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1.
J Pers Med ; 14(1)2024 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-38248810

RESUMEN

BACKGROUND: In recent years, increasingly complex ALI protocols involving specialized, albeit laboratory-specific media have been established, while at the same time, many studies compile the data of only a few ALI donors in spite of site-, protocol- and donor-specific differentiation. METHODS: We describe a simple morphology scoring protocol using histology material derived from epithelia grown on ALI inserts in parallel to other, more complex readouts. RESULTS: Among more than 100 ALI inserts derived from different donors, significant differences in layer score (p = 0.001) and goblet cell score (p = 0.002) were observed when ALI epithelia derived from explanted lung material were compared to trachea-derived ALI cultures. Cortisol withdrawal for the final 2 days of ALI cultures influenced goblet cell density (p = 0.001). CONCLUSIONS: While the histology score provides less resolution than FACS- or OMICs- based single cell analyses, the use of a subportion of the ALI epithelia grown on inserts makes it feasible to combine morphology assessment and other readouts of the same insert. This allows us to control for basic ALI morphology in research and personalized medicine settings in order to assess and, if desired, control for the impact of ALI culture protocols, site- and donor-specific influences on outcome of studies of ALI-derived epithelia.

2.
Front Pharmacol ; 14: 1114584, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36778025

RESUMEN

Background: Defects in expression, maturation or function of the epithelial membrane glycoprotein CFTR are causative for the progressive disease cystic fibrosis. Recently, molecular therapeutics that improve CFTR maturation and functional defects have been approved. We aimed to verify whether we could detect an improvement of CFTR protein expression and maturation by triple therapy with elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA). Methods: Rectal suction biopsies of 21 p.Phe508del homozygous or compound heterozygous CF patients obtained pre- and during treatment with ELX/TEZ/IVA were analyzed by CFTR Western blot that was optimized to distinguish CFTR glycoisoforms. Findings: CFTR western immunoblot analysis revealed that-compared to baseline-the levels of CFTR protein increased by at least twofold in eight out of 12 patients upon treatment with ELX/TEZ/IVA compared to baseline (p < 0.02). However, polydispersity of the mutant CFTR protein was lower than that of the fully glycosylated wild type CFTR Golgi isoform, indicating an incompletely glycosylated p.Phe508el CFTR protein isoform C* in patients with CF which persists after ELX/TEZ/IVA treatment. Interpretation: Treatment with ELX/TEZ/IVA increased protein expression by facilitating the posttranslational processing of mutant CFTR but apparently did not succeed in generating the polydisperse spectrum of N-linked oligosaccharides that is characteristic for the wild type CFTR band C glycoisoform. Our results caution that the lower amounts or immature glycosylation of the C* glycoisoform observed in patients' biomaterial might not translate to fully restored function of mutant CFTR necessary for long-term provision of clinical benefit.

3.
Hum Mol Genet ; 32(4): 543-550, 2023 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-36048831

RESUMEN

INTRODUCTION AND AIM: The signal transducer and activator of transcription 3 (STAT3) has been identified as one of the cystic fibrosis (CF) modifying genes. In this study, we aimed to assess the association between STAT3 genotype and CF patient survival over several decades and to investigate the effect of STAT3 inhibition on epithelial CFTR expression. METHODS: We analyzed the informative genetic marker STAT3Sat for its association with survival in 174 p.Phe508del-CFTR homozygous CF patients treated at the CF center in Hannover spanning birth cohorts from >3 decades (1959-1994). Furthermore, we treated two epithelial cell lines with STAT3 inhibitors and monitored changes of CFTR protein expression by western blot. RESULTS: Only for p.Phe508del-CFTR homozygous patients born prior to 1975, survival was significantly influenced by STAT3sat genotype (P = 0.023). The expression levels of STAT3 and CFTR positively correlated in epithelial cell lines (P = 0.01). CONCLUSIONS: Our results in different birth cohorts identified a time-dependent impact of STAT3 genotype on CF patients' survival and found that improved symptomatic treatment of later-born CF patients obviates STAT3's modifying influence. Consistent with our previous results, STAT3-specific inhibition resulted in increased CFTR expression in the epithelial cell line 16HBE14o-. Thus, care should be taken when CF-modifying genes are studied in cross-sectional cohorts as the impact of modifying genes might not be invariant in the light of changing therapeutic regimens.


Asunto(s)
Fibrosis Quística , Humanos , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Estudios Transversales , Genotipo
4.
Molecules ; 26(9)2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33946490

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial-mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.


Asunto(s)
Barrera Alveolocapilar/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Respiratoria/metabolismo , alfa 1-Antitripsina/metabolismo , Biomarcadores , Línea Celular , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , alfa 1-Antitripsina/genética
5.
BMJ Open Respir Res ; 7(1)2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33020115

RESUMEN

BACKGROUND: Nasal potential difference (NPD) and intestinal current measurements (ICM) are cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers recommended to make a diagnosis in individuals with inconclusive sweat test and CFTR genetics and a clinical suspicion for cystic fibrosis (CF) or CFTR-related disorder (CFTR-RD). METHODS: NPD and ICM were measured according to standard operating procedures of the European Cystic Fibrosis Society Diagnostic Network Working Group. RESULTS: We assessed 219 individuals by NPD or ICM who had been referred to our laboratory due to clinical symptoms suggestive of CF, but inconclusive sweat test and CFTR genetics (median age: 16.3 years, range 0.4 to 76 years). CF or CFTR-related disorder was diagnosed in 22 of 29 patients (76%) with a CFTR genotype of unknown or variable clinical significance and in 51 of 190 carriers (27%) of one (35/42) or no (16/148) identified CFTR mutation. If two CFTR sequence variants had been identified, the outcome of NPD and ICM was consistent with the classification of the CFTR2 database. Moreover, a suspected false-positive diagnosis of CF was confirmed in seven and withdrawn in eight patients. Of 26 individuals assessed by both NPD and ICM, eleven individuals exhibited discordant tracings of ICM and NPD, with one measurement being in the CF range and the other in the normal range. CONCLUSION: The majority of patients whom we diagnosed with CF or CFTR-RD by extended electrophysiology are carriers of the wild-type CFTR coding sequence on at least one of their CF alleles. The disease-causing genetic lesions should reside in the non-coding region of CFTR or elsewhere in the genome, affecting the regulation of CFTR expression in a tissue-depending fashion which may explain the large within-group variability of CFTR activity in the respiratory and intestinal epithelium seen in this group.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Adolescente , Adulto , Anciano , Biomarcadores , Niño , Preescolar , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Lactante , Persona de Mediana Edad , Valores de Referencia , Sudor , Adulto Joven
6.
Sci Rep ; 10(1): 22447, 2020 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-33384439

RESUMEN

SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA-protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o- cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients' nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.


Asunto(s)
Fibrosis Quística/genética , Susceptibilidad a Enfermedades , Canales Epiteliales de Sodio/genética , Genes Modificadores , Proteínas de Unión al ARN/metabolismo , Hermanos , Alelos , Empalme Alternativo , Sitios de Unión , Mapeo Cromosómico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Antecedentes Genéticos , Genotipo , Haplotipos , Humanos , Intrones , Masculino , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ADN
7.
Mol Genet Genomic Med ; 7(2): e00526, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30600599

RESUMEN

BACKGROUND: The impact of complex alleles on CFTR processing and function has yet not been investigated in native human tissue. METHODS: Intestinal current measurements (ICM) followed by CFTR immunoblot were performed on rectal biopsies taken from two siblings who are compound heterozygous for the CFTR mutations p.Phe508del and the complex allele p.[Arg74Trp;Val201Met;Asp1270Asn]. RESULTS: Normal and subnormal chloride secretory responses in the ICM were associated with normal and fourfold reduced amounts of the mature glycoform band C CFTR, respectively, consistent with the unequal clinical phenotype of the siblings. CONCLUSION: The combined use of bioassay and protein analysis is particularly meaningful to resolve the CFTR phenotype of "indeterminate" borderline CFTR genotypes on a case-to-case basis.


Asunto(s)
Potenciales de Acción , Colon/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fenotipo , Línea Celular , Células Cultivadas , Niño , Cloruros/metabolismo , Colon/citología , Colon/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Transporte Iónico , Masculino , Mutación Missense
8.
Biochim Biophys Acta ; 1859(11): 1411-1428, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27616356

RESUMEN

A challenging question in genetics is to understand the molecular function of non-coding variants of the genome. By using differential EMSA, ChIP and functional genome analysis, we have found that changes in transcription factors (TF) apparent binding affinity and dissociation rates are responsible for allele specific assembly or disruption of master TFs: we observed that NF-KBp50, NF-KBp65 and HIF1a bind with an affinity of up to 10 fold better to the C-allele than to the T-allele of rs7901656 both in vivo and in vitro. Furthermore, we showed that NF-KBp50, p65 and HIF1a form higher order heteromultimeric complexes overlapping rs7901656, implying synergism of action among TFs governing cellular response to infection and hypoxia. With rs7901656 on the FAS gene as a paradigm, we show how allele specific transcription factor complex assembly and disruption by a causal variant contributes to disease and phenotypic diversity. This finding provides the highly needed mechanistic insight into how the molecular etiology of regulatory SNPs can be understood in functional terms.


Asunto(s)
Alelos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , FN-kappa B/metabolismo , Polimorfismo de Nucleótido Simple , Receptor fas/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Cinética , Unión Proteica
9.
Eur J Hum Genet ; 21(6): 691-4, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23073314

RESUMEN

The manifestation of the monogenic disease cystic fibrosis results from the cystic fibrosis transmembrane conductance regulator (CFTR)-mediated basic defect defined as an altered chloride transport. An association study using contrasting endophenotypes was conducted with 17 markers to allow fine-mapping of a previously reported association signal within the CLCA gene cluster. Markers were analyzed for association with the manifestation of the basic defect in the patient population of the European CF Twin and Sibling Study composed of 101 families with a total of 171 patients. The manifestation of the basic defect was associated with markers rs11807298-rs6684219, encompassing the CLCA4 promoter (Praw=0.0013; Pcorr=0.0157). Refined analysis of the CLCA4 association signal among F508del homozygous CF patients who exhibit either no, CFTR-mediated or Ca(2+)-mediated residual chloride conductance revealed that allele distributions for markers rs11807298-rs113894048-rs6684219 differed significantly among these three patient groups. Our data strongly argue that CLCA4 modulates the capability to express residual chloride secretion in colonic tissue. The latter finding is in consistency with the now favored role of the CLCA proteins in signal transduction in epithelial cells.


Asunto(s)
Canales de Cloruro/genética , Fibrosis Quística/genética , Intestinos/patología , Polimorfismo de Nucleótido Simple/genética , Cloruros/metabolismo , Haplotipos/genética , Humanos , Mucosa Intestinal/metabolismo , Activación del Canal Iónico , Repeticiones de Microsatélite/genética , Familia de Multigenes , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN
10.
PLoS One ; 7(11): e50731, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23209821

RESUMEN

BACKGROUND/METHODS: The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years. RESULTS: Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient. CONCLUSION: The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.


Asunto(s)
Fibrosis Quística/microbiología , Epidemiología Molecular/métodos , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Estudios Transversales , Femenino , Genotipo , Humanos , Masculino , Pseudomonas aeruginosa/clasificación
11.
J Med Genet ; 48(1): 24-31, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20837493

RESUMEN

BACKGROUND: The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes. METHODS: Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed. RESULTS: Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01

Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/inmunología , Células Epiteliales/inmunología , Células Epiteliales/patología , Inflamación/genética , Inflamación/inmunología , Activación del Canal Iónico/fisiología , Alelos , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Ambiente , Estudios de Asociación Genética , Heterogeneidad Genética , Homocigoto , Humanos , Patrón de Herencia/genética , Transporte Iónico , Repeticiones de Microsatélite/genética , Modelos Genéticos
12.
Biochim Biophys Acta ; 1802(11): 1062-9, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20696241

RESUMEN

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.


Asunto(s)
Cloruros/metabolismo , Colon/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Mucosa Intestinal/metabolismo , Adolescente , Adulto , Niño , Colforsina/farmacología , Colon/patología , AMP Cíclico/farmacología , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glicosilación , Homocigoto , Humanos , Immunoblotting , Mucosa Intestinal/patología , Transporte Iónico/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Mutación , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Adulto Joven
13.
Hum Genet ; 127(4): 383-94, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20047061

RESUMEN

On 19q13, TGFB1 and the cystic fibrosis modifier 1 locus (CFM1) have been identified as modifiers of the course of the monogenic disease cystic fibrosis (CF). Recently, we have described a transmission disequilibrium at the microsatellite D19S197, localized between TGFB1 and CFM1. To map the corresponding molecular variants, we have selected informative SNP markers within a 600-kb area and compared two-marker-haplotype-distributions between phenotypically contrasting sib pair groups, intending to type only phylogenetically old markers by aiming for close-to-maximal polymorphism information content of the SNPs. Starting with a seed set of five SNPs that cover intermarker distances of up to 50 kb, we have iteratively added more SNPs to the map, until we could identify two genomic fragments of 3,289 and 2,052 bp for which pairs with contrasting phenotypes showed different haplotype distributions on the final 17-SNP-map (P(raw) = 0.0002, P(corr17SNPs) = 0.0106 and P(raw) = 0.0008, P(corr17SNPs) = 0.0469, respectively). Resequencing of these fragments of four unrelated individuals for each element showed that the mildly and severely affected pairs differ in seven SNPs and concordant pairs differ from discordant pairs in five SNPs. Annotation of these variants indicate that CEACAM6 and a regulatory element near the 3' end of CEACAM3 are associated with CF disease severity and intrapair discordance, respectively. While our approach was only guided by the markers' position, the involvement of genes from the CEACAM family in host defense and innate immunity designates these proteins as likely modifiers of the multi-organ disease cystic fibrosis which is known for its cytokine imbalance and pro-inflammatory phenotype.


Asunto(s)
Antígenos CD/genética , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular/genética , Cromosomas Humanos Par 19/genética , Fibrosis Quística/genética , Mapeo Cromosómico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Enfermedades en Gemelos/genética , Proteínas Ligadas a GPI/genética , Estudio de Asociación del Genoma Completo , Haplotipos , Homocigoto , Humanos , Polimorfismo de Nucleótido Simple , Elementos Reguladores de la Transcripción , Eliminación de Secuencia , Gemelos Monocigóticos
14.
Eur J Hum Genet ; 15(7): 774-8, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17406643

RESUMEN

Two entities localised within in a 5 Mb interval on 19q13, that is the transforming growth factor beta 1 (TGFbeta1) and the cystic fibrosis modifier 1, have been reported to modulate disease severity of cystic fibrosis (CF), albeit the designation of the risk allele for TGFbeta1 differs between studies. We have analysed genotyping data at seven microsatellite loci and four single nucleotide polymorphisms targeting the 19q13 area from 37 nuclear CF families with two affected offspring exhibiting extreme clinical phenotypes for indicators of transmission-ration distortion, maternal genetic or maternal non-genetic effects. Evidence for a transmission-ratio distortion was obtained at D19S112 (P=0.0304) near the recently characterised myotonic dystrophy locus myotonic dystrophy protein kinase (DMPK). Maternal and paternal genotype distributions were significantly different at rs1982073 (Leu10Pro at TGFbeta1) whereby all CF sibs heterozygous at rs1982073 inherited the Leu10 allele from their mother (P=0.000132) in our sibling panel. To ask whether the improved survival in CF over the last decades has any influence on TGFbeta1 allele frequencies, we analysed unrelated F508del homozygotes who were stratified by birth cohort. Sensitivity with respect to the survivor bias was reflected by significantly higher incidence of mild cystic fibrosis transmembrane conductance regulator mutation genotypes in the early born patient cohort (P=0.0169), and an allelic imbalance was also observed at TGFbeta1 (P=0.0664). In conclusion, the role of TGFbeta1 as a CF modulator, suggested from studies with a case-control setting, needs to be interpreted with caution unless family-based analysis is carried out to identify parental genetic and non-genetic effects.


Asunto(s)
Cromosomas Humanos Par 19/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Estudios de Cohortes , Frecuencia de los Genes , Humanos , Repeticiones de Microsatélite , Madres , Polimorfismo de Nucleótido Simple , Factor de Crecimiento Transformador beta1/genética
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