Prevention and Attenuation of Covid-19 with the BNT162b2 and mRNA-1273 Vaccines.

BACKGROUND: Information is limited regarding the effectiveness of the two-dose messenger RNA (mRNA) vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) in preventing infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and in attenuating coronavirus disease 2019 (Covid-19) when administered in real-world conditions. METHODS: We conducted a prospective cohort study involving 3975 health care personnel, first responders, and other essential and frontline workers. From December 14, 2020, to April 10, 2021, the participants completed weekly SARS-CoV-2 testing by providing mid-turbinate nasal swabs for qualitative and quantitative reverse-transcriptase-polymerase-chain-reaction (RT-PCR) analysis. The formula for calculating vaccine effectiveness was 100% × (1 - hazard ratio for SARS-CoV-2 infection in vaccinated vs. unvaccinated participants), with adjustments for the propensity to be vaccinated, study site, occupation, and local viral circulation. RESULTS: SARS-CoV-2 was detected in 204 participants (5%), of whom 5 were fully vaccinated (≥14 days after dose 2), 11 partially vaccinated (≥14 days after dose 1 and <14 days after dose 2), and 156 unvaccinated; the 32 participants with indeterminate vaccination status (<14 days after dose 1) were excluded. Adjusted vaccine effectiveness was 91% (95% confidence interval [CI], 76 to 97) with full vaccination and 81% (95% CI, 64 to 90) with partial vaccination. Among participants with SARS-CoV-2 infection, the mean viral RNA load was 40% lower (95% CI, 16 to 57) in partially or fully vaccinated participants than in unvaccinated participants. In addition, the risk of febrile symptoms was 58% lower (relative risk, 0.42; 95% CI, 0.18 to 0.98) and the duration of illness was shorter, with 2.3 fewer days spent sick in bed (95% CI, 0.8 to 3.7). CONCLUSIONS: Authorized mRNA vaccines were highly effective among working-age adults in preventing SARS-CoV-2 infection when administered in real-world conditions, and the vaccines attenuated the viral RNA load, risk of febrile symptoms, and duration of illness among those who had breakthrough infection despite vaccination. (Funded by the National Center for Immunization and Respiratory Diseases and the Centers for Disease Control and Prevention.).

SARS-CoV-2 Virus Dynamics in Recently Infected People-Data From a Household Transmission Study.

We used daily real-time reverse-transcription polymerase chain reaction (RT-PCR) results from 67 cases of SARS-CoV-2 infection in a household transmission study, conducted April 2020-May 2021, to examine the trajectory of cycle threshold (Ct) values, an inverse correlate of viral RNA concentration. Ct values varied across RT-PCR platforms and by participant age. Specimens collected from children and adolescents had higher Ct values and adults aged ≥50 years showed lower Ct values than adults aged 18-49 years. Ct values were lower on days when participants reported experiencing symptoms, with the lowest Ct value occurring 2-6 days after symptom onset.

Relationship of SARS-CoV-2 Antigen and Reverse Transcription PCR Positivity for Viral Cultures.

We assessed the relationship between antigen and reverse transcription PCR (RT-PCR) test positivity and successful virus isolation. We found that antigen test results were more predictive of virus recovery than RT-PCR results. However, virus was isolated from some antigen-negative and RT-PCR‒positive paired specimens, providing support for the Centers for Disease Control and Prevention antigen testing algorithm.

Interim Estimates of Vaccine Effectiveness of BNT162b2 and mRNA-1273 COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Health Care Personnel, First Responders, and Other Essential and Frontline Workers - Eight U.S. Locations, December 2020-March 2021.

Messenger RNA (mRNA) BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) COVID-19 vaccines have been shown to be effective in preventing symptomatic COVID-19 in randomized placebo-controlled Phase III trials (1,2); however, the benefits of these vaccines for preventing asymptomatic and symptomatic SARS-CoV-2 (the virus that causes COVID-19) infection, particularly when administered in real-world conditions, is less well understood. Using prospective cohorts of health care personnel, first responders, and other essential and frontline workers* in eight U.S. locations during December 14, 2020-March 13, 2021, CDC routinely tested for SARS-CoV-2 infections every week regardless of symptom status and at the onset of symptoms consistent with COVID-19-associated illness. Among 3,950 participants with no previous laboratory documentation of SARS-CoV-2 infection, 2,479 (62.8%) received both recommended mRNA doses and 477 (12.1%) received only one dose of mRNA vaccine.† Among unvaccinated participants, 1.38 SARS-CoV-2 infections were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) per 1,000 person-days.§ In contrast, among fully immunized (≥14 days after second dose) persons, 0.04 infections per 1,000 person-days were reported, and among partially immunized (≥14 days after first dose and before second dose) persons, 0.19 infections per 1,000 person-days were reported. Estimated mRNA vaccine effectiveness for prevention of infection, adjusted for study site, was 90% for full immunization and 80% for partial immunization. These findings indicate that authorized mRNA COVID-19 vaccines are effective for preventing SARS-CoV-2 infection, regardless of symptom status, among working-age adults in real-world conditions. COVID-19 vaccination is recommended for all eligible persons.

Association of variants in selected genes mediating host immune response with duration of Staphylococcus aureus bacteremia.

Host genetic variation may be a contributing factor to variability in Staphylococcus aureus bacteremia duration. We assessed whether 28 single nucleotide polymorphisms (SNPs) in seven genes (TLR2, TLR4, TIRAP, IRAK4, TRAF6, NOD2, and CISH) that mediate host immune response were associated with S. aureus bacteremia duration. Subjects included 158 patients with short-term (≤4 days) and 44 with persistent (>4 days) S. aureus bacteremia from an academic medical center. In single SNP analyses, the minor allele frequencies of three TIRAP SNPs (rs655540, rs563011, and rs8177376) were higher in persistent bacteremia (P < 0.05). A haplotype with all three minor alleles was also associated with persistent bacteremia (P = 0.037). The minor allele frequencies of four other TIRAP SNPs (rs8177342, rs4937114, rs3802813, and rs4937115) were higher in short-term bacteremia (P < 0.05), and a haplotype containing the four minor alleles was associated with short-term bacteremia (P = 0.045). All seven SNPs are located in binding sites for proteins or noncoding RNAs that regulate transcription. None of the associations remained statistically significant after adjustment for multiple comparisons. Further investigation is needed to understand how genetic variation in TIRAP and other host immune genes may influence the duration of S. aureus bacteremia.

Pilot screening study of targeted genetic polymorphisms for association with seasonal influenza hospital admission.

Host response to influenza is highly variable, suggesting a potential role of host genetic variation. To investigate the host genetics of severe influenza in a targeted fashion, 32 single nucleotide polymorphisms (SNPs) within viral immune response genes were evaluated for association with seasonal influenza hospitalization in an adult study population with European ancestry. SNP allele and genotype frequencies were compared between hospitalized influenza patients (cases) and population controls in a case-control study that included a discovery group (26 cases and 993 controls) and two independent, validation groups (1 with 84 cases and 4076 controls; the other with 128 cases and 9187 controls). Cases and controls had similar allele frequencies for variant rs12252 in interferon-inducible transmembrane protein 3 (IFITM3) (P > 0.05), and the study did not replicate the previously reported association of rs12252 with hospitalized influenza. In the discovery group, the preliminary finding of an association with a nonsense polymorphism (rs8072510) within the schlafen family member 13 (SFLN13) gene (P = 0.0099) was not confirmed in either validation group. Neither rs12252 nor rs8072510 showed an association according to the presence of clinical risk factors for influenza complications (P > 0.05), suggesting that these factors did not modify associations between the SNPs and hospitalized influenza. No other SNPs showed a statistically significant association with hospitalized influenza. Further research is needed to identify genetic factors involved in host response to seasonal influenza infection and to assess whether rs12252, a low-frequency variant in Europeans, contributes to influenza severity in populations with European ancestry.

Development and validation of a novel single nucleotide polymorphism (SNP) panel for genetic analysis of Blastomyces spp. and association analysis.

BACKGROUND: Single nucleotide polymorphism (SNP) genotyping is increasingly being utilized for molecular typing of pathogens and is cost-effective, especially for large numbers of isolates. The goals of this study were 1) to develop and validate a SNP assay panel for genetic analysis of Blastomyces spp., 2) ascertain whether microsatellite genotyping and the SNP genotyping with the developed panel resolve identical genetic groups, and 3) explore the utility of SNPs for examining phylogenetic and virulence questions in humans. METHODS: Three hundred sixty unique Blastomyces spp. isolates previously genotyped with microsatellite markers were genotyped with the MassARRAY® SNP genotyping system (Agena Bioscience™, San Diego, CA), for a custom panel of 28 SNPs. Clinical presentation data was analyzed for association with SNP variants. RESULTS: Three hundred twenty-three Blastomyces spp. isolates (90 %) were successfully genotyped by SNP analysis, with results obtained for at least 27 of 28 assays. For 99.7 % of isolates tested by both genotyping methods, microsatellite genetic group assignment correlated with species assignment based on internal transcribed spacer 2 (ITS2) genotyping, with Group 1 (Gr 1) being equivalent to B. gilchristii and Group 2 (Gr 2) being equivalent to B. dermatitidis. Thirteen isolates were genetic hybrids by one or both methods of genotyping and were difficult to assign to a particular genetic group or species. Fifteen SNP loci showed significantly different alleles in cases of pulmonary vs disseminated disease, at a p-value of <0.01 or less. CONCLUSIONS: This study is the largest genotyping study of Blastomyces spp. isolates and presents a new method for genetic analysis with which to further explore the relationship between the genetic diversity in Blastomyces spp. and clinical disease presentation. We demonstrated that microsatellite Gr 1 is equivalent to B. gilchristii and Gr 2 is equivalent to B. dermatitidis. We also discovered potential evidence of infrequent recombination between the two Blastomyces spp. Several Blastomyces spp. SNPs were identified as associated with dissemination or pulmonary disease presentation, but additional work is needed to examine virulence SNPs separately within B. dermatitidis and B. gilchristii.

Cone structure in subjects with known genetic relative risk for AMD.

PURPOSE: Utilize high-resolution imaging to examine retinal anatomy in patients with known genetic relative risk (RR) for developing age-related macular degeneration (AMD). METHODS: Forty asymptomatic subjects were recruited (9 men, 31 women; age range, 51 to 69 years; mean age, 61.4 years). Comprehensive eye examination, fundus photography, and high-resolution retinal imaging using spectral domain optical coherence tomography and adaptive optics were performed on each patient. Genetic RR scores were developed using an age-independent algorithm. Adaptive optics scanning light ophthalmoscope images were acquired in the macula extending to 10 degrees temporal and superior from fixation and were used to calculate cone density in up to 35 locations for each subject. RESULTS: Relative risk was not significantly predictive of fundus grade (p = 0.98). Only patients with a high RR displayed drusen on Cirrus or Bioptigen OCT. Compared to an eye with a grade of 0, an eye with a fundus grade equal to or greater than 1 had a 12% decrease in density (p < 0.0001) and a 5% increase in spacing (p = 0.0014). No association between genetic RR and either cone density (p = 0.435) or spacing (p = 0.538) was found. Three distinct adaptive optics scanning light ophthalmoscope phenotypical variations of photoreceptor appearance were noted in patients with grade 1 to 3 fundi. These included variable reflectivity of photoreceptors, decreased waveguiding, and altered photoreceptor mosaic overlying drusen. CONCLUSIONS: Our data demonstrate the potential of multimodal assessment in the understanding of early anatomical changes associated with AMD. Adaptive optics scanning light ophthalmoscope imaging reveals a decrease in photoreceptor density and increased spacing in patients with grade 1 to 3 fundi, as well as a spectrum of photoreceptor changes, ranging from variability in reflectivity to decreased density. Future longitudinal studies are needed in genetically characterized subjects to assess the significance of these findings with respect to the development and progression of AMD.

Antigen Test Performance Among Children and Adults at a SARS-CoV-2 Community Testing Site.

BACKGROUND: Performance characteristics of SARS-CoV-2 antigen tests among children are limited despite the need for point-of-care testing in school and childcare settings. We describe children seeking SARS-CoV-2 testing at a community site and compare antigen test performance to real-time reverse transcription-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Two anterior nasal specimens were self-collected for BinaxNOW antigen and RT-PCR testing, along with demographics, symptoms, and exposure information from individuals ≥5 years at a community testing site. Viral culture was attempted on residual antigen or RT-PCR-positive specimens. Demographic and clinical characteristics, and the performance of SARS-CoV-2 antigen tests, were compared among children (<18 years) and adults. RESULTS: About 1 in 10 included specimens were from children (225/2110); 16.4% (37/225) were RT-PCR-positive. Cycle threshold values were similar among RT-PCR-positive specimens from children and adults (22.5 vs 21.3, P = .46) and among specimens from symptomatic and asymptomatic children (22.5 vs 23.2, P = .39). Sensitivity of antigen test compared to RT-PCR was 73.0% (27/37) among specimens from children and 80.8% (240/297) among specimens from adults; among specimens from children, specificity was 100% (188/188), positive and negative predictive values were 100% (27/27) and 94.9% (188/198), respectively. Virus was isolated from 51.4% (19/37) of RT-PCR-positive pediatric specimens; all 19 had positive antigen test results. CONCLUSIONS: With lower sensitivity relative to RT-PCR, antigen tests may not diagnose all positive COVID-19 cases; however, antigen testing identified children with live SARS-CoV-2 virus.

Population based allele frequencies of disease associated polymorphisms in the Personalized Medicine Research Project.

BACKGROUND: There is a lack of knowledge regarding the frequency of disease associated polymorphisms in populations and population attributable risk for many populations remains unknown. Factors that could affect the association of the allele with disease, either positively or negatively, such as race, ethnicity, and gender, may not be possible to determine without population based allele frequencies.Here we used a panel of 51 polymorphisms previously associated with at least one disease and determined the allele frequencies within the entire Personalized Medicine Research Project population based cohort. We compared these allele frequencies to those in dbSNP and other data sources stratified by race. Differences in allele frequencies between self reported race, region of origin, and sex were determined. RESULTS: There were 19544 individuals who self reported a single racial category, 19027 or (97.4%) self reported white Caucasian, and 11205 (57.3%) individuals were female. Of the 11,208 (57%) individuals with an identifiable region of origin 8337 or (74.4%) were German.41 polymorphisms were significantly different between self reported race at the 0.05 level. Stratification of our Caucasian population by self reported region of origin revealed 19 polymorphisms that were significantly different (p = 0.05) between individuals of different origins. Further stratification of the population by gender revealed few significant differences in allele frequencies between the genders. CONCLUSIONS: This represents one of the largest population based allele frequency studies to date. Stratification by self reported race and region of origin revealed wide differences in allele frequencies not only by race but also by region of origin within a single racial group. We report allele frequencies for our Asian/Hmong and American Indian populations; these two minority groups are not typically selected for population allele frequency detection. Population wide allele frequencies are important for the design and implementation of studies and for determining the relevance of a disease associated polymorphism for a given population.

A LightCycler TaqMan assay for detection of Borrelia burgdorferi sensu lato in clinical samples.

Lyme disease (LD) is an infection caused by an ixodid tick-borne spirochete, Borrelia burgdorferi sensu lato. LD manifests itself as a multisystem inflammatory disease that affects the skin in its early localized stage and spreads to the joints, nervous system, heart, and, to a lesser extent, other organ systems in its later disseminated stages. If diagnosed and treated early with appropriate antibiotics, LD is almost always readily cured. Developing a highly sensitive and specific real-time polymerase chain reaction assay could be very useful in improving the diagnostic accuracy and decreasing turnaround time for results. We report the development of a LightCycler TaqMan assay targeting the OspA gene for clinical detection of B. burgdorferi sensu lato in various types of biologic samples. This assay was validated by testing a variety of clinical samples including cerebrospinal fluid, synovial fluid, skin biopsies, and blood and culture isolates from skin biopsies. The TaqMan testing results were 100% concordant with previously reported results. Reference strains representing isolates from other geographic regions were also successfully amplified. The developed assay is robust, is highly sensitive and specific for B. burgdorferi sensu lato, and is suitable for clinical detection of the bacterium in biologic samples.

Detection of ApoE E2, E3 and E4 alleles using MALDI-TOF mass spectrometry and the homogeneous mass-extend technology.

Apolipoprotein (Apo) E is one of the five main types of blood lipoproteins (A-E). It is synthesized primarily in the liver and brain and helps in transporting lipids from one place to another as well as facilitates the clearing of dietary fats, such as triglycerides, from the blood. The ApoE gene exists in three different forms: E2, E3 and E4. E3 is considered to be the normal form. Variants of the ApoE gene have been associated with various diseases. Developing an assay for the genotyping of ApoE variants for use both in clinical and large cohort based association settings would be extremely valuable and would require the use of a platform that has high-throughput capabilities and is highly accurate. Here we describe an assay for the simultaneous genotyping of the ApoE variants in a single bi-plex reaction and a single well using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the homogeneous mass-extend (hME) technology. The assay is robust, highly accurate and suitable for both clinical applications and for the genotyping of large disease cohorts. Moreover, the prevalence of ApoE variants in a cohort of Caucasians from the central Wisconsin area is outlined.

Blastomycosis in Children: An Analysis of Clinical, Epidemiologic, and Genetic Features.

BACKGROUND: Blastomyces spp. are endemic in regions of the United States and result in blastomycosis, a serious and potentially fatal infection. Little is known about the presentation, clinic course, epidemiology, and genetics of blastomycosis in children. METHODS: A retrospective review of children with blastomycosis confirmed by culture or cytopathology between 1999 and 2014 was completed. Blastomyces sp. isolates were genotyped by using microsatellite typing, and species were typed by sequencing of internal transcribed spacer 2 (its2). RESULTS: Of the 114 children with blastomycosis identified, 79% had isolated pulmonary involvement and 21% had extrapulmonary disease. There were more systemic findings, including fever (P = .01), poor intake (P = .01), elevated white blood cell count (P < .01), and elevated C-reactive protein level (P < .01), in children with isolated pulmonary disease than in children with extrapulmonary disease. Children with extrapulmonary disease had more surgeries (P = .01) and delays in diagnosis (P < .01) than those with isolated pulmonary infection. Of 52 samples genotyped, 48 (92%) were Blastomyces gilchristii and 4 (8%) were Blastomyces dermatitidis. CONCLUSION: This is the first large-scale study of the clinical, epidemiologic, and genetic features of blastomycosis in children. The majority of the children had isolated pulmonary disease with systemic findings. Patients with extrapulmonary disease were less likely to have systemic symptoms or additional laboratory evidence of infection, which made delays in diagnosis more common. More than 90% of the pediatric cases were caused by B gilchristii.

Genetic and Functional Associations with Decreased Anti-inflammatory Tumor Necrosis Factor Alpha Induced Protein 3 in Macrophages from Subjects with Axial Spondyloarthritis.

OBJECTIVE: Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is an anti-inflammatory protein implicated in multiple autoimmune and rheumatologic conditions. We hypothesized that lower levels of TNFAIP3 contributes to excessive cytokine production in response to inflammatory stimuli in axial spondyloarthritis (AxSpA). A further aim was to determine the immune signaling and genetic variation regulating TNFAIP3 expression in individual subjects. METHODS: Blood-derived macrophages from 50 AxSpA subjects and 30 healthy controls were assessed for TNFAIP3 expression. Cell lysates were also analyzed for NF-κB, mitogen-activated protein (MAP) kinase and STAT3 phosphorylation, and supernatants for cytokine production. Coding and regulatory regions in the TNFAIP3 gene and other auto-inflammation-implicated genes were sequenced by next-generation sequencing and variants identified. RESULTS: Mean TNFAIP3 was significantly lower in spondyloarthritis macrophages than controls (p = 0.0085). Spondyloarthritis subject macrophages correspondingly produced more TNF-α in response to lipopolysaccharide (LPS, p = 0.015). Subjects with the highest TNFAIP3 produced significantly less TNF-α in response to LPS (p = 0.0023). Within AxSpA subjects, those on TNF blockers or with shorter duration of disease expressed lower levels of TNFAIP3 (p = 0.0011 and 0.0030, respectively). TNFAIP3 expression correlated positively with phosphorylated IκBα, phosphorylated MAP kinases, and unstimulated phosphorylated STAT3, but negatively with LPS or TNF-α-stimulated fold induction of phosphorylated STAT3. Further, subjects with specific groups of variants within TNFAIP3 displayed differences in TNFAIP3 (p = 0.03-0.004). Nominal pQTL associations with genetic variants outside TNFAIP3 were identified. CONCLUSION: Our results suggest that both immune functional and genetic variations contribute to the regulation of TNFAIP3 levels in individual subjects. Decreased expression of TNFAIP3 in AxSpA macrophages correlated with increased LPS-induced TNF-α, and thus, TNFAIP3 dysregulation may be a contributor to excessive inflammatory responses in spondyloarthritis subjects.

Concordance between Research Sequencing and Clinical Pharmacogenetic Genotyping in the eMERGE-PGx Study.

There has been extensive debate about both the necessity of orthogonal confirmation of next-generation sequencing (NGS) results in Clinical Laboratory Improvement Amendments-approved laboratories and return of research NGS results to participants enrolled in research studies. In eMERGE-PGx, subjects underwent research NGS using PGRNseq and orthogonal targeted genotyping in clinical laboratories, which prompted a comparison of genotyping results between platforms. Concordance (percentage agreement) was reported for 4077 samples tested across nine combinations of research and clinical laboratories. Retesting was possible on a subset of 1792 samples, and local laboratory directors determined sources of genotype discrepancy. Research NGS and orthogonal clinical genotyping had an overall per sample concordance rate of 0.972 and per variant concordance rate of 0.997. Genotype discrepancies attributed to research NGS were because of sample switching (preanalytical errors), whereas the majority of genotype discrepancies (92.3%) attributed to clinical genotyping were because of allele dropout as a result of rare variants interfering with primer hybridization (analytical errors). These results highlight the analytical quality of clinically significant pharmacogenetic variants derived from NGS and reveal important areas for research and clinical laboratories to address with quality management programs.

Phenome-wide association studies (PheWASs) for functional variants.

The genome-wide association study (GWAS) is a powerful approach for studying the genetic complexities of human disease. Unfortunately, GWASs often fail to identify clinically significant associations and describing function can be a challenge. GWAS is a phenotype-to-genotype approach. It is now possible to conduct a converse genotype-to-phenotype approach using extensive electronic medical records to define a phenome. This approach associates a single genetic variant with many phenotypes across the phenome and is called a phenome-wide association study (PheWAS). The majority of PheWASs conducted have focused on variants identified previously by GWASs. This approach has been efficient for rediscovering gene-disease associations while also identifying pleiotropic effects for some single-nucleotide polymorphisms (SNPs). However, the use of SNPs identified by GWAS in a PheWAS is limited by the inherent properties of the GWAS SNPs, including weak effect sizes and difficulty when translating discoveries to function. To address these challenges, we conducted a PheWAS on 105 presumed functional stop-gain and stop-loss variants genotyped on 4235 Marshfield Clinic patients. Associations were validated on an additional 10 640 Marshfield Clinic patients. PheWAS results indicate that a nonsense variant in ARMS2 (rs2736911) is associated with age-related macular degeneration (AMD). These results demonstrate that focusing on functional variants may be an effective approach when conducting a PheWAS.

Development of a fingerprinting panel using medically relevant polymorphisms.

BACKGROUND: For population based biorepositories to be of use, rigorous quality control and assurance must be maintained. We have designed and validated a panel of polymorphisms for individual sample identification consisting of 36 common polymorphisms that have been implicated in a wide range of diseases and an additional sex marker. This panel uniquely identifies our biorepository of approximately 20,000 samples and would continue to uniquely identify samples in biorepositories of over 100 million samples. METHODS: A panel of polymorphisms associated with at least one disease state in multiple populations was constructed using a cut-off of 0.20 or greater confirmed minor allele frequency in a European Caucasian population. The fingerprinting assay was tested using the MALDI-TOF mass spectrometry method of allele determination on a Sequenom platform with a panel of 28 Caucasian HapMap samples; the results were compared with known genotypes to ensure accuracy. The frequencies of the alleles were compared to the expected frequencies from dbSNP and any genotype that did not achieve Hardy Weinberg equilibrium was excluded from the final assay. RESULTS: The final assay consisted of the AMG sex marker and 36 medically relevant polymorphisms with representation on each chromosome, encompassing polymorphisms on both the Illumina 550K bead array and the Affymetrix 6.0 chip (with over a million polymorphisms) platform. The validated assay has a P(ID) of 6.132 x 10(-15) and a Psib(ID) of 3.077 x 10(-8). This assay allows unique identification of our biorepository of 20,000 individuals as well and ensures that as we continue to recruit individuals they can be uniquely fingerprinted. In addition, diseases such as cancer, heart disease diabetes, obesity, and respiratory disease are well represented in the fingerprinting assay. CONCLUSION: The polymorphisms in this panel are currently represented on a number of common genotyping platforms making QA/QC flexible enough to accommodate a large number of studies. In addition, this panel can serve as a resource for investigators who are interested in the effects of disease in a population, particularly for common diseases.

A missense T (Brachyury) mutation contributes to vertebral malformations.

No major susceptibility genes for sporadically occurring congenital vertebral malformations (CVM) in humans have been identified to date. Body patterning genes whose mutants cause axial skeletal anomalies in mice are candidates for human CVM susceptibility. T (also known as Brachyury) and TBX6 are critical genes needed to establish mesodermal identity. We hypothesized that mutations in T and/or TBX6 contribute to the pathogenesis of human CVMs. The complete T and TBX6 coding regions, splice junctions, and proximal 500 bp of the promoters were sequenced in 50 phenotyped patients with CVM. Three unrelated patients with sacral agenesis, Klippel-Feil syndrome, and multiple cervical and thoracic vertebral malformations were heterozygous for a c.1013C>T substitution, resulting in a predicted Ala338Val missense alteration in exon 8. A clinically unaffected parent of each patient also harbored the substitution, but the variant did not occur in an ethnically diverse, 443-person reference population. The c.1013C>T variant is significantly associated with CVM (p < 0.001). Alanine 338 shows moderate conservation across species, and valine at this position has not been reported in any species. A fourth patient harbored a c.908-8C>T variant in intron 7. This previously unreported variant was tested in 347 normal control subjects, and 11 heterozygotes and 2 T/T individuals were found. No TBX6 variants were identified. We infer that the c.1013C>T substitution is pathogenic and represents the first report of an association between a missense mutation in the T gene and the occurrence of sporadic CVMs in humans. It is uncertain whether the splice junction variant increases CVM risk. TBX6 mutations do not seem to be associated with CVM. We hypothesize that epistatic interactions between T and other developmental genes and the environment modulate the phenotypic consequences of T variants.

Lack of evidence of WNT3A as a candidate gene for congenital vertebral malformations.

BACKGROUND: Prior investigations have not identified a major locus for vertebral malformations, providing evidence that there is genetic heterogeneity for this condition. WNT3A has recently been identified as a negative regulator of Notch signaling and somitogenesis. Mice with mutations in Wnt3a develop caudal vertebral malformations. Because congenital vertebral malformations represent a sporadic occurrence, linkage approaches to identify genes associated with human vertebral development are not feasible. We hypothesized that WNT3A mutations might account for a subset of congenital vertebral malformations. METHODS: A pilot study was performed using a cohort of patients with congenital vertebral malformations spanning the entire vertebral column was characterized. DNA sequence analysis of the WNT3A gene in these 50 patients with congenital vertebral malformations was performed. RESULTS: A female patient of African ancestry with congenital scoliosis and a T12-L1 hemivertebrae was found to be heterozygous for a missense variant resulting in the substitution of alanine by threonine at codon 134 in highly conserved exon 3 of the WNT3A gene. This variant was found at a very low prevalence (0.35%) in a control population of 443 anonymized subjects and 1.1% in an African population. CONCLUSION: These data suggest that WNT3A does not contribute towards the development of congenital vertebral malformations. Factors such as phenotypic and genetic heterogeneity may underlie our inability to detect mutations in WNT3A in our patient sample.