Mechanisms of Regulation of the CHRDL1 Gene by the TWIST2 and ADD1/SREBP1c Transcription Factors.

Setleis syndrome (SS) is a rare focal facial dermal dysplasia caused by recessive mutations in the basic helix-loop-helix (bHLH) transcription factor, TWIST2. Expression microarray analysis showed that the chordin-like 1 (CHRDL1) gene is up-regulated in dermal fibroblasts from three SS patients with the Q119X TWIST2 mutation. METHODS: Putative TWIST binding sites were found in the upstream region of the CHRDL1 gene and examined by electrophoretic mobility shift (EMSA) and reporter gene assays. RESULTS: EMSAs showed specific binding of TWIST1 and TWIST2 homodimers, as well as heterodimers with E12, to the more distal E-boxes. An adjoining E-box was bound by ADD1/SREBP1c. EMSA analysis suggested that TWIST2 and ADD1/SREBP1c could compete for binding. Luciferase (luc) reporter assays revealed that the CHRDL1 gene upstream region drives its expression and ADD1/SREBP1c increased it 2.6 times over basal levels. TWIST2, but not the TWIST2-Q119X mutant, blocked activation by ADD1/SREBP1c, but overexpression of TWIST2-Q119X increased luc gene expression. In addition, EMSA competition assays showed that TWIST2, but not TWIST1, competes with ADD1/SREBP1c for DNA binding to the same site. CONCLUSIONS: Formation of an inactive complex between the TWIST2 Q119X and Q65X mutant proteins and ADD1/SREBP1c may prevent repressor binding and allow the binding of other regulators to activate CHRDL1 gene expression.

Incidence and Mortality Rates for Childhood Acute Lymphoblastic Leukemia in Puerto Rican Hispanics, 2012-2016.

BACKGROUND: Acute lymphoblastic leukemia (ALL) accounts for 80% of all leukemias diagnosed in children. Although ALL age patterns are consistent across racial/ethnic groups, their incidence and mortality rates are highly variable. We assessed the age-standardized ALL incidence and mortality rates of Puerto Rican Hispanic (PRH) children and compared them with those of US mainland Hispanics (USH), non-Hispanic Whites (NHW), non-Hispanic Blacks (NHB), and Non-Hispanic Asian or Pacific Islanders (NHAPI). METHODS: Differences between racial/ethnic groups were assessed by estimating the standardized rate ratio (SRR) for 2010 to 2014. Secondary data analyses of the Puerto Rico Central Cancer Registry and the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) databases were performed for the 2001 to 2016 period. RESULTS: PRH children had 31% lower incidence rates than USH, but 86% higher incidence rates than NHB. In addition, the incidence trends of ALL increased significantly from 2001 to 2016 among PRH and USH, with 5% and 0.9% per year, respectively. Moreover, PRH have a lower 5-year overall survival (81.7%) when compared with other racial/ethnic groups. CONCLUSIONS: PRH children were found to have disparities in ALL incidence and mortality rates compared with other racial/ethnic groups in the US. Additional research is warranted to identify the genetic and environmental risk factors that may be associated with the disparities observed. IMPACT: This is the first study reporting the incidence and mortality rates of childhood ALL for PRH and making comparisons with other racial/ethnic groups in the US. See related commentary by Mejía-Aranguré and Núñez-Enríquez, p. 999.

Bioinformatic Characterization and Molecular Evolution of the Lucina pectinata Hemoglobins.

(1) Introduction: Lucina pectinata is a clam found in sulfide-rich mud environments that has three hemoglobins believed to be responsible for the transport of hydrogen sulfide (HbILp) and oxygen (HbIILp and HbIIILp) to chemoautotrophic endosymbionts. The physiological roles and evolution of these globins in sulfide-rich environments are not well understood. (2) Methods: We performed bioinformatic and phylogenetic analyses with 32 homologous mollusk globin sequences. Phylogenetics suggests a first gene duplication resulting in sulfide binding and oxygen binding genes. A more recent gene duplication gave rise to the two oxygen-binding hemoglobins. Multidimensional scaling analysis of the sequence space shows evolutionary drift of HbIILp and HbIIILp, while HbILp was closer to the Calyptogena hemoglobins. Further corroboration is seen by conservation in the coding region of hemoglobins from L. pectinata compared to those from Calyptogena. (3) Conclusions: Presence of glutamine in position E7 in organisms living in sulfide-rich environments can be considered an adaptation to prevent loss of protein function. In HbILp a substitution of phenylalanine in position B10 is accountable for its unique reactivity towards H2S. It appears that HbILp has been changing over time, apparently not subject to functional constraints of binding oxygen, and acquired a unique function for a specialized environment.

Expression Profiling Identifies TWIST2 Target Genes in Setleis Syndrome Patient Fibroblast and Lymphoblast Cells.

Background: Setleis syndrome (SS) is a focal facial dermal dysplasia presenting with bilateral temporal skin lesions, eyelash abnormalities and absent meibomian glands. SS is a rare autosomal recessive disorder caused by mutations in the TWIST2 gene, which codes for a transcription factor of the bHLH family known to be involved in skin and facial development. Methods: We obtained gene expression profiles by microarray analyses from control and SS patient primary skin fibroblast and lymphoblastoid cell lines. Results: Out of 983 differentially regulated genes in fibroblasts (fold change ≥ 2.0), 479 were down-regulated and 509 were up-regulated, while in lymphoblasts, 1248 genes were down-regulated and 73 up-regulated. RT-PCR reactions confirmed altered expression of selected genes. Conclusions: TWIST2 is described as a repressor, but expression profiling suggests an important role in gene activation as well, as evidenced by the number of genes that are down-regulated, with a much higher proportion of down-regulated genes found in lymphoblastoid cells from an SS patient. As expected, both types of cell types showed dysregulation of cytokine genes. These results identify potential TWIST2 target genes in two important cell types relevant to rare disorders caused by mutations in this bHLH gene.

Characterization of Histone Genes from the Bivalve Lucina Pectinata.

Lucina pectinata is a clam that lives in sulfide-rich environments and houses intracellular sulfide-oxidizing endosymbionts. To identify new Lucina pectinata proteins, we produced libraries for genome and transcriptome sequencing and assembled them de novo. We searched for histone-like sequences using the Lucina pectinata histone H3 partial nucleotide sequence against our previously described genome assembly to obtain the complete coding region and identify H3 coding sequences from mollusk sequences in Genbank. Solen marginatus histone nucleotide sequences were used as query sequences using the genome and transcriptome assemblies to identify the Lucina pectinata H1, H2A, H2B and H4 genes and mRNAs and obtained the complete coding regions of the five histone genes by RT-PCR combined with automated Sanger DNA sequencing. The amino acid sequence conservation between the Lucina pectinata and Solen marginatus histones was: 77%, 93%, 83%, 96% and 97% for H1, H2A, H2B, H3 and H4, respectively. As expected, the H3 and H4 proteins were the most conserved and the H1 proteins were most similar to H1's from aquatic organisms like Crassostrea gigas, Aplysia californica, Mytilus trossulus and Biomphalaria glabrata. The Lucina pectinata draft genome and transcriptome assemblies, obtained by semiconductor sequencing, were adequate for identification of conserved proteins as evidenced by our results for the histone genes.

Identification of Immunoglobulin E-Binding Proteins of the Xerophilic Fungus Aspergillus penicillioides Crude Mycelial Mat Extract and Serological Reactivity Assessment in Subjects with Different Allergen Reactivity Profiles.

BACKGROUND: Aspergillus penicillioides is a very common indoor xerophilic fungus and potential causative agent of respiratory conditions. Although people are constantly exposed to A. penicillioides, no proteins with allergenic potential have been described. Therefore, we aim to confirm allergic sensitization to A. penicillioides through reactivity in serological assays and detect immunoglobulin E (IgE)-binding proteins. METHODS: In an indirect ELISA, we compared the serological reactivity to A. penicillioides between subjects with specific IgE (sIgE) (group 1, n = 54) and no sIgE reactivity (group 2, n = 15) against commercial allergens. Correlations and principal component analysis were performed to identify associations between reactivity to commercial allergens and A. penicillioides. IgE-binding proteins in A. penicillioides were visualized using Western blotting (WB) in group 1. The IgE-binding proteins with the highest reactivity were analyzed by mass spectrometry and confirmed by transcript matching. RESULTS: There was no statistical significance (p = 0.1656) between the study groups in serological reactivity. Correlations between reactivity to A. penicillioides, dog epithelia, Aspergillus fumigatus, and Penicillium chrysogenum were observed. WB experiments showed 6 IgE-binding proteins with molecular weights ranging from 45 to 145 kDa. Proteins of 108, 83, and 56 kDa showed higher reactivity. Mass spectrometry analysis of these 3 proteins led to the putative identification of NADP-specific glutamate dehydrogenase and catalase B. This was confirmed with transcriptome analysis. CONCLUSIONS: These results provide evidence of the presence of potential allergenic components in A. penicillioides. Further analysis of the putatively identified proteins should reveal their allergenic potential.

Warfarin Anticoagulation Therapy in Caribbean Hispanics of Puerto Rico: A Candidate Gene Association Study.

Existing algorithms account for ~50% of observed variance in warfarin dose requirements after including common polymorphisms. However, they do not perform as well in populations other than Caucasians, in part because some ethno-specific genetic variants are overlooked. The objective of the present study was to identify genetic polymorphisms that can explain variability in warfarin dose requirements among Caribbean Hispanics of Puerto Rico. Next-Generation Sequencing of candidate genes CYP2C9 and VKORC1 and genotyping by DMET® Plus Assay of cardiovascular patients were performed. We also aimed at characterizing the genomic structure and admixture pattern of this study cohort. Our study used the Extreme Discordant Phenotype approach to perform a case-control association analysis. The CYP2C9 variant rs2860905, which was found in all the major haplotypes occurring in the Puerto Rican population, showed stronger association with warfarin sensitivity (<4 mg/day) than common variants CYP2C9*2 and CYP2C9*3. Although, CYP2C9*2 and CYP2C9*3 are separately contained within two of the haplotypes, 10 subjects with the sensitive phenotype were carriers of only the CYP2C9 rs2860905 variant. Other polymorphisms in CES2 and ABCB1 were found to be associated with warfarin resistance. Incorporation of rs2860905 in a regression model (R2 = 0.63, MSE = 0.37) that also includes additional genetics (i.e., VKORC1-1639 G>A; CYP2C9 rs1856908; ABCB1 c.IVS9-44A>G/ rs10276036; CES2 c.269-965A>G/ rs4783745) and non-genetic factors (i.e., hypertension, diabetes and age) showed better prediction of warfarin dose requirements than CYP2C9*2 and CYP2C9*3 combined (partial R2 = 0.132 vs. 0.023 and 0.007, respectively, p < 0.001). The genetic background of Puerto Ricans in the study cohort showed a tri-hybrid admixture pattern, with a slightly higher than expected contribution of Native American ancestry (25%). The genomic diversity of Puerto Ricans is highlighted by the presence of four different major haplotype blocks in the CYP2C9 locus. Although, our findings need further replication, this study contributes to the field by identifying novel genetic variants that increase predictability of stable warfarin dosing among Caribbean Hispanics.

Correction: Evolution of the Twist Subfamily Vertebrate Proteins: Discovery of a Signature Motif and Origin of the Twist1 Glycine-Rich Motifs in the Amino-Terminus Disordered Domain.

[This corrects the article DOI: 10.1371/journal.pone.0161029.].

Evolution of the Twist Subfamily Vertebrate Proteins: Discovery of a Signature Motif and Origin of the Twist1 Glycine-Rich Motifs in the Amino-Terminus Disordered Domain.

Twist proteins belong to the basic helix-loop-helix (bHLH) family of multifunctional transcriptional factors. These factors are known to use domains other than the common bHLH in protein-protein interactions. There has been much work characterizing the bHLH domain and the C-terminus in protein-protein interactions but despite a few attempts more focus is needed at the N-terminus. Since the region of highest diversity in Twist proteins is the N-terminus, we analyzed the conservation of this region in different vertebrate Twist proteins and study the sequence differences between Twist1 and Twist2 with emphasis on the glycine-rich regions found in Twist1. We found a highly conserved sequence motif in all Twist1 (SSSPVSPADDSLSNSEEE) and Twist2 (SSSPVSPVDSLGTSEEE) mammalian species with unknown function. Through sequence comparison we demonstrate that the Twist protein family ancestor was "Twist2-like" and the two glycine-rich regions found in Twist1 sequences were acquired late in evolution, apparently not at the same time. The second glycine-rich region started developing first in the fish vertebrate group, while the first glycine region arose afterwards within the reptiles. Disordered domain and secondary structure predictions showed that the amino acid sequence and disorder feature found at the N-terminus is highly evolutionary conserved and could be a functional site that interacts with other proteins. Detailed examination of the glycine-rich regions in the N-terminus of Twist1 demonstrate that the first region is completely aliphatic while the second region contains some polar residues that could be subject to post-translational modification. Phylogenetic and sequence space analysis showed that the Twist1 subfamily is the result of a gene duplication during Twist2 vertebrate fish evolution, and has undergone more evolutionary drift than Twist2. We identified a new signature motif that is characteristic of each Twist paralog and identified important residues within this motif that can be used to distinguish between these two paralogs, which will help reduce Twist1 and Twist2 sequence annotation errors in public databases.

Pathway Analysis using Gene-expression Profiles of HPV-positive and HPV-negative Oropharyngeal Cancer Patients in a Hispanic Population: Methodological Procedures.

OBJECTIVE: The incidence of oral cavity and pharyngeal cancer in Puerto Rican men is higher than it is in the men of any other ethnic/racial group in the United States of America (US). The information regarding the effect of the human papilloma virus (HPV) in the gene-expression profile among patients with this cancer is limited in Hispanic community. We aim to describe the methodology for future studies to identify the molecular networks for determining overrepresented signaling and metabolic canonical pathways, based on the differential gene-expression profiles of HPV+ and HPV- samples from patients with oropharyngeal squamous cell carcinoma in Puerto Rico. METHODS: We analyzed the RNA expression of 5 tissue samples from subjects diagnosed with oropharyngeal squamous cell carcinoma, 2 HPV+ and 3 HPV-, using Affymetrix GeneChips. The relative difference between the average gene expressions of the HPV+ and HPV- samples was assessed, based on the fold change (log2-scale). RESULTS: Our analysis revealed 10 up regulated molecules (Mup1, LRP1, P14KA, ALYREF, and BHMT) and 5 down regulated ones (PSME4, KEAP1, ELK3, FAM186B, and PRELID1), at a cutoff of 1.5-fold change. Ingenuity Pathway Analysis showed the following biological functions to be affected in the HPV+ samples: cancer, hematological disease, and RNA post-transcriptional modification. QRT-PCR analysis confirmed only the differential regulation of ALYREF, KEAP1, and FAM186B genes. CONCLUSION: The relevant methodological procedures described are sufficient to detect the most significant biological functions and pathways according to the HPV status in patients with oropharyngeal cancer in Puerto Rico.

Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes.

The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5'end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments.

A Novel Admixture-Based Pharmacogenetic Approach to Refine Warfarin Dosing in Caribbean Hispanics.

AIM: This study is aimed at developing a novel admixture-adjusted pharmacogenomic approach to individually refine warfarin dosing in Caribbean Hispanic patients. PATIENTS & METHODS: A multiple linear regression analysis of effective warfarin doses versus relevant genotypes, admixture, clinical and demographic factors was performed in 255 patients and further validated externally in another cohort of 55 individuals. RESULTS: The admixture-adjusted, genotype-guided warfarin dosing refinement algorithm developed in Caribbean Hispanics showed better predictability (R2 = 0.70, MAE = 0.72mg/day) than a clinical algorithm that excluded genotypes and admixture (R2 = 0.60, MAE = 0.99mg/day), and outperformed two prior pharmacogenetic algorithms in predicting effective dose in this population. For patients at the highest risk of adverse events, 45.5% of the dose predictions using the developed pharmacogenetic model resulted in ideal dose as compared with only 29% when using the clinical non-genetic algorithm (p<0.001). The admixture-driven pharmacogenetic algorithm predicted 58% of warfarin dose variance when externally validated in 55 individuals from an independent validation cohort (MAE = 0.89 mg/day, 24% mean bias). CONCLUSIONS: Results supported our rationale to incorporate individual's genotypes and unique admixture metrics into pharmacogenetic refinement models in order to increase predictability when expanding them to admixed populations like Caribbean Hispanics. TRIAL REGISTRATION: ClinicalTrials.gov NCT01318057.

Ocular Findings in Patients with the Hermansky-Pudlak Syndrome (Types 1 and 3).

PURPOSE: To describe and compare ocular findings in patients with Hermansky-Pudlak syndrome (HPS) type 1 and 3. METHODS: This is a retrospective case series of 64 patients with HPS from 1999 to 2009 evaluated at an outpatient private ophthalmologic clinic. Patients underwent genetic analysis of selected albinism (Tyrosine and P gene) and HPS genes (HPS-1 and HPS-3) by screening for common mutations and exon sequencing with DNA screening. Descriptive and non-parametric statistical analyses were carried out. RESULTS: Nearly 70% of the patients were homozygous for common Puerto Rican mutations leading to the HPS1 gene (16-BP DUP, 53.6%), while 30% had the 3904-BP DEL HPS3 gene mutation. Best corrected visual acuity (BCVA) was poorer in patients with type 1 HPS than in patients with type 3 HPS (p < 0.001), esotropia was more common among type 1 HPS patients (p < 0.018), while exotropia was more common among patients with type 3 HPS. Total iris transillumination was more common in patients with type 1 HPS and minimal iris transillumination in patients with type 3 HPS (p < 0.001). The maculae were translucent in patients with type 1 HPS, while patients with type 3 HPS had opaque maculae (p < 0.001). CONCLUSIONS: Patients with type 1 HPS had poorer BCVA, increased incidence of esotropia, lighter iris and macular appearance. In contrast, patients with type 3 HPS had more exotropia. In addition, to our knowledge this is the largest series type 3 HPS ever reported.

Pharmacogenetics of healthy volunteers in Puerto Rico.

Puerto Ricans are a unique Hispanic population with European, Native American (Taino), and higher West African ancestral contributions than other non-Caribbean Hispanics. In admixed populations, such as Puerto Ricans, genetic variants can be found at different frequencies when compared to parental populations and uniquely combined and distributed. Therefore, in this review, we aimed to collect data from studies conducted in healthy Puerto Ricans and to report the frequencies of genetic polymorphisms with major relevance in drug response. Filtering for healthy volunteers or individuals, we performed a search of pharmacogenetic studies in academic literature databases without limiting the period of the results. The search was limited to Puerto Ricans living in the island, excluding those studies performed in mainland (United States). We found that the genetic markers impacting pharmacological therapy in the areas of cardiovascular, oncology, and neurology are the most frequently investigated. Coincidently, the top causes of mortality in the island are cardiovascular diseases, cancer, diabetes, Alzheimer's disease, and stroke. In addition, polymorphisms in genes that encode for members of the CYP450 family (CYP2C9, CYP2C19, and CYP2D6) are also available due to their relevance in the metabolism of drugs. The complex genetic background of Puerto Ricans is responsible for the divergence in the reported allele frequencies when compared to parental populations (Africans, East Asians, and Europeans). The importance of reporting the findings of pharmacogenetic studies conducted in Puerto Ricans is to identify genetic variants with potential utility among this genetically complex population and eventually move forward the adoption of personalized medicine in the island.

Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.

A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.

Setleis syndrome: clinical, molecular and structural studies of the first TWIST2 missense mutation.

Setleis syndrome is characterized by bitemporal scar-like lesions and other characteristic facial features. It results from recessive mutations that truncate critical functional domains in the basic helix-loop-helix (bHLH) transcription factor, TWIST2, which regulates expression of genes for facial development. To date, only four nonsense or small deletion mutations have been reported. In the current report, the clinical findings in a consanguineous Turkish family were characterized. Three affected siblings had the characteristic features of Setleis syndrome. Homozygosity for the first TWIST2 missense mutation, c.326T>C (p.Leu109Pro), was identified in the patients. In silico analyses predicted that the secondary structure of the mutant protein was sustained, but the empirical force field energy increased to an unfavorable level with the proline substitution (p.Leu109Pro). On a crystallographically generated dimer, p.Leu109 lies near the dimer interface, and the proline substitution is predicted to hinder dimer formation. Therefore, p.Leu109Pro-TWIST2 alters the three dimensional structure and is unable to dimerize, thereby hindering the binding of TWIST2 to its target genes involved in facial development.

Pharmacogenetics of drug-metabolizing enzymes in US Hispanics.

Although the Hispanic population is continuously growing in the United States, they are underrepresented in pharmacogenetic studies. This review addresses the need for compiling available pharmacogenetic data in US Hispanics, discussing the prevalence of clinically relevant polymorphisms in pharmacogenes encoding for drug-metabolizing enzymes. CYP3A5*3 (0.245-0.867) showed the largest frequency in a US Hispanic population. A higher prevalence of CYP2C9*3, CYP2C19*4, and UGT2B7 IVS1+985 A>G was observed in US Hispanic vs. non-Hispanic populations. We found interethnic and intraethnic variability in frequencies of genetic polymorphisms for metabolizing enzymes, which highlights the need to define the ancestries of participants in pharmacogenetic studies. New approaches should be integrated in experimental designs to gain knowledge about the clinical relevance of the unique combination of genetic variants occurring in this admixed population. Ethnic subgroups in the US Hispanic population may harbor variants that might be part of multiple causative loci or in linkage-disequilibrium with functional variants. Pharmacogenetic studies in Hispanics should not be limited to ascertain commonly studied polymorphisms that were originally identified in their parental populations. The success of the Personalized Medicine paradigm will depend on recognizing genetic diversity between and within US Hispanics and the uniqueness of their genetic backgrounds.

Pharmacogenetic association study of warfarin safety endpoints in Puerto Ricans.

OBJECTIVE: This study was intended to determine the incidence rate of warfarin-related adverse events (e.g., bleeding) in Puerto Ricans and whether a genetic association between warfarin pharmacogenes and any of these adverse events was observed over the initiation period (i.e., the first 90 days of therapy). METHODS: We conducted an observational, retrospective cohort study of pharmacogenetic association in 122 warfarin-treated, male, Puerto Rican patients (69.9 +/- 9.6 years) from the Veterans Affair Caribbean Healthcare System (VACHS) who consented to participate. Genotyping was performed using the CYP2C9 and VKORC1 assays by Luminex. Event-free survival curves were estimated using the Kaplan-Meier method and analyzed by log-rank test. Cox regression models were constructed and hazard ratios (HR) calculated. RESULTS: Carriers of functional CYP2C9 and VKORC1 polymorphisms demonstrated a higher incidence rate of multiple adverse events (i.e., 5.2 vs. 1.0 cases per 100 patient-months; RR = 4.8, p = 0.12) than did wild types. A significant association was observed between multiple adverse events and carrier status (HR = 2.5; 95% CI: 1.0-6.3, p = 0.04). However, no significant associations between genotypes and individual outcomes over the first 90 days of therapy were found. CONCLUSION: The association of CYP2C9 and VKORC1 genotypes and risks for adverse events due to exposure to warfarin was examined for the first time in Puerto Ricans. Despite a lack of association with individual events in this study population, our findings revealed a potential utility of genotyping for the prevention of multiple adverse events during warfarin therapy.