RESUMO
Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) and is one of the major causes of sudden cardiac death (SCD). An exon-targeted gene sequencing strategy was used to investigate the association of functional variants in sarcomeric genes (MYBPC3, MYH7 and TNNT2) with severe LVH and other SCD-related risk factors in Brazilian HCM patients. Clinical data of 55 HCM patients attending a Cardiology Hospital (Sao Paulo city, Brazil) were recorded. Severe LVH, aborted SCD, family history of SCD, syncope, non-sustained ventricular tachycardia and abnormal blood pressure in response to exercise were evaluated as SCD risk factors. Blood samples were obtained for genomic DNA extraction and the exons and untranslated regions of the MYH7, MYBPC3 and TNNT2 were sequenced using Nextera® and MiSEq® reagents. Variants were identified and annotated using in silico tools, and further classified as pathogenic or benign according to the American College of Medical Genetics and Genomics guidelines. Variants with functional effects were identified in MYBPC3 (n = 9), MYH7 (n = 6) and TNNT2 (n = 4). The benign variants MYBPC3 p.Val158Met and TNNT2 p.Lys263Arg were associated with severe LVH (p < 0.05), and the MYH7 p.Val320Met (pathogenic) was associated with family history of SCD (p = 0.037). Increased risk for severe LVH was found in carriers of MYBPC3 Met158 (c.472 A allele, OR = 13.5, 95% CI = 1.80-101.12, p = 0.011) or combined variants (MYBPC3, MYH7 and TNNT2: OR = 12.39, 95% CI = 2.14-60.39, p = 0.004). Carriers of TNNT2 p.Lys263Arg and combined variants had higher values of septum thickness than non-carriers (p < 0.05). Molecular modeling analysis showed that MYBPC3 158Met reduces the interaction of cardiac myosin-binding protein C (cMyBP-C) RASK domain (amino acids Arg215-Ala216-Ser217-Lys218) with tropomyosin. In conclusion, the variants MYBPC3 p.Val158Met, TNNT2 p.Lys263Arg and MYH7 p.Val320Met individually or combined contribute to the risk of sudden cardiac death and other outcomes of hypertrophic cardiomyopathy.
Assuntos
Cardiomiopatia Hipertrófica , Morte Súbita Cardíaca , Hipertrofia Ventricular Esquerda , Variantes FarmacogenômicosRESUMO
Abstract Introduction: Sticky platelet syndrome (SPS) is a prothrombotic condition characterized by increased platelet aggregation that causes arterial and venous thrombosis. Its diagnosis is reached by identifying increased aggregation using low concentrations of adenosine diphosphate and epinephrine in platelet aggregation tests. Objectives: To identify common mutations through exome sequencing in two patients from the same family diagnosed with SPS and, thus, contribute to the molecular study of this disease. Materials and methods: Descriptive study. In January 2018, exome sequencing was performed in a 10-year-old patient treated at Fundación HOMI (Bogotá D.C., Colombia), index case, and in one of his adult first-degree relatives, both with a history of thrombotic disease and diagnosed with SPS. Exome sequencing was performed at the Complexo Hospitalario Universitario de Santiago de Compostela (Spain) using the SureSelect Clinical Research Exome V2 software by Agilent. Results: Exome sequencing led to detect genetic variants in both cases when compared with the reference sequence. The following variant was identified in the two samples: a cytosine to thymine transition at position c.236 (NM_000174.4) of the glycoprotein (GP)Ib-IX-V complex platelet membrane receptor, which causes a heterozygous transition of the amino acid threonine to isoleucine (i.e., a transition from hydrophilic amino acid to a hydrophobic amino acid) at position p. 79 of the extracellular leucine-rich repeat domain of GPIba subunit of the (GP)Ib-IX complex, involving a conformational change of the main receptor of ligands IB alpha, which might result in platelet hyperaggregation and thrombosis. This variant has not been described in patients with SPS to date. Conclusion: The mutation identified in both samples could be related to SPS considering the importance of glycoprotein IX in platelet function.
Resumen Introducción. El síndrome de plaqueta pegajosa (SPP) es una condición protrombótica caracterizada por un incremento de la agregabilidad plaquetaria que causa trombosis arterial y venosa. Su diagnóstico se realiza al identificar el aumento de la agregabilidad utilizando bajas concentraciones de adenosín difosfato y epinefrina en pruebas de agregación plaquetaria. Objetivos. Identificar mutaciones comunes mediante secuenciación del exoma en dos pacientes de una misma familia con diagnóstico de SPP y, de esta forma, contribuir al estudio molecular de esta enfermedad. Materiales y métodos. Estudio descriptivo en el que se realizó secuenciación del exoma en un paciente de 10 años atendido en la Fundación HOMI (Bogotá, Colombia), caso índice, y en uno de sus familiares adultos en primer grado, ambos con antecedente de enfermedad trombótica y diagnosticados con SPP. La secuenciación del exoma se realizó en el Complexo Hospitalario Universitario de Santiago de Compostela (España) con el programa SureSelect Clinical Research Exome V2 de Agilent. Resultados. En la secuenciación del exoma se detectaron variantes genéticas en ambos casos en comparación con la secuencia de referencia. En las muestras de ambos pacientes se identificó una variante heterocigota consistente en una transición de citosina a timina en la posición c.236 (NM_000174.4) que provoca el cambio del aminoácido treonina por isoleucina en la posición p.79 del dominio extracelular repetitivo rico en leucina (subunidad GPIba del complejo de la glicoproteína Ib-IX-V) y que podría provocar el cambio conformacional del receptor principal del ligando Ib alfa, así como hiperagregación plaquetaria y trombosis. Esta variante no ha sido descrita previamente en pacientes con SPP. Conclusión. La mutación identificada en las muestras estudiadas podría estar relacionada con el SPP considerando la importancia de la glicoproteína IX en las funciones plaquetarias.
RESUMO
Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) and is one of the major causes of sudden cardiac death (SCD). An exon-targeted gene sequencing strategy was used to investigate the association of functional variants in sarcomeric genes (MYBPC3, MYH7 and TNNT2) with severe LVH and other SCD-related risk factors in Brazilian HCM patients. Clinical data of 55 HCM patients attending a Cardiology Hospital (Sao Paulo city, Brazil) were recorded. Severe LVH, aborted SCD, family history of SCD, syncope, non-sustained ventricular tachycardia and abnormal blood pressure in response to exercise were evaluated as SCD risk factors. Blood samples were obtained for genomic DNA extraction and the exons and untranslated regions of the MYH7, MYBPC3 and TNNT2 were sequenced using Nextera® and MiSEq® reagents. Variants were identified and annotated using in silico tools, and further classified as pathogenic or benign according to the American College of Medical Genetics and Genomics guidelines. Variants with functional effects were identified in MYBPC3 (n = 9), MYH7 (n = 6) and TNNT2 (n = 4). The benign variants MYBPC3 p.Val158Met and TNNT2 p.Lys263Arg were associated with severe LVH (p < 0.05), and the MYH7 p.Val320Met (pathogenic) was associated with family history of SCD (p = 0.037). Increased risk for severe LVH was found in carriers of MYBPC3 Met158 (c.472 A allele, OR = 13.5, 95% CI = 1.80-101.12, p = 0.011) or combined variants (MYBPC3, MYH7 and TNNT2: OR = 12.39, 95% CI = 2.14-60.39, p = 0.004). Carriers of TNNT2 p.Lys263Arg and combined variants had higher values of septum thickness than non-carriers (p < 0.05). Molecular modeling analysis showed that MYBPC3 158Met reduces the interaction of cardiac myosin-binding protein C (cMyBP-C) RASK domain (amino acids Arg215-Ala216-Ser217-Lys218) with tropomyosin. In conclusion, the variants MYBPC3 p.Val158Met, TNNT2 p.Lys263Arg and MYH7 p.Val320Met individually or combined contribute to the risk of sudden cardiac death and other outcomes of hypertrophic cardiomyopathy.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Mutação , Cadeias Pesadas de Miosina/genética , Troponina T/genética , Brasil , Morte Súbita Cardíaca/etiologia , Ecocardiografia , Feminino , Estudos de Associação Genética , Septos Cardíacos/diagnóstico por imagem , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Análise de Sequência de DNARESUMO
Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.
Assuntos
Aspirina/administração & dosagem , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Inibidores da Agregação Plaquetária/administração & dosagem , Ticlopidina/análogos & derivados , Síndrome Coronariana Aguda/terapia , Idoso , Aspirina/farmacocinética , Aspirina/farmacologia , Ácidos Carboxílicos/metabolismo , Cromatografia Líquida/métodos , Clopidogrel , Citocromo P-450 CYP2C19/genética , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/métodos , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Polimorfismo Genético , RNA Mensageiro/metabolismo , Ácido Salicílico/metabolismo , Espectrometria de Massas em Tandem/métodos , Ticlopidina/administração & dosagem , Ticlopidina/farmacocinética , Ticlopidina/farmacologia , Regulação para CimaRESUMO
Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.
Assuntos
Masculino , Feminino , Humanos , Pessoa de Meia-Idade , Idoso , RNA , Tratamento FarmacológicoRESUMO
Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism...
Assuntos
Pacientes , Polimorfismo Genético , RNA MensageiroRESUMO
BACKGROUND: Genome-wide expression analysis using microarrays has been used as a research strategy to discovery new biomarkers and candidate genes for a number of diseases. We aim to find new biomarkers for the prediction of acute coronary syndrome (ACS) with a differentially expressed mRNA profiling approach using whole genomic expression analysis in a peripheral blood cell model from patients with early ACS. METHODS AND RESULTS: This study was carried out in two phases. On phase 1 a restricted clinical criteria (ACS-Ph1, n=9 and CG-Ph1, n=6) was used in order to select potential mRNA biomarkers candidates. A subsequent phase 2 study was performed using selected phase 1 markers analyzed by RT-qPCR using a larger and independent casuistic (ACS-Ph2, n=74 and CG-Ph2, n=41). A total of 549 genes were found to be differentially expressed in the first 48 h after the ACS-Ph1. Technical and biological validation further confirmed that ALOX15, AREG, BCL2A1, BCL2L1, CA1, COX7B, ECHDC3, IL18R1, IRS2, KCNE1, MMP9, MYL4 and TREML4, are differentially expressed in both phases of this study. CONCLUSIONS: Transcriptomic analysis by microarray technology demonstrated differential expression during a 48 h time course suggesting a potential use of some of these genes as biomarkers for very early stages of ACS, as well as for monitoring early cardiac ischemic recovery.
Assuntos
Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Proteínas de Fase Aguda/genética , Células Sanguíneas/metabolismo , Expressão Gênica , RNA Mensageiro/genética , Síndrome Coronariana Aguda/genética , Proteínas de Fase Aguda/metabolismo , Adulto , Biomarcadores/sangue , Células Sanguíneas/química , Diagnóstico Precoce , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/sangue , TranscriptomaRESUMO
Background: Genome-wide expression analysis using microarrays has been sed as a research strategy to discovery new biomarkers and candidate genes for a number of diseases. We aim to find new biomarkers for the prediction of acute coronary syndrome (ACS) with a ifferentially expressed mRNA profiling approach using whole genomic expression analysis in a peripheral blood cell model from patients with early ACS.Methods and results: This study was carried out in two phases. On phase 1 a restricted clinical criteria (ACS-Ph1, n = 9 and CG-Ph1, n = 6) was used in order to select potential mRNA biomarkers candidates. A subsequent phase 2 study was performed using selected phase 1 markers analyzed by RT-qPCR using alarger and independent casuistic (ACS-Ph2, n = 74 and CG-Ph2, n = 41). A total of 549 genes were found to be differentially expressed in the first 48 h after the ACS-Ph1. Technical and biological validation further confirmed that ALOX15, AREG, BCL2A1, BCL2L1, CA1, COX7B, ECHDC3, IL18R1, IRS2, KCNE1, MMP9, MYL4 and TREML4, are differentially expressed in both phases of this study.Conclusions: Transcriptomic analysis by microarray technology demonstrated differential expression during a 48 h time course suggesting a potential use of some of these genes as biomarkers for very early stages of ACS, as well as for monitoring early cardiac ischemic recovery.
Assuntos
Biomarcadores , Infarto do Miocárdio , Síndrome Coronariana Aguda , TranscriptomaRESUMO
BACKGROUND: The aim of this study was investigate the relationship between ABCB1 and ABCC3 gene expressions in peripheral blood cells (PBC) and the response to clopidogrel in patients with coronary arterial disease (CAD). METHODS: Twenty-six male CAD patients (50-70 years) under treatment with clopidogrel (75 mg/day) for at least 5 days were selected. Blood samples were obtained to evaluate platelet reactivity and ABCB1 and ABCC3 mRNA expression. Platelet reactivity was measured in P2Y12 Reaction Units (PRU) using VerifyNow. RNA was extracted from PBC and mRNA levels were measured by qPCR, using GAPD as a reference gene. RESULTS: Platelet response to clopidogrel was categorized in to PRU quartiles. Individuals with PRU values within the first quartile (Q1, <151 units) were considered good responders, while those who had PRU within the fourth quartile (Q4, PRU>260) were considered non-responders. ABCC3 was 1.7 times more expressed in Q4 than in Q1 PRU group (p=0.048). Moreover, CAD patients with low ABCC3 expression (Qe1, <2.5×10(-3)) had higher probability to have a good response to clopidogrel (OR: 18.00, 95%CI: 1.90-169.99, p=0.001). Univariate linear regression analysis demonstrated that low ABCC3 mRNA expression contributed with a reduction of 73 PRU in relation to the patients with expression value higher than 2.5×10(-3) (p=0.027). Neither ABCB1 mRNA levels nor clinical variables studied influenced PRU values. CONCLUSIONS: Low ABCC3 mRNA expression in peripheral blood cells is associated with increased clopidogrel response, but further studies are needed to describe the functional relationship of clopidogrel with the ABCC3.
Assuntos
Regulação da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ticlopidina/análogos & derivados , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Idoso , Clopidogrel , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ticlopidina/farmacologia , Ticlopidina/uso terapêutico , Resultado do TratamentoRESUMO
In the recent history of Colombia, two factors have contributed to change the population structure, the Spanish conquest and the slave trading promoted principally by Portugal, England and Spain. As a consequence the native population of Colombia has been reduced and mixed with the European and African arriving groups. To assess the male ancestry of the Cauca population, we have examined the frequency of the major Y-chromosome lineages by typing 30 Y-SNPs in two populations from this region: 105 Afro-Colombian individuals and 110 Caucasian-Mestizo individuals. Among the 33 haplogroups defined with the SNPs analysed, 15 haplogroups were detected, 10 of them being shared by both populations. In order to investigate how the level of admixture is, and to compare the genetic background with other neighbour populations, other South American samples previously published were used for comparative analysis.
Assuntos
Cromossomos Humanos Y , Genética Populacional , Polimorfismo de Nucleotídeo Único , População/genética , Alelos , População Negra/genética , Colômbia , Impressões Digitais de DNA , Frequência do Gene , Marcadores Genéticos , Genética , Geografia , Haplótipos , Humanos , Indígenas Sul-Americanos/genética , Masculino , Reação em Cadeia da Polimerase , Controle de Qualidade , População Branca/genéticaRESUMO
Allele frequencies and some forensic parameters for 12 autosomal microsatellites (CSF1PO, TPOX, THO1, VWA, D16S539, D7S820, D13S317, D5S818, F13A1, FESFPS, F13B, LPL) were estimated from three departments from Northwestern Colombia. The total number of samples analysed was 1045 individuals. Comparative analysis among the three studied departments and with other published Colombian populations were also performed and discussed.
Assuntos
Frequência do Gene , Genética Populacional , Repetições de Microssatélites , Colômbia , Impressões Digitais de DNA , Humanos , Reação em Cadeia da PolimeraseRESUMO
The European Consortium "High-throughput analysis of single nucleotide polymorphisms for the forensic identification of persons--SNPforID", has performed a selection of candidate Y-chromosome single nucleotide polymorphisms (SNPs) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y-Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups. A "Major Y-chromosome haplogroup typing kit" has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction. Allele genotyping was performed with a single base extension reaction (minisequencing) detected by CE. The validation of the multiplex was performed in a total of 1126 unrelated males distributed among 12 worldwide populations. The approach takes advantage of the specific geographic distribution of the Y-chromosome haplogroups and demonstrates the utility of binary polymorphisms to infer the origin of a male lineage.
Assuntos
Cromossomos Humanos Y/genética , Genética Populacional , Haplótipos , Polimorfismo de Nucleotídeo Único , África , Ásia , Primers do DNA , Europa (Continente) , Feminino , Humanos , Masculino , Filogenia , América do SulRESUMO
Allele frequencies for 19 autosomal STRs (F13A01, FESFPS, F13B, LPL, D5S818, D7S820, THO1, TPOX, VWA31, CSF1P0, D16S539, D13S317, D3S1358, D8S1179, FGA, PENTA D, PENTA E, D21S11, D18S51) were estimated from a sample between 364 and 400 unrelated individuals living in the northern department of Antioquia.
Assuntos
Frequência do Gene , Genética Populacional , Sequências de Repetição em Tandem , Colômbia , Impressões Digitais de DNA/métodos , Humanos , Reação em Cadeia da PolimeraseRESUMO
Eight Y-chromosome STRs were investigated in a male population sample from El Salvador. Complete Y-chromosomal STRs haplotypes were obtained in 121 individuals, among which 107 different haplotypes were observed. The two most common haplotypes were shared by approximately 4% of the sample, while 100 haplotypes were unique. The gene diversity was 0.9883 and the discrimination capacity was 0.8926. The combined Y-chromosome STR polymorphisms provide a powerful discrimination tool for routine forensic applications.
Assuntos
Cromossomos Humanos Y , Genética Populacional , Haplótipos , Sequências de Repetição em Tandem , Impressões Digitais de DNA/métodos , El Salvador , Variação Genética , Humanos , MasculinoRESUMO
Allele frequencies for eight short tandem repeats (STRs) (D5S818, D7S820, F13B, LPL, TH01, TPOX, VWA31 and CSF1PO) were estimated from a sample of 155 unrelated individuals living in different departments of the southwest of Colombia, Caquetá, Cauca, Huila, Nariño, Putumayo and Cauca Valley.