1 1 2 3 1 A Patient with Pontocerebellar Hypoplasia Type 6: Novel RARS2 Mutations, Comparison to 4 5 6 2 Previously Published Patients and Clinical Distinction from PEHO Syndrome 7 8 3 9 10 4 Viivi Nevanlinnaa,b, Svetlana Konovalovac, Berten Ceulemansd, Mikko Muonaa,1, Anni Laaria,c, Taru 11 12 5 Hilanderc, Katarin Gorskia,c, Leena Valannee, Anna-Kaisa Anttonena,f,g, Henna Tyynismaac, f, h, 13 14 a,c a,c,f,* 15 6 Carolina Courage , Anna-Elina Lehesjoki 16 17 7 18 19 8 aFolkhälsan Research Center, Helsinki, Finland 20 21 9 bFaculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland 22 23 c 24 10 Research Programs Unit, Molecular Neurology, University of Helsinki, Finland 25 26 11 dDepartment of Pediatric Neurology, Antwerp University Hospital, University of Antwerp, Belgium 27 28 12 eDepartment of Radiology, Hospital District of Helsinki and Uusimaa Medical Imaging Center, 29 30 13 31 University of Helsinki and Helsinki University Hospital, Helsinki, Finland 32 f 33 14 Department of Medical and Clinical Genetics, Medicum, University of Helsinki, Finland 34 35 15 gLaboratory of Genetics, Helsinki University Hospital, Helsinki, Finland 36 37 16 hNeuroscience Center, HiLIFE, University of Helsinki, Helsinki, Finland 38 39 17 1 40 Present address: Blueprint Genetics, Helsinki, Finland 41 42 18 *Corresponding author. Folkhälsan Research Center, Biomedicum Helsinki, Haartmaninkatu 8, 43 44 19 00290, Helsinki, Finland; tel: +358 2941 25072; email: [email protected] 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 61 62 20 Abstract 63 64 65 21 Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy 66 67 22 caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase 68 69 23 enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive 70 71 24 Encephalopathy with oedema, Hypsarrhythmia and Optic atrophy). The proband presented with 72 73 74 25 severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, 75 76 26 acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and 77 78 27 delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but 79 80 28 sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing 81 82 83 29 revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly 84 85 30 conserved amino acid and a frameshift variant with consequent degradation of the transcript 86 87 31 resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features 88 89 32 90 distinguishing the proband’s phenotype from PEHO syndrome were later appearance of hypotonia 91 92 33 and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with 93 94 34 more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO 95 96 35 syndrome patients. The study highlights the challenges in clinical diagnosis of patients with 97 98 36 99 neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI 100 101 37 findings. 102 103 38 104 105 39 Keywords 106 107 40 Pontocerebellar hypoplasia type 6, RARS2, PEHO syndrome, progressive cerebellar and cerebral 108 109 110 41 atrophy 111 112 113 114 115 116 117 118 119 3 120 121 42 Introduction 122 123 124 43 Pontocerebellar hypoplasia (PCH) is a group of neurodegenerative disorders with autosomal 125 126 127 44 recessive inheritance. Up to date 11 different subtypes have been described, with 17 causative 128 129 45 genes identified (van Dijk et al., 2018). Most of the subtypes are characterized by prenatal or 130 131 46 neonatal onset, global developmental delay and intellectual disability, microcephaly, hypoplasia 132 133 47 and variable atrophy of cerebellar cortex and/or brainstem. The specific neurological symptoms 134 135 136 48 and the severity of symptoms and brain loss vary between the subtypes (van Dijk et al., 2018). 137 138 49 Pontocerebellar hypoplasia type 6 (PCH6; MIM 611523) is a rare form of PCH first described in 139 140 141 50 2007 in three patients of a consanguineous Sephardic Jewish family (Edvardson et al., 2007). Since 142 143 51 then, altogether 32 patients in 18 families have been reported in the literature (for a detailed 144 145 52 summary of the patients and phenotypes, see Supplementary Table; Edvardson et al., 2007; 146 147 53 Rankin et al., 2010; Namavar et al., 2011; Glamuzina et al., 2012; Cassandrini et al., 2013; 148 149 150 54 Kastrissianakis et al., 2013; Joseph et al., 2014; Li et al., 2015; Lax et al., 2015; Nishri et al., 2016; 151 152 55 Alkhateeb et al., 2016; Ngoh et al., 2016; van Dijk et al., 2017; Luhl et al., 2016; Zhang et al., 2018). 153 154 56 Most PCH6 patients present with neonatal onset, hypotonia, microcephaly, seizures, severe 155 156 57 intellectual disability with lack of developmental milestones and progressive atrophy of cerebral 157 158 159 58 cortex, cerebellum and pons. The majority show a respiratory chain enzyme deficiency and 160 161 59 elevated lactate levels in blood or cerebrospinal fluid (CSF). Indeed, PCH6 may be distinguished 162 163 60 from the other PCH subtypes, which are highly variable clinically and neuroradiologically, by the 164 165 61 166 presence of elevated lactate concentration (van Dijk et al., 2018). 167 168 62 PCH6 is caused by biallelic mutations in RARS2, a nuclear gene that encodes the mitochondrial 169 170 63 arginine-tRNA synthetase enzyme (mtArgRS) (Edvardson et al., 2007). Aminoacyl-tRNA synthetases 171 172 64 play a crucial role in protein translation as they catalyze the specific attachment of an amino acid 173 174 175 176 177 178 4 179 180 65 (aminoacylation) to its cognate tRNA. MtArgRS participates in the synthesis of all 13 181 182 183 66 mitochondrial-encoded proteins by charging of mitochondrial tRNA-Arg, thus being an integral 184 185 67 part of mitochondrial protein translation machinery, participating in generation of complexes of 186 187 68 oxidative phosphorylation system, except complex II, which has a fully nuclear origin (Ibba and 188 189 69 Soll, 2000). 190 191 192 70 PCH6 shows clinically some resemblance to PEHO syndrome (Progressive Encephalopathy with 193 194 195 71 oedema, Hypsarrhythmia and Optic atrophy; MIM 260565), characterized by neonatal hypotonia, 196 197 72 profound psychomotor retardation, infantile spasms with hypsarrhythmia and atrophy of optic 198 199 73 disks (Salonen et al., 1991). Patients present with typical dysmorphic features, such as narrow 200 201 74 forehead, epicanthic folds, short nose and open mouth, and edema of the face and limbs (Somer, 202 203 204 75 1993). Neuroimaging findings include demyelination and progressive atrophy of the cerebellar 205 206 76 cortex, brainstem and optic nerves. In the cerebellum, the inner granular layer is nearly totally 207 208 77 absent and Purkinje cells are deformed and disaligned (Haltia and Somer, 1993). 209 210 211 78 PEHO syndrome is inherited autosomal recessively and was recently shown to be caused in Finnish 212 213 79 patients by a homozygous missense mutation c.92C>T; p.Leu31Ser in ZNHIT3, a gene encoding zinc 214 215 80 finger HIT domain-containing protein 3 (Anttonen et al., 2017). PEHO syndrome is enriched in the 216 217 218 81 Finnish population with an estimated incidence of 1:74 000 (Somer, 1993) and approximately 40 219 220 82 diagnosed patients. In other populations it is very rare, with less than 25 reported patients (Field 221 222 83 et al., 2003; Caraballo et al., 2011; Alfadhel et al., 2011) and only one patient with compound 223 224 84 225 heterozygous mutations in ZNHIT3 reported so far (Öunap et al., 2019). In the literature, patients 226 227 85 with symptoms closely resembling PEHO syndrome are more commonly reported. The clinical 228 229 86 presentation of patients with PEHO-like features, like those with PCH, is similar to that of PEHO 230 231 87 syndrome, but optic atrophy and typical neuroradiologic findings are usually absent or there is no 232 233 234 235 236 237 5 238 239 88 progression (Field et al., 2003; Longman et al., 2003; Chitty et al., 1996). Several genes underlying 240 241 242 89 phenotypes resembling PEHO have been described (Rankin et al., 2010; Anttonen et al., 2015; 243 244 90 Gawlinski et al., 2016; Langlois et al., 2016; Nahorski et al., 2016; Flex et al., 2016; Miyake et al., 245 246 91 2016; Zollo et al., 2017; Chitre et al., 2018). 247 248 249 92 We report a patient with the initial presenting features suggestive of PEHO syndrome with typical 250 251 93 dysmorphic features, epileptic spasms, optic atrophy and severe hypotonia, but in whom whole 252 253 254 94 genome sequencing revealed novel compound heterozygous mutations in RARS2. 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 6 297 298 95 Materials and methods 299 300 301 96 Patient and samples 302 303 304 97 The proband was clinically examined by B.C. in Antwerp and was referred to molecular genetic 305 306 98 analyses in Helsinki. DNA extracted from peripheral blood was obtained from the proband and 307 308 99 both parents. Primary fibroblast cultures from the proband were available for analyses of the gene 309 310100 product. 311 312 313101 An institutional review board at the Helsinki University Central Hospital approved the study. A 314 315102 written informed consent was obtained from the parents. 316 317103 318 319104 Sequencing of ZNHIT3 320 321 322105 The five coding exons of ZNHIT3 (NM_004773.3) were Sanger sequenced from genomic DNA of 323 324106 the proband (primer sequences available upon request). Exon 1 covering the c.8C>T, p.Ser3Leu 325 326107 variant was also sequenced in the parents. 327 328108 329 330 331109 Whole genome sequencing 332 333 334110 Library preparation for the genomic DNA sample was performed using KAPA Library Preparation 335 336111 Kit. The sample was sequenced in three lanes of an Illumina HiSeq2500 instrument with one lane 337 338 339112 having paired-end 250-bp reads and two lanes paired-end 10-bp reads. Sequence read alignment 340 341113 to human reference genome (GRCh37) and variant calling (Li et al., 2009) was done as described 342 343114 earlier with minor modifications (Sulonen et al., 2011). Called variants were annotated using 344 345115 ANNOVAR (Wang et al., 2010) and filtered using in-house scripts. DELLY (Rausch et al. 2012), which 346 347 348116 assesses split-read alignments and paired-end read information to detect structural variants was 349 350117 used to identify any copy number changes overlapping with the ZNHIT3 locus. Sanger sequencing 351 352 353 354 355 7 356 357 118 was performed from genomic DNA of the patient and the parents to validate the variants 358 359 360119 identified by whole genome sequencing and to test segregation of the variants in the family. 361 362120 Primer sequences are available upon request. 363 364121 365 366 367122 Sequencing of patient cDNA 368 369 370123 Patient fibroblasts were harvested, total RNA extracted (RNeasy plus mini kit, QIAGEN) and 371 372124 complementary DNA (cDNA) prepared (iScript cDNA synthesis kit, BioRad). Polymerase chain 373 374 125 reaction was performed using primers (sequences available upon request) binding to exons 8 and 375 376 377126 14 of RARS2 and the resulting 600-bp product covering the positions of the mutations in exons 10 378 379127 and 11 was sequenced using standard protocols. 380 381128 382 383 384129 Western blot analysis 385 386 387130 Protein extracts for the detection of mtArgRS, COXII or GAPDH were prepared by lysing fibroblasts 388 389131 in RIPA buffer (Cell Signaling Technology) containing protease inhibitors (Halt, Thermo Fisher 390 391 132 Scientific). After 10 min incubation on ice the samples were centrifuged at 14 000 g for 10 min (+4 392 393 394133 ⁰C). Proteins were separated by SDS-PAGE and transferred onto membranes. After blocking with 395 396134 5% milk in 0.1% TBS–Tween 20, the membranes were incubated with the corresponding primary 397 398135 antibodies: rabbit anti-human mtArgRS (1:1000, Biorbyt, orb374171), rabbit anti-human COXII 399 400 136 (1:500, GeneTex, GTX62145) or rabbit anti-human GAPDH (Cell Signaling Technology, 14C10). 401 402 403137 Reactive bands were detected using horseradish peroxidase-conjugated secondary antibodies 404 405138 (goat anti-rabbit or goat anti-mouse, 1:10 000, Life Technologies). Blots were imaged using the ECL 406 407139 western blotting substrate (Thermo Fisher Scientific) and Chemidoc XRS+ Molecular Imager (Bio- 408 409 140 Rad). Quantification of the band intensities was performed with the Image Lab Software (Bio-Rad). 410 411 412 413 414 8 415 416141 417 418 419142 Northern blot and aminoacylation assay 420 421 422143 Total RNA was extracted from cultured fibroblasts using Trizol reagent (Thermo Fisher scientific) 423 424 144 according to the manufacturer’s instructions. To preserve the aminoacylation state the final RNA 425 426 427145 pellet was re-suspended in 10mM NaOAc at pH 5.0. To investigate the aminoacylation status of 428 429146 mt-tRNAs, 4μg of RNA was separated on long (16cm length) 6.5% polyacrylamide gel (19:1 430 431147 acrylamide:bis-acrylamide) containing 8M urea in 0.1 NaOAc, pH 5.0. The fully deacylated tRNA 432 433 148 (dAc) was obtained by incubation of the control RNA at 75°C (pH 9.0) for 15 min. To determine mt- 434 435 Arg 436149 tRNA steady-state levels the samples were run on 10cm gel. Northern hybridization was 437 438150 performed with ϒ-32P labeled oligonucleotide probes: 5’-GAGTCGAAATCATTCGTTTTG-3’ for the 439 440151 mt-tRNAArg and 5’- GTGGCTGATTTGCGTTCAGT-3’ for the mt-tRNAAla. Radioactive signal was 441 442 152 detected by PhosphorImager plate using Typhoon scanner and quantified with the ImageQuant 443 444 445153 v5.0 software (GE Healthcare). 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 9 474 475 154 Results 476 477 478155 Clinical description 479 480156 The essential clinical features in our patient are summarized in Supplementary Table. The patient 481 482157 was the first child of non-consanguineous Belgian parents. Family history was unremarkable. He 483 484 158 was born at term after an uneventful pregnancy. Birth weight was 3.150 kg (-1 SD), length 50 cm (- 485 486 487159 1 SD) and head circumference 35 cm (-0.5 SD). After birth slight hypothermia occurred, leading to 488 489160 one day neonatal care, but otherwise physical examination was normal. Very early psychomotor 490 491161 milestones were reported normal, but at the age of 2 to 3 months lack of social interaction, late 492 493 162 visual contact and mild hypotonia were noted. No further developmental milestones were 494 495 496163 reached, he had no speech and showed no real social contact. The patient had no dysmorphic 497 498164 signs at birth, but later presented with bitemporal narrowing, high palate, open mouth, full 499 500165 cheeks, a tented upper lip (Fig. 1A) as well as mild edema of hands (Fig. 1B) and feet. Eye 501 502 166 503 examination showed no visual contact and a pale papilla on both eyes later progressing to optic 504 505167 atrophy. Due to feeding difficulties the child was tube fed. An acquired microcephaly was noted 506 507168 with occipitofrontal circumference (OFC) of 43 cm (-3.3 SD) at the age of 1 year and 46 cm (-3.7 508 509169 SD) at the age of 3 years. At the last clinical follow-up with 9 years of age, he presented as a 510 511 170 512 bedridden child with profound intellectual disability, axial hypotonia, spastic quadriplegia and 513 514171 significant seizure burden. 515 516172 First convulsions were witnessed at the age of 6 weeks with lateralized clonic movements of the 517 518173 face, followed by diminished consciousness and eye deviation to one side as well as bilateral clonic 519 520 174 521 movements of the body. It is unclear from the history whether these seizures were already 522 523175 present from birth. Convulsions evolved into therapy-resistant epilepsy with varying seizure types: 524 525176 complex focal seizures (with and without diminished consciousness) with myoclonic jerks and 526 527177 laughing, rhythmic clonic movements of one or both limbs and long-lasting eye deviations with 528 529 530 531 532 10 533 534 178 nystagmus. The patient suffered from daily seizures several times a day with isolated myoclonic 535 536 537179 spams and clusters in between. 538 539180 EEG studies at the age of one to 3 months showed normal background activity without any 540 541181 epileptic activity. Multifocal epileptic activity was seen from the age of 4 months and high voltage 542 543 182 slow background activity from the age of 5 months. The EEG did show some signs of 544 545 546183 hypsarrhythmia and could, because lack of total desynchronization, be described as a modified 547 548184 hypsarrhythmia. The last EEG recording, taken one day before the patient died, demonstrated a 549 550185 picture of status epilepticus with continuous multifocal epileptic activity. 551 552 186 Magnetic resonance imaging (MRI) was performed at the ages of 4.5 months and 7 years. At 4.5 553 554 555187 months (Fig. 1C,D), it showed severe cerebral atrophy, destruction of the thalami, and delayed 556 557188 myelination, whereas the cerebellum appeared normal in size. At 7 years (Fig. 1E-G), the 558 559189 cerebellar atrophy was prominent, and microcephaly masked some of the cerebral atrophy. The 560 561 190 562 pons was normal, and the myelination had reached almost a normal appearance. 563 564191 Thorough metabolic investigations were unremarkable, with the exception of an intermittently 565 566192 raised serum lactate up to 5.3 mmol/l (0.5-2 mmol/l) and an elevated lactate level in the CSF, up to 567 568193 2.8 mmol/l (<2.5 mmol/l). No abnormalities were seen in the muscle biopsy. 569 570 194 571 Prior genetic investigations including karyotype and microarray came out normal and 572 573195 mitochondrial DNA mutations were excluded. 574 575196 The patient died at the age of nearly 12 years due to a respiratory infection. 576 577197 578 579 580198 Molecular findings: RARS2 mutations and their consequence 581 582 583199 Given that the patient presented with symptoms overlapping with those reported in PEHO 584 585200 syndrome, his DNA was first Sanger sequenced to identify variants in the coding regions and splice 586 587 588 589 590 591 11 592 593 201 sites of ZNHIT3. A rare heterozygous c.8C>T, p.Ser3Leu (NM_004773.3) missense variant was 594 595 596202 identified, but the patient did not have other ZNHIT3 coding sequence variants. To identify any 597 598203 non-coding variants in ZNHIT3 locus, the patient was whole genome sequenced. Analysis for rare 599 600204 sequence variants in intronic or UTR regions of ZNHIT3, or up- or downstream to ZNHIT3 did not 601 602 205 identify a second variant. No copy number changes overlapping with the ZNHIT3 locus was 603 604 605206 identified. 606 607207 Analysis of the whole genome data was then expanded to all protein coding regions of the 608 609208 genome and splice sites. Whole genome sequence data was produced with mean sequencing 610 611 209 coverage of 24.48x, and 98.2%, 95.7% and 74.2% of the genome was covered at least 5x, 10x and 612 613 614210 20x, respectively. Analysis of the coding regions from the genome sequence data focused on rare 615 616211 heterozygous and potentially biallelic variants in established disease genes. Analysis of rare 617 618212 heterozygous variants did not yield any likely candidates explaining the patient’s disease. Analysis 619 620 213 621 of rare biallelic variants revealed two heterozygous variants in RARS2 (NM_020320.3; Fig. 2A and 622 623214 B; https://databases.lovd.nl/shared/individuals/00234052), a one-bp deletion in exon 10 causing a 624 625215 frameshift and premature termination of translation 16 codons downstream (c.795delA, 626 627216 p.Glu265Aspfs*16) and a missense variant, c.961C>T, p.Leu321Phe, in exon 11. There is one 628 629 217 630 heterozygous carrier for the c.961C>T, p.Leu321Phe variant in the gnomAD (Lek et al., 2016) 631 632218 database (v. 2.0; allele frequency 0.000004), whereas the frameshift variant is absent from the 633 634219 database. The leucine at position 321, located in the catalytic domain of RARS2, is highly 635 636220 conserved (Fig. 2B). In silico tools SIFT, PolyPhen-2 and MutationTaster predict the c.961C>T, 637 638 221 639 p.Leu321Phe substitution as deleterious. Sanger sequencing confirmed compound heterozygosity 640 641222 of the two mutations in the patient: the c.795delA frameshift mutation was inherited from the 642 643223 mother and the c.961C>T missense mutation from the father (Fig. 2A). 644 645224 The consequence of the RARS2 variants was studied on mRNA level in skin fibroblasts of the 646 647 648 649 650 12 651 652 225 patient. The frameshift variant in exon 10 resulting in a premature termination codon is predicted 653 654 655226 to be subjected to nonsense-mediated mRNA decay (NMD) and degradation of the transcript 656 657227 derived from the maternal allele. Indeed, sequencing of RARS2 cDNA revealed that at position 658 659228 c.961 only the paternal C>T variant was present (Fig. 2C). Western blot analysis of patient 660 661 229 fibroblasts revealed that the mtArgRS protein level was reduced to about 50 % of control level 662 663 664230 (Fig. 3A). Northern blot analysis of total RNA from fibroblasts suggested that the steady-state level 665 666231 of mitochondrial tRNAArg when compared to mitochondrial tRNAAla may be decreased in patient 667 668232 fibroblasts (Fig. 3B). In patient and control fibroblasts, aminoacylation analysis showed the 669 670 233 presence of only aminoacylated mt-tRNAArg, whereas deacylated mt-tRNAArg was not detected (Fig. 671 672 673234 3C). This finding is in agreement with the previous observation (Edvardson et al., 2007), suggesting 674 675235 that in cultured human fibroblasts uncharged mt-tRNAArg is not stable. 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 13 710 711 236 Discussion 712 713 714237 We describe a patient compound heterozygous for two novel pathogenic variants in RARS2, the 715 716238 gene associated with PCH6. The high conservation of the affected Leu321, the predicted 717 718239 deleteriousness of the Leu321Phe substitution combined with degradation of the transcript 719 720 240 derived from the allele with the frameshift variant strongly suggest that these variants are the 721 722 723241 underlying cause for PCH6 in the patient. 724 725242 The role of RARS2 in pontocerebellar hypoplasia is not fully understood with no clear genotype- 726 727243 phenotype correlations. It is though likely that the severity of the disease is dependent of the 728 729 244 amount of remaining aminoacylation activity (Konovalova and Tyynismaa, 2013). mtArgRS has a 730 731 732245 fundamental function in mitochondrial protein synthesis, so total loss-of-function mutations are 733 734246 likely to be lethal. Compatible with this notion, mice homozygous for a knock-out allele of Rars2 735 736247 are embryonic lethal (International Mouse Phenotyping Consortium; 737 738 248 739 http://www.mousephenotype.org/data/genes/MGI:1923596#section-associations). Considering 740 741249 the markedly reduced expression from the frameshift allele, the missense mutant allele is likely to 742 743250 retain some mtArgRS activity in our patient. It has been suggested that due to the leaky nature of 744 745251 the mutations, small amounts of protein synthesis is possible in most tissues, but in high energy 746 747 252 748 demanding cells, such as neurons, the reduced aminoacylation is not sufficient thus causing the 749 750253 symptoms of the disease (Edvardson et al., 2007). Low enzyme activity affects the development of 751 752254 the central nervous system already in utero as demonstrated by abnormal brain MRI findings in 753 754255 the neonatal period (e.g. Edvardson et al., 2007; Joseph et al., 2014; Lax et al., 2015). It is also 755 756 256 757 possible that the reduced aminoacylation of tRNA-Arg has bigger effect on specific neuronal types 758 759257 that causes the alterations in brain typical for PCH6. There is also evidence of particular uncharged 760 761258 tRNAs and amino acids working as potential signaling molecules (Dong et al., 2000; Wolfson et al., 762 763259 2016). Mitochondrial tRNA synthetases may also have non-canonical functions, similarly to their 764 765 766 767 768 14 769 770 260 cytosolic counterparts, in addition to their housekeeping function in protein synthesis, and these 771 772 773261 may contribute to the pathomechanisms. For example, mtArgRS was recently found to have a 774 775262 specific sub-mitochondrial localization in the membrane, which suggests that it also could have 776 777263 alternative functions (Gonzáles-Serrano et al., 2018). Regardless of the reason, this high tissue 778 779 264 specificity makes functional studies of the disease mechanism challenging. 780 781 782265 Including the present patient, 33 patients with PCH6 in 19 families have been described 783 784266 (Supplementary Table). An overview of the key clinical features in the patients is presented in 785 786267 Table 1. Most patients were normal at birth but presented with variable symptoms at early age 787 788 268 (hours to 9 months). First presenting features included hypotonia in 15/33 patients and seizures in 789 790 791269 16/33 patients. Other early symptoms were poor feeding, lethargy and apneic episodes. All 792 793270 patients were reported to have global developmental delay and the majority presented seizures, 794 795271 the onset varying from 9 hours to several months. Most seizures were intractable myoclonic or 796 797 272 798 tonic-clonic seizures, either focal, or multifocal or generalized. Other common features in the 799 800273 patients include progressive microcephaly, atrophy of cerebellum and cerebrum, as well as 801 802274 elevated lactate levels in blood or CSF. Notably, atrophy of pons was reported to be present in 803 804275 only 12 out of the 25 patients with reported MRI findings, indicating that pons can be normal in 805 806 276 807 PCH6 (Nishri et al., 2016). The phenotype in our patient is similar to that of previously published 808 809277 patients, and presents with all features listed in Table 1, except atrophy of the pons. Of note, as in 810 811278 at least three published patients (Ngoh et al., 2016; Zhang et al., 2018; Luhl et al., 2016), the 812 813279 serum lactate levels in our patient were intermittently raised. 814 815 280 816 Compatible with a previous report (Rankin et al., 2010), the initial clinical features in our patient 817 818281 including severe intellectual disability, epilepsy, optic atrophy, hypotonia, acquired microcephaly, 819 820282 mild edema of hands and feet, and dysmorphic features pointed to PEHO syndrome. Although the 821 822283 dysmorphic features raised the suspicion of the PEHO syndrome, they may, however, be non- 823 824 825 826 827 15 828 829 284 specific, as many of the dysmorphic facial features are associated with developing microcephaly, 830 831 832285 extreme floppiness, and edema (Somer, 1993). Contrary to findings in our patient, patients with 833 834286 PEHO syndrome do not show elevated lactate levels in blood or CSF and usually present with 835 836287 neonatal hypotonia (Anttonen et al., 2017). Importantly, the MRI findings in our patient (Fig. 1C-G) 837 838 288 were not typical for PEHO syndrome. The supratentorial atrophy was more severe than in a typical 839 840 841289 PEHO patient. Moreover, the myelination was not delayed to the degree seen in PEHO patients. 842 843290 Characteristic MRI findings including progressive cerebellar atrophy and dysmyelination are 844 845291 essential diagnostic criteria for PEHO syndrome (Anttonen et al., 2017). These typical findings are 846 847 292 often disregarded when suggesting a clinical PEHO diagnosis. 848 849 850293 851 852294 853 854295 Acknowledgements 855 856 296 857 We thank the family for their contribution to this study. We thank the Genome Aggregation 858 859297 Database (gnomAD) and the groups that provided exome and genome variant data to this 860 861298 resource. A full list of contributing groups can be found 862 863299 at http://gnomad.broadinstitute.org/about. This study was funded by the Folkhälsan Research 864 865 300 866 Foundation. 867 868301 869 870302 871 872303 Accession numbers 873 874 304 875 https://databases.lovd.nl/shared/individuals/00234052 876 877 878 879 880 881 882 883 884 885 886 16 887 888 305 Figure Titles and Legends 889 890 891306 Figure 1. Phenotypic features in the patient. 892 893307A) Facial features of the patient at 7 years of age. Note the open mouth, full cheeks, a tented 894 895308 upper lip and bitemporal narrowing. B) The hand shows edema. C) In a sagittal T1-weighted cranial 896 897 309 magnetic resonance image at the age of 4.5 months cerebellum (arrowhead) and pons (arrow) 898 899 900310 appear normal in size. D) T2-weighted axial image at 4.5 months shows cerebral atrophy. 901 902311 E & F) T2-weighted images of the patient at 7 years of age show microcephaly and widespread 903 904312 cerebral atrophy as well as severe cerebellar atrophy (arrowhead in E) with widened cerebellar 905 906 313 sulci (F). The pons (arrow in E) as well as the myelination appear normal. G) T2-axial slices at 7 907 908 909314 years also show atrophy and signal increase of the thalami (open arrowheads). 910 911315 912 913316 Figure 2. Two novel PCH6-associated mutations in the RARS2 gene. 914 915 317 916 A) Sanger sequencing chromatograms of the proband’s (P) and the parents’ genomic DNA showing 917 918318 the c.795delA variant inherited from the mother (M) and the c.961C>T variant inherited from the 919 920319 father (F). Positions of variants are indicated with arrowheads. B) A schematic picture of the exon- 921 922320 intron structure of RARS2 and the domain structure of the encoded protein (modified from 923 924 321 925 Gonzáles-Serrano et al., 2018) showing the locations of the identified mutations and high 926 927322 conservation of the leucine at position 321 affected by the missense substitution. C) Sanger 928 929323 sequencing chromatograms of the proband’s cDNA showing only the paternal c.961C>T variant 930 931324 (arrowhead) in exon 11 suggesting that the transcript derived from the maternal allele is 932 933 325 934 degraded. 11F denotes forward orientation sequence and 11R reverse orientation 935 936326 937 938327 Figure 3. Western blot, northern blot and aminoacylation analysis of the patient fibroblasts. 939 940 941 942 943 944 945 17 946 947 328A) Steady-state level of mtArgRS protein in patient (P) and control fibroblasts (C1, C2) detected by 948 949 950329 Western blot. Quantification of the Western blot analysis is shown in the right panel. GAPDH was 951 952330 detected as protein loading control. Data are presented as mean ± SD. B) Northern blot analysis of 953 954331 mt-tRNAArg levels in patient (P) and control (C1, C2) fibroblasts. Quantification of the northern blot 955 956 332 analysis is shown in the lower panel. Mitochondrial tRNAAla was detected as a loading control. 957 958 Arg 959333C) Aminoacylation assay of mt-tRNA in control (C1, C2) and patient (P) fibroblasts. Mitochondrial 960 961334 tRNAAla was detected as a loading control. dAC denotes the fully deacylated control tRNA. 962 963335 Experiments in B and C were carried out only once. 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 18 1005 1006 336 Table 1. Overview of clinical features in published PCH6 patients 1007 1008337 1009 1010 1011 Feature n/na 1012 1013 1014 1015 Global developmental delay 33/33 1016 1017 Epileptic seizures 24/24 1018 Microcephaly 20/27 1019 1020 MRI findings 1021 1022 Atrophy of cerebellum 22/25 1023 Atrophy of pons 12/25 1024 1025 Atrophy of cerebrum 18/25 1026 1027 Elevated lactate level in blood or CSF 19/23 1028 1029 Reduced respiratory chain enzyme activity 10/19 1030 Feeding difficulties 17/18 1031 1032 Dysmorphic features 6/8 1033 1034 1035338 1036 339 CSF – cerebrospinal fluid 1037 1038340 a 1039341 The features are variably reported in the patients. 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 19 1064 1065 342 References 1066 1067 1068343 Alfadhel, M., Yong, S.L., Lillquist, Y., Langlois, S., 2011. Precocious puberty in two girls with PEHO syndrome: 1069 1070344 A clinical feature not previously described. 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Table 1. Overview of clinical features in PCH6 patients

Feature n/na Global developmental delay 33/33 Epileptic seizures 24/24 Microcephaly 20/27 MRI findings Atrophy of cerebellum 22/25 Atrophy of pons 12/25 Atrophy of cerebrum 18/25 Elevated lactate level in blood or CSF 19/23 Reduced respiratory chain enzyme activity 10/19 Feeding difficulties 17/18 Dysmorphic features 6/8

CSF – cerebrospinal fluid aThe features are variably reported in the patients. 1 2 3 The first author of the manuscript and the corresponding author (if different) certify on honor, on 4 5 behalf of all co-authors, that they have been granted a permission to publish signed by the patient 6 himself (or by his legal representatives) for each patient whose facial features are identifiable in the 7 photographs illustrating this article. The authors maintain in their files a copy of this consent, which 8 will be forwarded to Elsevier in case of complaints or legal proceedings. 9 10 Figures for which a signed consent has been obtained: 11 12 Figure number Figure number Figure number Figure number 13 14 15 FIGURE 1 16 17 18 19 20 21 22 Name Forename 23 24 LEHESJOKI ANNA-ELINA 25 26

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59