Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5′ leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants.

The enriched libraries were sequenced by Illumina Novaseq 6000 genome sequencing analyzer platform (Illumina, San Diego, CA, USA).ES data annotation, filtering, and interpreting were done as previously described (1).
For F2, DNA was enriched for the complete coding regions of most genes of the human genome using a proprietary capture system developed by GeneDx for next generation sequencing with CNV calling.The enriched targets were simultaneously sequenced with paired end reads on an Illumina platform.
Bidirectional sequence reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSCS hg19.Using a custom-developed analysis tool (XomeAnalyzer) Ddata were filtered and analysed using XomeAnalyzer to identify sequence variants.

Respiratory chain activity assays and immunoblotting in fibroblasts
Respiratory chain complex activities were assessed in fibroblasts as described previously (2).Extraction of proteins and subsequent SDS-PAGE and immunoblotting was also performed on patient fibroblasts, as previously described (3), using primary antibodies against GAPDH (Proteintech, Rosemont, IL, USA:60004-1-Ig) and PRORP (Proteintech:20959-1-AP).

Mutagenesis of PRORP
The vector pET-28b(+) containing the PRORP cDNA sequence from amino acid 46 was mutagenised, as described previously (4).Oligonucleotides designed for mutagenesis are listed in Table S1, with altered bases highlighted.Following transformation of the mutagenesis reaction, individual colonies were cultured overnight, and plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany).Successful mutagenesis was confirmed with Sanger sequencing using petup and T7 terminator primers (Eurofins Genomics, Ebersberg, Germany).

Production of mitochondrial pre-tRNA transcripts
The pre-tRNA Ile from plasmid phI2 (6) was prepared by XbaI digestion of the plasmid, then in vitro transcription using the T7 RiboMAX Express Large Scale RNA Production System according to the   (B) The location of the residue Tyr365.Substitution to histidine results in a similarly bulky side chain but could potentially remove a hydrogen bond.Mapping Tyr365 onto the known mtRNase P structure 7 shows that this residue is not located in the vicinity of the active site or any interaction regions.
(C) The location of the residue Thr387.This residue is located at the interaction site with TRMT10C.Its substitution to alanine may alter mtRNase P complex formation.

(D)
The location of the residue Ala414.Substitution to valine will result in a bulkier sidechain and a possible clash with Leu426.This may result in altered interactions with the 5' end of the tRNA substrate.Recent structural studies have revealed that the adjacent residue Lys415 contacts the acceptor arm and leader nucleotides of the pre-tRNA substrate, helping to position the tRNA substrate into the active site 7 .This observation suggests that variants in this region of the metallonuclease domain could impair proper positioning and cleavage of pre-tRNAs.

Figure S1 :Figure S2 :
Figure S1: Additional clinical data for affected individuals in all families.(A) Axial T2-weighted brain MR images taken of individual F1 II-1, exhibiting multifocal leukoencephalopathy and cerebellar atrophy (particularly in the vermis), respectively.(B) Hormone profile for individual F1 II-1.(C) Audiogram for individual F2 II-1, highlighting severe to profound bilateral sensorineural hearing loss in the proband with increasing severity at higher frequencies.Hearing level in the left ear is represented by blue crosses, and the right ear with red circles.Designed using the AudGen online tool (version 0.6.3)(https://audsim.com/audgenJS/audgenjs.html).(D) MR images of individual F3 II-3.A -Axial FLAIR showing bilateral symmetrical hyperintensities in the globus pallidus and putamen nuclei, and mildly in the head of the caudate nuclei.B -Sagittal T1 highlighting a short corpus callosum with a thin body.C (DWI) + D (ADC) -Shows bilateral symmetrical restricted diffusion in the globus pallidus, most prominent in the posterior parts.May be resultant of hypoxic ischaemic injury.(FLAIR -fluid-attenuated inversion recovery, DWI -diffusion-weighted image, ADC -apparent diffusion coefficient).

Figure S3 :
Figure S3: Schematic illustrating the mitochondrial tRNA processing assay.Recombinant MRPP1, MRPP2 and PRORP (WT or patient variant) were purified, then reconstituted to form the mtRNase P complex in vitro.PRORP variants can diminish mtRNase P activity, resulting in reduced 5'-end pre-tRNA processing, which is visualised on a denaturing urea gel to compare mtRNase P efficacy.Created with BioRender.com.

Figure S4 :
Figure S4: Structural analysis of PRORP variants based on the known mtRNase P complex.(A) Cryo-electron microscopy structure of the mtRNase P complex (PDB:7ONU) consisting of a SDRC51 tetramer, a TRMT10C monomer, and a PRORP monomer engaged with a tRNA Tyr substrate.The individual domains of PRORP are labelled.Inset shows a zoom-in view of the metallonuclease domain of PRORP.The reported variants are labelled and highlighted in chartreuse.

Table S3 : Comparison of clinical phenotypes observed with all reported PRORP variants to date
. NR = Not reported, HH = hypergonadotropic hypogonadism, POI = primary ovarian insufficiency.