ABSTRACT
The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.
Subject(s)
Introns , RNA, Messenger , Humans , Male , Introns/genetics , RNA, Messenger/genetics , Vacuolar Proton-Translocating ATPases/genetics , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/diagnosis , Congenital Disorders of Glycosylation/pathology , Mutation , Whole Genome Sequencing , Exome Sequencing , Sequence Analysis, RNA , Intellectual Disability/genetics , Intellectual Disability/diagnosis , Intellectual Disability/pathology , Child , RNA Splicing/genetics , Child, PreschoolABSTRACT
The gold-standard diagnostic test for peroxisomal disorders (PDs) is plasma concentration analysis of very long-chain fatty acids (VLCFAs). However, this method's time-consuming nature and limitations in cases which present normal VLCFA levels necessitates alternative approaches. The analysis of C26:0-lysophosphatydylcholine (C26:0-LPC) in dried blood spot samples by tandem-mass spectrometry (MS/MS) has successfully been implemented in certain newborn screening programs to diagnose X-linked adrenoleukodystrophy (ALD). However, the diagnostic potential of very long-chain LPCs concentrations in plasma remains poorly understood. This study sought to evaluate the diagnostic performance of C26:0-LPC and other very long-chain LPCs, comparing them to VLCFA analysis in plasma. The study, which included 330 individuals affected by a peroxisomal ß-oxidation deficiency and 407 control individuals, revealed that C26:0- and C24:0-LPC concentrations demonstrated the highest diagnostic accuracy (98.8% and 98.4%, respectively), outperforming VLCFA when C26:0/C22:0 and C24:0/C22:0 ratios were combined (98.1%). Combining C24:0- and C26:0-LPC gave the highest sensitivity (99.7%), with ALD females exhibiting notably higher sensitivity compared with the VLCFA ratio combination (98.7% vs. 93.5%, respectively). In contrast, C22:0-LPC exhibited suboptimal performance, primarily due to its low sensitivity (75%), but we identified a potential use to help distinguish between ALD and Zellweger spectrum disorders. In summary, MS/MS analysis of plasma C24:0- and C26:0-LPC concentrations represents a rapid and straightforward approach to diagnose PDs, demonstrating superior diagnostic accuracy, particularly in ALD females, compared with conventional VLCFA biomarkers. We strongly recommend integrating very-long chain LPC plasma analysis in the diagnostic evaluation of individuals suspected of having a PD.
Subject(s)
Adrenoleukodystrophy , Lysophosphatidylcholines , Infant, Newborn , Female , Humans , Tandem Mass Spectrometry , Adrenoleukodystrophy/diagnosis , Neonatal Screening/methods , Biomarkers , Fatty Acids, Nonesterified , Fatty AcidsABSTRACT
Objetivo. El objetivo de este trabajo fué evaluar, mediante un estudio multicéntrico, la imprecisión y la veracidad de un elevado número de procedimientos de medida en el nuevo sistema analítico BioSystems BA 400(R). Material y método. El estudio de la imprecisión se llevó a cabo siguiendo recomendaciones establecidas y utilizando sueros control con 2 concentraciones distintas. El estudio de la veracidad se ha realizado mediante la comparación de los procedimientos de medida del nuevo sistema con los utilizados habitualmente en los centros evaluadores. Resultados. Los resultados obtenidos para la imprecisión interdiaria con el nuevo analizador han sido en general excelentes en relación a los errores máximos permitidos. Se han encontrado algunas diferencias no despreciables y estadísticamente significativas entre los distintos procedimientos de medida, que son debidas a diferencias en el mensurando en algunos casos (transaminasas, inmunoanálisis) y a diferencias en los calibradores en otros. Conclusiones. La evaluación ha demostrado las excelentes prestaciones de precisión y veracidad del sistema. El nuevo analizador proporciona resultados en muestras de pacientes que son equivalentes a los obtenidos con otros analizadores (Olympus AU5400 y AU2700, Roche Cobas C711 y Siemens ADVIA 2400, 1800 y BNII) (AU)
Background. The purpose of the study was a multicentre evaluation of the imprecision and of the trueness of a wide variety of measurement procedures with the new analytical system BioSystems BA 400(R). Methods. The imprecision study was performed following established recommendations and using control sera with two different concentrations. The trueness was studied by means of a comparison of the measurement procedures of the new analyser with those of routine use in the evaluating centres. Results. The results obtained for the between-day imprecision with the new analyser have been in general excellent in relation to the maximum allowed errors. Several differences that are not worthless and that are statistically significant have been found between the measurement procedures. The differences are due to measurand differences in some cases (transaminases, immunoanalysis), and to the calibration in other. Conclusion. The evaluation study has demonstrated the excellent performance of the system regarding precision and trueness. The results obtained for patient samples with the new analyzer are equivalent to those obtained with other analyzers (Olympus AU5400 y AU2700, Roche Cobas C711 y Siemens ADVIA 2400, 1800 y BNII) (AU)