CRISPR/Cas9-based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders.

The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

An update on the molecular analysis of classical galactosaemia patients diagnosed in Spain and Portugal: 7 new mutations in 17 new families

Background and objectives: Classical galactosaemia is an inherited metabolic disorder due to mutations in the galactose-1-phosphate uridytransferase gene (GALT). We previously reported molecular analysis of 83 Spanish and Portuguese unrelated galactosaemic patients. Here we present the results of another seventeen unreported affected individuals. Material and methods: DNA from patients was PCR-amplified and sequenced following standard protocols. Results: Twelve patients diagnosed in Spain were studied. We detected five alleles carrying p.Q188R, accounting for 21%. Other six alleles (25%) were identified with the mutation p.K285N. We also identified six novel mutations: p.Q9X, c.328+2T>C, p.I170N, p.C180F, p.V233L and p.P257L. Taking into account all the Spanish galactosaemic diagnosed patients, mutation p.Q188R is still the most frequent mutation identified (44.4%). In five new Portuguese patients, five alleles p.Q188R were detected, representing 50%. One novel mutation (p.F171C) was identified. Conclusions: Our results confirm our previous observations that p.Q188R is the most frequent mutation in Iberian Peninsula galactosaemic patients (49%), and that Portuguese and Spanish genotypes differ (AU)

Fundamento y objetivo: La galactosemia clásica es una enfermedad metabólica hereditaria debida a mutaciones en el gen de la galactosa-1-fosfato uridiltransferasa (GALT). Nuestro grupo ya publicó los resultados del análisis molecular de 83 pacientes galactosémicos de España y Portugal. Aquí se presentan los resultados de una nueva serie de pacientes. Material y método: El ADN de los 17 pacientes estudiados se amplificó por PCR y se secuenció siguiendo métodos ya descritos. Resultados: Se analizó a 12 doce pacientes diagnosticados en España y detectamos 5 alelos con la mutación p.Q188R (21%). Otros 6 alelos (25%) resultaron portadores de la mutación p.K285N. También se identificaron 6 mutaciones nuevas:p.Q9X,c.328+2T4C, p.I170N, p.C180F, p.V233L y p.P257L. Considerando a todos los pacientes diagnosticados en España, la mutación p.Q188R sigue siendo la mutación mas frecuente (44,4%). En 5 pacientes portugueses se detectaron 5 alelosp.Q188R(50%) y se identificó una nueva mutación (p.F171C). Conclusiones: Estos resultados confirman las observaciones previas, en las cuales se mostraba que la mutación más frecuente en la península Ibérica es p.Q188R(49%), pero que los genotipos españoles y portugueses difieren entre sí (AU)

An update on the molecular analysis of classical galactosaemia patients diagnosed in Spain and Portugal: 7 new mutations in 17 new families.

BACKGROUND AND OBJECTIVES: Classical galactosaemia is an inherited metabolic disorder due to mutations in the galactose-1-phosphate uridyltransferase gene (GALT). We previously reported molecular analysis of 83 Spanish and Portuguese unrelated galactosaemic patients. Here we present the results of another seventeen unreported affected individuals. MATERIAL AND METHODS: DNA from patients was PCR-amplified and sequenced following standard protocols. RESULTS: Twelve patients diagnosed in Spain were studied. We detected five alleles carrying p.Q188R, accounting for 21%. Other six alleles (25%) were identified with the mutation p.K285N. We also identified six novel mutations: p.Q9X, c.328+2T>C, p.I170N, p.C180F, p.V233L and p.P257L. Taking into account all the Spanish galactosaemic diagnosed patients, mutation p.Q188R is still the most frequent mutation identified (44.4%). In five new Portuguese patients, five alleles p.Q188R were detected, representing 50%. One novel mutation (p.F171C) was identified. CONCLUSIONS: Our results confirm our previous observations that p.Q188R is the most frequent mutation in Iberian Peninsula galactosaemic patients (49%), and that Portuguese and Spanish genotypes differ.