[Phenotypic and molecular characterization of a Colombian family with phenylketonuria].

INTRODUCTION: Phenylketonuria is a metabolic disorder characterized by severe neurological involvement and behavioral disorder, whose early diagnosis enables an effective treatment to avoid disease sequelae, thus changing the prognosis. Objective: To characterize a family with phenylketonuria in Colombia at clinical, biochemical and molecular levels. Materials and methods: The population consisted of seven individuals of a consanguineous family with four children with suggestive symptoms of phenylketonuria. After signing an informed consent, blood and urine samples were taken for colorimetric tests and high performance liquid and thin layer chromatographies. DNA extraction and sequencing of the 13 exons of the PAH gene were performed in all subjects. We designed primers for each exon with the Primer 3 software using automatic sequencing equipment Abiprism 3100 Avant. Sequences were analyzed using the SeqScape, v2.0, software. Results: We described the clinical and molecular characteristics of a Colombian family with phenylketonuria and confirmed the presence of the mutation c.398_401delATCA. We established a genotype-phenotype correlation, highlighting the interesting clinical variability found among the affected patients despite having the same mutation in all of them. Conclusions: Early recognition of this disease is very important to prevent its neurological and psychological sequelae, given that patients reach old age without diagnosis or proper management.

Caracterización fenotípica y molecular de una familia colombiana con fenilcetonuria / Phenotypic and molecular characterization of a Colombian family with phenylketonuria

Introducción. La fenilcetonuria es un trastorno metabólico caracterizado por un compromiso neurológico grave y por alteraciones del comportamiento. Su diagnóstico temprano permite establecer un tratamiento efectivo que evita las secuelas y modifica el pronóstico. Objetivo. Caracterizar a una familia con fenilcetonuria en Colombia, a nivel clínico, bioquímico y molecular. Materiales y métodos. Se estudió una población de siete individuos de una familia consanguínea en la que cuatro hijos presentaban signos clínicos sugestivos de fenilcetonuria. Una vez firmado el consentimiento informado, se tomaron muestras de sangre y orina para las pruebas colorimétricas, la cromatografía de capa fina y la cromatografía líquida de alta eficacia. Se extrajo el ADN y se secuenciaron los 13 exones del gen PAH de todos los sujetos estudiados. Se diseñaron iniciadores para cada exón con el programa Primer 3; la secuenciación automática se hizo con el equipo Abiprism 3100 Avant y, el análisis de las secuencias, con el programa SeqScape v2.0. Resultados. Se describieron las características clínicas y moleculares de una familia colombiana con fenilcetonuria en la que se identificó la mutación c.398_401delATCA; se presentó una correlación fenotipo-genotipo con una interesante variabilidad clínica entre los afectados, a pesar de tener la misma mutación. Conclusiones. Es importante el reconocimiento temprano de esta enfermedad para evitar sus secuelas neurológicas y psicológicas, pues los pacientes llegan a edades avanzadas sin diagnóstico ni tratamiento adecuados.

Introduction: Phenylketonuria is a metabolic disorder characterized by severe neurological involvement and behavioral disorder, whose early diagnosis enables an effective treatment to avoid disease sequelae, thus changing the prognosis. Objective: To characterize a family with phenylketonuria in Colombia at clinical, biochemical and molecular levels. Materials and methods: The population consisted of seven individuals of a consanguineous family with four children with suggestive symptoms of phenylketonuria. After signing an informed consent, blood and urine samples were taken for colorimetric tests and high performance liquid and thin layer chromatographies. DNA extraction and sequencing of the 13 exons of the PAH gene were performed in all subjects. We designed primers for each exon with the Primer 3 software using automatic sequencing equipment Abiprism 3100 Avant. Sequences were analyzed using the SeqScape, v2.0, software. Results: We described the clinical and molecular characteristics of a Colombian family with phenylketonuria and confirmed the presence of the mutation c.398_401delATCA. We established a genotype-phenotype correlation, highlighting the interesting clinical variability found among the affected patients despite having the same mutation in all of them. Conclusions: Early recognition of this disease is very important to prevent its neurological and psychological sequelae, given that patients reach old age without diagnosis or proper management.

Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants.

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Transient evoked oto-acoustic emission screening in newborns in Bogotá, Colombia: a retrospective study.

OBJECTIVE: The aim of this study was to investigate the characteristics and performance of transient evoked oto-acoustic emission (TEOAE) hearing screening in newborns in Colombia, and analyze all possible variables and factors affecting the results. MATERIALS AND METHODS: An observational, descriptive and retrospective study with bivariate analysis was performed. The study population consisted of 56,822 newborns evaluated at the private institution, PREGEN. TEOAE testing was carried out as a pediatric hearing screening test from December 2003 to March 2012. The database from PREGEN was revised, and the protocol for evaluation included the same screening test performed twice. Demographic characteristics were recorded and the newborn's background was evaluated. Basic statistics of the qualitative and quantitative variables, and statistical analysis were obtained using the chi-square test. RESULTS: Of the 56,822 records examined, 0.28% were classed as abnormal, which corresponded to a prevalence of 1 in 350. In the screened newborns, 0.08% had a major abnormality or other clinical condition diagnosed, and 0.29% reported a family history of hearing loss. A prevalence of 6.7 in 10,000 was obtained for microtia, which is similar to the 6.4 in 10,000 previously reported in Colombia (database of the Latin-American Collaborative Study of Congenital Malformations - ECLAMC). Statistical analysis demonstrated an association between presenting with a major anomaly and a higher frequency of abnormal results on both TEOAE tests. CONCLUSIONS: Newborns in Colombia do not currently undergo screening for the early detection of hearing impairment. The results from this study suggest TEOAE screening tests, when performed twice, are able to detect hearing abnormalities in newborns. This highlights the need to improve the long-term evaluation and monitoring of patients in Colombia through diagnostic tests, and to provide tests that are both sensitive and specific. Furthermore, the use of TEOAE screening is justified by the favorable cost: benefit ratio demonstrated in many countries worldwide.

The importance of fundus eye testing in rubella-induced deafness.

OBJECTIVE: The purpose of this study was to establish a new approach to improve detection of deafness due to rubella. METHODS: Colombian institutes for the deaf were visited by a medical team to perform in all enrolled individuals an ophthalmological examination with emphasis in fundus eye by a retina specialist. In cases where ocular alterations compatible with CRS were found, a medical interview by a clinical geneticist analyzing pre-and postnatal history and a thorough medical examination was done. RESULTS: A total of 1383 deaf institutionalized individuals were evaluated in 9 Colombian cities in the period of 2005 to 2006, finding a total of 463 positive cases for salt-and-pepper retinopathy (33.5%), in which rubella could be the etiology of deafness. Medellin, Cartagena, Bucaramanga and Barranquilla were the cities with the highest percentage of Congenital rubella, corresponding to 22.8% of analyzed population. The analysis performed on cases in which reliable prenatal history was obtained in a second appointment (n=88) showed association between positive viral symptoms during pregnancy and salt-and-pepper retinopathy in 62.5% of cases, while both (retinopathy and viral symptoms) were absent in 29.5% of cases; showing a correlation in 92% of cases. CONCLUSIONS: The frequency of deafness by rubella obtained by this study is significantly high compared with previous Colombian studies and with international reports. It was possible to correlate the antecedent of symptoms during pregnancy with the presence of salt-and-pepper retinopathy in this deaf population when reliable prenatal history was available, therefore eye testing with emphasis in fundus examination is a good indicator of rubella induced deafness. We propose a new approach in the search of deafness causes, based on a thorough ophthalmologic examination in all deaf people.

Allele-specific impairment of GJB2 expression by GJB6 deletion del(GJB6-D13S1854).

Mutations in the GJB2 gene, which encodes connexin 26, are a frequent cause of congenital non-syndromic sensorineural hearing loss. Two large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854), which truncate GJB6 (connexin 30), cause hearing loss in individuals homozygous, or compound heterozygous for these deletions or one such deletion and a mutation in GJB2. Recently, we have demonstrated that the del(GJB6-D13S1830) deletion contributes to hearing loss due to an allele-specific lack of GJB2 mRNA expression and not as a result of digenic inheritance, as was postulated earlier. In the current study we investigated the smaller del(GJB6-D13S1854) deletion, which disrupts the expression of GJB2 at the transcriptional level in a manner similar to the more common del(GJB6-D13S1830) deletion. Interestingly, in the presence of this deletion, GJB2 expression remains minimally but reproducibly present. The relative allele-specific expression of GJB2 was assessed by reverse-transcriptase PCR and restriction digestions in three probands who were compound heterozygous for a GJB2 mutation and del(GJB6-D13S1854). Each individual carried a different sequence variant in GJB2. All three individuals expressed the mutated GJB2 allele in trans with del(GJB6-D13S1854), but expression of the GJB2 allele in cis with the deletion was almost absent. Our study clearly corroborates the hypothesis that the del(GJB6-D13S1854), similar to the larger and more common del(GJB6-D13S1830), removes (a) putative cis-regulatory element(s) upstream of GJB6 and narrows down the region of location.

Screening program for Waardenburg syndrome in Colombia: clinical definition and phenotypic variability.

A screening program to detect Waardenburg syndrome (WS) conducted between 2002 and 2005, among 1,763 deaf individuals throughout Columbia identified 95 affected individuals belonging to 95 families, giving a frequency of 5.38% of WS among the institutionalized deaf population. We confirmed the clinical diagnosis of WS in the 95 propositi and, through the family evaluation, we also identified 45 non-institutionalized affected relatives. Audiologic, ophthalmologic, and genetic studies were performed to confirm the diagnosis. Following the classification of the WS consortium, based on the Waardenburg Index (WI), to define the type of WS. We classified 62.1% of the propositi as WS2 and 37.9% as WS1. We present here the results of the study of clinical manifestations, analyzing the presence, severity, and symmetry of clinical findings among this affected population. Overall, among the 95 propositi, in addition to sensorineural deafness in all, the most frequent features were broad nasal root (58.9%), a first degree relative affected (37.9%), heterochromia irides (36.8%), skin hypopigmentation (31.6%), white forelock (28.0%), intense blue iris (27.4%), synophrys (12.6%), premature graying (10.5%), ptosis of the eyelids (9.5%), and hypoplasia alae nasi (1.1%). The majority of individuals had normal psychomotor development (87%), while the remaining 13% had developmental delay. Among the latter, 9.4% corresponded to WS2 and 3.6% to WS1. Our data confirm an interesting inter- and intrafamilial variability in the phenotypic manifestations as well as extremely variable expression.