other immunologic diseases.

The long-term clinical outcomes of Lyme disease. A population-based retrospective cohort study.

Lyme borreliosis is a tick-borne infection caused by the spirochete Borrelia burgdorferi [1-3]. The disease usually begins with erythema migrans accompanied by viral-like or meningitis-like symptoms. Weeks later meningitis, facial palsy, atrioventricular nodal block, or migratory musculoskeletal pain may develop, followed months to years later by episodes of frank arthritis, encephalopathy, polyneuropathy, or acrodermatitis [4]. Lyme disease is now the most common vector-borne disease in the United States; nearly 50 000 patients have been diagnosed with it since 1982 [5]. Musculoskeletal and neurologic sequelae may occur from Lyme disease. Some of the late consequences of Lyme disease, such as oligoarticular arthritis, axonal polyneuropathy, or active encephalopathy, are thought to be caused by persistent spirochetal infection and are amenable to antibiotic treatment [6-8]. Other syndromes such as persistent arthritis, fibromyalgia, subtle joint pain, or mild encephalopathy do not improve with antibiotic treatment, suggesting a mechanism other than active infection [9-12]. We studied persons residing in an endemic coastal area of Massachusetts who were previously infected with B. burgdorferi in the early 1980s [13]. They contracted Lyme disease while the clinical syndromes and optimal antibiotic therapies were still evolving, which offered a natural experiment for the identification of risk factors for Lyme disease sequelae. We ascertained the prevalence of persistent symptoms in unselected patients with a history of Lyme disease; ascertained their rheumatologic, neurologic, and health status outcomes; and identified potential risk factors for these long-term sequelae. Methods Participants In February l991, we did a follow-up analysis of residents of Argylla Road in Ipswich, Massachusetts, an endemic coastal area for Lyme disease. The incidence and clinical course of Lyme disease among residents of this area have been reported previously [13]. Participants were recruited by calling consecutive households located in the Argylla Road area, the epicenter of infection, to ask if they would be interested in enrolling in a study about Lyme disease in their area. Potential participants were told that the study involved a history, physical examination, and serologic analysis for Lyme disease. Information about whether a person ever had a previous diagnosis of Lyme disease was obtained and used to assign tentative status (with or without Lyme disease) for study participants. We recruited participants until we had 50 tentative persons with Lyme disease and 50 tentative controls. Once the potential Lyme disease group was filled, calls were made consecutively to fill the potential control group. Residents 18 years of age or older were invited to participate in the study. This protocol was approved by the Brigham & Womens Hospital Committee for the Protection of Human Subjects. Confirmation of Lyme Disease For inclusion in the Lyme group, persons needed a previous diagnosis of Lyme disease by a physician and needed to fulfill the Centers for Disease Control and Prevention (CDC) criteria for Lyme disease (a history of physician-documented erythema migrans or a late manifestation of Lyme disease confirmed by a positive Lyme serologic test result, or both [14]). This information was obtained through patient interview and then medical record review to determine if patients fulfilled criteria for Lyme disease. Previous study records, local physician reports, and previous serologic test results were available for confirmation of Lyme disease. Persons without a previous clinical history of Lyme disease were classified as controls. The status of the participants (with or without Lyme disease) was determined independent of the clinical assessment, using a protocol that did not include any outcome data. Assessment of Clinical Outcomes A blinded investigator determined outcomes in a standardized manner independent of Lyme disease status. All patients completed a standardized questionnaire, had electrocardiography, and had a neuropsychological battery of tests. The questionnaire included data on demographics, comorbidity, education, review of systems, medications, memory and cognitive function, and the Short Form-36 health status measure (a reliable, previously validated measure of physical, psychological, social, and role functions [15]). A physical examination was done by one observer blinded to Lyme disease status. It included a joint examination (the American College of Rheumatology Glossary examination) that measured swelling and pain through passive range of motion [16] and a neurologic evaluation of strength and deep tendon reflexes, light touch, and vibration sensation with a 128-Hz tuning fork (at the elbow, wrist, fibula, and ankles). Pain and swelling indices from the joint examination (the American College of Rheumatology Glossary examination) were summed and recorded as a global score. A vibration test result of a distal gradient was considered present if the participant reported diminished vibratory sensation at a distal compared with proximal site. Each participant had an electrocardiographic study that was interpreted blindly by a cardiologist uninvolved with the clinical assessment. All outcomes were determined by one investigator who had no knowledge of whether participants were in the Lyme or control groups. The neuropsychological battery of tests measured immediate and delayed verbal memory, attention, conceptualization, fine motor dexterity, and perceptual discrimination. Tests included the California Verbal Learning Test [17], Wechsler Memory Scale (visual reproduction and verbal paired associates subtests [18]), Shipley abstraction subtest [19], Stroop test [20], Trailmaking test [21], and Purdue Pegboard Test [22]. The California Verbal Learning Test measures verbal memory. Participants are asked to learn a list of 16 words during five trials; recall on the fifth trial is recorded (trial 5). This is then followed by a distracter list. The original list is recalled after the distracter list is learned (short recall) and then recalled again after a 20-minute delay (long recall). This is a challenging test of memory for patients with superior premorbid experience. Normative values are available for young and elderly adults [17]; the range of normal is between 11 and 15 words for trial 5 and is between 10 and 15 words for the long-recall subtest for persons between 45 and 54 years of age. A clinically significant change in the California Verbal Learning Test would be recalling 4 more words or 4 fewer words. All tests were administered according to published procedures. Test scores were transformed into standard scores calculated from published, age-corrected normative data. Participants with a score of 2 or more SDs from age-adjusted means were considered impaired. All results were reviewed by a neuropsychologist (RK) who was not involved with the participants evaluation, to determine those patients who were in need of further clinical evaluation. Participants with swelling or pain (joint examination test result), evidence of a distal gradient (vibration test result) or persistent symptoms of paresthesias in an extremity, or impairment on two or more neurocognitive tests were sent for further clinical evaluation. Nine patients were evaluated at the Lyme disease clinic at Tufts-New England Medical Center and 4 were evaluated by other neurologist or rheumatologist consultants to determine if these abnormal screening test results were accompanied by objective findings. This evaluation included lumbar puncture, electrophysiologic studies, magnetic resonance imaging, detailed neuropsychological tests [8], joint radiographs, or arthrocentesis. Serologic Evaluation All patients had serologic testing after the history and examination. Serum samples were stored at 70C and were tested for IgG antibodies to B. burgdorferi by indirect enzyme-linked immunosorbent assay (ELISA [23]); for IgM, IgG, and IgA antibodies to the spirochete by antibody-capture enzyme immunoassays [24]; and for the pattern of IgG reactivity to spirochetal polypeptides by Western blotting. In general, Western blot reactivity varied with the degree and duration of dissemination of Lyme disease. For example, patients with early localized infection or erythema migrans might react to only 2 to 8 B. burgdorferi polypeptides, those with meningitis might react to at least 8 to 14 polypeptides, and those with arthritis or late central nervous system disease might react to as many as 18 to 25 polypeptides (Berardi VP. Personal communication). The isolate used for antigen preparations was the B. burgdorferi G39/40 strain obtained through low passage [24]. Indirect ELISA titers greater than 400 and ELISA capture ratios (sample optical density/control optical density) of 1.0 or more were considered as increased test results. Western blot reactivity to five or more B. burgdorferi-specific polypeptides indicated previous infection [25]. Silver Stain Method The Dieterle silver impregnation stain used was a modification made by one of us (PHD) in 1985 [26]. This standard approach has yielded a constant clean yellow background of cerebral cortex sections with no silver impregnation of anatomic neural processes and dendrites. Spirochetes are easily seen as black to blue-black cells against the yellow tissue. Specificity for nonstaining of normal tissue fibers (procollagen, elastin, basement membrane material, and neural dendrites and filaments) and documentation of the cytologic structure of Borrelia spirochete strains were further tested in a large extended study [27]. Controls routinely used in each stain assay consisted of NP40 strain that was injected into human normal breast tissue removed for cosmetic surgery and was paraffin-embedded in the usual manner (negative control), and rat gonad tissue infected with the Reiter strain of treponemal spirochetes (positive c

The natural history of exercise-induced anaphylaxis: survey results from a 10-year follow-up study.

BACKGROUND Exercise-induced anaphylaxis (EIA) is a unique physical allergy that is triggered by exertion, the clinical spectrum and modifying factors of which have been previously studied. At the time of initial description, it was postulated that other factors contributed to this disorder. OBJECTIVE We sought to determine the clinical course and potential modifying factors in EIA. METHODS In 1993, we conducted a cross-sectional analysis of 671 individuals with exercise-associated symptoms for more than a decade using a validated 75-item questionnaire. Subjects met criteria for EIA if they had anaphylactic symptoms, including hypotension or upper airway obstruction, urticaria, or angioedema with physical exertion but without a passive increase in core body temperature. RESULTS Of 365 (54%) questionnaire respondents, 279 (87%) met criteria for EIA (199 females and 80 males). At the time of study entry, subjects with EIA (mean age, 37.5 years; range, 13 to 77 years) had an average of 10.6 years of symptoms, which were most frequently triggered by aerobic activities such as jogging or brisk walking (78% and 42%, respectively). On average, subjects reported that the frequency of attacks had decreased (47% of subjects) or stabilized (46% of subjects) since onset. One hundred (41%) subjects reported being completely free of attacks in the past year. Subjects reduced their attacks by avoiding exercise during extremely hot or cold weather (44%), avoiding ingestion of certain foods before exercise (37%), and restricting exercise during their allergy season (36%) or humid weather (33%). The most common pharmacologic agents used to manage symptoms were H1 antagonists (56%) and/or epinephrine (31%). However, 28% used no treatment at all. CONCLUSION EIA is an episodic condition in which the frequency of attacks tends to stabilize or decrease over time. Improvement appears to result from individual modification of exercise and avoidance of known environmental and ingestible precipitants.

A Comparison of Patient Characteristics and Outcomes in Selected European and U.S. Rheumatoid Arthritis Registries

PURPOSE Randomized controlled trials (RCTs) have demonstrated the efficacy of biologic agents in the treatment of rheumatic diseases. However, results from RCTs may not be generalizable to clinical practice because of their strict inclusion and exclusion criteria. Assessment of safety using RCT data also is limited by short duration of follow-up and relatively small sample sizes, which generally preclude analysis of longer term outcomes and rare adverse events. In rheumatology, various observational cohorts and registries have been created to complement information obtained from RCTs, some with the primary purpose of monitoring effectiveness and safety of biologic agents. Most registries are either drug based or disease based. These registries include patients with a variety of rheumatic diseases including RA. METHODS To provide a qualitative comparison of selected U.S. and European rheumatoid arthritis (RA) biologics registries and cohorts including ARTIS, BIOBADASER, BSRBR, BRASS, CLEAR, CORRONA, NDB, RABBIT, SCQM, and VARA. RESULTS A careful comparison of these registries, as provided in this article, can provide a basis for understanding the many similarities and differences inherent in their design, as well as societal context and content, all of which can significantly impact their results and comparisons across registers. SUMMARY The increasing use of biologic agents for treatment of rheumatic diseases has raised important questions about cost, safety, and effectiveness of these agents. The unique and variable features of patient populations and registry designs in Europe and the U.S. provide valuable and complementary data on comparative effectiveness and safety of biologic agents to what can be derived from RCTs.