The Genetic Landscape and Epidemiology of Phenylketonuria

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si lo hubiere, y los autores pertenecientes a la UAMPhenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]–1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066−11G>A (IVS10−11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066−11G>A];[1066−11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcomeThis work was funded in part by the Fundacio´n Isabel Gemio-Fundacion La Caixa (LCF/PR/PR16/11110018), the Regional Government of Madrid (CAM, B2017/BMD3721), and by NIH, United States grants R01DK117916 and R01NR016991

Dysregulated cell homeostasis and miRNAs in human iPSC-derived cardiomyocytes from a propionic acidemia patient with cardiomyopathy

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategie

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMLife-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseasesThis work was supported by grants of Fondazione Telethon Italy (to N.B.‐P.), MIUR (PRIN2017 to N.B.‐P.), of the Swiss National Science Foundation (grant 320030_176088 to J.H.), of the US National Institutes of Health grants (R21NS091654 and R01NS100979 both to G.S.L.), of the Spanish Ministry of Science and Innovation (PID2019-105344RB-I00/AEI/10.13039/501100011033 toL.R.D. and E.R.), and by a Wellcome Trust Investigator Award (110061to D.M.F.v.A.

Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells

The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defectThis book is based upon work from COST Action DARTER (CA 17103), supported by COST (European Cooperation in Science and Technology)

Functional analysis of novel variants identified in cis in the PCCB gene in a patient with propionic acidemia

Next-generation sequencing has improved the diagnosis of inborn errors of metabolism, allowing rapid confirmation of cases detected by clinical/biochemical studies or newborn screening. The challenge, however, remains for establishing the pathogenicity of the identified variants, especially for novel missense changes or small in-frame deletions. In this work we report a propionic acidemia patient exhibiting a severe neonatal form with coma and hyperammonaemia. Genetic analysis identified the previously described pathogenic PCCB variant p.R512C in the maternal allele and two novel PCCB variants in cis in the paternal allele, p.G246del and p.S322F. Expression analysis in a eukaryotic system confirmed the deleterious effect of the novel missense variant and of the one amino acid deletion, as they both exhibited reduced protein levels and reduced or null PCC activity compared to the wild-type construct. Accordingly, the double mutant resulted in no residual activity. This study increases the knowledge of the genotype-phenotype correlations in the rare disease propionic acidemia and highlights the necessity of functional analysis of novel variants to understand their contribution to disease severity and to accurately classify their pathogenic status. In conclusion, two novel PCCB pathogenic variants have been identified, expanding the current mutational spectrum of propionic acidemiaPID2019-105344RB-I00, PID2022-137238OB-I0

Identification of 34 novel mutations in propionic acidemia: Functional characterization of missense variants and phenotype associations

Propionic acidemia (PA) is caused by mutations in the PCCA and PCCB genes, encoding α and β subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Up to date, >200 pathogenic mutations have been identified, mostly missense defects. Genetic analysis in PA patients referred to the laboratory for the past 15 years identified 20 novel variants in the PCCA gene and 14 in the PCCB gene. 21 missense variants were predicted as probably disease-causing by different bioinformatics algorithms. Structural analysis in the available 3D model of the PCC enzyme indicated potential instability for most of them. Functional analysis in a eukaryotic system confirmed the pathogenic effect for the missense variants and for one amino acid deletion, as they all exhibited reduced or null PCC activity and protein levels compared to wild-type constructs. PCCB variants p.E168del, p.Q58P and p.I460T resulted in medium-high protein levels and no activity. Variants p.R230C and p.C712S in PCCA, and p.G188A, p.R272W and p.H534R in PCCB retained both partial PCC activity and medium-high protein levels. Available patients-derived fibroblasts carriers of some of these mutations were grown at 28 °C or 37 °C and a slight increase in PCC activity or protein could be detected in some cases at the folding-permissive conditions. Examination of available clinical data showed correlation of the results of the functional analysis with disease severity for most mutations, with some notable exceptions, confirming the notion that the final phenotypic outcome in PA is not easily predictedWe thank the following physicians/clinicians for sending samples for genetic analysis: Dr. Wilson (Auckland, New Zealand), Dr. Parini (Rome, Italy), Dr. Vilaseca (Barcelona, Spain), Dr. Gockay (Istanbul, Turkey), Dr. Al Sannaa (Saudi Arabia), Dr. Pedrón (Madrid, Spain), Dr. Savvapoulou (Tesalonica, Greece), Dr. Martínez-Pardo (Madrid, Spain), Dr. Lama (Madrid, Spain), Dr. Lemes (Montevideo, Uruguay), Dr. Van Calcar (Madison, USA), Dr. Pintos (Badalona, Spain), Dr. Laszlo (Szeged, Hungary), Dr. Kuijtmans (Nijmegen, The Netherlands), Dr. Schatz (München, Germany), Dr. EL Khateeb (Jordan), Dr. de las Heras (Baracaldo, Spain), Dr. Miñana (Buenos Aires, Argentina). The technical assistance of A. Sánchez is gratefully acknowledged. This work was supported by Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (grant number SAF2016-76004-R). Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Arece

Genes and variants underlying human congenital lactic acidosis—from genetics to personalized treatment

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, los autores pertenecientes a la UAM y el nombre del grupo de colaboración, si lo hubiereCongenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group’s experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system’s workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system’s use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this conditio

Estudio del mecanismo de inserción en membrana de la Penicillin Binding Protein 3 de "Escherichia coli"

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-10-199