At roughly 4 months old, the child was sent to the emergency department of our hospital due to spit-up and cyanosis at home. He had gone into a sudden respiratory and cardiac arrest. After rescue, the child's complexion gradually turned rosy, and his spontaneous heartbeat was restored. Considering that the child's breathing was unstable after successful cardiopulmonary resuscitation, advanced life support treatment was still needed, so he was transferred to PICU under the condition of tracheal intubation and resuscitation bag positive pressure ventilation for further monitoring and treatment.
Later, despite the use of non-invasive respiratory support, the child still had repeated apneas, accompanied by mental fatigue, drowsiness, and sudden hypotension. The lactic acid was always high, fluctuating at 5-12.9 mmo/L (normal range: 0.5-2.2 mmol/L), showing a progressive increase.
A brain magnetic resonance imaging (MRI) examination was performed and revealed a bilateral hyperintensity of basal ganglia (Figure 1A). Hydrocephalus was suggested by a moderately enlarged ventricle system, centered on each side of the ventricle and a widened extracerebral space in the temporal part of both sides. The patient's lactate peak was distinctly discernible as an inverted doublet at TE = 144 ms by single voxel magnetic resonance spectroscopy (MRS) of the left basal ganglia (Figure 1B). Multiple basic symmetrical abnormal signals, combined with MRS can be seen in the bilateral basal ganglia, brainstem, thalamus, frontal lobe, parietal lobe, bilateral limbic lobe, and temporal cortex, suggesting mitochondrial encephalopathy. Finally, the proband died due to respiratory failure after the parents ceased treatment.
The peripheral blood samples were obtained from the patient and his family members after informed consent was obtained. Whole Exome Sequencing (WES) was performed to analyze the coding exons and the exon-intron boundaries of protein-coding genes by the Novogene Bioinformatics Institute (Tianjin, China). The American College of Medical Genetics and Genomics (ACMG) guidelines were also used to classify this variant. The 3D models of the normal and mutant protein are predicted by the I-TASSER server1 and visualization was carried out by PyMOL.
RNA sequencing was conducted by the Novogene Bioinformatics Institute (Beijing, China). Preparation of sample is followed by the RNA library preparation. RNA library is formed by polyA capture (or rRNA removal) and reverse transcription of cDNA. Illumina PE150 technology is employed to sequence the sample and the final stage involves the bioinformatics analysis.
Quadricep muscle and skin biopsies were obtained from the patient. Age- and gender-matched specimens of normal human muscle and skin were obtained from a patient during surgery. For the use of biomedical studies, fibroblasts were cultured and harvested from the biopsies.
We preformed reverse transcription-polymerase chain reaction (RT-PCR) on mRNA extracted from the patient's fibroblasts in order to evaluate the potential effect of this variant on splicing. The cDNA obtained was amplified by PCR, using a set of primers: the forward primer aligning on exon 10 (5'-CAGCAACTGGACCATGGAAA-3') and the reverse primer aligning on exon 12 (5'-GCTCTGCATTTACCCGGAAG-3') of AIFM1 gene. The PCR products were gel-purified and ligated into the pMD19-T vector and transformed into E. coli DH5alpha. The colonies were picked and screened by PCR, and the positive clones were sent for sequencing (Sangon Biotech, Shanghai, China).
The relative mRNA expression levels of AIFM1 in the fibroblasts of the patient and control normal individual were compared by real-time quantitative polymerase chain reaction (RT-qPCR). Four pairs of primers were designed to detect the mRNA level of AIFM1. Each experiment was performed in triplicate, and expression data were normalized to beta-actin as an internal reference gene. The 2-DeltaDeltaCt method was used for quantification by comparing the Ct values.
Fibroblasts were trypsinied, centrifuged at 1000 g for the duration of 5 min, followed by solubilizing in RIPA buffer with protease inhibitors. For muscle tissues, ice-cold lysis buffer was rapidly added to the samples, followed by homogenizing with an electric homogenizer and rinsing the blade twice with another 2 x 300 muL lysis buffer. The samples were then kept at 4 C for 2 h with continual agitation. Immunoblot analysis was performed with the ECL-chemiluminescence kit in accordance with manufacturer's protocol. A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used. The HRP-conjugated Affinipure Goat Anti- Rabbit IgG (H + L) was used as secondary antibody (Proteintech, Illinois, IL, United States, Cat. No. SA00001-2).
For immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized in 0.25% Triton X-100. Subsequently, cells were blocked and immunostained with antibodies in 5% goat serum and treated with 4',6-Diamidino-2-phenylindole (DAPI) staining solution (Beyotime Biotechnology, Shanghai, China) before mounting. Anti-TOMM20 (Millipore, Bedford, MA, United States) was used at 1/200 dilution for mitochondria localization. Images were visualized using Nikon Eclipse Ti-S Phase Contrast Fluorescence Inverted Microscope (Nikon, Tokyo, Japan).
The level of NAD+ in fibroblasts was determined using NAD+ /NADH assay kit (Beyotime, S0175, Shanghai, China) with WST-8 according to the manufacturer's instructions. Human Cytochrome C Oxidase (COX) of cell culture supernatant were measured using the Human COX ELISA kit (RX105095H, Ruixin Biotech, Quanzhou, Fujian, China) according to instruction of the manufacturer.