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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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
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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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| Sumario: | 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. |
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