Bleeding assessment in female patients with the Hermansky-Pudlak syndrome-A case series.

INTRODUCTION: The Hermansky-Pudlak syndrome (HPS) is an autosomal recessive rare disorder characterized by oculocutaneous albinism, bleeding diathesis, chronic granulomatous colitis and/or pulmonary fibrosis. HPS is the most common single-gene disorder in Puerto Rico with a prevalence of 1:1,800 in the Northwest of the island. Risk of menorrhagia and post-partum hemorrhage (PPH) in cases of women with HPS have been described in the medical literature, but data regarding comprehensive description of bleeding diathesis remains lacking. For this reason, we aim to identify bleeding events using the International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT), a standardized quantitative tool that translates the range of severity of bleeding symptoms into a cumulative bleeding score (BS). OBJECTIVE: To use the ISTH-BAT in HPS in order to describe bleeding symptoms and allow for comparison with other inherited bleeding disorders. METHODS: Puerto Rican females and adult participants with HPS based on genetic linkage were enrolled. The ISTH-BAT was administered and results were identified using descriptive statistical analysis. RESULTS: Questionnaire answers of twelve women with HPS-1 and HPS-3 were evaluated. Participants' mean BS was HPS-1 (11.4) and HPS-3 (8.0) Participants with HPS-1 and HPS-3 reported abnormal bleeding events that presented during dental extractions, menorrhagia, surgical interventions, gastrointestinal, oral cavity and post-partum. Patients with history of pulmonary fibrosis (PF) showed a higher mean bleeding score than those who had no history of PF. CONCLUSIONS: Female patients with HPS type 1 and 3 experienced abnormal bleeding events according to the ISTH-BAT bleeding score. Bleeding medications were inconsistently used and varied independently from healthcare professionals. The benefits of this study were to understand the history of bleeding complications in patients with HPS type 1 and 3 using an international validated system. The results of this study will help design strategies to improve the care we provide to this population.

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.

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.

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.

A divalent interaction between HPS1 and HPS4 is required for the formation of the biogenesis of lysosome-related organelle complex-3 (BLOC-3).

Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1-249 and 506-700 are required for binding to HPS4; the middle portion of HPS1 (residues 250-505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340-528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles.

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.

Homozygous nonsense mutations in TWIST2 cause Setleis syndrome.

The focal facial dermal dysplasias (FFDDs) are a group of inherited developmental disorders in which the characteristic diagnostic feature is bitemporal scar-like lesions that resemble forceps marks. To date, the genetic defects underlying these ectodermal dysplasias have not been determined. To identify the gene defect causing autosomal-recessive Setleis syndrome (type III FFDD), homozygosity mapping was performed with genomic DNAs from five affected individuals and 26 members of the consanguineous Puerto Rican (PR) family originally described by Setleis and colleagues. Microsatellites D2S1397 and D2S2968 were homozygous in all affected individuals, mapping the disease locus to 2q37.3. Haplotype analyses of additional markers in the PR family and a consanguineous Arab family further limited the disease locus to approximately 3 Mb between D2S2949 and D2S2253. Of the 29 candidate genes in this region, the bHLH transcription factor, TWIST2, was initially sequenced on the basis of its known involvement in murine facial development. Homozygous TWIST2 nonsense mutations, c.324C>T and c.486C>T, were identified in the affected members of the Arab and PR families, respectively. Characterization of the expressed mutant proteins, p.Q65X and p.Q119X, by electrophoretic mobility shift assays and immunoblot analyses indicated that they were truncated and unstable. Notably, Setleis syndrome patients and Twist2 knockout mice have similar facial features, indicating the gene's conserved role in mammalian development. Although human TWIST2 and TWIST1 encode highly homologous bHLH transcription factors, the finding that TWIST2 recessive mutations cause an FFDD and dominant TWIST1 mutations cause Saethre-Chotzen craniocynostosis suggests that they function independently in skin and bone development.

Redundant or separate entities?--roles of Twist1 and Twist2 as molecular switches during gene transcription.

Twist1 and Twist2 are highly conserved members of the Twist subfamily of bHLH proteins responsible for the transcriptional regulation of the developmental programs in mesenchymal cell lineages. The regulation of such processes requires that Twist1 and Twist2 function as molecular switches to activate and repress target genes by employing several direct and indirect mechanisms. Modes of action by these proteins include direct DNA binding to conserved E-box sequences and recruitment of coactivators or repressors, sequestration of E-protein modulators, and interruption of proper activator/repressor function through protein-protein interactions. Regulatory outcomes of Twist1 and Twist2 are themselves controlled by spatial-temporal expression, phosphoregulation, dimer choice and cellular localization. Although these two proteins are highly conserved and exhibit similar functions in vitro, emerging literature have demonstrated different roles in vivo. The involvement of Twist1 and Twist2 in a broad spectrum of regulatory pathways highlights the importance of understanding their roles in normal development, homeostasis and disease. Here we focus on the mechanistic models of transcriptional regulation and summarize the similarities and differences between Twist1 and Twist2 in the context of myogenesis, osteogenesis, immune system development and cancer.

Ocular findings in patients with oculocutaneous albinism type ia with G47D tyrosinase gene mutation in Puerto Rico: a case report.

UNLABELLED: Previous studies have suggested that the G47D mutation leads patients to develop Oculocutaneous albinism (OCA) type IA. This mutation has been described in the Canary Islands. Historically, there has been a migration from the Canary Islands to some regions of Puerto Rico. OBJECTIVE: To report on the ocular findings of two Puerto Rican patients with OCA IA due to the G47D Tyrosinase gene mutation. PATIENT AND FINDINGS: Two unrelated patients with OCA underwent a comprehensive eye examination and were referred for genetic analysis. Patients had almost total iris transillumination, clear lenses, foveal hypoplasia with transparent maculae, and albinotic mid peripheries. Both patients had nystagmus, and only one patient had strabismus. CONCLUSIONS: Patients with the G47D muta- tion leading to OCA IA have poor visual acuities and poorly pigmented phenotypic ophthalmic findings. Further studies comparing ocular findings in patients th several mutations leading to OCA IA are warranted. To our knowledge this is the first report on ocular findings in Puerto Rican patients with OCA type IA with the rare G47D mutation.

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.

Prediction of warfarin dose reductions in Puerto Rican patients, based on combinatorial CYP2C9 and VKORC1 genotypes.

BACKGROUND: The influence of CYP2C9 and VKORC1 polymorphisms on warfarin dose has been investigated in white, Asian, and African American populations but not in Puerto Rican Hispanic patients. OBJECTIVE: To test the associations between genotypes, international normalized ratio (INR) measurements, and warfarin dosing and gauge the impact of these polymorphisms on warfarin dose, using a published algorithm. METHODS: A retrospective warfarin pharmacogenetic association study in 106 Puerto Rican patients was performed. DNA samples from patients were assayed for 12 variants in both CYP2C9 and VKORC1 loci by HILOmet PhyzioType assay. Demographic and clinical nongenetic data were retrospectively collected from medical records. Allele and genotype frequencies were determined and Hardy-Weinberg equilibrium (HWE) was tested. RESULTS: Sixty-nine percent of patients were carriers of at least one polymorphism in either the CYP2C9 or the VKORC1 gene. Double, triple, and quadruple carriers accounted for 22%, 5%, and 1%, respectively. No significant departure from HWE was found. Among patients with a given CYP2C9 genotype, warfarin dose requirements declined from GG to AA haplotypes; whereas, within each VKORC1 haplotype, the dose decreased as the number of CYP2C9 variants increased. The presence of these loss-of-function alleles was associated with more out-of-range INR measurements (OR = 1.38) but not with significant INR >4 during the initiation phase. Analyses based on a published pharmacogenetic algorithm predicted dose reductions of up to 4.9 mg/day in carriers and provided better dose prediction in an extreme subgroup of highly sensitive patients, but also suggested the need to improve predictability by developing a customized model for use in Puerto Rican patients. CONCLUSIONS: This study laid important groundwork for supporting a prospective pharmacogenetic trial in Puerto Ricans to detect the benefits of incorporating relevant genomic information into a customized DNA-guided warfarin dosing algorithm.

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.

Prevalence of a loss-of-function mutation in the proton-coupled folate transporter gene (PCFT-SLC46A1) causing hereditary folate malabsorption in Puerto Rico.

OBJECTIVE: To determine whether subjects of Puerto Rican heritage are at increased risk for a specific mutation of the proton-coupled folate transporter (PCFT) causing hereditary folate malabsorption (HFM). STUDY DESIGN: Three percent of the births in Puerto Rico in 2005, with additional regional oversampling, were screened for the prevalence of the c.1082G>A; p.Y362_G389 del PCFT gene mutation. Six new subjects of Puerto Rican heritage with the clinical diagnosis of HFM were also assessed for this mutation. RESULTS: Six subjects of Puerto Rican heritage with the clinical diagnosis of HFM were all homozygous for the c.1082G>A; p.Y362_G389 del PCFT mutation. Three heterozygote carriers were identified from the 1582 newborn samples randomly selected from births in Puerto Rico in 2005. The carrier frequency for the mutated allele was 0.2% island-wide and 6.3% in Villalba. CONCLUSION: These findings are consistent with a common mutation in the PCFT gene causing HFM that has disseminated to Puerto Ricans who have migrated to mainland United States. Because prompt diagnosis and treatment of infants with HFM can prevent the consequences of this disorder, newborn screening should be considered in high-risk populations and physicians should be aware of its prevalence in infants of Puerto Rican ancestry.

Asymptomatic child heterozygous for hemoglobin S and hemoglobin Pôrto Alegre.

Hemoglobin Pôrto Alegre (PA) is a rare hemoglobin resulting from a mutation in ß9(A6)Ser → Cys. We describe an asymptomatic Puerto Rican female with combined heterozygosity for Hb PA and Hb S. Since birth, she has maintained normal hemoglobin, bilirubin, LDH levels, and reticulocyte count. Peripheral smear evaluation has revealed normal erythrocyte morphology with no changes suggestive of hemolysis. We conclude that the presence of Hb PA does not increase the risk of red blood cell sickling in patients who carry the Hb S mutation.

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.

Newborn screening for hermansky-pudlak syndrome type 3 in Puerto Rico.

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by albinism, mucocutaneous bleeding, and storage of ceroid material in macrophages. Patients who are not easily identified by physical characteristics (mostly HPS-3 patients) may have hemorrhagic complications with trauma or surgery. OBJECTIVE: To determine the prevalence of HPS-3 in Puerto Rican newborns using DNA pooling technique. DESIGN/METHODS: Twelve percent of annual Puerto Rican births were tested randomly by polymerase chain reaction for the HPS-3 mutation, using pooled DNA extracted from dried blood samples. RESULTS: HPS-3 mutation was detected in 75 samples. Two newborns were found to be homozygous. Carrier frequency was 1:85 (1.18%). CONCLUSIONS: The HPS-3 carrier frequency found (1.18%) justifies universal newborn screening in Puerto Rico. DNA pooling reduces time and labor in newborn screening thus facilitating early diagnosis and treatment of children with HPS-3 and the provision of genetic counseling to parents and relatives.

Factors controlling the reactivity of hydrogen sulfide with hemeproteins.

Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H(2)S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H(2)S toxicity. Single point mutations at E7, B10, and E11 were introduced into the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild-type recombinant proteins were first evaluated using the well-known ferrous CO and O(2) derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O(2) binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, -PheB10Val, -PheB10Leu, and -E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H(2)S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H(2)S complexes, which show reduction of heme iron as judged by the appearance of the nu(4) oxidation state marker at 1356 cm(-1), indicative of heme-Fe(II) species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting the strongest H-bonding interactions. Overall, the results presented here show that (a) H(2)S association is regulated by external kinetic barriers, (b) H(2)S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction, (c) at high H(2)S concentrations, reduction of the ferric center dominates, and (d) reduction of the heme is also enhanced in those HbI mutants having polar distal environments.

Prevalence of combinatorial CYP2C9 and VKORC1 genotypes in Puerto Ricans: implications for warfarin management in Hispanics.

Polymorphisms in the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes significantly alter the effective warfarin dose. We determined the frequencies of alleles, single carriers, and double carriers of single nucleotide polymorphisms (SNPs) in the CYP2C9 and VKORC1 genes in a Puerto Rican cohort and gauged the impact of these polymorphisms on warfarin dosage using a published algorithm. A total of 92 DNA samples were genotyped using Luminex x-MAP technology. The polymorphism frequencies were 6.52%, 5.43% and 28.8% for CYP2C9 *2, *3 and VKORC1-1639 C>A polymorphisms, respectively. The prevalence of combinatorial genotypes was 16% for carriers of both the CYP2C9 and VKORC1 polymorphisms, 9% for carriers of CYP2C9 polymorphisms, 35% for carriers of the VKORC1 polymorphism, and the remaining 40% were non-carriers for either gene. Based on a published warfarin dosing algorithm, single, double and triple carriers of functionally deficient polymorphisms predict reductions of 1.0-1.6, 2.0-2.9, and 2.9-3.7 mg/day, respectively, in warfarin dose. Overall, 60% of the population carried at least a single polymorphism predicting deficient warfarin metabolism or responsiveness and 13% were double carriers with polymorphisms in both genes studied. Combinatorial genotyping of CYP2C9 and VKORC1 can allow for individualized dosing of warfarin among patients with gene polymorphisms, potentially reducing the risk of stroke or bleeding.