2.1 Editorial policies and ethical considerations

This study was approved by the Centre for DNA Fingerprinting and Diagnostics institutional bioethics committee (approval no. IEC 40/2021 dated 7th April 2021).

2.2 Whole mitochondrial genome sequencing analysis

The entire mitochondrial genome (16.5 kb) was amplified from whole blood DNA by long range PCR using PrimeSTAR GXL DNA polymerase (Takara #R050A) with following primers: (1) Set1_F: 5′-GGCTTTCTCAACTTTTAAAGGATA-3′, Set1_R: 5′-TTTATGGGGTGATGTGAGCC-3′; (2) Set2_F: 5′-AACCAAACCCCAAAGACACC-3′, Set2_R: 5′- GCCTCCGATTATGATGGGTAT-3′ and (3) Set3_F: 5′-GCAACCTTCTAGGTAACGACC-3′, Set3_R: 5′-GTGGTAAGGATGGGGGGAATTA-3 to obtain overlapping fragments of lengths 4958, 5585 and 6365 bp respectively. The fragments were pooled in equimolar proportion and library preparation was done using QIAseq FX DNA Library Kit (Qiagen # 180479) according to the manufacturer's protocol. For each sample, paired-end 150 bp reads with average 2500× coverage were obtained. Sequencing reads quality was analyzed using FastQC. Sequencing data was mapped and then aligned to mitochondrial reference genome rCRS (NC_012920.1) using Burrows-Wheeler aligner version 0.7 (BWA-MEM). Mutect2 from Genome Analysis Tool Kit (GATKv.4.1) and Mutserve were used for variant calling. Variants obtained were annotated using the web interface of the MITOMASTER tool. Candidate variants were prioritized by considering the following parameters: (1) All the coding and tRNA variants, (2) variants with allele frequency less than 0.01 in reference populations, such as gnomAD, HelixMTdb, and Mitomap. (3) variants with a heteroplasmy ratio above 5%. (4) deleterious variants potentially affecting the protein and tRNA structure and functions were prioritized based on damage prediction tools MitoTIP and APOGEE.

2.3 WES analysis

Exome capture was done using the Agilent SureSelectXT V5 exome capture kit (Agilent, USA). The library was sequenced to mean 110X coverage on the Illumina HiSeq2000 sequencing platform. The sequences obtained were aligned to the human reference genome (GRCh37/hg19) using the BWA (Burrows-Wheeler algorithm) program and analyzed using Picard and GATK-Lite toolkit to identify variants in the whole exome relevant to clinical indication. Variant annotation was performed using Annovar for location and predicted function. Gross filtering was done using gnomAD (≤0.01 MAF), 1000 genomes (≤0.01 MAF), EVS (≤0.01 MAF), ExAC (≤0.01 MAF) dbSNP, and in-house exome databases. Clinically relevant mutations were annotated using published variants in literature and a set of variant databases including ClinVar, OMIM, and HGMD. Only non-synonymous, splice site, nonsense, and frameshift variants found in the coding regions were used for clinical interpretation. Silent variations that do not result in any change in amino acid in the coding region were excluded. The variant region in proband, father, and mother were confirmed by Sanger sequencing using ABI 3130 Genetic analyzer (Applied Biosystems, USA).

2.4 Quantitative PCR (qPCR)

The qPCR was done to quantify allele copy number in proband, father, mother, and control. 25 ng of genomic DNA and 0.5 pmol CA5A specific primers (CA5A_RT_F: 5′-GCTGGGATTACTGGACCTACGC-3′ and CA5A_RT_R: 5′-TTACGGGCACGGCTCACC-3′) were used. The qPCR reactions were performed using the HiMedia Insta Q96 real-time PCR system and the amplifications were done using the SYBR Green PCR Master Mix (ABI #A25742). The thermal cycling conditions were composed of an initial denaturation step at 95°C for 2 min followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The experiments were carried out in triplicates for each data point. The relative quantification of gene copy number was determined using the 2^−ΔΔCt method. The G6PDH gene with the following primers: G6PDH-F: 5′-TCTTCATCACCACAGAGAACTTGC-3′ and G6PDH-R: 5′-GACCTGGAAGTCACTGGGCA-3′ was used as an internal control for data normalization.

2.5 Long range polymerase chain reaction (LR-PCR)

Long-range PCR was performed to identify CA5A deletions using the following primers: CA5A-del-F: 5′-TCCCATTCTAGCTGCCTGAGGA-3′ and CA5A-del-R: 5′-AGACGAATGCCCTGTTCCTGTACC-3′. The PCR conditions: an initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 15s extension at 68°C for 6 min followed by a final extension at 68°C for 10 min.

3.1 Clinical features

The proband was a 3-day-old neonate, born via vaginal delivery to a 32-year-old mother out of non-consanguineous marriage. Father was 35 years old with no significant contributory family history. The neonate was born with a birth weight of 3100 g. She cried immediately at birth not requiring any resuscitation and was discharged home on day 2 of life on breastfeeds. The baby was on exclusive breastfeeds at the time of discharge. On day 3 of life, the neonate was noted to be lethargic and was not accepting breastfeeds. There were also seizures noted, which were multifocal-clonic occurring multiple times. She was referred to the Neonatal Intensive Care Unit (NICU) with complaints of poor feeding, lethargy, and seizures on the day of life 4. At the time of admission, the neonate was having status epilepticus and was stuporous. Vitals signs at the time of admission: temperature: 36.5°C, HR-170 bpm, CFT- 4 s, Peripheral pulses- low volume and poorly felt. RR- 52 bpm, SpO₂–92%. These findings were suggestive of shock. Weight at the time of admission was 2655 g with a cumulative 14% weight loss. The head circumference was 34 cm and length 48 cm (normal). There were no dysmorphic features, and no evidence of hepatosplenomegaly, and the rest of the systemic exam including the cardiac examination was normal. Biochemical tests were suggestive of metabolic acidosis with respiratory alkalosis, elevated ammonia, and lactate levels (Table 1). Neonate was stabilized with normal saline fluid bolus followed by intravenous fluids. For further control of shock inotropes (Inj. Dopamine and Inj. Adrenaline were added). She was mechanically ventilated for worsening hemodynamic stability. For the control of seizures, neonate required Inj. Levetericetam, Inj. Phenytoin and Inj. Lorazepam.

After initial hemodynamic stabilization and control of seizures, neonate was started on mother milk feeds via a nasogastric tube which was gradually incremented and there was no vomiting or episodes of feed intolerance. Neonate was extubated to CPAP support on DOL-10 and subsequently was off oxygen support by DOL-15. Neurologically neonate remained abnormal throughout NICU stay with axial and appendicular hypotonia and poor suck. Neonate was discharged on DOL-40 on cup and spoon feeds and continues to be on Syp. Leveritecetam, Syp. Phenytoin and early intervention.

3.2 Molecular findings

The clinical symptoms of the patient were suggestive of mitochondrial involvement. Hence, we performed a whole mitochondrial genome sequencing of the proband, which revealed no pathogenic or likely pathogenic variant(s). This prompted us to proceed with whole exome sequencing (WES). WES identified a homozygous pathogenic variant (NM_001739.2: c.721G>A: p.Glu241Lys) in exon 6 of the CA5A gene (Figure S1). Interestingly, the variant turned out to be heterozygous on targeted Sanger sequencing (Figure 1a). Upon reanalyzing the exome data, it was found that the sequencing reads also mapped to two highly identical regions (Figure S2) outside the CA5A gene, indicating the presence of a related genomic region that was initially overlooked and later found to be a pseudogene. Two pseudogenes of CA5A were identified on chromosome 16 having a high sequence similarity of almost 95% (Figure 1b). These pseudogenes having high sequence similarity co-amplified along with the target CA5A region, complicating Sanger sequencing and erroneously indicating the variant as heterozygous. By repeating Sanger sequencing with primers specifically designed for CA5A, thereby preventing co-amplification of pseudogenes, the variant was conclusively identified as homozygous in the proband (Figure 1c). Familial segregation analysis concerning the homozygous variant prompted suspicions, pointing towards the potential existence of a microdeletion in the proband's alternate allele which was confirmed using qPCR (Figure 1d). The proband and father were found to harbor a 4078 bp microdeletion (NM_001739.2: c.619-3420_c.774 + 502del4078bp) in the CA5A gene which spans regions of intron 5 and 6, and whole exon 6 (Figure 1e). The deletion breakpoints were identified by long-range PCR (Figure 1f) followed by Sanger sequencing. Therefore, it was confirmed that the proband harbors a pathogenic missense variant in one allele inherited from mother and a microdeletion in the other allele inherited from the father.