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. 2019 Nov 1;8(11):1811.
doi: 10.3390/jcm8111811.

Genes and Variants Underlying Human Congenital Lactic Acidosis-From Genetics to Personalized Treatment

Irene Bravo-Alonso  1 , Rosa Navarrete  2 , Ana Isabel Vega  3 , Pedro Ruíz-Sala  4 , María Teresa García Silva  5 , Elena Martín-Hernández  6 , Pilar Quijada-Fraile  7 , Amaya Belanger-Quintana  8 , Sinziana Stanescu  9 , María Bueno  10 , Isidro Vitoria  11 , Laura Toledo  12 , María Luz Couce  13 , Inmaculada García-Jiménez  14 , Ricardo Ramos-Ruiz  15 , Miguel Ángel Martín  16 , Lourdes R Desviat  17 , Magdalena Ugarte  18 , Celia Pérez-Cerdá  19 , Begoña Merinero  20 , Belén Pérez  21 , Pilar Rodríguez-Pombo  22
Affiliations

Genes and Variants Underlying Human Congenital Lactic Acidosis-From Genetics to Personalized Treatment

Irene Bravo-Alonso et al. J Clin Med. .

Abstract

Congenital 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 condition.

Keywords: RNA analysis; antisense therapy for mitochondrial disorders; clinical-exome sequencing; congenital lactic acidosis; healthcare; metabolomics datasets; mitochondrial dysfunction; mitochondrial morphology.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Figures

Figure 1
Figure 1
Aberrant splicing of FARS2 in Pt15. (A) Diagram of the human FARS2 gene. Red stars depict the location of nucleotide variants identified. (B) Agarose gel showing the results of reverse transcription polymerase chain reaction (RT-PCR) amplifications in control (CT) and patient (Pt) fibroblasts.
Figure 2
Figure 2
Aberrant splicing of GFM1 in Pt16. (A) Diagram of GFM1 cDNA with primers (arrows) used to amplify the complete coding region in two overlapping (F1 and F2) fragments. Agarose gel showing the results of RT-PCR amplifications in control (CT) and patient (Pt) fibroblasts. (B) Cloning of F1 and F2 PCR products and Sanger sequencing of regions around the nucleotide variations detected. (C) Distribution of reads. Data represent the percentage of GFM1 transcript reads with exon 16 skipped (stripped bars) and full length (filled bars). Read numbers were 41,758 for Pt16, 13,581 for CT1 and 16,718 for CT2.
Figure 3
Figure 3
Antisense morpholino oligonucleotide-based pseudoexon skipping efficacy. (A) Diagram of the pseudoexon insertion caused by c.689+908G>A and the predicted effect of the antisense morpholino oligonucleotide (AON). Inset showing location and sequence of the 25mer AON. (B) Representative image of the RT-PCR product from mutant (Pt16) and wild type (CT) cells, non-transfected (-) and transfected with 10 μM of non-target control (#); or in the presence of different concentrations (0 to 30 μM) of GFM1-specific AON. (C) EFG1 rescue upon treatment with 10 μM of non-target control (#) or GFM1-specific AON.
Figure 4
Figure 4
Bioenergetics of congenital lactic acidosis (CLA)-patients’ fibroblasts. (A) Oxygen consumption rates. The data shown are for ATP-production-dependent maximal respiration (Rmax) and spare capacity (spare). Results are expressed as fold over the control concentrations and are the mean ± SD of 3–5 wells from n = 2–3 independent experiments. Control values are the means of two different control cell lines. (B) Flow cytometry analysis of mitochondrial mass (Mitotracker green) and membrane potential (TMRM staining) in the absence/presence of carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Results are the means of three independent experiments. (C) Western blots for representatives of all five respiratory complexes. Anti-MTOC1, anti-SDHA and anti-citrate synthase were also included. (D) Electron microscopy images showing defects of mitochondrial ultrastructure and cristae organization in patient fibroblasts. Mitochondrial length was analysed in control (CT) and patient (Pt) fibroblasts. Mitochondrial enlargement is expressed as the aspect ratio (major/minor mitochondrial axis ratio). Student t test (* p < 0.05; ** p < 0.01; *** p < 0.001).
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