Pathogenicity in POLG syndromes : DNA polymerase gamma pathogenicity prediction server and database

Con en s lis s a ailable a ScienceDi ec BBA Clinical jou nal homepage: www.else ie .com/loca e/bbaclin Pa hogenici y in POLG synd omes: DNA polyme ase gamma pa hogenici y p edic ion se e and da abase Anssi Nu minen a , G ego y A. Fa num b , Lau ie S. Kaguni a,b,⁎ a Ins i u e o Biosciences and Medical Technology, Uni e si y o Tampe e, Tampe e, Finland b Depa men o Biochemis y and Molecula Biology and Cen e o Mi ochond ial Science and Medicine, Michigan S a e Uni e si y, Eas Lansing, MI, USA ARTICLE INFO Keywo ds: POLG synd ome DNA polyme ase gamma Mi ochond ial diso de Pa hogenici y p edic ion Pa ien da abase Mu a ion da abase ABSTRACT DNA polyme ase gamma (POLG) is he eplica i e polyme ase esponsible o main aining mi ochond ial DNA (m DNA). Diso de s ela ed o i s unc ionali y a e a majo cause o mi ochond ial disease. The clinical spec um o POLG synd omes includes Alpe s-Hu enloche synd ome (AHS), childhood myoce eb ohepa opa hy spec um (MCHS), myoclonic epilepsy myopa hy senso y a axia (MEMSA), he a axia neu opa hy spec um (ANS) and p og essi e ex e nal oph halmoplegia (PEO). We ha e collec ed all publicly a ailable POLG- ela ed pa ien da a and analyzed i using ou pa hogenic clus e ing model o p o ide a new esea ch and clinical ool in he o m o an online se e . The se e e alua es he pa hogenici y o bo h p e iously epo ed and no el mu a ions. The e a e cu en ly 176 unique poin mu a ions epo ed and ound in mi ochond ial pa ien s in he gene encoding he ca aly ic subuni o POLG, POLG. The mu a ions a e dis ibu ed nea ly uni o mly along he leng h o he p ima y amino acid sequence o he gene. Ou analysis shows ha mos o he mu a ions a e ecessi e, and ha he epo ed dominan mu a ions clus e wi hin he polyme ase ac i e si e in he e ia y s uc u e o he POLG enzyme. The POLG Pa hogenici y P edic ion Se e (h p://polg.bmb.msu.edu) is a ge ed a clinicians and scien is s s udying POLG diso de s, and aims o p o ide he mos cu en a ailable in o ma ion ega ding he pa hogenici y o POLG mu a ions. 1. In oduc ion DNA polyme ase gamma is he enzyme esponsible o eplica ing and main aining mi ochond ial DNA ( e iewed in [15,16]). The unc- ional, holoenzyme o m o POLG is a he e o ime consis ing o a ca aly ic subuni (POLGA) and a dime ic accesso y subuni (POLGB). POLGA ca ies h ee ca aly ic ac i i ies: 5′−3′DNA polyme ase, 3′−5′ exonuclease and 5′-deoxy ibose phospha e lyase. POLGB enhances DNA binding, ca alysis and holoenzyme p ocessi i y. Mu a ions in POLG a e associa ed wi h a wide ange o human diso de s ha exhibi sha ed and p og essi e pheno ypes ( e iewed in [46]). The POLG diso de s ange om p ena ally- a al condi ions and se e e in an ile onse diso de s, such as Alpe s disease (Alpe s-Hu enloche synd ome), o milde , la e-onse condi ions such as p og essi e ex e nal oph halmo- plegia, and a e desc ibed collec i ely as POLG synd omes. POLG mu a ions a e ela i ely a e, wi h an es ima ed ca ie equency o 1/100 indi iduals in he Wes e n wo ld [11]. Mos a e ecessi e, and symp oms ypically mani es only in compound he e o- zygous pa ien s. Al hough he symp oms and se e i y o he condi ions a y widely, all sha e a ew common ea u es: hey la gely affec issues wi h high ene gy demand, such as in he ne ous sys em, muscle and li e , and hey a e all p og essi e condi ions ha show di ec co ela- ion be ween age o onse and he se e i y o he condi ion [9]. To da e, 176 unique POLG missense mu a ions in mi ochond ial pa ien s ha e been epo ed in he li e a u e. Typically, POLG pa ien s ca y ei he homozygous o compound he e ozygous POLG mu a ions, hough some dominan pa hogenic mu a ions exis . We ha e p e iously mapped he mu a ions on o he e ia y s uc u e o POLGA in he apo- enzyme o m (PDB ID: 3IKM), and shown ha he pa hogenic mu a ions clus e in o fi e dis inc egions ha we ha e e med Alpe s clus e s 1–5 ([6,9],Fig. 1). Fu he mo e, we epo ed ha he clus e mapping o POLG mu a ions shows a di ec geno ype-pheno ype ela ionship [9]. Clus e 1 consis s o he polyme ase ac i e si e o POLGA and i s en i ons. Clus e 2 includes esidues o he ups eam DNA binding h p://dx.doi.o g/10.1016/j.bbacli.2017.04.001 Recei ed 15 Feb ua y 2017; Recei ed in e ised o m 11 Ap il 2017; Accep ed 12 Ap il 2017 ⁎ Co esponding au ho a : Depa men o Biochemis y and Molecula Biology, Michigan S a e Uni e si y, Eas Lansing, MI, USA. E-mail add ess: [email p o ec ed] (L.S. Kaguni). Abb e ia ions: AHS, Alpe s-Hu enloche synd ome; ANS, A axia neu opa hy spec um; IP, In insic p ocessi i y subdomain o POLGA space -domain; MCHS, Childhood myoce eb ohepa opa hy spec um; MEMSA, Myoclonic epilepsy myopa hy senso y a axia; PDB ID, Fou -cha ac e iden ifica ion code o a p o ein s uc u e in he RSCB PDB da abase; PEO, P og essi e ex e nal oph halmoplegia; POLG, DNA polyme ase gamma; POLGA, Ca aly ic subuni o DNA polyme ase gamma; POLGB, Accesso y subuni o DNA polyme ase gamma; PNF, Pu a i ely non- unc ional enzyme; SNP, Single nucleo ide polymo phism/non-pa hogenic mu a ion BBA Clinical 7 (2017) 147–156 A ailable online 18 Ap il 2017 2214-6474/ © 2017 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/). MARK channel, mu a ions in which a e likely o al e DNA binding affini y and he o e all p ocessi i y o he holoenzyme. Though he unc ionali y o Clus e 3 emains unde e mined, i s loca ion in he s uc u e sugges s ha i may play a ole in pa i ioning he 3′-end o he DNA p ime s and be ween he polyme ase and exonuclease ac i e si es. Mu a ions in Clus e 3 a e likely o affec bo h DNA binding affini y and ca aly ic ac i i y. A ecen s udy has also sugges ed ha his egion may be in ol ed in unc ional in e ac ion wi h he m DNA helicase [31]. Clus e 4 con ains he ewes esidues and is loca ed a he in e ace o he ca aly ic subuni wi h he dis al accesso y subuni . This in e ac- ion has no been cha ac e ized ex ensi ely, bu i is implica ed o se e an impo an ole in enhancing enzyme p ocessi i y [26]. Residues in Clus e 5 lie wi hin he in insic p ocessi i y (IP) subdomain o he space domain o he ca aly ic subuni , and based on mu a ional analysis a e sugges ed o be in ol ed in p o ein-p o ein in e ac ions [23]. The fi e Alpe s clus e s ep esen bo h s uc u al and unc ional modules o POLG and a e hus ho spo s o pa hogenic mu a ions. Con e sely, mu a ions ha lie ou side o he clus e s a e subs an ially less likely o be pa hogenic. Ou clus e ing model demons a es ha he age o onse o nume ous symp oms co ela es s ongly wi h he se e i y o he synd ome and he ange o symp oms ha a e mani es (see Fig. 5, [9]). Fo example, he mos se e e o m o POLG synd ome, MCHS ( ypical age o onse om bi h o h ee yea s), includes pa ien s ha p esen wi h symp oms such as hypo onia, de elopmen al delay, gas oin es inal dys unc ion and hepa opa hy. Pa ien s diagnosed wi h AHS ( ypical age o onse om a ew mon hs a e bi h o 16 yea s) sha e mos o hese symp oms, bu a e also mo e likely o expe ience epilep ic seizu es. MEMSA pa ien s ( ypical age o onse 13 o 25 yea s o age) a e in mos cases diagnosed wi h myopa hies, neu opa hies and a axia. Pa ien s diagnosed wi h ANS ( ypical age o onse om 15 o middle-age) show a wide a ie y in hei symp oms, including symp- oms om bo h he MEMSA and PEO pa ien g oups. Finally, pa ien s wi h PEO ( ypical age o onse om 30 yea s and abo e) ep esen he leas se e e end o he spec um, and p esen wi h symp oms such as diplopia and p osis. 2. Resul s 2.1. POLG pa hogenici y p edic ion se e We ha e ga he ed all a ailable pa ien case epo s iden i ying POLG mu a ions om public sou ces and c ea ed an online da abase o p o iding easy access o hese da a. The da abase and an online se e , known as he “POLG Pa hogenici y P edic ion Se e ,”a e a ailable a h p://polg.bmb.msu.edu. Fo each pa ien case epo , we ha e colla ed he mu a ions iden ified, he epo ed age o onse o he symp oms mani es ed, he clinical desc ip ion o he pa ien s and sou ce o he da a. Pa ien gende and in o ma ion on adminis a ion o alp oic acid is also included whe e a ailable. The cu en da abase con ains 681 pa ien case epo s, wi h 176 unique missense mu a ions and 215 unique combina ions o pa hogenic mu a ions (Table 1). The la ge majo i y o pa ien cases (546, 80.2%) iden i y ei he compound he e ozygous o homozygous POLG mu a ions. The emaining 135 cases (19.8%) epo he e ozygous a ian s ha a e hus pu a i ely-dominan mu a ions. In combina ion wi h ou pa hogenic clus e ing model [6,9], he pa ien da a has enabled us o make s a is ical p edic ions abou he se e i y and age o onse o symp oms o pa ien s wi h bo h known and no el mu a ions. The se e ea u es a mu a ion que y in e ace whe e he use can en e he POLG mu a ions iden ified in a pa ien (Fig. 2). The se e hen displays he clus e mapping o he inpu mu a ions and shows any exis ing pa ien cases. The s a is ical in o ma ion also includes he allelic combina ions in which he mu a ions ha e been epo ed. I o he membe s o he amily a e no a ailable o analysis, his in o ma ion may be help ul in de e mining whe he he inpu Fig. 1. S uc u al model o he POLG holoenzyme in complex wi h p ime - empla e DNA. In he h ee-dimensional e na y complex o POLG, he pa hogenic clus e s in he ca aly ic co e, POLGA, o m fi e dis inc unc ional egions [6]. Uppe panel, he s uc u e modeled a e PDB s uc u e 4ZTU [39] illus a es he pa hogenic clus e s o POLGA colo ed as g een o Clus e 1, yellow o Clus e 2, ed o Clus e 3, blue o Clus e 4 and cyan o Clus e 5. Lowe panel, subclus e s a e dis ibu ed h oughou he p ima y amino acid sequence o he POLGA polypep ide. Subclus e s a e assigned o each con inuous block on he p ima y amino acid sequence. The accesso y subuni s, POLGB, a e illus a ed in ligh g ey (p oximal subuni ) and da k g ey (dis al subuni ). (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.) A. Nu minen e al. BBA Clinical 7 (2017) 147–156 148 mu a ions occu in cis o in ans. Finally, based on he exis ing cases wi h simila clus e -mapping mu a ions, he se e displays an indica o o he mos p obable age o onse , which can be used as he basis o a diagnosis/p ognosis o a pa ien . The se e also displays he symp- oms ound in pa ien s wi h simila clus e combina ions o mu a ions. An al e na e and powe ul use o he da a is he classifica ion o mu a ions as likely non-pa hogenic a ian s (SNPs). The se e p o ides access o he pa ien and mu a ion da a in a ious ways (Fig. 3). Fo example, he pa ien cases can be iewed based on hei sou ce e e ence, mu a ions, clus e combina ions, age o onse , mu a ed esidue numbe o closes known mu a ion (ei he in p ima y sequence o h ee-dimensional s uc u e). The se e also ea u es da a compila ion pages, displaying he equencies o age o onse and epo ed symp oms o all exis ing clus e combina ions. The symp oms a e displayed in wo ways: as epo ed in he o iginal case epo s, and g ouped in o b oade ca ego ies based on affec ed issue ypes (as pe [9]). Addi ionally, hough hey a e no used di ec ly in he s a is ical p edic ions o pa hogenici y, he se e includes p ima y sequence-based pa hogenici y p edic ions om PON-P2 [28], and also he mos cu en popula ion exome sequencing da a on POLG om he ExAC p ojec (Table S1; [7]). 2.2. Adjus ing he clus e bounda ies The ecen ly published POLG holoenzyme e na y s uc u e makes i possible o iden i y all o he POLGA esidues ha in e ac di ec ly wi h he DNA ligand. O he 56 esidues shown o in e ac wi h DNA, only six map ou side o ou p e iously defined pa hogenic clus e s. Because hese 56 esidues ha e a clea ole in he unc ionali y o POLG o bind and posi ion he DNA ligand, hey ha e a high isk o p oducing dele e ious effec s i mu a ed. By ex ending sligh ly he bo de s o ou o ou subclus e s: 2B, 496–517 (p e iously 497–517); 2D, 752–769 (p e iously 752–767); 1F, 1098–1138 (p e iously 1104–1138); and 3C, 795–807 (p e iously 804–807), hese esidues a e accommoda ed wi hin he defined high- isk loca ions o mu a ions in he POLG s uc u e. Full clus e defini ions as defined p e iously based on biochemical, s uc u al and gene ic s udies [9] a e included as supple- men a y Table S2. A majo diffe ence be ween he c ys al s uc u es o he apo- holoenzyme (PDB ID: 3IKM; [19]) and holoenzyme e na y complex (PDB ID: 4ZTU; [39]) is he posi ioning o he egion cons i u ing pa hogenic Clus e 3D ( esidues 1047–1096; Fig. 4). We ha e p e- iously e med his egion he pa i ioning loop. Clus e 3D ca ies se e al known pa hogenic mu a ions including p.S1080I, p.I1079L, p.C1077G, p.P1073L, p.G1076V and p.R1096C. In he apoenzyme s uc u e, he ip o his long looping egion ex ends wi hin ~5 Å o he polyme ase ac i e si e, p o uding inwa d om he su ace o he enzyme. In con as , in he holoenzyme e na y complex s uc u e, he Table 1 Con en s o he POLG Pa hogenici y P edic ion Se e and dis ibu ion o mu a ions. The majo i y o pa ien cases epo compound he e ozygous mu a- ions. To al numbe o pa ien cases 660 In an ile onse cases 181 Childhood onse cases 103 Ju enile onse cases 85 Adul onse cases 284 Unknown age o onse cases 7 Unique missense mu a ions 176 Unique pa hogenic mu a ion combina ions 215 Compound he e ozygous pa ien s 323 Homozygous pa ien s 152 He e ozygous pa ien s 128 Repo s e e enced 182 Fig. 2. The mu a ion que y in e ace o he POLG Pa hogenici y P edic ion Se e . The se e p o ides s a is ical p edic ions o age o onse , and shows he ypical symp oms o pa ien s wi h mu a ions ha map wi hin pa hogenic clus e s 1–5. In o ma ion abou allelic configu a ion o mu a ions can be help ul in cases o which pedig ees a e no a ailable. The se e also p o ides di ec access o clinical desc ip ions o pa ien cases ha ha e simila mu a ions. A. Nu minen e al. BBA Clinical 7 (2017) 147–156 149 same egion o he loop closes o he polyme ase ac i e si e in he apoenzyme s uc u e is on he su ace o he polyme ase domain a a dis ance o > 20 Å om he polyme ase ac i e si e. Though no discussed by Yin and collabo a o s [19,39], he c ys al s uc u e in he p oximi y o Clus e 3D is appa en ly highly diso de ed, and is missing wo s e ches o ~30 amino acids, esidues 993–1024 and 317–340, sugges ing ha he s uc u al e alua ion o his egion will emain a subs an ial challenge. In he apo-holoenzyme s uc u e, mos o he pa hogenic mu a ions in Clus e 3D a e loca ed be ween he polyme ase and exonuclease domains, sugges ing ha modula ion o he exonuclease o polyme ase a io is a possible unc ion o his egion [6]. In addi ion, in he holoenzyme s uc u e e na y complex, he 5′- end o he empla e s and o he DNA ligand exi s he polyme ase domain a a si e in which in e ac ion wi h Clus e 3D esidues appea s likely. A ecen s udy by Qian e al. [31] epo ed ha pa hogenic mu a ion p.P1073L, loca ed in clus e 3D, does no affec ei he he polyme ase o exonuclease ac i i ies o POLG, bu exhibi s dele e ious effec s in an assay in which mi ochond ial DNA helicase unc ion is equi ed, sugges ing a possible unc ional in e ac ion be ween hem. Clea ly, u he in es iga ion o he s uc u e and unc ion o his egion is wa an ed. Fig. 3. Si e na iga ion wi hin he POLG Pa hogenici y P edic ion Se e . The se e p o ides access o colla ed da a by a ious sea ch in e aces, and p o ides s a is ical p edic ions o onse o symp oms based on he pa hogenic clus e ing model. Fig. 4. Posi ioning o Clus e 3D in he POLG apo-holoenzyme as compa ed o he holoenzyme e na y complex. Clus e 3D ( esidues 1047–1096, ed su ace ep esen a ion) is loca ed in a subs an ially diffe en posi ion be ween he apo-holoenzyme (panel A, PDB ID: 3IKM) and holoenzyme e na y complex (panel B, PDB ID: 4ZTU). The eposi ioning o Clus e 3D appea s unlikely o ep esen a con o ma ional change. Ra he , his egion o he e ia y s uc u e is likely o con ain diso de ed egions, ende ing difficul i s s uc u al e alua ion. (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.) A. Nu minen e al. BBA Clinical 7 (2017) 147–156 150 2.3. Common mu a ions in he pa ien da a Th ee mu a ions ep esen he mos commonly epo ed POLG mu a ions: p.A467T (c.1399G/A, 228 cases), p.W748S (c.2243G/C, 176 cases) and p.G848S (c.2542G/A, 64 cases). These map o h ee diffe en clus e s (p.G848S, Clus e 1; p.A467T, Clus e 2; p.W748S, Clus e 5). They ha e been epo ed in all possible compound he e o- zygous combina ions as well as in homozygous o m, making a compa a i e analysis o hei obse ed pheno ypes possible (Table 2). p.A467T and p.W748S ha e also been epo ed in he e ozygous pa ien s in eigh and wo cases, espec i ely. p.W748S is commonly ound in cis wi h ano he mu a ion, p.E1143G. p.E1143G was assessed p e iously as non-pa hogenic and po en ially compensa o y [3,30]. Ou analysis co obo a es his assessmen as simila ages o onse a e epo ed among pa ien s homozygous o p.W748S (21 yea s, 30 cases) and p.W748S + p.E1143G (22 yea s, 35 cases). Fu he mo e, p.E1143G maps ou side o he defined pa hogenic clus e s. A compa ison o he h ee mu a ions shows ha p.G848S is associa ed wi h he mos pa hogenic pheno ypes. When ound in compound he e ozygous o m wi h p.A467T, he a e age age o onse is 1.7 yea s ( ± 2.0 yea s, in an ile). By con as , in compound he e o- zygous pa ien s ca ying p.G848S and p.W748S, he a e age age o onse is 5.7 yea s ( ± 2.6 yea s, childhood). A single pa ien ca ying homozygous p.G848S has been epo ed wi h an age o fi e yea s a he ime o examina ion [40]. Mu a ion p.G848S has been cha ac e ized biochemically o ha e a subs an ially educed DNA-binding affini y and e y low DNA polyme ase ac i i y [18]. A possible explana ion o he lack o addi ional homozygous p.G848S pa ien case epo s is ha mos o hem esul in p ena al mo ali y. Pa ien s ca ying mu a ions p.A467T and p.W748S show simila ages o onse : he a e age age o onse o homozygous pa ien s is 19.9 yea s ( ± 13.7) o p.A467T and 21.4 ( ± 10.4) yea s o p.W748S. These da a co obo a e he findings epo ed in a ecen s udy on he ex ensi e clinical he e ogenei y o homozygous p.A467T pa ien s [32]. In e es ingly, compound he e ozygous cases wi h geno- ype p.A467T/p.W748S mani es la e , a ~25 yea s o age, sugges ing ha enzymes ca ying dele e ious mu a ions in diffe en clus e s could ha e compensa o y p ope ies. The he e ozygous cases o p.A467T (fi e cases) and p.W748S ( wo cases) ep esen a small ac ion o known ca ie s. The ExAC genome mapping p ojec epo s global popula ion equencies o 0.052% o p.A467T and 0.083% o p.W748S [7]. Mo eo e , in independen s udies, p.A467T was ound in he Belgian popula ion a a pa icula ly high equency o 0.6% [43] and likewise, p.W748S was ound in he Finnish popula ion a a equency o 0.8% [12]. By compa ison, he equency o p.W748S in he Finnish popula- ion is 0.57% in he ExAC da ase . These da a alone a gue ha a dominan s a us o hese mu a ions is unlikely in he absence o o he gene ic o en i onmen al ac o s p edisposing he pa ien s o a mi o- chond ial diso de . Biochemical s udies show ha in compa ison o he wild ype POLG, he p.A467T mu an enzyme exhibi s mode a e o subs an ially educed DNA binding affini y esul ing in simila ly educed polyme ase p oces- si i y [25]. The p.W748S mu an enzyme was shown in one s udy o exhibi subs an ially educed DNA binding affini y and polyme ase p ocessi i y [3], whe eas ano he epo ed enzyma ic p ope ies simila o he wild ype enzyme [30]. A summa y o he biochemical cha ac e iza ion o hese mu a ions is included as Table S3. 2.4. Mu a ions yielding pu a i ely non- unc ional POLG Mu a ions ha in oduce ameshi s, p ema u e s op codons, exon skipping o la ge dele ions a e likely o inac i a e POLG unc ion en i ely and/o impac i s olding, subuni in e ac ion o s abili y. S uc u al pe u ba ions would likely ende he enzyme subjec o cellula u no e . We ha e g ouped oge he on he se e mu a ions ha would esul in a pu a i ely non- unc ional (PNF) POLG. Se e al o hem ha e been cha ac e ized biochemically, co obo a ing such a non- unc ional s a us, hough some may e ain limi ed DNA binding capabili y o o he pa ial unc ionali y [17,34]. Pa ien s wi h com- pound he e ozygous PNF mu a ions ha ca y he h ee mos com- monly epo ed POLG mu a ions ypically mani es wi h e y se e e, in an ile onse condi ions. Fo example, 14 pa ien s wi h a compound he e ozygous p.A467T/PNF geno ype mani es ed symp oms a an a e age age o onse o 1.5 yea s. Fo ou epo ed pa ien s wi h a p.W748S/PNF geno ype, he a e age age o onse is 1.7 yea s. No pa ien s ha e been epo ed wi h p.G848S/PNF mu a ions. These da a a gue ha he enzyme le el p oduced om he single, pa ially- unc ional allele is a c ucial ac o in hese pa ien s, as compa ed wi h he mo e mode a e pheno ypes exhibi ed by he compound he e o- zygous and homozygous cases in which pA467T, p.W748S o p.G848S a e p esen . Se e al excep ions ha e been epo ed. A single pa ien ca ying A467T + E873X/E873X (A467T + PNF/PNF) su i ed un il Table 2 Common POLG mu a ions. The h ee mos commonly epo ed POLG mu a ions, p.A467T, p.W748S and p.G848S, ha e been epo ed in all possible compound he e ozygous combina ions, as well as in homozygous o m. He e ozygous case epo s a e a e o p.A467T and p.W748S, and he e a e none o p.G484S. Compa a i ely, p.G848S appea s o occu in mo e se e e cases, and p.W748S consis en ly shows a sligh ly milde pheno ype in compa ison o p.A467T. Mu a ion Clus e s A e age age o onse (yea s) S anda d de ia ion Numbe o epo ed cases W748S + E1143G/w 5 + SNP/–59.0 4.0 2 (1 ou lie a ) A467T/w 2/–42.8 11.2 5 (1 ou lie b ) W748S/W748S 5/5 21.4 10.4 27 (1 ou lie c ) W748S + E1143G/W748S + E1143G 5 + SNP/5 + SNP 22.1 11.1 35 A467T/A467T 2/2 19.9 13.7 45 G848S/G848S 1/1 5.0 0.0 1 W748S/A467T 5/2 28.7 11.2 27 W748S + E1143G/A467T 5 + SNP/2 21.4 11.7 15 A467T/W748S + K561M 2/5 + 2 0.01 0.0 1 A467T/G848S 2/1 1.7 2.0 22 W748S/G848S 5/1 5.7 2.6 8 A467T/PNF 2/–1.5 1.0 14 (1 ou lie d ) W748S/PNF 5/–1.7 1.3 5 W748S + Q497H + E1143G/ W748S + Q497H + E1143G 5 + 2 + SNP/5 + 2 + SNP 19.0 4.0 2 W748S + Q497H + E1143G/A467T 5 + 2 + SNP/2 17.0 0.0 1 a Sa zi e al. [47], pa ien 36 (id: 404). [h p://www.ncbi.nlm.nih.go /pubmed/17452231]. b Galassi e al. [48], (id: 409). [h p://www.ncbi.nlm.nih.go /pubmed/18504126]. c Tzoulis e al. [49], (id: 552). [h p://www.ncbi.nlm.nih.go /pubmed/24841123]. d Ma ikainen e al. [50], (id: 666). [h p://www.ncbi.nlm.nih.go /pubmed/27111573]. A. Nu minen e al. BBA Clinical 7 (2017) 147–156 151 10 yea s o age [27]. One possible explana ion sugges ed by he au ho s o he epo is ha he p ema u e s op codons a e leaky, and some unc ional enzyme is s ill p oduced by he PNF allele. Simila ly, Roos e al. [34] epo ed wo b o he s wi h a compound he e ozygous geno ype p.T914P/c.3104 + 3A > T (Clus e 1 mu a ion/splice si e mu a ion). Molecula analysis showed ha skipping o POLG exon 19 caused by c.3104 + 3A > T was incomple e, such ha some wild ype POLG was s ill p oduced, leading o an amelio a ed pheno ype wi h he pa ien s mani es ing symp oms a 20 and 50 yea s o age. Simila molecula analyses a e wa an ed o de e mine which specific PNF mu a ions migh s ill p oduce some wild ype enzyme. PNF mu a ions a e all ecessi e, and he PNF ca ying enzymes a e likely incapable o compe ing o DNA binding wi h wild ype POLG a he m DNA eplica ion o k. This hypo hesis is co obo a ed by he p esence o asymp oma ic ca ie s in he pedig ees o pa ien epo s. As such, he PNF enzymes also se e o es ablish an app oxima e baseline o assessing he se e i y and dominan e sus ecessi e s a us o a mu a ion. Because PNF enzymes a e pu a i ely incapable o con ibu ing o m DNA eplica ion, a mu a ion ha is p esen in an indi idual in compound he e ozygous o m wi h a PNF mu a ion is likely o be a ecessi e mu a ion ha p oduces an enzyme ha is o some ex en capable o m DNA eplica ion. This assessmen suppo s a ecessi e s a us o wo o he mos common mu a ions, p.A467T and p.W748S, because hey ha e been ound in compound he e ozygous o m wi h PNF mu a ions. The pa ien da a co obo a es he biochem- ical e idence ha p.G848S is only ma ginally mo e p ocessi e han a PNF enzyme [18], and hus a p.G848S/PNF geno ype is no iable. The pa ien da a suppo s a model in which each dele e ious POLG mu a ion affec s he o e all capaci y o he enzyme o eplica e and main ain m DNA. Al hough ecessi e mu a ions may exe dele e ious effec s on enzyme unc ionali y, symp oms a e no mani es du ing he li e ime o an indi idual because a single wild ype allele is ypically sufficien . In bo h compound he e ozygous and homozygous cases, each mu a ion may educe he o e all cellula capaci y o main ain m DNA, such ha he mos ene gy-demanding and mi ochond ia-dense issues a e affec ed. The pa ien da a show ha homozygous mu a ions a e simila o compound he e ozygous mu a ions, such ha each allele wi h one o mo e dele e ious mu a ions ende s he condi ion mo e se e e as aging p og esses. Mo eo e , mu a ions affec ing diffe en c i ical unc ionali ies (and mapping o diffe en clus e s) o POLG may show ei he compensa o y o exace ba ing effec s. The pa ien s a us in he fi s wo- o- h ee yea s o li e appea s o be a c i ical de e minan . The a e age su i al ime o pa ien s ha mani es wi h POLG synd omes unde he age o h ee is on a e age unde en mon hs wi h a maximum o fi e yea s a e he ime o onse o symp oms, as e idenced by 144 pa ien cases in which bo h he age o onse and age o dea h we e epo ed (Fig. 5). The use o alp oic acid o ea pa ien s wi h epilep ic seizu es may accoun o sho e li e imes epo ed a he mo e se e e end o POLG synd omes, because ea men may lead o hepa o oxici y and acu e li e ailu e [44]. Effec s o en i onmen al and cellula s ess ac o s such as in ec- ions, unheal hy li es yle, malnu i ion, sleep dep i a ion and o he condi ions could no be con olled in he pa ien da a e alua ed in his s udy. In addi ion o he inconsis en epo ing o pa ien da a (age o onse /age a examina ion/age o dea h) o explain he high s anda d de ia ion seen in he da a, i is likely ha o he cu en ly uniden ified gene ic ac o s con ibu e o disease p og ession. In homozygous cases, he possible effec s o consanguini y could also no be con olled. 2.5. Dominan POLG mu a ions The POLG Pa hogenici y P edic ion Se e con ains 50 unique missense mu a ions ha ha e been epo ed as he e ozygous POLG mu a ions in a o al o 131 indi idual pa ien s. Pa ien case epo s a e a ailable o examina ion h ough he mu a ion que y and pa ien da a access in e aces on he se e , and a lis o all pa hogenic he e ozygous POLG mu a ions is also a ailable wi h hype links o he indi idual pa ien case epo s. Based on he clinical da a and in he absence o o he explaining ac o s, hese 50 mu a ions a e en a i ely assigned as pa hogenic wi h a dominan inhe i ance mode. We ha e assessed he p obabili y ha hey a e he oo cause o he symp oms desc ibed in he epo ed pa ien cases by aking in o accoun he ull pa ien da ase and hei pa hogenic clus e assignmen s. We ha e subdi ided he 50 he e ozygous mu a ions in o h ee g oups as ollows. Mu a ions in pa ien case epo s ha a e no ound a an ele a ed equency in a ailable popula ion da a and ha e been cha ac e ized as pu a i ely-dominan in biochemical s udies a e ca e- go ized as mos likely dominan . Fo 14 he e ozygous mu a ions, only a single pa ien case has been epo ed, and addi ional da a a e equi ed o confi m a pu a i ely-dominan s a us. Mu a ions ound in he e o- zygous pa ien cases wi hou any o he explana o y ac o s o he condi ion o he pa ien s a e ca ego ized as ha ing an unclea dominan pa hogenici y. Mu a ions in which he case epo indica ed hey a e likely no he oo cause o he condi ion o he pa ien ha e been ca ego ized in he leas likely dominan g oup. We ha e also aken in o accoun biochemical da a and epo ed asymp oma ic ca ie s when assigning mu a ions in o his las g oup. Ou classifica ion o he 50 he e ozygous POLG mu a ions is discussed below, and is indica ed on he lis a ailable on he POLG Pa hogenici y P edic ion Se e . The majo i y o he mos -likely dominan pa hogenic mu a ions su ound he polyme ase ac i e si e in he ca aly ic subuni , POLGA (Fig. 6). The esidues in his egion a e in ol ed in incoming nucleo ide selec i i y, base pai ing wi h he empla e DNA s and and inco po a- ion o he incoming deoxynucleo ides in o he p ime DNA s and. These a e he c i ical s eps in DNA s and syn hesis, and de ec s can impac bo h he efficiency and fideli y o mi ochond ial DNA eplica- ion and lead o s alling o he DNA eplica ion o k. Replica ion s alling can lead o m DNA deple ion, and esul in base subs i u ion, ameshi and dele ion mu a ions and e o s in nucleo ide inco po a ion [37]. No ably, i was shown in a mu ine model ha mi ochond ia can be ela i ely ole an o poin mu a ions, and a 500- old inc ease in poin mu a ions did no limi he li espan o he animals [45]. This would a gue ha nei he he educed nucleo ide selec i i y no de ec i e exonucleoly ic edi ing obse ed o some pu a i ely-dominan mu a- ions a e likely he sole con ibu o s in dominan pa hogenici y. In ha ega d, exonucleoly ic p oo eading has been epo ed o inc ease he fideli y o POLG 20–100 old depending on he assay used Fig. 5. A e age li espan a e onse o symp oms. POLG synd omes a e p og essi e condi ions, and age o onse co ela es di ec ly wi h he se e i y o he disease. Pa ien da a we e analyzed in cases o which bo h he age o onse o symp oms and age o dea h we e epo ed, and included 144 pa ien cases. The use o alp oic acid o ea pa ien s wi h epilep ic seizu es may accoun o sho ened li espans in he da a in he mo e se e e cases o POLG synd omes. A. Nu minen e al. BBA Clinical 7 (2017) 147–156 152 [14,21,22,29]. I also appea s possible ha p oo eading ac i i y is no he mos c i ical unc ion o he exonuclease domain; some mu a ions in he exonuclease domain esul in educed nucleo ide polyme iza ion a he han de ec s in p oo eading [38], and i has been shown ecen ly ha exonucleolysis by POLGA is equi ed o p oducing ci cula double- s anded DNA [26]. Though all mu a ions ha a e mos likely o be dominan a e loca ed a ound he polyme ase ac i e si e, no all mu a ions in close p oximi y o i a e likely o be dominan . These include p.S433C and p.R1187W. The e is only a single case epo o p.S433C wi h an unspecified age o onse , and he mo he o he pa ien was epo ed o be an asymp oma ic ca ie [13]. p.R1187W was epo ed in wo cases in which he amily his o ies do no suppo an assignmen o dominan inhe i ance [33,35]. Th ee mu a ions, p.G848S, p.T851A and p.R852C, loca ed in a highly conse ed β-hai pin s uc u e ( esidues 844–856) ha lies be ween he polyme ase and exonuclease domains, we e epo ed as he e ozygous in one in 66, one in ou and one in 16 he e ozygous pa ien cases, espec i ely. This egion o he POLG s uc u e is impo an o mispai ecogni ion [39], and is likely o be in ol ed in acili a ing co ec DNA binding nea he polyme ase ac i e si e, hus affec ing bo h he polyme iza ion a e and fideli y o he enzyme. Because he e a e many asymp oma ic ca ie s o each o he h ee mu a ions, hey a e ca ego ized as unlikely-dominan mu a ions. Mo e- o e , hey appea in he a ailable popula ion da a wi h equencies o 0.016% o p.G848S, 0.008% o p.T851A and 0.0066% o p.R852C [7]. Though unlikely dominan , hey a e none heless highly pa hogenic when ound in compound he e ozygous o m. Mu a ions ha lie nea he polyme ase ac i e si e and a e ca ego - ized as ha ing an unclea dominan s a us include p.D930N, p.H945L and p.R953C. Only a single pa ien case has been epo ed o p.H945L wi h ma ching symp oms and age o onse ypical o dominan POLG mu a ions, bu wi h no a ailable amily his o y [4]. p.R953C has been epo ed as a he e ozygous mu a ion in only one pa ien case o ou , wi hou any amily backg ound [24] and is epo ed a a popula ion equency o 0.0016% in he ExAC da abase [7]. p.D930N has been desc ibed as exhibi ing dominan -like pa hogenici y in a yeas model, wi hou any co obo a i e pa ien da a [1]. The mos commonly epo ed POLG mu a ion p esen ing wi h a dominan inhe i ance pa e n is p.Y955C, wi h 39 pa ien case epo s. The onse o symp oms o hese pa ien s is in adul hood wi h an a e age age o 35 ( ± 14.5) yea s. Residue Y955 is loca ed nea he polyme ase ac i e si e and maps wi hin pa hogenic Clus e 1. I s loca ion in he POLG s uc u e and he exis ing biochemical li e a u e suppo a c i ical ole in o ien ing and base pai ing o he co ec nucleo ide wi h he empla e DNA s and [5]. Nucleo ide polyme iza- ion by he a ian POLG is educed and misinco po a ion a e is ele a ed; kine ic analysis documen s up o 1300- old educ ion in nucleo ide inco po a ion fideli y [5]. Typical symp oms o pa ien s ca ying p.Y955C include PEO, muscle weakness and a ious neu olo- gical symp oms. Indeed, he mos commonly epo ed symp oms o pa ien s ca ying pu a i ely-dominan mu a ions a e PEO/p osis and muscle weakness, epo ed in 44% and 23% o he he e ozygous pa ien s, espec i ely (Fig. 7). The biochemical cha ac e iza ions epo ed o da e o he pu a- Fig. 6. He e ozygous POLG mu a ions. Panel A, model o he ca aly ic subuni o POLG illus a ing mu a ions ha ha e been epo ed o be he e ozygous in he POLG Pa hogenici y P edic ion Se e da abase. Mu a ions o which subs an ial co obo a i e e idence indica es dominan inhe i ance a e shown in ed sphe es, and su ound he polyme ase ac i e si e (black a ow). Yellow sphe es indica e mu a ions o which he e is insufficien da a o a gue dominan s a us. G een sphe es depic mu a ions leas likely o be dominan . The colo s a us is no indica i e wi h espec o pa hogenici y in compound he e ozygous o m. Two o he mos common pa hogenic POLG mu a ions, p.A467T and p.W748S, a e shown in cyan sphe es. Panel B, close-up o he ac i e si e o he polyme ase domain o POLGA (black a ow). The pu a i ely-dominan mu an esidues ( ed sphe es) in e ac wi h he incoming dNTP (o ange), empla e DNA o he ca aly ic Mg-ions (black sphe es). A ho spo o hese is on he O-helix, in which e e y esidue acing he polyme ase ac i e si e has been epo ed in a he e ozygous POLG pa ien . (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.) Fig. 7. The mos commonly epo ed symp oms o he e ozygous POLG pa ien s. Symp oms in o de o commonness in he e ozygous pa ien s include p osis, p og essi e ex e nal oph halmoplegia (PEO), muscle weakness, Pa kinson's disease and emo s. A. Nu minen e al. BBA Clinical 7 (2017) 147–156 153 i ely-dominan mu a ions [2,3,10,18, 25,30], combined wi h he pa ien case epo s, highligh he ac ha he mos c i ical p ope ies o dominan pa hogenic mu a ions in POLG appea o be se e ely- educed polyme ase ac i i y wi h sufficien DNA binding affini y o compe e wi h wild ype enzyme a he m DNA eplica ion o k (Table S3). 3. Discussion We epo he de elopmen o an online POLG Pa hogenici y P edic ion Se e . The se e ep esen s a comp ehensi e, in e ac i e da abase ha complemen s and ex ends subs an ially he online Human DNA Polyme ase gamma Mu a ion Da abase (h p:// ools.niehs.nih. go /polg/). POLG da a and pa ien cases a e accessible in nume ous ways as desc ibed unde Resul s, and addi ional con en is p o ided by including analysis by he PON-P2 algo i hm [28] and ExAC popula ion equency da a [7]. Mos no ably, he POLG Pa hogenici y P edic ion Se e inco po a es ou p e iously desc ibed clus e ing model o pa hogenici y p edic ion, which was de i ed by e alua ion o biochem- ical, s uc u al and gene ic da a on POLG and he ela ed amily A g oup DNA polyme ases, bac e ial DNA polyme ase I and bac e ioph- age T7 DNA polyme ase [6,9]. Because mos POLG synd omes esul om he incidence o compound he e ozygous mu a ions, he clus e ing analysis o pai s o mu a ions based on he analysis o cu en ly a ailable mu a ions and mu a ional pai s gi es insigh in o he likely pa hogenici y o no el pai s, and o p e iously-iden ified alleles in combina ion wi h no el alleles. As such, we belie e he p edic ion ool will be widely applicable as one pa ame e in he diagnosis o mi ochond ial diso de s. No ewo hy challenges and/o limi a ions o he da ase include he a ying me hods and a ie y o es s employed in he examina ion o pa ien s ha ha e been epo ed and in pa icula , in publica ions p io o he gene alized use o DNA sequencing o e alua e he spec um o possible genes and gene ic causes o mi ochond ial diso de s. Addi ionally, age o onse may no be epo ed as he fi s onse o symp oms; a he , he age o he pa ien a he ime o examina ion has some imes been epo ed. In iew o hese challenges, we highligh he impo ance o whole exome analysis (see o example [41]) and o epo ing pa ien cases wi h ull pedig ees and de ailed desc ip ions o age o onse o symp oms. Though ho ough clinical examina ion may no be possible o he immedia e ela i es o he pa ien s, a desc ip ion and o e all assessmen o hei wellbeing would p o e help ul. Finally, i is impo an o documen he co-exis ence o po en ially non- pa hogenic polymo phisms such ha any impac hey migh ha e in combina ion wi h a pa hogenic mu a ion can be assessed app op ia ely. Issues ela ed o bo h in insic gene ic and biochemical pa ame e s mus be e alua ed u he as new pa ien da a on pa hogenic mu a ions eme ges. A p esen , he da ase o a ailable mu a ions is cha ac e ized by he o e ep esen a ion o he h ee common pa hogenic mu a ions p.A467T, p.W748S and p.G848S ha a e p esen in composi e in 71% o he 681 case epo s. Whe eas he ela i e equency o new a ian s epo ed will likely emain simila , hei absolu e numbe will inc ease wi h s eamlined epo ing on an in e na ional scale. The POLG Pa hogenici y P edic ion Se e ea u es a con ac o m ha can be used o poin ing ou e o s in he exis ing da a, no i ying he da abase adminis a o s o new publica ions and eques ing confiden ial analysis o a e POLG mu a ions. A po en ial sho coming o he pa hogenic clus e ing p o ocol is a di ec eflec ion o he in insic s uc u al and biochemical p ope ies o POLG and indeed, o all enzymes. Whe eas a new a ian allele mapping o a pa hogenic clus e is much mo e likely o be pa hogenic han one ha lies ou side o a clus e , no all amino acid esidues wi hin a clus e o subclus e ha e a simila likelihood and/o le el o pa hogenici y. This inhe en p ope y is dependen s ongly on bo h he biochemical unc ion o he specific amino acid and he chemical na u e o he amino acid change induced by he mu a ion. Fo example, an amino acid ha is c ucial o POLG unc ion such as he ca aly ic esidues in he pol mo i s A, B and C ha a e conse ed among amily A DNA polyme ases and which lie in POLG subclus e s 1D, 1E and 1F, espec i ely, may be ei he unde ep esen ed o absen in he da ase because amino acid subs i u ions a e no ole a ed in li e indi iduals. On he o he hand, conse a i e amino acid changes wi hin clus e - mapping esidues may show no clinical pheno ypes. In such si ua ions, use o he esidue b owse unc ion o he se e will p o ide ele an in o ma ion o u he e alua ion. None heless, in he ‘big pic u e’ sense, i is clea ha combina ions o mu a ions mapping o he pol domain clus e 1 in combina ion wi h hose mapping in ei he he DNA- binding channel clus e 2 o he pu a i e p o ein: p o ein in e ac ion clus e 5 cons i u e he bulk o he pa ien cases (96) mani es ing in an ile onse , and in ol e one o mo e o he mos common mu a ions epo ed. Addi ionally, nine o 10 pu a i ely-dominan mu a ions ha we ha e classified as mos -likely dominan (56 o 57 pa ien cases epo ed) map wi hin he ca aly ic subclus e s o he pol domain, 1D and 1E. This includes o example, he pa hogenic mu a ion p.Y955C [10,20,42]. The single mu a ion mapping ou side o clus e 1 is P765T in clus e 2; hough i s loca ion in he POLG s uc u e sugges s a high likelihood o dominance, he e is only a single case epo and no exis ing biochemical da a. Biochemical cha ac e iza ion o mu a ions in conjunc ion wi h he pa ien da a sugges s ha mu a ions ha impai nucleo ide polyme - iza ion by POLG wi hou affec ing DNA binding ca y he highes isk o dominan inhe i ance. The low numbe o epo ed pa ien cases o some o he pu a i ely-dominan mu a ions and lack o consis en es ing and epo ing in pa ien pedig ees obscu es he e alua ion o a likely dominan s a us. Based on he a ailable gene ic, biochemical and clinical da a in ou da abase we ha e e-e alua ed he s a us o 49 pu a i ely-dominan mu a ions and di ided hem in o h ee classes. We sugges ha he 10 mu a ions ha all wi hin he mos -likely dominan g oup be conside ed s ongly by clinicians as a oo cause o mi ochon- d ial dys unc ion. I is well documen ed ha he symp oms and issue specifici y o pa hogenici y in pa ien s suffe ing om POLG synd omes a ies widely, e en among pa ien s ca ying he same POLG mu a ions (see o example [32]). None heless ou analysis shows a clea consis ency in age o onse ha does no diffe wi h gende . We also obse ed an appa en ly s onge co ela ion o symp oms wi hin amilies. This obse a ion highligh s he likelihood ha o he gene ic componen s a e con ibu o y and he impo ance o whole exome analysis in he e alua ion o POLG synd omes, and o mi ochond ial diso de s in gene al o iden i y such componen s. Effo s such as Mi ochond ial Disease Sequence Da a Resou ce (MSeqDR) a e unde way o add ess he issue o nuclea gene ic modifie s [8,36]. Fu u e effo s should also add ess he possible con ibu ions o m DNA gene ic backg ound. A p esen , ou unde s anding o he disease e iology o POLG synd omes oge he wi h he cu en ly a ailable diagnos ic ools should be beneficial in amily planning o couples unde going gene ic es ing, e en hose wi h no amilial his o y o mi ochond ial diso de s. 4. Ma e ials and me hods Pa ien da a deposi ed in o he da abase and used in his s udy a e anonymous and collec ed om publicly a ailable jou nal a icles. The sou ce o each pa ien case is s a ed and a ailable on he se e . The da a in he li e a u e is epo ed in a non-s anda dized o m and comp ises a a ie y o diffe en es s, examina ions and de ails. The se e ca ego izes each case based on he age o onse o he fi s symp oms epo ed in o he ollowing age g oups: in an ile, < 3 yea s; childhood, 3–13 yea s; ju enile, 13–20 yea s; o adul , ≥20 yea s. Fo epo s in which only he age o he pa ien a he ime o examina ion is epo ed, his age was used. I no age o he pa ien is epo ed, he age g oup is indica ed as “unknown.” Symp oms we e ca ego ized based only on he diagnoses p o ided A. Nu minen e al. BBA Clinical 7 (2017) 147–156 154 in he epo . Fo condi ions such as Alpe s synd ome, i migh be he case ha nea ly all o he known symp oms o his condi ion ha e been epo ed in a pa ien case. Howe e , i he diagnosis o Alpe s has no been epo ed di ec ly, he case has no been deposi ed as an Alpe s en y. Fo o he condi ions such as a axia, we ha e accep ed e ms such as “mo emen diso de ,”“a axic gai ,”“gai dis u bance”and “gai uns eadiness”as being synonymous. The ou pu o p edic ions o pa hogenici y p o ided by he mu a ion que y in e ace o he se e a e based on he pa hogenic clus e ing model and s a is ics o exis ing pa ien cases wi h simila clus e - mapping mu a ions. The bo de s o he subclus e s ha e been defined by a ailable pa ien da a, s uc u al in o ma ion and biochemical s udies o mu a ions [6]. The s a is ical p edic ions eflec di ec ly he con en s o he da abase, and can be efined as new cases a e added. T anspa ency Documen The T anspa ency documen associa ed wi h his a icle can be ound, in online e sion. Acknowledgmen s This wo k was suppo ed by Na ional Ins i u es o Heal h G an GM45295 o L.S.K. Anssi Nu minen was suppo ed by he Uni e si y o Tampe e. Appendix A. Supplemen a y da a Supplemen a y da a o his a icle can be ound online a h p://dx. doi.o g/10.1016/j.bbacli.2017.04.001. Re e ences [1] E. Ba uffini, R. Ho a h, C. Dallabona, B. Cze min, E. Laman ea, L. Bindoff, ... T. Lodi, P edic ing he con ibu ion o no el POLG mu a ions o human disease h ough analysis in yeas model, Mi ochond ion 11 (1) (2011) 182–190, h p://dx. doi.o g/10.1016/j.mi o.2010.09.007. [2] S.S.L. Chan, M.J. Longley, W.C. Copeland, The common A467T mu a ion in he human mi ochond ial DNA polyme ase (POLG) comp omises ca aly ic efficiency and in e ac ion wi h he accesso y subuni , J. Biol. Chem. 280 (36) (2005) 31341–31346, h p://dx.doi.o g/10.1074/jbc.M506762200. [3] S.S.L. Chan, M.J. Longley, W.C. Copeland, Modula ion o he W748S mu a ion in DNA polyme ase gamma by he E1143G polymo phismin mi ochond ial diso de s, Hum. Mol. Gene . 15 (23) (2006) 3473–3483, h p://dx.doi.o g/10.1093/hmg/ ddl424. [4] M. Delgado-al a ado, P. De, H. Jiménez-u bie a, B. Gago, A. Gabilondo, B. Bo ns ein, M.C. Rod íguez-o oz, Pa kinsonism, cogni i e defici and beha iou al dis u bance caused by a no el mu a ion in he polyme ase gamma gene, J. Neu ol. Sci. 350 (1–2) (2015) 93–97, h p://dx.doi.o g/10.1016/j.jns.2015.02.011. [5] P.A. Es ep, K.A. Johnson, Effec o he Y955C mu a ion on mi ochond ial DNA polyme ase nucleo ide inco po a ion efficiency and fideli y, Biochemis y 50 (29) (2011) 6376–6386, h p://dx.doi.o g/10.1021/bi200280 . [6] L. Eu o, G.A. Fa num, E. Palin, A. Suomalainen, L.S. Kaguni, Clus e ing o Alpe s disease mu a ions and ca aly ic de ec s in biochemical a ian s e eal new ea u es o molecula mechanism o he human mi ochond ial eplicase, Pol γ, Nucleic Acids Res. 39 (21) (2011) 9072–9084, h p://dx.doi.o g/10.1093/na /gk 618. [7] ExAC, Exome Agg ega ion Conso ium (ExAC) b owse , Re ie ed om h p://exac. b oadins i u e.o g, (2015). [8] M.J. Falk, L. Shen, M. Gonzalez, J. Leipzig, M.T. Lo , A.P.M. S assen, ... L.J. Wong, Mi ochond ial Disease Sequence Da a Resou ce (MSeqDR): a global g ass- oo s conso ium o acili a e deposi ion, cu a ion, anno a ion, and in eg a ed analysis o genomic da a o he mi ochond ial disease clinical and esea ch communi ies, Mol. Gene . Me ab. 114 (3) (2015) 388–396, h p://dx.doi.o g/10.1016/j.ymgme.2014. 11.016. [9] G.A. Fa num, A. Nu minen, L.S. Kaguni, Mapping 136 pa hogenic mu a ions in o unc ional modules in human DNA polyme ase γes ablishes p edic i e geno ype–- pheno ype co ela ions o he comple e spec um o POLG synd omes, Biochim. Biophys. Ac a, Bioene g. 1837 (7) (2014) 1113–1121, h p://dx.doi.o g/10.1016/j. bbabio.2014.01.021. [10] M.A. G aziewicz, M.J. Longley, R.J. Biens ock, M. Ze iani, W.C. Copeland, S uc u e- unc ion de ec s o human mi ochond ial DNA polyme ase in au osomal dominan p og essi e ex e nal oph halmoplegia, Na . S uc . Mol. Biol. 11 (8) (2004) 770–776, h p://dx.doi.o g/10.1038/nsmb805. [11] A.H. Hakonen, G. Da idzon, R. Salemi, L.A. Bindoff, G. Van Goe hem, S. Dimau o, ... A. Suomalainen, Abundance o he POLG disease mu a ions in Eu ope, Aus alia, New Zealand, and he Uni ed S a es explained by single ancien Eu opean ounde s, Eu . J. Hum. Gene . 15 (7) (2007) 779–783, h p://dx.doi.o g/10.1038/sj.ejhg. 5201831. [12] A.H. Hakonen, S. Heiskanen, V. Ju onen, I. Lappalainen, P.T. Luoma, M. Ran amaki, ... A. Suomalainen, Mi ochond ial DNA polyme ase W748S mu a- ion: a common cause o au osomal ecessi e a axia wi h ancien Eu opean o igin, Am. J. Hum. Gene . 77 (3) (2005) 430–441, h p://dx.doi.o g/10.1086/444548. [13] G. Hudson, M. Deschaue , R.W. Taylo , M.G. Hanna, D. Fialho, A.M. Schae e , L.P. He, E. Blakely, D.M. Tu nbull, P.F. Chinne y, POLG1, C10ORF2, and ANT1 mu a ions a e uncommon in spo adic p og essi e ex e nal oph halmoplegia wi h mul iple mi ochond ial DNA dele ions, Neu ology 66 (2006) 1439–1441, h p://dx. doi.o g/10.1212/01.wnl.0000210486.32196.24 h ps://www.ncbi.nlm.nih.go / pubmed/16682683. [14] A.A. Johnson, K.A. Johnson, Fideli y o nucleo ide inco po a ion by human mi ochond ial DNA polyme ase, J. Biol. Chem. 44613 (512) (2001) 38090–38096, h p://dx.doi.o g/10.1074/jbc.M106045200. [15] L.S. Kaguni, DNA polyme ase γ, he mi ochond ial eplicase, Annu. Re . Biochem. 73 (1) (2004) 293–320, h p://dx.doi.o g/10.1146/annu e .biochem.72.121801. 161455. [16] L.S. Kaguni, M.T. Oli ei a, S uc u e, unc ion and e olu ion o he animal mi ochond ial eplica i e DNA helicase, C i . Re . Biochem. Mol. Biol. 9238 (2016) 1–12, h p://dx.doi.o g/10.3109/10409238.2015.1117056 h ps://www.ncbi.nlm. nih.go /pubmed/26615986. [17] M. Kaliszewska, J. K uszewski, B. Kie daszuk, A. Kos e a-P uszczyk, M. Nojszewska, A. Łusakowska, ... K. Tońska, Yeas model analysis o no el polyme ase gamma a ian s ound in pa ien s wi h au osomal ecessi e mi ochond ial disease, Hum. Gene . 134 (9) (2015) 951–966, h p://dx.doi.o g/10.1007/s00439-015-1578-x. [18] R. Kasi iswana han, M.J. Longley, S.S.L. Chan, W.C. Copeland, Disease mu a ions in he human mi ochond ial DNA polyme ase humb subdomain impa se e e de ec s in mi ochond ial DNA eplica ion, J. Biol. Chem. 284 (29) (2009) 19501–19510, h p://dx.doi.o g/10.1074/jbc.M109.011940. [19] Y.S. Lee, W.D. Kennedy, Y.W. Yin, S uc u al insigh in o p ocessi e human mi ochond ial DNA syn hesis and disease- ela ed polyme ase mu a ions, Cell 139 (2) (2009) 312–324, h p://dx.doi.o g/10.1016/j.cell.2009.07.050. [20] W. Lewis, B.J. Day, J.J. Kohle , S.H. Hosseini, S.S.L. Chan, E.C. G een, ... W.C. Copeland, Dec eased m DNA, oxida i e s ess, ca diomyopa hy, and dea h om ansgenic ca diac a ge ed human mu an polyme ase gamma, Lab. In es . 87 (4) (2007) 326–335, h p://dx.doi.o g/10.1038/labin es .3700523. [21] M.J. Longley, D.W. Mosbaugh, P ope ies o he 3′ o 5′exonuclease associa ed wi h po cine li e DNA polyme ase γ: Subs a e specifici y, p oduc analysis, inhibi ion, and kine ics o e minal excision, J. Biol. Chem. 36 (1991) 24702–24711. h ps:// www.ncbi.nlm.nih.go /pubmed/1662214. [22] M.J. Longley, D. Nguyen, T.a. Kunkel, W.C. Copeland, The fideli y o human DNA polyme ase γwi h and wi hou exonucleoly ic p oo eading and he p55 accesso y subuni , J. Biol. Chem. 276 (42) (2001) 38555–38562, h p://dx.doi.o g/10.1074/ jbc.M105230200. [23] N. Luo, L.S. Kaguni, Mu a ions in he space egion o D osophila mi ochond ial DNA polyme ase affec DNA binding, p ocessi i y, and he balance be ween Pol and Exo unc ion, 280 (2005), pp. 2491–2497, h p://dx.doi.o g/10.1074/jbc. M411447200 h ps://www.ncbi.nlm.nih.go /pubmed/1662214. [24] P. Luoma, A. Melbe g, J.O. Rinne, J.A. Kaukonen, N.N. Nupponen, R.M. Chalme s, ... A. Suomalainen, Pa kinsonism, p ema u e menopause, and mi ochond ial DNA polyme ase γmu a ions: clinical and molecula gene ic s udy, Lance 364 (9437) (2004) 875–882, h p://dx.doi.o g/10.1016/S0140-6736(04)16983-3. [25] P.T. Luoma, N. Luo, W.N. Lösche , C.L. Fa , R. Ho a h, J. Wanschi z, ... A. Suomalainen, Func ional de ec s due o space - egion mu a ions o human mi ochond ial DNA polyme ase in a amily wi h an a axia-myopa hy synd ome, Hum. Mol. Gene . 14 (14) (2005) 1907–1920, h p://dx.doi.o g/10.1093/hmg/ ddi196. [26] B. Macao, J.P. Uhle , T. Siibak, X. Zhu, Y. Shi, W. Sheng, ... M. Falkenbe g, The exonuclease ac i i y o DNA polyme ase γis equi ed o liga ion du ing mi o- chond ial DNA eplica ion, Na . Commun. 6 (2015) 7303, h p://dx.doi.o g/10. 1038/ncomms8303. [27] R.K. Na iaux, K.V. Nguyen, POLG mu a ions associa ed wi h Alpe s' synd ome and mi ochond ial DNA deple ion, Ann. Neu ol. 55 (5) (2004) 706–712. [28] A. Ni oula, S. U olagin, M. Vihinen, PON-P2: p edic ion me hod o as and eliable iden ifica ion o ha m ul a ian s, PLoS One 10 (2) (2015) 1–17, h p://dx.doi.o g/ 10.1371/jou nal.pone.0117380. [29] M.W. Olson, L.S. Kaguni, 3′-5′exonuclease in D osophila mi ochond ial DNA polyme ase, J. Biol. Chem. 32 (1992) 223136–223142. h ps://www.ncbi.nlm.nih. go /pubmed/1429661. [30] E.J.H. Palin, A. Lesonen, C.L. Fa , L. Eu o, A. Suomalainen, L.S. Kaguni, Func ional analysis o H. sapiens DNA polyme ase γspace mu a ion W748S wi h and wi hou common a ian E1143G, Biochim. Biophys. Ac a 6 (2010) 545–551, h p://dx.doi. o g/10.1016/j.bbadis.2010.02.003 h ps://www.ncbi.nlm.nih.go /pubmed/ 20153822. [31] Y. Qian, J.L. Zieh , K.A. Johnson, Alpe s disease mu a ions in human DNA polyme ase gamma cause ca aly ic de ec s in mi ochond ial DNA eplica ion by dis inc mechanisms, F on . Gene . 6 (2015) 1–11, h p://dx.doi.o g/10.3389/ gene.2015.00135 h ps://www.ncbi.nlm.nih.go /pubmed/25914719. [32] S. Rajakulend an, R.D.S. Pi cea hly, J.W. Taanman, H. Cos ello, M.G. Sweeney, C.E. Woodwa d, ... S. Rahman, A clinical, neu opa hological and gene ic s udy o homozygous A467T POLG- ela ed mi ochond ial disease, PLoS One 11 (1) (2016) 1–16, h p://dx.doi.o g/10.1371/jou nal.pone.0145500. [33] J. Reichenbach, R. Schube , R. Ho à h, J. Pe e sen, N. Fü e e , E. Malle, ... S. Zielen, Fa al neona al-onse mi ochond ial espi a o y chain disease wi h T cell A. Nu minen e al. BBA Clinical 7 (2017) 147–156 155