Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy

Vol.:(0123456789) 1 3 Jou nal o Neu ology (2019) 266:353–360 h ps://doi.o g/10.1007/s00415-018-9137-8 ORIGINAL COMMUNICATION Recessi e PYROXD1 mu a ions cause adul -onse limb-gi dle- ype muscula dys ophy Ma kusT.Sainio1· SallaVälipakka2· B unoRinaldi3· HelenaLapa o1· Ande sPae au4· SimoOjanen1· Vi giniaB ilhan e1· ManuJokela5,6· SannaHuo inen7· Ma iAu anen8· JohannaPalmio6· Syl ieF ian 3· EmilYlikallio1,8· Bja neUdd6,9· HennaTyynismaa1,10 Recei ed: 23 Augus 2018 / Re ised: 9 No embe 2018 / Accep ed: 21 No embe 2018 / Published online: 4 Decembe 2018 © The Au ho (s) 2018 Abs ac Objec i e To desc ibe adul -onse limb-gi dle- ype muscula dys ophy caused by biallelic a ian s in he PYROXD1 gene, which has been ecen ly linked o ea ly-onse congeni al myo ib illa myopa hy. Me hods Whole exome sequencing was pe o med o adul -onse neu omuscula disease pa ien s wi h no molecula diag- nosis. Pa ien s wi h PYROXD1 a ian s unde wen clinical cha ac e iza ion, lowe limb muscle MRI, muscle biopsy and spi ome y. A yeas complemen a ion assay was used o de e mine he biochemical consequences o he gene ic a ian s. Resul s We iden i ied ou pa ien s wi h biallelic PYROXD1 a ian s. Th ee pa ien s, who had symp om onse in hei 20s o 30s, we e homozygous o he p e iously desc ibed p.Asn155Se . The ou h pa ien , wi h symp om onse a age 49, was compound he e ozygous o p.Asn155Se a ian and p e iously unknown p.Ty 354Cys. All pa ien s p esen ed wi h a LGMD- ype pheno ype o symme ic muscle weakness and was ing. Symp oms s a ed in p oximal muscles o he lowe limbs, and p og essed slowly o in ol e also uppe limbs in a p oximal-p edominan ashion. All pa ien s emained ambulan pas he age o 60. They had es ic i e lung disease bu no ca diac impai men . Muscle MRI showed s ong in ol emen o an e ola e al high muscles. Muscle biopsy displayed ch onic myopa hic changes. Yeas complemen a ion assay demons a ed he p.Ty 354Cys mu a ion o impai PYROXD1 oxido educ ase abili y. Conclusion PYROXD1 a ian s can cause an adul -onse slowly p og essi e LGMD- ype pheno ype. Keywo ds Limb-gi dle muscula dys ophy· PYROXD1· Myopa hy· Exome sequencing Elec onic supplemen a y ma e ial The online e sion o his a icle (h ps ://doi.o g/10.1007/s0041 5-018-9137-8) con ains supplemen a y ma e ial, which is a ailable o au ho ized use s. *Henna Tyynismaa [email p o ec ed] 1Resea ch P og ams Uni , Molecula Neu ology, Uni e si y o Helsinki, Helsinki, Finland 2Folkhälsan Ins i u e o Gene ics, Medicum, Uni e si y o Helsinki, Helsinki, Finland 3Depa men o Molecula andCellula Gene ics, CNRS, GMGM-UMR7156, Uni e si é de S asbou g, S asbou g, F ance 4Depa men o Pa hology, HUSLAB andUni e si y o Helsinki, Helsinki, Finland 5Di ision o Clinical Neu osciences, Tu ku Uni e si y Hospi al, Uni e si y o Tu ku, Tu ku, Finland 6 Depa men o Neu ology, Neu omuscula Resea ch Cen e , Uni e si y Hospi al andUni e si y o Tampe e, Tampe e, Finland 7 Depa men o Pa hology, Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Tampe e, Finland 8 Clinical Neu osciences, Neu ology, Uni e si y o Helsinki andHelsinki Uni e si y Hospi al, Helsinki, Finland 9 Neu ology Depa men , Vasa Cen al Hospi al, Vaasa, Finland 10 Depa men o Clinical andMedical Gene ics, Uni e si y o Helsinki, Helsinki, Finland 354 Jou nal o Neu ology (2019) 266:353–360 1 3 In oduc ion Inhe i ed diso de s o he skele al muscles a e classi ied based on clinical, his opa hological and gene ic ea u es. Limb-gi dle muscula dys ophy (LGMD) is a gene ically he e ogeneous g oup o muscula dys ophies wi h au o- somal inhe i ance cha ac e ized by slowly p og essi e degene a ion o p oximal limb muscles. In many ypes o LGMD also o he muscles, e.g., dis al muscles, espi a o y muscle and he hea may be a ec ed. Inhe i ance can be ei he dominan (LGMD1) o ecessi e (LGMD2), and o da e a leas 34 LGMD disease genes a e known [1–3]. Dis ibu ion o muscle in ol emen , age a onse and a e o p og ession show g ea a iabili y, which a e unde - sco ed by he ex ensi e gene ic di e si y. Biallelic PYROXD1 gene mu a ions we e iden i ied o unde lie congeni al myopa hy in nine pa ien s om i e di e en amilies [4]. These pa ien s had he onse o mus- cle weakness be ween bi h and 8yea s wi h slow dis- ease p og ession, and muscle pa hology consis en wi h myo ib illa myopa hy. All pa ien s we e s ill ambulan a he ime o s udy, he oldes pa ien being 31yea s o age. PYROXD1 encodes a nuclea -cy oplasmic oxido e- duc ase, wi h a cu en ly unknown exac unc ion in cel- lula edox egula ion [4]. Pa ien s om ou amilies had a mu a ion causing p.Asn155Se amino acid change, which was shown o impai educ ase ac i i y in a complemen- a ion assay o yeas lacking glu a hione educ ase [4]. Ano he pa ien wi h homozygous p.Asn155Se a ian was ecen ly epo ed, p esen ing wi h p og essi e muscle weakness s a ing a he age o 9yea s and leading o loss o ambula ion a he age o 37yea s [5]. He e we desc ibe addi ional ou pa ien s om h ee amilies wi h ecessi e PYROXD1 a ian s. In con as o he p e ious epo , ou pa ien s had he disease onse in adul hood and ha e now eached 60yea s o age, allowing e alua ion o he na u al his o y o PYROXD1 associa ed disease. Ma e ials andme hods Pa ien s The pa ien s in his s udy a e om h ee amilies o Finn- ish o igin wi h non-consanguineous pa en s (Fig.1). Pa ien s; pa ien 1 (P1, Family 1), pa ien 2 (P2, Family 2), and siblings pa ien 3 and 4 (P3 and P4, Family 3), we e s udied in coho s o undiagnosed neu omuscula disease pa ien s. In addi ion o de ailed clinical neu ological exam- ina ions, all pa ien s unde wen elec oneu omyog aphy (ENMG) in es iga ions, muscle biopsy, lowe limb muscle MRI, spi ome y/ espi a o y assessmen and measu emen o se um c ea ine kinase (CK) alues (Table1). The MR images o pa ien P4 had been pe o med a long ime ago a age 45yea s and he e o e only he w i en adiology epo was a ailable o e iew. Whole-body MRI had been pe o med in wo pa ien s (P1 and P2). Muscle biopsies we e his ochemically s ained wi h hae- ma oxylin & eosin (H&E), Gomö i ich ome, nico inamide adenine dinucleo ide e azolium educ ase (NADH-TR) and combined cy och ome oxidase (COX)/succina e dehyd o- genase (SDH). The ollowing immunohis ochemical s ain- ings we e pe o med o P1 and P3: MyHC double s aining [Myosin Hea y Chain, Slow, Myosin Hea y Chain, A4.74 ( as )], p62, myo ilin and desmin. Fu he mo e, biopsies om P1 and P2 we e s ained o sa colemmal p o eins dys- ophin 1–3, dys e lin, sa coglycan-alpha, dys oglycan- alpha and ca eolin-3 in addi ion o me osin and eme in. Fo P2, only a chi al his ological and his ochemical s ainings we e a ailable, and hus MyHC, p62, myo ilin o desmin immunohis ochemical s ainings we e no possible. All subjec s in his s udy ha e p o ided w i en consen o he use o clinical da a and ma e ial. The s udy was app o ed by Helsinki Uni e si y Hospi al and Tampe e Uni e si y Hospi al e hics boa ds. DNA sequencing Fo P1 and P2, whole exome sequencing (WES) was pe - o med a he Finnish Ins i u e o Molecula Medicine (FIMM). B ie ly, 150ng o gDNA was agmen ed wi h a Co a is E220 e olu ion ins umen (Co a is). Sample lib a - ies we e p ocessed acco ding o SeqCapEZ Lib a y SR (Roche Nimblegen) manual. NimbleGen cap u e was pe - o med acco ding o NimbleGen SeqCap EZ Exome Lib a y SR Use ’s Guide. Sequencing was pe o med wi h Illumina HiSeq2500 sys em in Rapid mode using HiSeq Rapid 2 ki s (Illumina). Reads we e hen aligned o he GRCh37 e e - ence genome wi h he BWA (0.6.2), and he mpileup om he SAMTOOLS (1.4) package was used o a ian calling. Fig. 1 Family pedig ees and he iden i ied PYROXD1 a ian s 355Jou nal o Neu ology (2019) 266:353–360 1 3 Table 1 Clinical ea u es o he pa ien s FVC o ced i al capaci y, MIP maximum inspi a o y p essu e, MEP maximum expi a o y p essu e, PCF peak cough low a Age a dea h due o espi a o y insu iciency and pneumonia Family 1 2 3 3 Pa ien P1 P2 P3 P4 Gende , age Male, 65yea s Male, 65yea s Male, 70yea s Female, 70yea sa E hnici y, consanguini y Finnish, no Finnish, no Finnish, no Finnish, no PYROXD1 a ian s c.464A > G (p.Asn155Se , ch 12: g.21605064A > G), c.1061A > G (p. Ty 354Cys, ch 12:g. 12: 21615741A > G) Hom, c.464A > G (p.Asn155Se , ch 12: g.21605064A > G) Hom, c.464A > G (p.Asn155Se , ch 12: g.21605064A > G) Hom, c.464A > G (p.Asn155Se , ch 12: g.21605064A > G) Onse /p og ession 49yea s, slowly p og essi e 10 yea s, slowly p og essi e 30yea s, slowly p og essi e 33yea s, slowly p og essi e Age on las comp ehensi e examina- ion, se e i y 63yea s, ambulan wi hou aids, help om bo h hands when ising om chai 64yea s, ambulan wi h wo s icks, kypho ic pos u e and a ophic uppe back muscles 70yea s, ambulan wi h 1–2 s icks o 200m, se e e p oximal uppe and lowe limb weakness 70yea s, wheelchai bound (66yea s) Res ic i e lung disease Yes, FVC 54%(63 yea s) Yes, episodic dyspnea and FVC 40% (64yea s) Yes, FVC 67% wi h se e ely educed MIP, MEP and PCF al- ues (70yea s) Yes, FVC 42% (59yea s) and 30% (68yea s) Dis al and p oximal uppe limb s eng hs P ox. 4/5 Dis . 5/5 P ox. 2/5 Dis . 4/5 P ox. 3/5, 2/5 Dis . 4/5 P ox. 4/5, 2/5 Dis . 3.5/5 Hip lexion s eng h 3/5 2/5 1/5 2/5 Knee lexion/ex ension Flex. 4/5 Ex . 4/5 Flex. 3/5 Ex . 2/5 Flex. 3/5 Ex . 2/5 Ex . 2/5 Dis al lowe limb s eng hs Ankle plan a and do sal lexion 4/5 (64yea s) Ankle do sal lexion 3/5, plan a lexion 5/5 (65yea s) Ankle plan a and do sal lexion 3/5 (70yea s) Ankle plan a and do sal lexion 5/5 (45yea s) Spinal and uncal muscles Mode a e olume loss A ophic uppe back muscles Neck lexo , spinal and abdominal muscle weakness Neck lexo and abdominal muscle weakness EMG P oximal and dis al muscle abno mal mo o uni po en ials Myopa hic changes Myopa hic changes in p oximal and pa aspinal muscles Myopa hic changes in p oximal muscles Neu og aphy No mal n.a No mal No mal MRI Symme ic a ophy and a y eplace- men in all lowe limb muscles, mild p oximal a ophy in uppe limbs and signi ican a ophy in muscles o he pec o al gi dle Symme ic a ophy and a eplace- men in all muscle compa men s. In he lowe limbs, a ophy was ound a he uni o mly, whe eas in he uppe limbs i was ela i ely mo e ad anced in do sal compa ed o en al muscles (61yea s) Wide sp ead a y eplacemen in glu eal, high and lowe leg mus- cles (65yea s) Mos se e e a y eplacemen in semi endinosus, sa o ius, g acilis. and gas ocnemius medialis muscles (45yea s) Biopsy Dys ophic Dys ophic Dys ophic Dys ophic CK No mal 288–340 U/l No mal No mal Acylca ni ine p o ile No mal n.a n.a n.a Ca diac ul asound No mal No mal (61yea s) No mal (66yea s) n.a 356 Jou nal o Neu ology (2019) 266:353–360 1 3 Fo amily 3 (P3, P4 and hei una ec ed b o he ), WES was pe o med essen ially by he same me hod, bu lib a y p epa a ion was done using KAPA Hype lib a y p epa a ion Ki (Kapa Biosys ems, Wilming on, Ma, USA) and SeqCap EZ MedExome assay (Roche Nimblegen) was used o a ge en ichmen . Sange sequencing was pe o med wi h p ime s spe- ci ic o PYROXD1 exon 5 (CAG TGG GAA AGT GAG ATT CATTT and ATT ACG GAT TCC ACA AGA GCT) and exon 10 (CCA TGG AAA TTC AGC TCA GGT and AAC AAC TGT GCT AGC TTC CT). Plasmids, s ains, media, andme hods o yeas cells The human PYROXD1 and PYROXD1-p.Ty 354Cys cDNAs we e cloned by he Ga eway® (In i ogen) me hod in o pDONR221 en y ec o and hen ecombined in o yeas des ina ion ec o s (Addgene;[6]) o ob ain pAG415- p omGPD-PYROXD1 (pSF371) and pAG415-p omGPD- PYROXD1-p.Ty 354Cys (pSF501) plasmids. The pAG415 is a low-copy numbe CEN plasmid bea ing he cons i u- i e GPD (glyce aldehyde-3-phospha e dehyd ogenase) p o- mo e and he LEU2 auxo ophic ma ke o selec ion o he ans o man s on SC-Leu medium. Plasmid sequences we e e i ied (GATC Bio ech). The Saccha omyces ce e isiae wild- ype BY4742 (MATα leu2Δ0 u a3Δ0 his3Δ0 lys2Δ0) e e ence s ain and he gl 1∆ (MATα leu2Δ0 u a3Δ0 his3Δ0 lys2Δ0 gl 1::KanMX) mu an s ain we e used. Yeas cells we e ans o med using he modi ied li hium ace a e me hod [7]. The indica ed yeas s ains we e g own a 30°C o mid-exponen ial g ow h phase in syn he ic comple e (SC) medium SC-Leu: 0.67% yeas ni ogen base (YNB) wi hou amino acids, 2% glucose and he app op ia e—Leu d op- ou mix, o main ain he plasmid. These p ecul u es we e used o inocula e he ich medium YPD: 1% yeas ex ac , 2% pep one, 2% glucose, o he oxida i e s ess medium YPD + H2O2: 1% yeas ex ac , 2% pep one, 2% glucose, 3mM H2O2, and he g ow h o he yeas cells was analysed a 30°C unde agi a ion in liquid medium by measu ing he OD a 600nm o e ime, wi h measu emen s e e y 10min o e 17h. Fo wes e n blo analysis, o al yeas p o ein ex ac s we e p epa ed by NaOH lysis o 1.5 OD600nm uni o yeas cells, ollowed by ichlo oace ic acid (TCA) p ecipi a ion and he pelle was esuspended in 50µl o 2X Laemmli bu e plus T is Base. Samples we e incuba ed 5min a 37°C p io wes e n-blo analysis by 8% SDS-PAGE, ollowed by ans- e on a ni ocellulose blo ing memb ane (Ame sham™ P o an™ 0.45µm NC) and immunoblo ing wi h abbi polyclonal an i-PYROXD1 (1/500, R3500) an ibodies [4] using s anda d p ocedu es. Images we e acqui ed wi h he ChemiDoc Touch Imaging Sys em (Bio-Rad). Resul s Clinical ea u es o P1 Pa ien 1 (Family 1) is a male who had been p e iously diagnosed wi h Méniè e’s disease, bu had been o he - wise heal hy. He had slowly p og essi e p oximal muscle weakness combined wi h back pain s a ing a he age o 49yea s (Table1). Be o e his his s eng hs had been no - mal, and he epo ed no p oblems wi h doing spo s as a child o young adul . A lumba disk he nia ion had been ope a ed a he age o 51yea s. A he age o 60yea s, he was e e ed o neu ologic consul a ion because o an inciden al MRI inding o a ophy and a eplace- men symme ically in he pa a e eb al lowe back mus- cles. The amily his o y was nega i e o neu omuscula disease. On clinical examina ion a he age o 63yea s, he was ambulan wi hou aids, bu needed o use bo h hands when ising om a chai o climbing s ai s. Mode a e olume loss was no ed in he uppe pa aspinal muscles, bu o h- e wise he e was no e iden a ophy. Dis ally uppe limb s eng hs we e no mal, p oximally sligh ly educed on bo h sides. In he lowe limbs, hip lexion and knee lex- ion/ex ension s eng hs we e dec eased on bo h sides. Dis- al lowe limb s eng hs we e wi hin no mal limi s. The e was no acial o bulba weakness. Elec omyog aphy (EMG) o bo h p oximal and dis al muscles in all limbs showed abno mal mo o uni po en- ials (MUPs), ei he small polyphasic o la ge and pa ially polyphasic. Ne e conduc ion s udies showed no mal con- duc ion eloci ies and senso y and mo o ampli udes. Plasma c ea ine kinase (CK), acylca ni ine p o ile and ca diac ul asound we e no mal. Spi ome y showed e i- dence o mode a e es ic i e lung disease. High esolu- ion CT o he ho ax showed nodula pa enchymal change consis en wi h pulmona y sa coidosis. He was placed on an inhaled glucoco icoid. Clinical ea u es o P2 In his la e eens be o e he age o 20yea s, pa ien 2 (Fam- ily 2) needed suppo om his hands o climb s ai s and he had ouble unning. Ne e heless, he played oo ball a he age o 35yea s, and s a ed equi ing walking aids only a he age o 54yea s. On examina ion a he age o 64yea s he walked wi h wo s icks, and sho dis ances wi hou aids. He had kypho ic pos u e and uppe back muscles we e no ed as a ophic. The e was sligh p osis and acial weakness bila e ally. Limb s eng h was dec eased p edominan ly p oximally 357Jou nal o Neu ology (2019) 266:353–360 1 3 in a symme ic ashion. A m ele a ion was less han 90° and elbow ex ension was se e ely dec eased. Dis al hand muscle s eng h was be e p ese ed. In he lowe limbs, hip lexion, knee ex ension and ankle do si lexion/plan- a lexion we e se e ely a ec ed, while knee lexion was mode a ely educed. EMG showed myopa hic changes. Plasma CK was sligh ly inc eased. Ca diac ul asound a age 61yea s was no mal. He had episodic dyspnea and educed espi a o y unc ion (age 64yea s), bu so a has no equi ed en ila- o suppo . The pa ien ’s b o he had also been diagnosed wi h mus- cle disease and died o pneumonia a he age o 43yea s. Bo h pa en s and wo olde sis e s we e heal hy. Clinical ea u es o pa ien s P3 andP4 The p oband P3 and his sis e P4 (Family 3) we e asymp- oma ic un il ea ly hi ies when hey no iced weakness in hei p oximal lowe limbs. They had di icul ies ising om squa o chai s. Few yea s la e muscle weakness was also epo ed in p oximal uppe limbs. The p og es- sion was e y slow. A he age o 70yea s he p oband was ambulan wi h s icks o 200m bu ising om low chai was impossible. Gai was waddling wi h hype ex- ended knees. He could ele a e his a ms less han 90°. His pos u e was no mal al hough spinal and abdominal muscles we e weak wi h mild neck lexo weakness. Also dis al lowe limb muscles we e weak (ankle plan a and do sal lexion g ade 3). Scapula winging o acial weak- ness was no obse ed. He had hoa seness in his oice bu no o he bulba symp oms. He had had wo s okes (le hemisphe e aged 61yea s and igh hemisphe e 66yea s), which migh con ibu e o he symp oms al hough acu e hemiplegic symp oms had esol ed. The e was se e e sym- me ical muscle a ophy in he an e io pa o he high, p oximal uppe limbs and shoulde gi dle. He unde wen echoca diog aphy wi h no mal esul s. Mild espi a o y insu iciency and weak cough s eng h was obse ed in pulmona y unc ion es s. The sis e , P4, s a ed o use olla o a he age o 65yea s and wheelchai aged 66yea s. She could no ex end he knees and hip lexion was weak as well as neck lexo s and abdominal muscles. She had asymme ical uppe limb weakness bo h p oximally and dis ally wi h le side mo e se e ely a ec ed. No bulba symp oms we e p esen . She had se e e p og essi e espi a o y insu i- ciency and she died om pneumonia aged 70yea s. The e we e no ca diac symp oms; echoca diog aphy was no pe - o med. Bo h siblings had no mal CK le els. EMG showed myopa hic changes especially in he p oximal uppe and lowe limbs. Muscle MRI andbiopsy Muscle MRI in all pa ien s (P1, P2, P3 and P4) showed an unusual pa e n o a y dys ophic changes wi h o al o nea - o al eplacemen o glu eus maximus, sa o ius, g a- cilis and quad iceps (Fig.2a–c). Simila o he o iginal epo on PYROXD1- ela ed myopa hy [4], ec us emo is was less a ec ed han he as i muscles. In he dis al lowe limbs, gas ocnemii we e he mos a ec ed muscles in pa ien s P2, Fig. 2 Muscle MRI and biopsies. MRI was pe o med o a indi- idual P1 a age 60, b indi idual P2 a age 59, and c indi idual P3 a age 65. Glu eus maximus, quad iceps, sa o ius and g acilis mus- cles we e mos se e ely a ec ed in all pa ien s, al hough he mos p oximal pa s o he ec us emo is muscles we e ela i ely spa ed o e en hype ophic. An e io high muscles we e always mo e se e ely a ec ed han he hams ings and adduc o compa men s. A e y peculia pa e n o a y degene a ion was obse ed in pa ien s P2 and P3, whe e only he ou e pa s o he an e io and la e al com- pa men muscles we e eplaced by a . In pa ien P1, he e was an unusual c escen -shaped a y-degene a ion in ol ing he inne pa s o as us la e alis and medialis muscles. Muscle biopsy ( as us la - e alis) om d indi idual P2 and e indi idual P1 showed a dys ophic pa e n, whe e indi idual P2 had an almos end-s age pa hology wi h se e e a ophy, ib osis and a y in il a ion. Scale ba s 100µm 358 Jou nal o Neu ology (2019) 266:353–360 1 3 P3 and P4, while all lowe leg muscles we e only mildly and di usely a ec ed in pa ien P1. In wo pa ien s (P2 and P3), a e y peculia pa e n o a y degene a ion was obse ed, whe e he ou e pa s o he an e io and la e al compa - men muscles we e eplaced by a , bu no he inne po ions (Fig.2b, c). No signi ican STIR edema was de ec ed in any muscle, which is consis en wi h a ela i ely inac i e and ch onic degene a i e p ocess. Muscles o he uppe gi dle and he o so displayed di use a y degene a i e changes o a mild-mode a e deg ee, while he muscles o he o ea m and hand we e minimally a ec ed o en i ely spa ed (no shown). Muscle biopsies om he as us la e alis o P2 (Fig.2d) and P1 (Fig.2e) showed ch onic myopa hic changes wi h a ophy, a in il a ion and s ong ibe size a iabili y, mul- iple in e nalized nuclei, and wi hou signi ican myo ib illa pa hology. Immunohis ochemis y o sa colemmal mem- b ane-associa ed p o eins, me osin and eme in was no mal. S ainings o p62, myo ilin and desmin in P1 showed no myo ib illa ea u es. The i s biopsies o he siblings, P3 and P4, we e pe - o med 30yea s ago and we e no a ailable o u he analy- sis. The mo phological changes we e epo ed o be dys- ophic wi hou speci ic ea u es. Pa ien 3 unde wen a new biopsy om ibialis an e io muscle a he age o 70. The muscle pa hology showed ex ensi e end-s age dys ophic changes. No ibe ype p edominance was no ed. Immuno- his ochemical s aining o myo ilin e ealed a ew posi i e cy oplasmic inclusions in sca e ed a ophic muscle ibe s (Supplemen a y Fig.1). Howe e , no desmin- o p62-posi- i e p o ein agg ega es we e ound. Gene ic indings The pa ien s’ pedig ees sugges ed ecessi ely inhe i ed disease (Fig.1). Thus, he exome sequencing da a we e il e ed o homozygous o compound he e ozygous a i- an s ha induce damaging changes o amino acid sequence, ha e popula ion equency o less han 0.001 in he o al popula ion and Finnish sub-popula ion in ExAC a ian da abase, CADD-sco es o 20 o g ea e , and a e p esen in less han 1% o an in-house da abase wi h 429 samples (P1 and P2) o less han 4% o a sepa a e in-house da a- base o 63 samples (P3 and P4). Fo P2, P3 and P4, he homozygous a ian c.464A > G p.Asn155Se in PYROXD1 (ENST00000240651.9) caugh ou a en ion because o i s ecen associa ion wi h myopa hy [4]. In he p e ious s udy, his a ian was iden i ied in ou ou o i e s udied amilies as homozygous ( wo amilies) o compound he e ozygous ( wo amilies), and i is p esen in a ian da abases as a low equency Eu opean a ian always in he e ozygous s a e. Acco ding o he GnomAD da abase, i s allele equency is sligh ly highe in he Finnish popula ion (1.186 × 10−4) han elsewhe e. The una ec ed b o he in he amily 3 was a he e ozygous ca ie o he p.Asn155Se a ian . Pa ien 1 was compound he e ozygous o he p.Asn155Se and a p e iously unknown a ian c.1061A > G p.Ty 354Cys. The la e a ian is ound in ou he e ozy- gous indi iduals in he GnomAD da abase wi h a equency o 1.444 × 10−5. The mu a ed y osine is loca ed in he N- e minal end o he py idine nucleo ide oxido educ ase domain o PYROXD1 (Fig.3a), and he amino acid is e o- lu iona ily conse ed (Fig.3b). Seg ega ion o he a ian s was in es iga ed in he amily o pa ien 1, and he a ec ed indi idual was he only one ca ying bo h mu a ions. The p.Asn155Se allele was inhe i ed ma e nally and he p.Ty 354Cys pa e nally. The PYROXD1‑p.Ty 354Cys isde ec i e inoxida i e s ess esis ance inyeas cells Humaniza ion o Saccha omyces ce e isiae yeas cells can be used o be e unde s and and/o o iden i y he cellula ole o a human p o ein [8, 9]. Indeed, he human PYROXD1 cDNA was shown o escue he oxida i e s ess de ec asso- cia ed wi h he yeas glu a hione oxido educ ase gl 1∆ dele- ion mu an s ain [4]. Fu he mo e, he p.Asn155Se a i- an was shown o ha e impai ed escue abili y in he yeas complemen a ion assay [4]. We es ed he e he e ec o he new p.Ty 354Cys a ian using he same yeas gl 1∆ s ain. The gl 1∆ mu an cells we e ans o med by emp y plasmid (pAG415) o by plasmids bea ing ei he PYROXD1 wild- ype cDNA, o he mu an allele p.Ty 354Cys. Exp ession o he human cDNAs was con olled by wes e n blo analysis wi h an i-PYROXD1 an ibody, showing ha he wild- ype and he mu an o m o PYROXD1 we e exp essed in wo di e en clones (cl1 and cl2) o yeas cells (Fig.3c). We obse ed ha he yeas glu a hione educ ase gl 1∆ mu an had a s ong g ow h de ec when g own in he p es- ence o hyd ogen pe oxide (Fig.3d). As shown be o e, he exp ession o he human PYROXD1 cDNA was able o com- plemen he g ow h de ec o he gl 1∆ mu an cells [4], indica ing ha PYROXD1 has an oxido educ ase ac i i y in i o in yeas cells. Compa ed o wild- ype PYROXD1, he p.Ty 354Cys a ian had a lowe g ow h a e in he p esence o H2O2, showing ha his new pa ien mu a ion impai s he oxido educ ase ac i i y o PYROXD1 in i o in yeas cells (Fig.3d). Discussion We epo he e new pa ien s wi h ecessi e PYROXD1 a i- an s unde lying myopa hy pheno ypes. In con as o he congeni al myopa hy cases desc ibed p e iously [4, 5], ou pa ien s had a conside ably la e disease onse and lacked 359Jou nal o Neu ology (2019) 266:353–360 1 3 signi ican myo ib illa pa hology. In ac , a ew myo ilin- posi i e inclusions we e only obse ed in he end-s age degene a ed muscle o one o ou pa ien s. The clinical pic- u e o ou pa ien s was consis en wi h adul -onse , slowly p og essi e LGMD ype disease. Reasons o he clea di - e ence in disease onse be ween he pa ien s o Tu kish [4] and Sudanese o A ab [5] ances y, and he Finnish pa ien s o his s udy, who we e homozygous o he p.Asn155Se a ian , a e specula i e a his poin bu may in ol e he pop- ula ion-speci ic gene ic backg ound o some en i onmen al ac o s ha a e ele an o he egula ion o PYROXD1 oxido educ ase ac i i y. Muscle MRI may help o dis inguish PYROXD1 myopa- hy om o he myopa hies, as ou pa ien s showed an unu- sual p e e ence o an e ola e al high muscles. O e all, he clinical cou se o he disease appea s ela i ely benign, wi h p ese a ion o ambula ion pas he age o 60. Respi a o y impai men is a conce n, howe e , as all ou pa ien s had de eloped espi a o y muscle weakness and he b o he o P2, who had been a ec ed by a e y simila kind o p o- g essi e muscle weakness, died om a espi a o y in ec- ion a age 43yea s. Also a 21-yea -old pa ien desc ibed by O’G ady e al. had es ic i e lung disease [4]. Regula moni o ing o pulmona y unc ion is he e o e manda ed in PYROXD1 myopa hy. Fo una ely, he e appea s o be no ca diac in ol emen . I is impo an o no e ha he aged indi iduals in his s udy did no ha e e idence o neu opa hy. While wo a ec ed indi iduals aged 26 and 29yea s desc ibed by O’G ady e al. had an axonal neu opa hy [4], ou inding sugges s ha neu opa hy is no a uni e sal consequence o PYROXD1 a ian s e en wi h ad ancing age. An impo an ea u e o no e is axial myopa hy, which was p ominen in P1, and also p esen in 8/9 o O’G ady’s pa ien s. I may be o in e es o look o PYROXD1 a ian s in indi iduals p esen ing wi h pu e axial myopa hy, he gene ics o which emains incomple ely s udied [10]. Th ee o ou ou pa ien s we e homozygous o he p e iously desc ibed p.Asn155Se , whe eas one was com- pound he e ozygous o he same a ian and he p e i- ously undesc ibed p.Ty 354Cys. Bo h o hese a ian s a e ound in di e en popula ions acco ding o exome and genome da abases, sugges ing ha addi ional pa ien s will p obably be iden i ied. The p.Asn155Se change appea s o be a common pa hogenic PYROXD1 a ian , as mos desc ibed pa ien s ca y i in one o bo h alleles. Howe e , Fig. 3 PYROXD1 a ian s iden i ied in his s udy and he unc ional cha ac e iza ion o p.Ty 354Cys in a yeas complemen a ion assay. a A schema ic o PYROXD1 wi h unc ional domains and he mis- sense a ian s o pa ien s o his s udy. b E olu iona y conse a ion o Ty 354. c Wes e n blo o gl 1Δ yeas cells ans o med wi h yeas exp ession ec o s emp y (pAG415) o bea ing he PYROXD1 wild- ype (WT) o p.Ty 354Cys (Y354C) unde he con ol o a cons i u- i e p omo e , wo di e en clones (cl1 and cl2) we e analyzed. The black a ows indica e he PYROXD1 p o ein and he loading con- ol. d The indica ed yeas cells we e g own a 30°C in ich medium (YPD) con aining H2O2 (3 mM) o induce an oxida i e s ess. The g ow h cu es o he di e en s ains we e de e mined by measu ing he cell g ow h (OD a 600nm) o e ime (h) 360 Jou nal o Neu ology (2019) 266:353–360 1 3 he associa ed myopa hy can ha e highly a iable ages o onse . Ou pa ien , P1, who was compound he e ozygous o he p.Ty 354Cys a ian oge he wi h p.Asn155Se had he highes age o onse o all pa ien s, sugges ing ha p.Ty 354Cys migh impai he unc ion o PYROXD1 less han he p.Asn155Se a ian . In conclusion, ou s udy shows ha PYROXD1 should be conside ed as a causa i e gene also in la e -onse unsol ed myopa hies, which esemble LGMD. Acknowledgemen s Open access unding p o ided by Uni e si y o Helsinki including Helsinki Uni e si y Cen al Hospi al. We hank he amilies in pa icipa ion on his s udy. Rii a Leh inen is acknowledged o echnical assis ance. We hank Se gio Buenes ado Se ano (E asmus s uden , Uni e si é de S asbou g), Tom Schelche and Syl ain Deba d o help wi h he yeas expe imen s, and Jocelyn Lapo e (IGBMC, Illki ch, F ance) o sha ing he abbi polyclonal an i-PYROXD1 an i- body. We acknowledge he exome cap u e and sequencing pe o med by he Ins i u e o Molecula Medicine Finland FIMM, Technology Cen e, and Uni e si y o Helsinki, and CSC—IT Cen e o Science, Finland, o compu a ional esou ces. Funding This s udy was unded by he Academy o Finland pHeal h p ojec ‘Neu ogenomics in ou ine diagnos ics o child en and adul s: impac on pa ien ca e and cos -e ec i eness’ o HT and MA, and by CNRS and Uni e si é de S asbou g o BR and SF. Compliance wi h e hical s anda ds Con lic s o in e es The au ho s decla e ha hey ha e no con lic o in e es . E hical S anda ds All subjec s in his s udy ha e p o ided w i en con- sen o he use o pa ien da a and ma e ial. The s udy was app o ed by Helsinki Uni e si y Hospi al and Tampe e Uni e si y Hospi al e hics boa ds. Open Access This a icle is dis ibu ed unde he e ms o he C ea- i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i eco mmons .o g/licen ses/by/4.0/), which pe mi s un es ic ed use, dis ibu- ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. Re e ences 1. Nig o V, Sa a ese M (2014) Gene ic basis o limb-gi dle muscula dys ophies: he 2014 upda e. Ac a Myol 33:1–12 2. Liewluck T, Milone M (2018) Un angling he complexi y o limb- gi dle muscula dys ophies. Muscle Ne e 58(2):167–177 3. Vissing J (2016) Limb gi dle muscula dys ophies: classi ica- ion, clinical spec um and eme ging he apies. Cu Opin Neu ol 29:635–641 4. 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