Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. a dnie.W i odlnaeteMC2peoyeadgv vdneo h lnclsgicneof significance clinical the of the evidence within give variant and inframe Method phenotype novel, MOCS2 whom the in delineate variant. patient, novel to another the aim present We to identified. like its was we implicates which paper, what (ORFs) this 2003), deficiency. frames (Leimkuhler, In MOSC synthase reading of MPT open diagnostics of overlapping molecular subunits (Reiss, bicistronic, two and 2.7.7.75) the is by and functioning MOCS2B, 2.10.1.1 MOCS2 encoded EC and (GPHN; proteins MOCS2A 2011). gephyrin encode by Hahnewald, by MPT and Z of Reiss moiety precursor dithiolene 1999; of the to synthesis molybdenum of involving: ment process, the multistep by a MOCS1 oxidase however, encoded sulfite is, is molybdenum of biosynthesis developmen- (MOSC2) are activity which global cofactor decreased 1.17.3.2), with Molybdenum from and form 1.17.1.4 results milder EC activity. disorder (XDH; a their early The dehydrogenase for with to xanthine cofactor-dependent presented lead and 2019). may 1.8.3.1) date) (Arican, which EC to seizures (SUOX; complications, described first without neurologic attention cases delay severe medical three show tal to children (only hy- dis- came Affected It rarely with metabolic accompanying death, dysmorphism. 1980). recessive spasticity, craniofacial (Johnson, autosomal muscular and 1980 levels an neonatal-onset, in sulfite is of urinary #252160) seizures elevated intractable pouricemia, (MOCODB, by B characterized type order deficiency cofactor Molybdenum treatment including disease, Introduction rare this on of knowledge use current the resulting into with pathomechanism insight level potential molecular original Moreover, a giving complex. on significance presented, options. synthase consequences clinical was mutation molybdopterin Its defect human possible identified. molecular the the genotype was from of of disease gene analysis structure the MOCS2 and crystal delineate the available history to within the medical variant aimed the inframe paper by novel, Our supported whom levels date. was in sulfite to patient, characterized urinary reported another elevated disorder were presenting hypouricemia, metabolic patients by with Thirty-five recessive accompanying autosomal dysmorphism. spasticity, rare craniofacial muscular a and neonatal-onset, is of #252160) seizures (MOCODB, intractable B by type deficiency cofactor Molybdenum Abstract 2020 26, May 4 3 2 1 Kusmierska Jezela-Stanek gene Aleksandra MOCS2 the in (c.472_477del) variant Novel nttt fMte n Child and Technology Mother of University of Silesian Institute No2 Centre, Hospital Biotechnology Provincial Group, Clinical Tunneling Queen the Institute Jadwiga Diseases Saint Lung and Tuberculosis National 630) ovrino rcro omlbotrn(P)b P ytae(OS) attach- (MOCS2), synthase MPT by (MPT) molybdopterin to Z precursor of conversion (603707), 4 n oat Sykut-Cegielska Jolanta and , 1 iodBlaz Witold , 2 ru Gora Artur , 4 1 MOCS2 ee oaie ncrmsm q12 Its 5q11.2. chromosome on localized gene, 3 agraaBochenska Malgorzata , 2 Katarzyna , MOCS2 gene Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. oMr ee.Proial etes rig n yeepei eeosre,a ela plpi seizures, epileptic as reflexes, well grasping as weak bilaterally, observed, head sign were pasty, Babinski hyperekplexia pale, no and sides, was crying, both restless, skin on Periodically the the reflex examination, reflex. did Physically, neurological sole Moro the and no tube. no In contact negative, appeared. nasogastric was eye hyperreflexia a test tendon no impo- observed; traction with with were an spasticity fed but clearly limb presented lower eyes, was and still 97 his He above but opened eyes. still reactive, was boy his more circumference the with became times, gradually follow At child not activity. the motor muscle life, spontaneous hospitalization, of verished the appeared. month contractures During second joints effect. the knee In good and initially elbow with and levetiracetam increased, treatment uncontrolled, gradually the still extremities. tone were to upper seizures in extremities introduced Since especially the areflexia. additionally tone, by of was muscle accompanied movements was increased spontaneous tension and Abnormal muscular the pedaling, Reduced flaccid. waving, Within nonrhythmic and observed. as inactive still observed, were epileptic became in were desaturation, polymorphic child drops implementation, with the with phenobarbital sometimes days, the Convulsions disorders, following Despite period. breathing life. myoclonus, perinatal of brea- limb day complicated and seizures, first seizures, further in the epileptic crying on deterioration disorders, congenital occurred abnormal birth, tension of saturation an muscular the diagnosis with sucking), pectus subsequent after of difficulties with feet, minutes (lack observed thing large 30 feeding were desaturation in At fingers, in Difficulties nar- region. overlapping drops pneumonia. bitemporal lumbosacral and grunting cheeks, and condition, long general eyelids puffy auricles), the the with dysplastic of features philtrum, swelling coarse long excavatum, (as eyes, dysmorphism deep-set child’s facial The rowing, sister. negative. revealed 2-yr-old were healthy, examination diseases a Physical metabolic has for proband and history the neurological family was unrelated; and The mother are noted. childhood, the cord parents were early pregnancy, umbilical hiccups During in in fetal condition. deaths acidosis levothyroxine; child’s metabolic miscarriages, with the mild treated stabilize hypothyroidism, and circumference to with 65% head used diagnosed of 3960g, was saturation weight ventilation noted; birth Mechanical were with blood. tone pregnancy muscle polyhydramnios, of increased to and week (due skin, 38th section Caesarean in 97 by CTG) (> born in 37cm birth, anomalies second rate pregnancy, heart second from fetal born was boy The course Clinical webserver and PredictSNP aligned the history were by and Proband’s examined structures 158 were Protein at subunit substitutions catalytic synthase of 2017). and 2014). molybdopterin effects (Bendl, (Rose, 2016) the potential Bank in The (Kopec, Schr¨odinger positions LLC]. Data [PyMol 5MPO, 159 software Protein ID: PyMol the using (PDB from analyzed complex downloaded synthase were molybdopterin 2008) human from synthase of variantmolybdopterin structure the crystal of The sequencing Sanger patient. analysis 2019). the structures annotation, The for (Seo, Crystal calling, performed Variant described 2009). was 10-fold. previously February sequencing than exome in as higher by coverage accessed performed identified 99.2% GRCh37; were included Human with assembly prioritization analyses Twist 100-fold, data genome was and a sequencing coverage (NCBI using genome of sequence Raw depth captured sequenced USA). reference mean were were CA, regions the 22,000) Diego, genomic to San captured (approximately sequencing alignment (Illumina, The patient’s genes exome USA). platform the NovaSeq CA, human from Whole Francisco, a isolated all San using DNA Bioscience, of parents. (Twist genomic Kit exons patient’s using Exome Korea), All the Core South from (Seoul, blood. Inc obtained 3billion, whole was in performed testing was genetic (WES) for consent Informed analysis Molecular th ecnie.I h pa cl,h eevd771/0pit.Irglrbetig aegrey pale breathing, Irregular points. 7/7/10/10 received he scale, Apgar the In percentile). tpyoocsaureus Staphylococcus th ecnie n rmnn rna uue eentd h xa hypotonia axial The noted. were sutures cranial prominent and percentile, opee ihpeusrZ(D D QE (Daniels, 2QIE, ID: (PDB Z precursor with complexed 2 Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. al .Gntcrsl forptet oecaueo h ain olwn h udlnso h Human the of protein guidelines a the as NP_004522.1 following and variant sequence the cDNA of reference a Nomenclature as sequence. patient. NM_004531.4 molybdopterin using our of Society of Variation subunit Genome result catalytic the Genetic of 2014). 2. 159 (Brendl, Table and server 158 PredictSNP the position by at in obtained mutations variant complex of a synthase effect predicted of the identification of the results to led WES result Genetic 1 Figure Proband. our of assessment imagine and laboratory of results Abnormal 1. Table 1. Table was in child summarized are the These condition, clinical assessment. worsening imagine and and Laboratory severe introdu- substitution hospice. of later requiring home was Because Hypothyroidism a treatment. acid diagnosed. to the valproic transferred also in levetiracetam, was included and levothyroxine was propranolol phenobarbital with tachycardia, to to addition Due in ced. Thus, myoclonic. mainly te lvtdlvl ftooi n NT-proBNP, and troponin of levels elevated non-restrictive cardiomyopathy, hypertrophic tendency a with changes localized - period neonatal other ECHO fluid EEG cerebellar dilated and hypoplasia cerebellar structure brain vague - birth after day 3rd Result 1) Figure positive birth, after day (5th MR brain detectable not umol/L <1.00 US brain dipstick Sulfite homocysteine/serum acid/serum uric Test MOSC2 3 hypothyroidism burst-suppression pattern continuous localized almost left-sided life– or of right- month 2nd generalize to 0.36) - index lateral (Evans the ventricle within widened (after symmetrically ventricular lobe system supratentorial frontal the left the bleeding?) of the grooves within the FLAIR of and one enhanced images of T1-dependent band in discrete signal a the region in temporal spaces fluid left cerebral enlarged slightly spaces hydrocephalus low-pressure brain and disseminated necrosis extensive - the regions and subcortical hemispheres the within disseminated porencephaly extensively life– 2nd of bilaterally month around pallidus areas globus hyperechogenic and and tegmentum side right prevalence the with on frontal symmetrically the cortex within parietal of lesions and week malacic 4th extensive sinus – sagittal life the in slightly flows index) accelerated both Evans of (normal corners ventricles occipital lateral the of widening marginal ee-c4247e Tbe2 up.1.The 1). Suppl. 2, (Table c.472_477del - gene Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. upr hssaeet h e18 y19aelclzda h n ftels ei ftectltcsubunit, catalytic to the of complex helix synthase last the molybdopterin of human end the the at of localized structure are Lys159 crystal Leu158, the The of statement. this analysis support an performed homozygous causative. have the as We account suggest into clinical we function. taking child’s Additionally, variant, protein The this disease. College disrupt guideline. the of This American with (AMP) and state the (GenomAD). consistent Pathology of proteins highly Molecular cohorts recommendation however, for of were, the population Association length features to and large according (ACMG) the the Genetics significance change Medical uncertain in can of as frequency region classified low is non-repeat sequence. variant extremely protein-coding the This an the in unknown in with deletion of frameshift reported inframe variant change been not Sulfite a has does revealed positive clinical which It It and 52396264, initial homocysteine, chromosome5 analysis. This from sequencing serum ’CTTTTA’ whole-exome low the feature. in very in phenotypic verified acid, significance suggestive finally uric was most serum results, the undetectable dipstick were by images supported difficulties, brain diagnosis, feeding to epilepsy, and us refractory prompted neonatal-onset, dysmorphism, That while cofactor, patients descriptions. facial molybdenum previous 35 of the deficiency with encompassed the a consistent toward in are group test features identified presented clinical variants and The pathogenic history proband’s different (2019). Our 29 al. and etiology et molecular Arican proven by to with encephalopathy. due reviewed rapidly deficiency hypoxic-ischemic extensively by cofactor recently as molybdenum characterized of been misdiagnosed disease, characteristics molecular sometimes recessive and damage, autosomal clinical The neurological ultra-rare severe an and is progressive deficiency cofactor molybdenum The Discussion participate 2B). they (Figure the 2 binding of C-terminus, Figure of Z comparison the and end precursor the with the human Moreover, for together essential at 2A). from and is (Figure located synthases residue binding subunit, are molybdopterin subunit the residues and carrier of of Lys159 Leu158 sulfur structures C-terminus and synthase The Leu158 the molybdopterin The to molyb- the subunit. of close in subunit. catalytic consists helix, catalytic synthase heterodimer last the molybdopterin Each the in and synthase heterodimers. occur molybdopterin subunit two deletion Human of carrier Lys159 deletion. constituted sulfur native the dimer the synthase of a of consequences dopterin from as the analysis structure crystallized on the the was light however, with shed complex deletion; Z comparison described precursor and carrying with synthase protein complexed molybdopterin the the of of structure significance conformation crystal unknown no of variant is VUS There database; gnomAD the in analysis genomes Structural on based is frequency Allele * ies oydnmcfco ecec (autosomal B deficiency cofactor Molybdenum VUS homozygous - deletion inframe p.(Leu158_Lys159del) NP_004522.1: Chr5(GRCh38):g.53100435_53100440del c.472_477del NM_004531.3: nomenclature Variant Disease Classification frequency* Allele Zygosity type Variant Level: Protein Level: gDNA Level: cDNA nomenclature Variant Gene MOSC2 ee h ooyosvratc4247e on c.472_477del variant homozygous The gene. 4 recessive) MOCS2 tpyoocsaureus Staphylococcus MOCS2 ugss htteLys159 the that suggests, h MOCS2 the MOCS2 tpyoocsaureus Staphylococcus eee nucleotides deletes gene. ain have variant Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. ail,J . ubes .M,Rjgpln .V,&Shnei,H 20) rsa tutr fa in of role structure its Crystal and mechanism (2008). transfer H. sulfur Schindelin, its & into V., insight muta- K. deficiency. disease-related Rajagopalan, cofactor complex: molybdenum of M., Z prediction M. synthase-precursor Wuebbens, for molybdopterin Damborsky, N., classifier & J. consensus J., Brezovsky, Daniels, accurate J., Zendulka, and D., robust E. Wieben, PredictSNP: MOCS2 tions. A., to Pavelka, N. O., Due (2014). Dundar, Salanda, Deficiency J. Olgac J., Cofactor & Stourac, Molybdenum O., J., Bendl, Oztekin, of A., Characteristics Ersen, Molecular S., and Yilmaz, Mutations. Clinical Bozkaya The O., Kirbiyik, (2019). P., Gencpinar, P., Arican, authors. References the from request on enzymatic available Children’s Data presented the on Genetics, suggestion Medical Statement and Availability of comments Data Department valuable the her from for data. Ciara Warsaw, El˙zbieta molecular in and Dr. Institute thanks Health to Memorial like is authors formation The heterodimer the chemical challenging a if more but much Acknowledgment that be limited, described would perhaps binding speculate, The subunit be carrier can could in reverse. changes We binding poor. the to Z whereas is precursor precursor, shows distorted. the prognosis abnormal analysis of by structural be modification the caused preliminary can when the deficiency and the that or subunit, preserved, picture processes catalytic the metabolic clinical potential of in cavity unclear two (NGS), c.472_- binding sequencing with - the variant next-generation modifies cases inframe diagnosis, mutation novel, genetic in the rapid of especially for pathogenicity need of diseases, evidence the structural emphasize and We clinical precursor 477del. give we the paper, disturbed, this In mutation mostly considered. any is be that functionality to indicates has which clearly study verify, Conclusions another report further to the binding, suggest However, but subunit may carrier possible, consequences. or which is has binding analysis formation, the area deletion complex this in no significant binding have in active 2), variant in could changes the c.472_477del (Suppl. deletion in shown described of and have the result pathogenicity heterodimer Thus, can of of one. and the catalytic C-terminus deletion mechanism both, the the the to of for that subunit folding probable carrier consequences the highly sulfur the on is of consequences It affinity lysine. have second C-terminus. will the the it of at Moreover, position located can the situation is binding modify the residue Z can case, crucial precursor residues this in two the this in (123 However, that and residue expect, modified. lysine binding, already second could Z The be one can complicated. lysine, (Subramanian, affinity more crucial protein-ligand is specificity the the enzyme-substrate replaces however, the the one of occur, the function modifies second still of the and replacement the repossess (similar mutation Tyr314 Since the preserved where Luckily, Tyr55Ala oxidase, 2014). be the Lys161. acid can D-amino by by role in Lys159 eliminated it’s example and thus Tyr55, for Ala160, observed and was by Lys161, acid human replaced by amino be replaced of key will be structure Leu158 should crystal the Lys159 mutation, deleted the the of of functionality. alignment consequence dual In The ( has of Z. synthase of Leu116 precursor molybdopterin one to the the corresponds the of and Lys159 region with subunit This synthase carrier C-terminus. sulfur molybdopterin the the of binds aa It 13 the before just LScmuainlbiology computational PLoS eiti neurology Pediatric , 99 Biochemistry .aureus S. , 10 55.https://doi.org/10.1016/j.pediatrneurol.2019.04.021 55–59. , 1,e034.https://doi.org/10.1371/journal.pcbi.1003440 e1003440. (1), tpyoocsaureus Staphylococcus sesnilrsdefrpoe idn ftepeusrZ. precursor the of binding proper for residue essential is ) , 47 2,6566 https://doi.org/10.1021/bi701734g 615–626. (2), 5 .aureus S. MOCS2 n 6 nhmn sesnilfrprecursor for essential is human) in 166 and opee ihpeusrZrflcsthat reflects Z precursor with complexed ee srslsfo predictSNP from results As gene. Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. variant-c-472_477del-in-the-mocs2-gene solvent legend.docx facilitated Figure’s through specificity substrate file (DAAO) Hosted oxidase acid Schaap, D-amino & Modulating V., access. Santos, genetic Dos utility (2018). suspected Martins clinical J. 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J., & type deficiency Reiss, N., cofactor genetics Cohen, molybdenum human in R., mutations of R. Mechanistic and journal structure Mendel, ican genomic B., (2003). gene: Stallmeyer, synthase R. C., B dopterin R. Dorche, group J., Mendel, in Reiss, identified & mutants V., of K. characterization deficiency. and of Rajagopalan, https://doi.org/10.1074/jbc.M303092200 cofactor reaction structure molybdenum A., synthase Crystal of J. molybdopterin patients (2016). Araujo, human W.W. of A., Yue, studies & Freuer, C., S., Bountra, Burgess-Brown,Leimkuhler, A., 10.2210/pdb5mpo/pdb E., DOI: Edwards, Williams, complex C., A.E., synthase Arrowsmith, Oberholzer, molybdopterin F., human C., Strain-Damerell, Delft, (1980). F., von K. Fitzpatrick, N., S. H., Wadman, Bailey, & J., Kopec, A., dehydroge- F. xanthine cofactor. America and Beemer, molybdenum oxidase of M., the sulfite States lacking Duran, of United patient deficiencies V., a combined K. in metabolism: nase Rajagopalan, molybdenum R., of W. errors Waud, Inborn L., J. Johnson, lSone PloS , 13 6,e189.https://doi.org/10.1371/journal.pone.0198990 e0198990. (6), vial at available , 77 6,31–79 https://doi.org/10.1073/pnas.77.6.3715 3715–3719. (6), , 64 3,7671 https://doi.org/10.1086/302296 706–711. (3), https://authorea.com/users/325910/articles/453893-novel- h ora fbooia chemistry biological of Journal The rceig fteNtoa cdm fSine fthe of Sciences of Academy National the of Proceedings 6 uli cd research acids Nucleic , 278 2) 26127–26134. (28), , 45 D) D271– (D1), Amer- Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. 7 Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. 8 Posted on Authorea 26 May 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159050459.99592045 | This a preprint and has not been peer reviewed. Data may be preliminary. 9