Phenylalanine hydroxylase deficiency in Mexico: genotype phenotype correlations, BH 4 responsiveness and evidence of a founder effect

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1 Clin Genet 2015: 88: Printed in Singapore. All rights reserved Short Report 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Phenylalanine hydroxylase deficiency in Mexico: genotype phenotype correlations, BH 4 responsiveness and evidence of a founder effect Vela-Amieva M, Abreu-González M, González-del Angel A, Ibarra-González I, Fernández-Lainez C, Barrientos-Ríos R, Monroy-Santoyo S, Guillén-López S, Alcántara-Ortigoza MA. Phenylalanine hydroxylase deficiency in Mexico: BH 4 responsiveness and evidence of a founder effect. Clin Genet 2015: 88: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 The mutational spectrum of the phenylalanine hydroxylase gene (PAH)in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH 4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype phenotype correlations and genotype-based prediction of BH 4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5g>t beingthe most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1a>t and c.969+6t>c. The genotype phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH 4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH 4 testing and therapy. Conflict of interest The authors have declared no conflicting interests. M. Vela-Amieva a, M. Abreu-González b, A. González-del Angel c, I. Ibarra-González d, C. Fernández-Lainez a, R. Barrientos-Ríos c, S. Monroy-Santoyo a, S. Guillén-López a and M.A. Alcántara-Ortigoza c a Laboratorio de Errores Innatos del Metabolismo y Tamiz, Instituto Nacional de Pediatría, Secretaría de Salud, México, b Programa de Maestría y Doctorado en Ciencias Biológicas, Facultad de Ciencias, Universidad Nacional Autónoma de México, México D.F., México, c Laboratorio de Biología Molecular, Departamento de Genética Humana, Instituto Nacional de Pediatría, Secretaría de Salud, México, and d Unidad de Genética de la Nutrición, Instituto de Investigaciones Biomédicas, UNAM Instituto Nacional de Pediatría, Secretaría de Salud, México Key words: hyperphenylalaninemia phenylketonuria PKU tetrahydrobiopterin Corresponding author: Miguel Ángel Alcántara-Ortigoza, Laboratorio de Biología Molecular, Departamento de Genética Humana, Instituto Nacional de Pediatría, Secretaría de Salud. Av. IMAN #1, piso 9, Col. Insurgentes-Cuicuilco, Delegación Coyoacán, México D.F , México. Tel.: +52 (55) ext fax: +52 (55) malcantaraortigoza@gmail.com Received 29 April 2014, revised and accepted for publication 16 June

2 Phenylalanine hydroxylase deficiency in Mexico Hyperphenylalaninemia (HPA) and phenylketonuria (PKU) (OMIM ) are caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). About 851 variants have been described in the PAH gene, with c.1222c>t and c g>a being the most common worldwide according to the BIOPKUdb (BIOPKU database, Blau N and Yue W, and Perez B, The mutational spectrum of PAH in Mexico is poorly known; the only study that has been conducted, showed the presence of the c g>a in 5/14 alleles (1). There is some evidence that PKU in Mexico has a predominance of cases occurring in the Middle West of the country, mainly in the state of Jalisco, where a possible founder effect has been suggested (2). The PAH genotype, as a predictor of metabolic phenotype, has come to play an important role in assessing the potential responsiveness to treatment with sapropterin dihydrochloride, a synthetic formulation of the active naturally cofactor for PAH, tetrahydrobiopterin (BH 4 ) (3). In this report, we present the mutational spectrum of the PAH gene in 48 unrelated Mexican PKU/HPA patients and report genotype phenotype correlations and genotype-based prediction to BH 4 -responsiveness. We also analyzed PAH gene mini-haplotypes associated with the most prevalent and new variants in order to establish their ancestral origin. Materials and methods Subjects Forty-eight Mexican PKU/HPA unrelated patients and their families from 19/32 states of the country were included and written informed consent was obtained. Patients were assigned to four phenotype categories according to literature (4) and were further categorized into two groups: those diagnosed by newborn screening (NBS) and those diagnosed based on clinical symptoms. Genotype analysis Genomic DNA was isolated by standard procedures. PAH variants were identified using direct automated DNA sequencing of the 13 coding exons and flanking regions as previously described (5); c.60+5g>t and c.969+6t>c were analyzed in silico using the Human Splicing Finder software ( Genotype phenotype correlations and genotype-based predictions of BH 4 -responsiveness On the basis of available evidence, BH 4 responsiveness prediction was made comparing identical genotypes or by single alleles reported on BIOPKUdb or literature, assigning the category Yes or No when a favorable response (decrease of 30% of blood Phe levels) was evident or not respectively. Thus, patients were assigned as candidates for BH 4 testing (+) when: (i) genotype was previously reported as responder; (ii) at least one allele was assigned as responder and (iii) cases with no documented evidence of responsiveness. Patients whose alleles showed no response were considered as non-candidates ( ). Ancestral origin by mini-haplotype analysis In order to assign the ancestral origin of the three most common and the two new variants found in the studied population, we performed a mini-haplotype analysis in probands and their parents when possible, with the polymorphisms BglII, PvuII, MspI and XmnI and the VNTR site at the 3 region of the PAH gene. The numbering system used was previously established (6). Results Of 48 patients, 33 (68.7%) were classic PKU, 4 (8.3%) moderate, 8 (16.7%) mild, and 3 (6.3%) benign HPA. Twenty-six patients were diagnosed by NBS. Within this group, 14 patients (54%) were classified as classic PKU, 1 (3.8%) as moderate, 8 (30.7%) as mild, and 3 (11.5%) as benign HPA. The remaining 22 patients were diagnosed based on clinical symptoms (average age at diagnosis, 39.5 months). Nineteen of these patients (86.4%) were classic PKU and 3 (13.6%) moderate HPA. Genotype analysis The mutational spectrum included 34 different variants (32 single-base substitutions and 2 frameshift deletions). Missense variants represented 58.8%, splicing and nonsense variants comprised 26.4 and 8.8%, respectively (Table 1). The most frequent variant, identified in 20/96 (20.8%) of the PAH alleles was c.60+5g>t, followed by c g>a and c.1162g>a, each of which represented 8.3% (8/96) of total alleles (Table 1). We found 41 different genotypes, most of them were observed in a single case, however, the c.[60+5g>t];[60+5g>t], c.[60+5g>t];[1162g>a] and c.[60+5g>t];[441+5g>t] genotypes were identified more than once; 9 patients were homozygous and 39 compound heterozygous (Table 2, 7 10). Familial segregation analysis was confirmed in 45 of our patients and only in three cases both parents were not available. Carrier state was proved in six of nine homozygous patients progenitors; however, in two of the three remaining families, the parents were consanguineous. Two new variants were found: c.1a>t and c.969+6t>c. These were absent in 105 non-related healthy controls and are not enlisted in the Exome Variant Server ( The genotypes, c.[1a>t];[1a>t], c.[1a>t];[722g>a] and c.[969+6t>c];[ g>a] were observed in a classic, in a mild, and in a moderate PKU patients, respectively. The in silico analysis showed that c.60+5g>t abolished the recognition of the natural splice donor site of intron 1 (MaxEnt CV: 55.4%), and the evaluation for c.969+6t>c reported a decrease in the affinity value for recognition of the natural splicing donor site (MaxEnt CV: 37.4%). 63

3 Vela-Amieva et al. Table 1. Characterization of PAH gene mutations identified in 48 Mexican patients Alleles (n = 96) and frequency (%) Sequence variation a Protein variant Location Variation type Domain PAH reported activity (%) b Associated haplotype (number of linked alleles) 20 (20.8) c.60+5g>t p.? In. 1 Splice (15) 8 (8.3) c.1162g>a p.(val388met) Ex. 11 Missense Catalytic (2) 8 (8.3) c g>a p.? In. 10 Splice (1); 5 or 6 (1) 7 (7.3) c.441+5g>t p.? In. 4 Splice 0 5 (5.2) c.1045t>c p.(ser349pro) Ex. 10 Missense Catalytic 1 3 (3.1) c.838g>a p.(glu280lys) Ex. 7 Missense Catalytic 2 c.508c>g p.(his170asp) Ex. 5 Missense Catalytic 43 c.782g>a p.(arg261gln) Ex. 7 Missense Catalytic 44 c.1169a>g p.(glu390gly) Ex. 11 Missense Catalytic 62 c g>a p.? In. 12 Splice 0 2 (2.1) c.809g>a p.(arg270lys) Ex. 7 Missense Catalytic 0 c.754c>t p.(arg252trp) Ex. 7 Missense Catalytic 0 c.722g>a p.(arg241his) Ex. 7 Missense Catalytic 23 c.439c>t p.(pro147ser) Ex. 4 Missense Catalytic NR c.842+1g>a p.? In. 7 Splice NR c.781c>t p.(arg261 * ) Ex. 7 Nonsense Catalytic 0 c.912g>a p.(gln304=) Ex. 8 Splice Catalytic 0 1 (1) c.472c>t p.(arg158trp) Ex. 5 Missense Catalytic NR c.728g>a p.(arg243gln) Ex.7 Missense Catalytic 14 c.194t>c p.(ile65thr) Ex. 3 Missense Regulatory 33 c.1042c>g p.(leu348val) Ex. 10 Missense Catalytic 35 c.204a>t p.(arg68ser) Ex. 3 Missense Regulatory 68 c.941c>a p.(pro314his) Ex. 9 Missense Catalytic NR c.1157a>g p.(tyr386cys) Ex.11 Missense Catalytic NR c.673c>a p.(pro225thr) Ex. 6 Missense Catalytic NR c.907t>g p.(ser303ala) Ex. 8 Missense Catalytic NR c.727c>t p.(arg243 * ) Ex. 7 Nonsense Catalytic 0 c.1127dela p.(asn376ilefs * 24) Ex. 11 Frameshift Catalytic NR c.969+6t>a p.? In. 9 Splice NR c.526c>t p.(arg176 * ) Ex. 6 Nonsense Catalytic 0 c t>c p.? In. 12 Splice NR c.165delt p.(phe55leufs * 6) Ex. 2 Frameshift Regulatory 0 3 (3.1) c.1a>t p.(met1?) Ex. 1 Missense Regulatory NEW 1.7 (2) 1 (1) c.969+6t>c p.? In. 9 Splice NEW 5 or 6 (1) Ex., exon; In., intron; NR, not reported. a Based on: NG_ RefSeqGene. b PAH activity was recorded as described in PAHvdb (PAHvdb, Blau N and Yue W, and Perez B Genotype phenotype correlation and genotype-base predictions of BH 4 responsiveness The genotype phenotype correlation was found to be concordant in 34/48 patients (70.8%) and discordant in 5 (10.4%). In the remaining nine patients (18.8%), the theoretical phenotype has not yet been established, thus the genotype phenotype correlation was not determined. The genotype-based prediction to BH 4 -responsiveness was 41.7% and estimation of candidates to BH 4 testing based on genotype was 47.9% (Table 2). Ancestral origin by mini-haplotype analysis We found 41.6% (40/96) of alleles with a geographical origin in the Middle West region of Mexico, called El Bajío, comprising the states of Jalisco, Guanajuato, Michoacán and Aguascalientes. Chi-squared analysis showed statistical significance in the c.60+5g>t frequency alleles among the native patients from El Bajío region compared with those born in the rest of the country (17 vs 3, respectively, p < %). The four homozygous patients for this variant came from this region. The haplotypic background could be characterized in only 15/20 alleles with c.60+5g>t and all of them were found to carry the haplotype 4.3. The five remaining haplotypes linked to c.60+5g>t alleles were not characterized because of non-informative genotypes in cases and their parents. Variant c.1162g>a was associated with haplotype 4.3 and the c g>a with haplotypes 11.7 and 5 or 6 (not assigned because of lack of EcoRV assay data). The new c.1a>t variant in a homozygous patient showed an association with the 1.7 haplotype. The other new variant, c.969+6t>c was linked to haplotype 5 or 6 (Table 1). 64

4 Phenylalanine hydroxylase deficiency in Mexico Table 2. Genotype phenotype correlations and genotype-based prediction of BH 4 responsiveness in 48 Mexican PKU patients a Number of patients Allele 1 Allele 2 Reported phenotype Observed phenotype Prediction of BH 4 responsiveness BH 4 test candidate Reference allele 1/ allele 2 Identical genotype reported on BIOPKUdb 1 c.1169a>g c.1169a>g Mild-benign Mild Yes + 1 c.1169a>g c.1045t>c Benign HPA/mild Mild Yes + 1 c.508c>g c.60+5g>t Benign HPA Benign HPA Yes + 1 c.1042c>g c.194t>c Mild/classic Classic Yes + 1 c.1045t>c c.439c>t Classic Classic Yes + 1 c g>a c.781c>t Mild/classic Classic No 1 c g>a c.1045t>c Classic Classic No 1 c.441+5g>t c.441+5g>t Classic Classic No 1 c.912g>a c.912g>a Classic Classic No 1 c.1162g>a c g>a Mild/classic Classic No 1 c.782g>a c.781c>t Moderate Classic b No 1 c.60+5g>t c g>a Classic Classic No 1 c g>a c.526c>t Classic Classic No 1 c g>a c.809g>a Classic Classic No 1 c.838g>a c.838g>a Mild/classic Classic No 1 c.165delt c.441+5g>t Classic Classic Uncertain c + At least one allele has been reported as BH 4 responsive 1 c.728g>a c.722g>a Mild Mild Yes/Yes + 7, 8/3, 7, 8 1 c.204a>t c.782g>a NR Mild Yes/Yes + 3, 7, 8/7, 8 1 c.782g>a c.439c>t NR Classic Yes/Yes + 7, 8/8 1 c.1162g>a c.60+5g>t Moderate Classic b Yes/No + 7, 8/4 1 c.1162g>a c g>a Moderate Classic b Yes/No + 7, 8/7, 8 3 c.1162g>a c.60+5g>t Moderate Moderate Yes/No + 7, 8/4 1 c.508c>g c g>a NR Mild Yes/No + 8/7, 8 1 c.508c>g c.842+1g>a NR Moderate Yes/No + 7/10 1 c.809g>a c.941c>a NR Mild No/Yes + 7, 8/3 1 c.1162g>a c.969+6t>a NR Benign HPA Yes/NR + 7, 8/NR 1 c.1162g>a c t>c Moderate Classic b Yes/NR + 7, 8/NR 1 c.722g>a c.1a>t NEW Mild Yes/NEW + 3, 7, 8/NEW Both alleles have been reported as BH 4 non-responders 4 c.60+5g>t c.60+5g>t Classic Classic No/No 3/3 2 c.60+5g>t c.441+5g>t Classic Classic No/No 4/10, 11 1 c.441+5g>t c.842+1g>a Classic Classic No/No 10, 11/9 1 c.60+5g>t c g>a Classic Classic No/No 4/7, 8 1 c.1045t>c c.472c>t Classic Classic No/No 7, 8/BIOPKUdb 1 c.60+5g>t c.727c>t Classic Classic No/No 4/7, 8 1 c.60+5g>t c.754c>t Classic Classic No/No 4/7, 8 1 c.60+5g>t c.838g>a Classic Classic No/No 4/7, 8 1 c.441+5g>t c.1045t>c Classic Classic No/No 10, 11/7, 8 BH 4 response not determined 1 c g>a c.969+6t>c NR Mild No/NR + 3/NEW 1 c.1a>t c.1a>t NEW Classic NEW/NEW + NEW/NEW 1 c g>a c.1127dela Classic Classic No/NR + 3/NR 1 c.754c>t c.1157a>g Classic Classic No/NR + 7, 8/NR 1 c.673c>a c.907t>g Mild Benign HPA b No/NR + 11/NR NR: not reported; +, candidate for BH 4 testing;, non-candidate for BH 4 testing. a New variants are shown in bold type. b Discordant genotype phenotype correlation. c Uncertain: for this genotype BIOPKUdb reports two responder cases and two other non-responders. Discussion We found a predominance of classic PKU phenotype in this study; however it may be biased due to the presence of high proportion of patients detected by symptoms instead of being detected by newborn screening, where the number of HPA patients detected would be higher. The mutational spectrum of PAH in Mexican PKU/HPA patients showed high heterogeneity. Unlike European populations, where the 10% are splicing PKU alleles (12), the Mexican population studied here exhibited a high proportion of such variants (26.4%). This difference is because of the high frequency (20.8%) of the c.60+5g>t variant (Table 1), which was described 65

5 Vela-Amieva et al. initially as a null mutation in the European population (13); owing to its low incidence worldwide, there are no reports of its effect on the splicing process. It is known that variants affecting the +5 position could decrease the affinity of the natural splicing sites in human genes (14), an observation that is in agreement with our in silico analyses. Therefore, c.60+5g>t has been classified as severe (15) and the classic PKU phenotype documented in our homozygous patients supports this classification. The Human Gene Mutation Database ( hgmd.org/) estimates that 0.6% of single-base variants involve the translational initiation codon, such as the c.1a>t allele identified herein; an expression analysis of the previously described c.1a>g, revealed no enzymatic activity or immunoreactive protein (16). Accordingly, the expected phenotype for c.1a>t would presumably be severe; consistent with this, of the three alleles found, two were present in homozygous form in a patient who exhibited a classic PKU phenotype. The other was present as compound heterozygous in a patient with mild PKU, probably conditioned by c.722g>a allele, which possesses 23 25% residual activity and was previously identified in three homozygous patients who had been classified with benign HPA and a mild PKU phenotype (BIOPKUdb), one of whom was found to respond favorably to BH 4. The in silico analysis of the second new variant c.969+6t>c, appears to slightly decrease the affinity for the natural splicing donor site, so it predicts normal mature transcripts amounts could be diminished (17). In agreement with this, the c.969+6t>c variant even in combination with the severe c g>a allele, was associated with a mild phenotype; although transfection mini-gene studies should be necessary to confirm its deleterious effect. We found a genotype phenotype discordance of 10.4%. It is especially remarkable in the patients harboring the c.1162g>a allele (3/5), (Table 2). It has been suggested that this discrepancy could be because c.1162g>a affect interactions at the subunit interface (7), induce negative interallelic complementation depending on the accompanying allele and by other non-genetic factors (15, 17). Our results show an estimation of BH 4 -responders of 41.7% however, despite the value of genotype-based prediction of BH 4 responsiveness, the only precise method for determining patients responsiveness to the drug is the BH 4 loading test, this is particularly important in patients with only one responder allele and in those with new allele variants (Table 2). According to the PAH database (PAHdb, mcgill.ca), c.60+5g>t accounts for <0.35% of PAH variants worldwide, but in 1.36% of alleles from southern Spain (4). The mutational spectrum identified here clearly revealed a differential geographical distribution, in which c.60+5g>t was significantly more prevalent in El Bajío compared to the rest of the country. Furthermore, c.60+5g>t has been linked with the 4.3 haplotype in Danish and Spanish populations (13, 18) and in this study it is also in linkage disequilibrium with the 4.3 haplotype (Table 1). These data could suggest that c.60+5g>t in Mexican patients was not an independent mutational event, but rather that its origin could be traced to the Iberian Peninsula. This interpretation is consistent with the settlement history of Mexico, especially by individuals from southern Spain, who colonized El Bajío, particularly the state of Jalisco ( Los Altos region) and its surroundings, during the XVI century (19). These results support a founder effect and/or genetic drift for c.60+5g>t but not for the Mediterranean c g>a, as was previously suggested (2). Regarding the haplotypes associated with newly identified variants, the patient with c.[969+6t>c]; [ G>A] genotype bore haplotype 5 or 6, although the latter is commonly linked with the c g>a allele in the Mediterranean population (PAHdb). Variant c.1a>t was linked to 1.7 haplotype, which is associated with other pathogenic PAH variants (18). In conclusion, the mutational spectrum of PAH gene in Mexican PKU/HPA patients exhibited high heterogeneity, with c.60+5g>t being the most common variant, very likely attributable to a founder effect and/or genetic drift. We also reported two new variants, c.969+6t>c and c.1a>t, associated with a mild and severe phenotype, respectively. The overall genotype phenotype correlation was 70.8%, and the genotype-based prediction of BH 4 -responder patients was 41.7%. 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