Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires
Introduction: Hyperphenylalaninemia is a biochemical phenotype with a spectrum ranging from classical phenylketonuria (cPKU) to benign hyperphenylalaninemia (HPA). It is mainly caused by deleterious biallelic variants in Phenylalanine Hydroxylase gene (PAH). This enzyme metabolizes Phenylalanine (Ph...
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7181 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7181.dir/7181.PDF |
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I28-R145-HWA_7181 |
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dspace |
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Universidad de Buenos Aires |
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I-28 |
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R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
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PKU PAH Diagnóstico molecular Ciencias de la vida |
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PKU PAH Diagnóstico molecular Ciencias de la vida Esnaola Azcoiti, María Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
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PKU PAH Diagnóstico molecular Ciencias de la vida |
| description |
Introduction: Hyperphenylalaninemia is a biochemical phenotype with a spectrum ranging from classical phenylketonuria (cPKU) to benign hyperphenylalaninemia (HPA). It is mainly caused by deleterious biallelic variants in Phenylalanine Hydroxylase gene (PAH). This enzyme metabolizes Phenylalanine (Phe) into Tyrosine (Tyr) in a reaction that involves O2, non-heme iron and tetrahydrobiopterin (BH4) cofactor. If not treated early, patients develop severe mental retardation. There is a group of patients who retain some degree of enzymatic activity and may respond to BH4 supplementation. Identifying these patients is important because BH4 treatment may allow to slightly reduce diet restriction improving their quality of life. Genotyping has become a useful tool to either design the diet accurately or to consider other treatment options now available. Other genes involved in BH4 synthesis and recycling, and DNAJC12 that codes for a co-chaperone of PAH can cause hyperphenylalaninemia to a lesser extent (1-2% of cases). These diagnoses must be considered if no variants in PAH are found.
Aim: to efficiently implement a molecular diagnostic strategy for PAH related disorders in patients detected by neonatal screening. Genotype-phenotype correlation analysis of our cohort of patients.
Methods: Twenty-eight patients (8 female, 20 male) median age 12 years, with clinical and biochemical diagnosis of HPA were included. According to their tolerance to phenylalanine they were classified as classic PKU (cPKU) (n:11), moderate PKU (moPKU) (n: 7), mild PKU (mPKU) (n:4) or HPA (n:6). Twenty-one patients were studied by Sanger sequencing of PAH exons and intronic flanking regions. Six patients underwent molecular diagnosis by NGS (TruSightOne, Illumina). Variants were classified according to ACMG and ClinGen criteria and information available in BIOPKU database. Parents and siblings were studied to assess segregation for all prioritized variants. Phenotypic predictions were performed using allele phenotypic values (APVs) and genotypic phenotype values (GPVs) as proposed by Garbade et al.
Results: Twenty-six different already reported variants were found. Sixty-one % were missense variants, 15% splicing variants, 12% little frameshift deletions and 4% in frame deletions and CNV. 66% of the alleles presented variants in the catalytic region, 18% in oligomerization domain and 14% in regulatory domain. Most frequent variants were c.1066-11G>A (intron 10: 21%), p.V388M (exon 11: 7,7%), p.R261Q (exon 7: 7,7 %), and p.R158Q (exon 5: 7,7 %). Only one patient was homozygous (variant c.1066-11G>A) and 2 unrelated patients shared the same genotype ([c.1066-11G>A];[p.Arg261Gln]). 65% of all variants were distributed along exons 11, 12 and 7 and their flanking regions. No variants were found in exons 1, 4, 8 or 13. Genotype-phenotype correlation obtained was 100% for cPKU, 40% for moPKU and 83% for HPA.
Conclusions: We were able to design a strategy for molecular diagnosis of PAH deficient patients. PCR amplification and Sanger sequencing of exons 11, 12, 7, 3, 2, 5, 6 y 10 accounting for 96,5% of variants found is the starting point, followed by the rest of the exons if variants are not found. If the case is still unsolved, MLPA should be performed to search for CNVs. NGS sequencing should be used only when PAH variants have been ruled out and genes involved in biopterin metabolic pathways and DNJAC12 need to be tested. This work also allowed us to fully characterize our cohort confirming the allelic heterogeneity of PKU patients, and to establish genotype-phenotype correlations that will be important to perform a personalized follow-up, treatment and genetic counseling for the patients and their families. |
| author2 |
Ropelato, María Gabriela |
| author_facet |
Ropelato, María Gabriela Esnaola Azcoiti, María |
| format |
Tesis de maestría Tesis de maestría acceptedVersion |
| author |
Esnaola Azcoiti, María |
| author_sort |
Esnaola Azcoiti, María |
| title |
Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
| title_short |
Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
| title_full |
Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
| title_fullStr |
Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
| title_full_unstemmed |
Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires |
| title_sort |
diagnóstico molecular de trastornos relacionados con la fenilalanina hidroxilasa (pah) en un hospital público de la ciudad de buenos aires |
| publisher |
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica |
| publishDate |
2022 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7181 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7181.dir/7181.PDF |
| work_keys_str_mv |
AT esnaolaazcoitimaria diagnosticomoleculardetrastornosrelacionadosconlafenilalaninahidroxilasapahenunhospitalpublicodelaciudaddebuenosaires |
| _version_ |
1840330188833947648 |
| spelling |
I28-R145-HWA_71812025-08-01 Introduction: Hyperphenylalaninemia is a biochemical phenotype with a spectrum ranging from classical phenylketonuria (cPKU) to benign hyperphenylalaninemia (HPA). It is mainly caused by deleterious biallelic variants in Phenylalanine Hydroxylase gene (PAH). This enzyme metabolizes Phenylalanine (Phe) into Tyrosine (Tyr) in a reaction that involves O2, non-heme iron and tetrahydrobiopterin (BH4) cofactor. If not treated early, patients develop severe mental retardation. There is a group of patients who retain some degree of enzymatic activity and may respond to BH4 supplementation. Identifying these patients is important because BH4 treatment may allow to slightly reduce diet restriction improving their quality of life. Genotyping has become a useful tool to either design the diet accurately or to consider other treatment options now available. Other genes involved in BH4 synthesis and recycling, and DNAJC12 that codes for a co-chaperone of PAH can cause hyperphenylalaninemia to a lesser extent (1-2% of cases). These diagnoses must be considered if no variants in PAH are found. Aim: to efficiently implement a molecular diagnostic strategy for PAH related disorders in patients detected by neonatal screening. Genotype-phenotype correlation analysis of our cohort of patients. Methods: Twenty-eight patients (8 female, 20 male) median age 12 years, with clinical and biochemical diagnosis of HPA were included. According to their tolerance to phenylalanine they were classified as classic PKU (cPKU) (n:11), moderate PKU (moPKU) (n: 7), mild PKU (mPKU) (n:4) or HPA (n:6). Twenty-one patients were studied by Sanger sequencing of PAH exons and intronic flanking regions. Six patients underwent molecular diagnosis by NGS (TruSightOne, Illumina). Variants were classified according to ACMG and ClinGen criteria and information available in BIOPKU database. Parents and siblings were studied to assess segregation for all prioritized variants. Phenotypic predictions were performed using allele phenotypic values (APVs) and genotypic phenotype values (GPVs) as proposed by Garbade et al. Results: Twenty-six different already reported variants were found. Sixty-one % were missense variants, 15% splicing variants, 12% little frameshift deletions and 4% in frame deletions and CNV. 66% of the alleles presented variants in the catalytic region, 18% in oligomerization domain and 14% in regulatory domain. Most frequent variants were c.1066-11G>A (intron 10: 21%), p.V388M (exon 11: 7,7%), p.R261Q (exon 7: 7,7 %), and p.R158Q (exon 5: 7,7 %). Only one patient was homozygous (variant c.1066-11G>A) and 2 unrelated patients shared the same genotype ([c.1066-11G>A];[p.Arg261Gln]). 65% of all variants were distributed along exons 11, 12 and 7 and their flanking regions. No variants were found in exons 1, 4, 8 or 13. Genotype-phenotype correlation obtained was 100% for cPKU, 40% for moPKU and 83% for HPA. Conclusions: We were able to design a strategy for molecular diagnosis of PAH deficient patients. PCR amplification and Sanger sequencing of exons 11, 12, 7, 3, 2, 5, 6 y 10 accounting for 96,5% of variants found is the starting point, followed by the rest of the exons if variants are not found. If the case is still unsolved, MLPA should be performed to search for CNVs. NGS sequencing should be used only when PAH variants have been ruled out and genes involved in biopterin metabolic pathways and DNJAC12 need to be tested. This work also allowed us to fully characterize our cohort confirming the allelic heterogeneity of PKU patients, and to establish genotype-phenotype correlations that will be important to perform a personalized follow-up, treatment and genetic counseling for the patients and their families. Fil: Esnaola Azcoiti, María. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Ropelato, María Gabriela Esnaola Azcoiti, María 2022-09-07 Introducción: La hiperfenilalaninemia se debe fundamentalmente a variantes deletéreas bialélicas en el gen PAH que codifica para la fenilalanina hidroxilasa (PAH). Esta enzima es la encargada de metabolizar la fenilalanina (Phe) y transformarla en tirosina (Tyr) mediante una reacción que utiliza O2, Fe no-hemo y el cofactor tetrahidrobiopterina. El espectro clínico varía desde la fenilcetonuria clásica (PKUc) hasta la hiperfenilalaninemia (HFA) pasando por PKU moderada (PKUm) y PKU leve (PKUl). Si los pacientes no son tratados a tiempo con dieta de restricción pueden desarrollar dishabilidad intelectual severa. Existe un grupo de pacientes en los que la enzima PAH retiene algo de actividad, que puede responder al tratamiento de suplementación con el cofactor tetrahidrobiopterina (BH4) que actuaría en estos casos estabilizando la forma tetramérica funcional de PAH. Es importante identificar a estos pacientes ya que la incorporación de BH4 al tratamiento puede permitirles realizar una dieta menos restrictiva y mejorar su calidad de vida. El genotipado es una herramienta útil para predecir fenotipo metabólico, diseñar una dieta adecuada para cada paciente y considerar la opción de otros tratamientos posibles. Otras causas de hiperfenilalaninemia minoritarias (1-2% de los casos) pueden ser alteraciones en los genes que codifican para las enzimas involucradas en las vías de síntesis de novo y de reciclaje del cofactor BH4 y en el gen DNAJC12 que codifica para una proteína co-chaperona que interviene en la función de la PAH y otras enzimas del metabolismo de aminoácidos aromáticos. Estos diagnósticos deben ser considerados en caso de no encontrar variantes en PAH. Objetivos: Implementar una estrategia eficiente para el diagnóstico molecular de pacientes con hiperfenilalaninemia detectados por pesquisa neonatal. Realizar la genotipificación de nuestra cohorte de pacientes y evaluar la relación genotipo-fenotipo. Sujetos y métodos: se estudiaron 28 pacientes (8 mujeres, 20 hombres) con una mediana de edad de 12 años con diagnostico bioquímico de hiperfenilalaninemia (11 PKUc, 7 PKUm, 4 PKUl y 6 HFA). En 22 pacientes se realizó la PCR y posterior secuenciación Sanger de los 13 exones del gen PAH y sus regiones intrónicas adyacentes. Seis pacientes se estudiaron mediante la técnica de secuenciación de nueva generación utilizando el kit de exoma clínico TruSightOne (Illumina). Las variantes halladas tanto por Sanger como por NGS fueron clasificadas de acuerdo con los criterios de ACMG, ClinGen y la información disponible en la base de datos BIOPKU. Los familiares directos de los pacientes fueron estudiados para determinar la segregación de las variantes halladas. Las predicciones fenotípicas se realizaron utilizando el sistema de allele phenotypic values (APVs) y genotypic phenotype values (GPVs) diseñado por Garbade y colaboradores. Resultados: Los 28 pacientes pudieron ser genotipificados. Se encontraron 26 variantes diferentes todas previamente reportadas. El 61% fueron variantes missense, el 15% variantes de splicing, 12% pequeñas deleciones con corrimiento del marco de lectura y 4% deleciones sin corrimiento y CNV. El 64% de los alelos presentaron variantes en la región catalítica, el 18% en el dominio de oligomerización y el 14% en el dominio regulatorio. La c.1066-11G>A (intrón 10: 21,4%), p.Val388Met (exón 11: 7,1%), p.Arg261Gln (exón 7: 7,1 %) y p.Arg158Gln (exón 5: 7,1%) fueron las variantes más frecuentes. El 65% de las variantes halladas en los pacientes se distribuyeron entre los exones 11, 12 y 7 y sus correspondientes regiones intrónicas adyacentes. No se encontraron variantes en los exones 1, 4, 8 o 13. Se hallaron 27 genotipos diferentes de los cuales 7 no han sido reportados en BIOPKU. Un solo paciente fue homocigota y sólo 2 pacientes compartieron genotipo ([c.1066-11G>A];[p.Arg261Gln]). Se obtuvo una correlación genotipo fenotipo del 100% para los PKUc, del 40% para el grupo PKUm y PKUl y del 83% para HFA. Discusión y conclusiones: Este trabajo nos permitió poner a punto las técnicas de PCR y secuenciación Sanger, NGS (kit TruSightOne) y MLPA para el diagnóstico molecular de desórdenes de la PAH y establecer un algoritmo de estudio que inicia con la amplificación por PCR y secuenciación por Sanger de los exones 11, 12, 7, 3, 2, 5, 6 y 10 en los cuales se encontró el 96,5% de las variantes, siguiendo por el resto de los 13 exones en caso de no completar el diagnóstico, y por el estudio de MLPA para la búsqueda de CNVs en los casos aún inconclusos. La técnica de NGS queda para una última instancia en caso de no hallar variantes en PAH para pesquisar defectos en los genes de las enzimas de las vías de las Biopterinas y DNAJC12. Por otra parte, pudimos determinar el genotipo de los pacientes de nuestra cohorte, establecer las correlaciones genotipo fenotipo y articular de esta manera los resultados del laboratorio de biología molecular con el seguimiento, tratamiento y consejo genético a los pacientes y sus familias. application/pdf Spécola, Norma Adamo, Ana Alonso, Cristina PKU PAH Diagnóstico molecular spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Magíster de la Universidad de Buenos Aires en Biología Molecular Médica Diagnóstico molecular de trastornos relacionados con la Fenilalanina Hidroxilasa (PAH) en un Hospital Público de la Ciudad de Buenos Aires info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7181 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7181.dir/7181.PDF |