Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n T604gn htatrRAslcn ymngn assay minigene by Zhang splicing Ruixiao RNA alter ATP6V1B1 that SLC4A1, gene the ATP6V0A4 in and variants exonic eight of Identification h r-RAslcn rcs,i a ensgetdta pt 0 feoi ainsicuigaltypes all including variants in exonic of signals 50% splicing potentially to auxiliary up to and that considered suggested core were been of AS) has importance or it (DS the process, past, sites given splicing the Currently, pre-mRNA splice In the acceptor exons. processing. join or and transcriptional donor introns structure remove alter affecting secondary site accurately mutations RNA acceptor to spliceosome, intronic the splice the silencers or 3’ only and and and components factors (ESEs/ISEs) splicing (5’ss), regulatory with enhancers site other cooperate splicing donor as pre- exonic/intronic well splice track, in as (5’ polypyrimidine (ESSs/ISSs), sites signals site, splice splicing branch conserved the of and the (3’ss)), number U6, include large signals and splicing a U5 The recognize U4, U2, can removal ribonucleoprotein intron U1, that large stimulate (snRNPs) proteins, a and ribonucleoproteins spliceosome, non-snRNP mRNA nuclear the is associated small by which other catalyzed exons, 5 is many of by connection Splicing constituted the expression. complex and gene introns (RNP) of of mRNAs removal process translatable the important Mature involving an splicing, introns. called pre-mRNAs protein- segments by the insertion generated from non-coding are by transcribed interrupted are are cells introns regions, eukaryotic coding and most exons of of genes composed coding (Pre-mRNAs) RNAs messenger Precursor effective an is analysis our minigene To that Introduction highlight vitro suggest both. results in and These or variants level on mRNA site, genes. dRTA-related splicing the splicing the of at classic effects variants in variants the the exonic evaluating exonic of of for of effects recognition tool splicing the the assessing pre-mRNA with of on interference importance study or the first the ESSs, is of this generation knowledge, and ESEs of disruption among result, c.1571C a asso- (c.1765C As variants SLC4A1 ATP6V0A4, assay. of in splicing minigene SLC4A1, distributed consequences and alter variants c.370C the tools can in 8 bioinformatics determine variants variants, variants predictive to candidate exonic with was 15 with of combined the study types splicing associated This pre-mRNA all on disease that process. dRTA with splicing evidence tubular ciated growing pre-mRNA rare the is affecting there a Currently, elements, is regulatory genes. (dRTA) WDR72 acidosis or FOXI1 tubular ATP6V1B1, renal distal Primary Abstract 2021 7, February 5 4 3 2 1 Shi Xiaomeng RC-aaSliv el oeez Hospital Sofferenza della University Sollievo Qingdao IRCCS-Casa of Hospital Affiliated District The Jimo of Hospital People’s University Fudan Group Hospital Municipal Qingdao > > ,pAg2Tp c.484G p.Arg124Trp; T, ,pPo2Luadc.1572G and p.Pro524Leu T, 1 1 ecn Guo Wencong , eigChen Zeqing , > ,pGu6*adc.1102G and p.Glu162* T, > ,pPo2Po eeietfidt euti hl rpr feo kpigb either by skipping exon of part or whole in result to identified were p.Pro524Pro) A, 2 4 iigSong Qijing , ahaLang Yanhua , [1] [2,3] xn,cnitn ftotria nrnltdrgosadprotein- and regions untranslated terminal two of consisting Exons, . . 3 > a Wang Sai , 1 ,pGu6Ls n T604gns(c.322C genes ATP6V0A4 and p.Glu368Lys) A, 1 rn Bottillo Irene , > ,pAg8Cs,APVB(c.368G ATP6V1B1( p.Arg589Cys), T, 1 hyn Liu Zhiying , 5 n eigShao Leping and , 1 inzogZhao Xiangzhong , [4,5] l hs factors these All . > ,p.Gly123Val; T, > 1 ,p.Gln108*; T, 4 , Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n/ratvto fnwsts eal hehlswr efre o l nlss ainsta aif the satisfy generation that the Variants predict analyses. to all and for sites performed consensus were acceptor thresholds on 3’ Default variants or sites. of donor new effect 5’ of putative classic ( activation the on and/or BDGP variation identify presence of of and the effects Analysis ESSs), investigate software potential Online and to motifs. variants. (ESEs performed regulatory the sequences was of splicing regulatory umd.be/HSF3) each splicing of http://www. potential effect 3.1, splicing of (version the Finder predict Splice to out Human carried was analysis literatures Bioinformatics and 2019) (April, Database c.904C Mutation and Gene (p.Pro137Ser) Human the from collected in variants point All Analyses Silico In and codon. Collection start for Variant translation the sequence of ( position Society Variation first cDNA Genome the Human the the of on guidelines based and 012188.4) is numbering ATP6V1B1 variant DNA dRTA- described previously Nomenclature Variant the of effects Methods and the minigene Materials into a using insight primary splicing with pre-mRNA gain associated on variants to genes nonsense pathogenic was and technology. of RNA missense study variants including and exonic variants this DNA pathogenic in both related of splicing at of purpose cases effect few the The a dRTA. on in studies only few but to are level, There performed genome was the analysis variants at protein most However, and variants. mRNA levels in all on 29 effects of including the (101/201) assess 50% 2019.4), about Professional for been (HGMD, have account variants variants Database gene of dRTA-related in Mutation different number 201 73 Gene The technology of , Human sequencing total individuals. a high-throughput the far, of unaffected in So application and described the sequencing. affected with parallel in large-scale rapidly resulting on variations increase diseases, based to rare genetic expected related of of is variants thousands diagnosis clinical these of the identification in used the widely now in been has analysis may sequence DNA which subunits, A4 transmembrane in patients. variants encode dRTA that 20 the in which reported V-ATPase H of literatures the epithelia in exons, of sac involved 23 subunit endolymphatic includes be B1 reabsorption and the the and duct for codifies 7q33-34 collecting which responsible chromosome renal exons, primarily the 14 is of which of cells protein, total 3 a of band with composed or chromosome exchanger AE1 chloride–bicarbonate as HCO the known protein The of also membrane trait. a acids, (AR) encodes amino acidosis recessive and 911 metabolic or exons gap (AD) 20 contains dominant anion 17q21, autosomal serum in tubular an normal variants impaired either with in by as nephrolithiasis associated resulting characterized and are nephron, disease nephrocalcinosis, patients distal hypokalemia, dRTA tubular to the rare progresses in a often ions that is (dRTA) hydrogen acidosis of disrupt may tubular secretion elements, renal human regulatory distal in splicing diseases Primary alter various that cause deletions) and pathway or splicing insertions the small and nonsense, (missense, [16] ATP6V1B1 3 . - ope ihteuiayeceino chloride of excretion urinary the with coupled GnakNM (GenBank WDR72 + rnlcto rtasotado sebyo h H the of assembly and/or transport or translocation 4in 94 , SLC4A1 GnakNM (GenBank > ATP6V0A4 pAg0Tp in (p.Arg302Trp) T 001692.3), , ATP6V1B1 FOXI1 8783.Tevratnmnltr a ecie codn othe to according described was nomenclature variant The 182758.3). in 2 , ATP6V0A4 [13] , FOXI1 ATP6V0A4 SLC4A1 , ATP6V1B1 WDR72 http://www.fruitfly.org http://varnomen.hgvs.org 2 GnakNM (GenBank n in 3 and , [14] [6,7] ATP6V0A4 [9] , and htwsietfidi u rvosstudies previous our in identified was that h gene the ; FOXI1 . WDR72 ATP6V1C2 [10,11] SLC4A1 , 020632.2), SLC4A1 WDR72 the ; and a mlydt eemnt the determinate to employed was ) mn hm missense/nonsense them, Among . ATP6V1B1 + -ATPase [8] ATP6V1B1 gene ATP6V0A4 h atmjrt fprimary of majority vast The . ee oae nchromosome on located gene, ,wt ubrn trigat starting numbering with ), and [15] GnakNM (GenBank [15,17,18] FOXI1 [12] eefudi primary in found were ATP6V1C2 sfudo h 2p13 the on found is nadto,recent addition, In . xetc.409C except , ee,transmitted genes, eei oae on located is gene GnakNM (GenBank α- intercalated 000342.3), eewere gene SLC4A1 [19] > . T - Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eta piigvrat ihntrebsso h o ’edo h xn rpeitdta ffc splicing minigene affect further that for predicted selected or were exon, the silencers) of of Po- end software. creation 3’ bioinformatics or ’or the enhancers by 5 analysis of the for of (elimination collected bases elements were three regulatory of gene, within ATP6V1C2 20 variants of splicing including 1 tential variants, WDR72, of different 3 113 FOXI1, of total USA). two-tailed A Software, the GraphPad using 6.02, (Version analyzed value Results band]) P Prism were 3 upper A GraphPad results + (n=3). by band SEM The test [lower represent software. ANOVA bars / One-way SPSS Error band (lower or using as t-test performed calculated Student’s was was (%) analysis exclusion Statistical exon of percentage The target The analyzed J. were analysis transcripts Image all Statistical software then by and quantified China), (CWBIO, was Kit signal Extraction sequencing. band Gel 72 by a each using and at purified were gel, step cycler. bands agarose DNA thermal elongation 1.5% USA) a final on CA, a City, electrophoresis Foster 94 by Biosystem, of followed cycles (Applied and 33 9700 were a conditions 50 in Thermal in Japan) (TaKaRa, follows: Polymerase as 1 DNA Japan), performed (TaKaRa, was Buffer vector-specific reaction PrimerSTAR following amplification the (5’-TCTGAGTCACCTGGACAACC- PCR SD6 (5’-ATCTCAGTGGTATTTGTGAGC-3’). minigenes, The SA2 primer transfected primer forward the reverse a a from amplification: and RT-PCR transcripts Carlsbad, 3’) for of Corporation, used (Invitrogen pattern were 2 Transcriptase primers the from Reverse evaluate synthesized II To was Superscript CA). cDNA with RT-PCR for First-strand transcription used and patterns. reverse USA) primed (Invitrogen, splicing reagent the TRIzol using confirm extracted was to RNA total incubation, h 24 instructions. After manufacturer’s the 80% to Minigenes to according of USA) 70% RT-PCR CA, approximately Carlsbad, OPTI-MEM to (Invitrogen, using grow 2000 2 (MUT)) with Lipofectamine pSPL3-Mutation to fetal transfected and and plate then 10% pSPL3-WT were 37 culture pSPL3-control, containing Cells (empty at 12-well medium. medium minigenes free mg/L) DMEM to antibiotic (100 in an transferred streptomycin in cultured were confluence and were cells U/L), cells transfection, (100 T) before penicillin 293 (FBS), (HEK serum T bovine 293 kidney coli epithelial E. Cells Human into T transformed 293 further HEK were Mutagenesis vectors of manufacturer. Transfection constructed the All by 2. instructed PLUS Table. Mutagenesis as SUP DH5 Site-Directed at USA) GeneArt demonstrated Massachusetts, the were through Scientific, primers variant plasmid The Fisher pSPL3 WT NheI: 1). (Thermo vector the TGGAGCˆTCGAG; Table. in System splicing (XhoI: (SUP introduced PP5 sites the was using up- enzyme fragment change into restriction of target sequence cloned each NheI nucleotides for and were designed Minigene 50-200 XhoI were sequence, A. the approximately Primers AATTTGˆCTAGC). linking intronic 1 by primers downstream Figure flanked specific at and exon, using shown interested sequence vector involving intronic trapping alleles stream exon reported (WT) pSPL3 previously variants, wild-type the as candidate the on the described based of were assay process constructions splicing splicing minigene the a on using effect the investigate of To exons. creation of or enhancers ends of 3’ Constructions elimination or Minigene 5’ assay: the splicing minigene to further close for and selected silencers; be will criteria following α optn el Tkr,Sia aa) olwdb cenn ycnetoa agrsequencing. sanger conventional by screening by followed Japan), Shiga, (Takara, cells competent ° μ o 0scns 58 seconds, 30 for C fec rmr 0.8 primer, each of M < .5wscniee ttsial significant. statistically considered was 0.05 ° o 0mnts h C rdcswr eaae by separated were products PCR The minutes. 10 for C SLC4A1 [20,21] 3 rey o ahvrain h rget with fragments the variation, each for Briefly, . 9of 39 , ° o 0scns n 72 and seconds, 30 for C μ MdTs n 0.5 and dNTPs, lM μ ATP6V1B1 oue 2 volume, l μ nvitro in lsi N nec ru of group each in DNA plasmid g μ fttlRAb random- by RNA total of g μ 8of 48 , ° na5 CO 5% a in C fcN,10 cDNA, of l nlsswsperformed was analysis μ rmrTRHS PrimerSTAR L ATP6V0A4 ° o 0seconds, 90 for C ® 2 μ n day One . IMedium f5 of L × of 2 , 100. × Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. cr fteW ’slc iefo .6t .4wt DP diinly nlsso S otovariants two to HSF of analysis Additionally, BDGP. with the 0.94 reduces c.370C to marginally Variant 0.96 variant 1). this from (Table that site demonstrated 0.76 splice was 3’ to and WT 5 0.96 the exon from of of site nucleotide score +3 splice the 3’ at WT substitutions the of score of 5 c.368G variant missense Presumed plasmids. WT the with compared c.368G of Variants variants variations 3.2.1 these sequences Therefore, of analysis. outcome sequencing Splicing by 3.2 confirmed splicing. further pre-mRNA was affect This not c.1437C did 2A). variant (Figure generated containing and constructs minigenes sites WT ESE the potential of four of bioinformatic inactivated 13 analysis variant using exon However, this sites that ESSs. ESS indicated new potential analysis four two HSF of of generation results the The and 1). c.1564G ESEs variant two missense of Presumed HSF. disruption tool the to lead to predicted c.1437C Variant splicing reading pre-mRNA open alter the c.1437C alter of not Variants 3.1.2 14- does exon bp the 174 of amounts of the loss P that the Sequencing 43.93%, revealed in c.1765C HEK293T 2A). transfected of (Figure resulting of the bp transcript 14 from 14 263 skipping exon extracted of exon cDNA of band of absence contained smaller Analysis The frame. product a 14. and splicing exon bp both larger contain 400 in not detected of the were band that bands larger showed electrophoresis a different analysis minigene: two mutant that revealed and assay WT minigene of the of c.1765C result of The effect cells. (c.1765C the mutant investigate To and 1). (c.1765C) (CTG Table WT at the ESSs shown overlapping bold, is two nucleotide ( generate (affected ESEs four also eliminates but only ) not variant the that predicted c.1765C Variant plasmids. c.1765C of Variant variations 3.1.1 sequences of outcome technique. Splicing experimental 3.1 of limitation site-directed to through due generated minigene successfully pSPL3-WT minigenes variants c.1251C candidate except all mutagenesis, template, the as minigenes WT (pSPL3- 15 Ex6), (pSPL3- (pSPL3- 5 exons in of the 3 analyses, more follows: silico sing is as in was generation these of study ESSs c.2257C results and this the c.1251C disruption in From and ESEs enrolled ATP6V0A4). variants of and candidate number ATP6V1B1 that c.1564G for effects these total combined 7 of (the had SLC4A1, distribution ESE variants for selected broke 6 Some and splicing. than ESS affect variants generate such both whether determine to assay ATP6V1B1 SLC4A1 > > ATP6V1B1 )a hw nTbe1 n iue1BD ieetcnrlmngnswr eeae compri- generated were minigenes control Different B-D. 1 Figure and 1, Table in shown as T) n c.1765C and A < SLC4A1 .5,sgetn httevratc.1765C variant the that suggesting 0.05), > Tsqecso xn 3(pSPL3- 13 exons of sequences WT )ad5in 5 and G) iifrai nlsswt DPdmntae htti ain rsial eue the reduces drastically variant this that demonstrated BDGP with analysis Bioinformatic . > > ATP6V0A4 pAg8Cs,lctda uloiepsto 3 rmte3 n feo 4 was 14, exon of end 3’ the from -36 position nucleotide at located (p.Arg589Cys), T pCs7Tp,lctda uloiepsto 6fo h ’edo xn1,was 13, exon of end 5’ the from +6 position nucleotide at located (p.Cys479Trp), G ATP6V1B1 eutdi TPRpout htmthdi iewt hs eeae yterespective the by generated those with size in matched that products RT-PCR in resulted x3,and Ex13), > > > ) in 6 T), pAg8Cs nue kpigo xn1 oprdwt h WT the with compared 14 exon of skipping induced (p.Arg589Cys) T pGy2Vl andc.370C (p.Gly123Val) T ATP6V0A4 > > x5,ad2 (pSPL3- 20 and Ex15), > in G pCs7Tp n c.1564G and (p.Cys479Trp) G eesgicnl nrae ihtoeo h Tpamd(30%versus (83.03% plasmid WT the of those with increased significantly were T x) (pSPL3- 6 Ex5), ATP6V0A4 > ATP6V1B1 ATP6V1B1 pGy2Vl scue ytefis uloiesbttto nexon in substitution nucleotide first the by caused is (p.Gly123Val) T > (c.52C )mngn eecetdadwr rnfce eaaeyit 293T into separately transfected were and created were minigene T) > feos3(pSPL3- 3 exons of pGu2Ls slctd13b ptemo xn1 (Table 13 exon of upstream bp 133 located is (p.Glu522Lys) A (c.368G > SLC4A1 n c.2257C and ,c.322C T, T ATP6V1B1 C TGCTG GC, ATP6V0A4 4 > SLC4A1 itre h omlslcn nvto(iue2D). (Figure vitro in splicing normal the disturbed T ATP6V1B1 > ,C.370C T, x3 n 4(pSPL3- 14 and Ex13) C > > CAT G GCAAGT, > pAg2Tp e oeo skipping 5 exon to led (p.Arg124Trp) T ,c.1571C T, x) 1(pSPL3- 11 Ex6), in T x0 epciey ihtecorresponding the With respectively. Ex20) > T ATP6V0A4 ATP6V0A4 pGu2Ls neo 3ddnot did 13 exon in (p.Glu522Lys) A > ybonomtc nlsswt HSF with analysis bioinformatics by ) ,c.481G T, SLC4A1 > C ,C.1572G T, CA TG GCAA, > pAg2Tp eutfrom result (p.Arg124Trp) T x) (pSPL3- 6 Ex3), > SLC4A1 a o nrdcdit the into introduced not was ATP6V1B1 > ,c.484G A, hra h mle did smaller the whereas , npemN splicing, pre-mRNA on T > SLC4A1 n c.1564G and G > ,c.2190C A, C Ex14), C,TGCTG GCA, > ,c.1102G T, x1,ad13 and Ex11), (c.1437C ATP6V0A4 ATP6V1B1 > and G > in A > > G, C A Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ain c.322C Variant the disrupt that nucleotides c.322C of 83 Variant variations of 3.3.1 sequences loss of the outcome in result Splicing would 3.3 transcript mutant (P the 2.60% Quantitative in of frame. 11. 11 WT reading exon exon with compared excluding of frame the transcript skipping reading amplicon of the the open larger products was the RT-PCR the broke smaller the c.1102G that that the that from showed transcript showed found and products products both two 11, two were of of exon analysis bp sequencing containing experiments 236 Direct transcript and splicing 2B). the bp (Figure was minigene 346 mutant of splicing, and products minigene mRNA two WT affected result, a variant As this carried. whether (A verify site ESS to one create (CACA and ESEs seven analysis broke minigene to the through c.1102G splicing Variant affect not did c.1102G it Variant ESSs, 3.2.3 two produce and ESEs 60.35% (P five was destroy transcripts to full HSF nucleotides, the c.481G variant 140 to although has addition, 6 In 6 exon exon lacking Because c.484G variant transcript 2B). nonsense the of (Figure that of rate 6 frame exon the reading by of that (P the produced mutated splicing revealed disrupt band The the single products would analysis. with the RT-PCR 6 to sequencing one exon corresponded by shorter of one (1.22%) a loss larger mRNA and a truncated transcripts: WT, mature a transcripts the two different and two produced in mRNA also, resulted minigene mature minigene, WT a The HEK293T. to in pattern corresponding spicing the analyzed and c.484G pSPL3, or WT the either inserted (CGAG site donor (Table protein nonfunctional AGAT, and truncated a produce (CCCGAG to the predicted However, is which 1). ((p.Glu162*), codon stop TAG reading open c.484G the Variant changing without c.484G level variant protein of between Nonsense the skipping connection 3.2.2 at 6 abnormal exon acids the in amino in resulting 26 c.368G result site, of variants would splicing loss of transcript the both mutant together, to Taken the leading frame. in 4, and 5 increase 3 significant exon a exons of was Deletion there that mutant c.370C 2D). showed of the HEK293T Figure transcript from contrast, prepared 5-skipping In cDNA exon exons. of of Analysis of pSPL3 products. 5’ exons WT two and c.370C the respectively variant to by 3’ bp, of flanked the result 368 5 only the and exon included whereas, bp contained one 263 fragment smaller c.368G of of the larger lane fragments while the vector, different that pSPL3 showed 2 c.368G the sequencing demonstrated variants Direct lane of 2B). WT effect (Figure splicing the the that determine showed To analysis ESSs. three generated c.370C both they that predicted > < 0.05). indicated minigene mutant the of pattern splicing the together, findings these Taking 2D). Figure 0.05, > T ,amngn otiigeo n akn nrncsqecswsue.Tersl fRT-PCR of result The used. was sequences intronic flanking and 5 exon containing minigene a T, GT) eie,HFas rdce htti ain ol as h ciaino cryptic a of activation the cause would variant this that predicted also HSF Besides, AGATG). G > ,CCGAG A, > > eeldoeuiu rgeto 6 pcrepnigt kpigo xn9i h mRNA; the in 9 exon of skipping to corresponding bp 263 of fragment unique one revealed T > fteitra oiino xn6in 6 exon of position internal the of T ,a osnevrat(.l18)lctda uloiepsto 3 rmte5 n of end 5’ the from +31 position nucleotide at located (p.Gln108*) variant nonsense a as T, nsilico in dnie tncetd oiin4 feo 1in 11 exon of 43 position nucleotide at identified A T GT.T ute lrf h mato ain c.484G variant of impact the clarify further To AGAT). > > pGn0* eutdi kpigo xn6. exon of skipping in resulted (p.Gln108*) T G nlssb S otaeidctdta c.484G that indicated software HSF by analysis .l38y)rsle nprilsipn feo 11. exon of skipping partial in resulted p.Glu368Lys) ( A A > CCGAG , G GG orsodn oanwhRP1bnigst Tbe1.I order In 1). (Table site binding hnRNPA1 new a to corresponding AGGG) nue xn6skipping. 6 exon induces T ,CACA A, > -uae xn6 ln ihtenab nrnsqecs notevector the into sequences, intron nearby the with along 6, exon T-mutated > > > .l12)ldt h kpigo xn6 exon of skipping the to led p.Glu162*) ( T pGu6Ls ls oc.484G to close (p.Glu161Lys) A eeldtodffrn lcrpoei ad hs ie correspond sizes whose bands electrophoresis different two revealed T > G oprdwt h Tpamd 2.9 ess47% P 4.78%, versus (28.19% plasmids WT the with compared T G G CGAG AG, ATP6V1B1 > G ACA AG, > lee ekyslcn eutn na95%o h aberrant the of 9.51% a in resulting splicing weakly altered A n c.370C and T ATP6V1B1 5 ATP6V0A4 G G ATP6V1B1 . ,GAG A, GG CA AGGG, > rknpoe eonto fteacceptor the of recognition proper broken T lesaGGcdnfrGut premature a to Glu for codon GAG a alters G . rncit uniaieaayi fthe of analysis Quantitative transcript. G,btas rae w Ss(G ESSs two creates also but AG), > G ATP6V1B1 a lopeitdb h software the by predicted also was T G,CA AGG, > < > o nydsut v ESEs five disrupts only not T .5 iue2) naddition, In 2D). Figure 0.05, nteslcn rcs,we process, splicing the on T G eepeitdb HSF by predicted were AGGG, . nvitro in G AGGGA) eealso were > < and T 0.05, T Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. steatrto fpemN piig sn iifraistos oa f1 igebs ainsin variants single-base 15 of variants these total of a some tools, of bioinformatics effect Using pathogenic splicing. the identified that pre-mRNA often are assumed of variants, SLC4A1 we synonymous alteration or Herein, nonsense the dRTA. missense, primary is to lead with to patients predicted in are which functionally of to many the tool gene, simple in relatively substitutions and reliable Single-base effective, due an detect as splicing to confirmed was potential difficult assay assay are minigene pathway mRNAs decay the and mRNA on unavailable, variations, nonsense-mediated is based are sequence the it of individual of cases, Currently, activation many affected pathogenicity the level. In the the to sequences. DNA from regulatory assess the splicing samples to the at RNA affecting performed variants however, only by be caused performed should abnormalities missense, splicing is analysis as especially RNA analysis elements classified that gene been RNA believed when have important widely variants dinucleotide or other site synonymous sites altering causing splice or splice by conserved splicing, nonsense new also the creating pre-mRNA outside but by variants AS), in or exonic Traditionally, or sites) defects (DS binding to sites silencers lead or splice enhancers classic will that (splicing disrupting variations diseases exons by many genetic and only so many introns, not introns of and severity of exons the definition of modifying recognition correct accurate the on nucleotides. break 94 depends of would splicing loss transcript pre-mRNA the mutant Normal to the due in frame 15 reading exon open of Discussion the skipping of the the alteration Besides, are the 2D). amplicons in products significant Figure result smaller a respectively, all was c.1571C the 0, of there versus in and that sequencing 84.48% revealed 15-skipping transcripts Direct HEK293T exon 2C). exons-included from of prepared (Figure the increase cDNA respectively were of bp, Analysis amplicons transcripts. 357 larger exons-excluded fragment and one the bp demonstrated that 263 minigene lane pro- showed WT of a splicing The fragments used the different. different also were showed Additionally, two contains minigenes we variation. results that variants, WT bp after analysis and two 357 RT-PCR analyzed mutant of of sequences. the be (CC effect intronic by sites not splicing produced surrounding donor could ducts and the WT it 15 examine the whereas exon break to 0.8, containing to together, the is predicted Taken that HSF 15 splicing. demonstrated with BDGP intron affecting variant with of site this analysis splice site of Bioinformatic donor c.1572G 15. donor analysis WT variant exon the Synonymous the in 1). of of substitution (Table score nucleotide score BDGP the last with reduces the 0.68 variant G to this the 0.8 that from demonstrated was 15 intron site, of splice 3’ the of stream c.1571C 15. variant exon Missense of protein skipping the in at c.1571C resulted 98-139) (p.Pro524Pro) (residues variant generation. acids ESSs amino and 42 3.3.2Missense destruction c.322C of ESEs variant loss of Therefore, pSPL3 the because frame. 5’ in reading skipping and result open 9 corresponding 3’ would the bp the altering transcript 263 only without mutant of assay,level included the (1.78%) in minigene one fragment in 6 by smaller smaller 6 exon a vector the exon of revealed mutant while contained skipping also vector, the fragment incorrect vector pSPL3 of WT larger to the that the the of However, from exons that (24.22%). detected two showed exons by were results flanked bp sequencing 6 263 Direct exon and 2C). bp (Figure 389 respectively of demons- fragments HSF software two The ly, 1). (Table protein nonfunctional GTTA and truncated c.322C a that generate trated to predicted is 6, exon C ,AGTTA A, , ATP6V1B1 > o nydsrydsxEE ( ESEs six destroyed only not T C ,btas nedr he Ss(TA ESSs three engenders also but ), [25] and ATP6V0A4 > hc a enetnieyvldtdi u rvosstudies previous our in validated extensively been has which , ATP6V0A4 pPo2Lu asdb usiuin tte- uloieo xn1 down- 15 exon of nucleotide -2 the at substitutions by caused (p.Pro524Leu) T SLC4A1 ATP6V0A4 xn1,weesbt fmtn c.1571C mutant of both whereas 15, exon > pPo2Lu n yoyosvratc.1572G variant synonymous and (p.Pro524Leu) T eewr eetdadtse ihamngn sa.Teevariants These assay. minigene a with tested and selected were gene > n c.1572G and T , ATP6V1B1 xetframtr rncit(iue2) h kpigof skipping The 2D). (Figure transcript mature a for expect [4] ti urnl la htteevrain ffc splicing affect variations these that clear currently is It . C 6 GA,TA AGGAA, > , ATP6V0A4 ihtecnrlpamd(.4 ess0and 0 versus (3.74% plasmid control the with A T G,TTA AGG, [24] otntl,afntoa piigassay splicing functional a Fortunately, . C , GA TA AGGA, > FOXI1 T pGn0* assprilexon partial causes (p.Gln108*) T G AGTTA AG, [4,5,22,23] > G n c.1572G and T , > TAA,ms probably most GTAATA), WDR72 pPo2Po affected (p.Pro524Pro) A C . GA,TTA AGGAA, [20,21,26] T and ) Consequent- A). > generated A . ATP6V0A4 ATP6V1C2 C AG, > A Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ffiinyo h ’sD r3s So ycetn e piests eunevrain htocri the in respectively occur 3’ss, that and variations 5’s Sequence GT recognize invariant sites. to the recognition splice spliceosome because the new the aberration, decreasing c.370C splicing creating for by cause and by required either will or are splicing exons AG AS normal flanking dinucleotides and 3’ss affect AG may or and AS), DS GT or conserved 5’ss (DS the sites of splice efficiency classical to c.368G dRTA, near with this or associated consequence, variants a exonic three As categorize in H protein. to of us truncated 7 activity allows a assay and transport of Minigene the 5 production abolish exons the or of H and reduce ligation apical acids may the and amino and of Subsequently, motifs 42 skipping. A4 ESSs of 6 subunit and lack exon mutated ESEs a causing related in vitro influenced result in would it splicing that normal showed generation the results the disturbed and Our c.484G decay. sites 13. to ESE mRNA exon Similar functional nonsense-mediated in hnRNPA1. seven codon binds of 25th which disruption the site, the ATP6V0A4 at ESS Moreover, to termination functional HSF. due premature a with probably and of motifs is frameshift this ESS The subsequent that two site. a hypothesize of ESS in We gain functional in resulting a the 11, codon creating and exon 22th and/or ESE lack the site five ESE at of c.1102G functional termination variant loss a missense premature the affecting and predicted by frameshift probably analysis c.484G subsequent mRNA bioinformatics for the a assay in with splicing 7 inclusion minigene exon 6 the phenotype and exon between prevents p.Glu162*, correlation mutation, it of nonsense elements as loss exonic predicted the the alter was in by stop mutations occasionally nonsense demonstrated by premature can induced be skipping a variants can generate nonsense which pathway, genotype that to and splicing found considered the have generally affect studies and is of many phenotype substitution, However, a We single-nucleotide to codon. frame. leading a reading and variant, protein open Nonsense encoded the the change of c.1765C morphogenesis not containing abnormal did transcript in dRTA. which this a resulting 14, that generated 14, revealed exon only functional exon lacking analysis not lacking and minigene it vitro transcripts structural that in of in hypothesized Further part ESEs/ESSs. resulting loading of membrane, produced defective proportion variant the cell the or the in protein, imbalance to AE1 significant of transferring membrane the after in cell variant the structure the abnormalities to of advanced transported forms the protein completely other pathogenic two AE1 to of and be the failure region, to of to transmembrane confirmed regions lead 6th been may transmembrane have the 7th p.Arg589Ser) of (p.Arg589His, cytoplasm the site the and same of 6th apex the the between at domain and intracellular the in located the inhibit regulate inheritance or to dominant promote coordinating thus can proportion factors, c.1765C ESS, the Variant protein exons and different in of recruiting imbalance ESEs splicing by correct significant including sites the a splicing sequences, causing surrounding regulatory by of splicing splicing recognition c.1765C pre-mRNA Many of affect ESEs/ESSs. they variants of to that four found shown have were of exon, them PCR of quantitative 8 c.1102G Consequently, by sequencing. cells, analysis DNA HEK293T either Transcript and into to RT-PCR transfected ligated by plasmids were skipping. generate Minigene constructs analyzed exon ESSs), The was cause strength. of constructed. mRNA site creation were splice the sequences or of and genomic ESEs reduction mutant significant of a or (disruption induce WT sequences or regulatory sites splicing splice new modify to predicted were ATP6V1B1 > in A > a ntal enda osnevrat(.l18)laigt nual RAdet the to due mRNA unable an to leading (p.Gln108*) variant nonsense as defined initially was in T [30,31] [28-30] c.1571C , ATP6V1B1 > pAg8Cs in (p.Arg589Cys) T ATP6V1B1 h euto h’ eerhhseuiae h ealdmlclrmcaim fexon of mechanisms molecular detailed the elucidated has research Zhu’s of result The . nti td,oln otaeHFwsue opeitta c.1765C that predict to used was HSF software online study, this In . [28] > ntepeiu td,teAg0 a osdrda uainhtpt hc is which hotspot, mutation a as considered was Arg509 the study, previous the In . > n c.1572G and T pGu6Ls in (p.Glu368Lys) A [27] n c.322C and , hrfr,a mrprEE S ai ilpeetcretpeN splicing. preRNA correct prevent will ratio ESS ESE/ improper an Therefore, . novn h rtad+ uloieo xn5 epciey eeinitially were respectively, 5, exon of nucleotide +3 and first the involving + APpoenwuddsrytefis oooia oano cytoplasmic of domain topological first the destroy would protein -ATP SLC4A1 > > in A in T DMD ATP6V0A4 ATP6V1B1 eewsrpre ocuedT ntefr fautosomal of form the in dRTA cause to reported was gene ATP6V0A4 gene 7 + -ATPase. [30] lognrtdapr faern rncit that transcripts aberrant of part a generated also nti otx,vratc.484G variant context, this In . sslcn uain hs ainslctdat located variants These mutation. splicing as , ee oae nteitra oiino the of position internal the in located gene, > in T > > ATP6V1B1, in T u lopoue transcript a produced also but T [29,30] [1,2,25] SLC4A1 ainsa .Ag8 site Arg589 p. at Variants . ainsc.368G Variants . ain c.322C variant > > > c.481G , n c.370C and T > in T ol as a cause could T hwdthat showed T ATP6V1B1 > and T > in T > > T T Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. sncsayt netgt h piigipc falsnnmu usiuin oeaut hi clinical their c.368G evaluate of to product it substitutions PCR c.1765C that however, synonymous the highlight variants, all which 8 of Jourdy, variant Among Yohann impact synonymous by splicing as assay the Just minigene investigate a significance splicing. to using the abnormal necessary mutations from to splicing is But as lead perspective. 94 Significantly, reclassified may protein of were a 16. substitutions loss exon from these a in benign in view, 13th c.1572G results be of the 15 to at point exon considered and termination of mRNA’s generally 3.74% premature skipping are approximately and The substitutions increasing frameshift respectively. synonymous by subsequent WT, a splicing with with normal compared nucleotides altered exclusion 15 certainly exon variants two 84.48% these that c.1572G that showed that revealed further Such analysis predicted BDGP HSF respectively. Our But (p.Pro524Pro), sites. variant splicing classic synonymous of and c.1571C recognition the (p.Pro524Leu) reduces missense usually substitution as 5. identified exon were the 15, of skipping the c.1571C caused Variants certainly they c.368G that variants revealed of mutations Analysis two respectively. p.Arg124Trp, c.370C and p.Gly123Val and mutations missense as reported c.1571C rmsit h eann RAi aaigdet h uae mn cdcag,wihcudrsl in result could c.1437C which of to change, decay. variants due acid mRNA codons addition, amino mediated the of In mutated nonsense of termination the skipping or premature to the polypeptide or to due truncated domains due damaging important protein, deleterious is some non-functional is mRNA probably of transcript remaining they part the the of so of frameshift, part loss exon, some cause corresponding the would missing while which transcript exon, effect: the damaging was double product a RT-PCR have one that except product hssuywsspotdb rnsfo h aua cec onaino hn (81873594). China of Foundation Science Natural the from grants by supported was study This interests competing no Funding have they that declare authors The study interests This Competing minigene specimens, skipping. kidney exon or applicable. in Not RNA resulting materials patients’ dRTA, and DS, the the data in 5’ obtain of level a to Availability mRNA tool. the failure and valuable at the AS a mutations be of ’ point interfere may exon condition 3 assay or of the of ESSs, effect under the produce site be assessing and especially splicing should of ESEs which importance the disrupt the variations, of either emphasized splicing variants recognition These as the variants account. synonymous with into in or pathogenicity variants nonsense their exonic missense, taken of presumed analysis previously extensive 8 an performed have ATP6V0A4 expression we surface sensitivity cell H high minigenes conclusion, the certain or detect our In and a AE1 of cDNA had after the into software variants results activity with these online However, transport inconsistent introduce the ion not site. was of did and which we splicing results Also, splicing, the of specificity. pre-mRNA that low strength indicating affect and recognition software, not of of did results variants change predicted these the that cause demonstrated to assay or HSF of c.2190C > > > > eutdi h euto frcgiinsrnt fdnrslcn ie hl c.1572G while site, splicing donor of strength recognition of reduction the in resulted T n c.1572G and T in G fthe of A > [2] soitdwt RAuigbonomtc ol n iigns hssuyalwdt reclassify to allowed study This mini-genes. and tools bioinformatics using dRTA with associated nBG hwdardcini h cr fte3 piest.Temngn nlsso these of analysis minigene The site. splice 3’ the of score the in reduction a showed BDGP in T . ATP6V0A4 > > ATP6V0A4 n c.1572G and T in T SLC4A1 > in A eefudt nunesronigEE n Ssmtf yteassessment the by motifs ESSs and ESEs surrounding influence to found were , nti td rdcdteaern rncit h v yoyosvariants synonymous five the transcript, aberrant the produced study this in > ATP6V0A4 > n c.1564G and G c.370C , > etoe h Tdnrsts osqety h iieeanalysis minigene the Consequently, sites. donor WT the destroyed A in A ATP6V0A4 + > APs uain,wihrqiefrhrstudy. further require which mutations, -ATPase ,c.481G T, ee tl rdcdoetasrp ihtesz fteWT the of size the with transcript one produced still gene, > > in T in A 8 > oae tte- n h atncetd fexon of nucleotide last the and -2 the at located , ATP6V1B1 n c.1102G and T SLC4A1 c.481G , a nqetasrp akn xn5, exon lacking transcript unique a was > in A > in A ATP6V1B1 SLC4A1 ATP6V1B1 , n c.322C and , ATP6V1B1 n c.52C and > i not. did A > > and > T, T, T Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 1]PlzoV rvnaoA ehrciF ta.Tegntcadciia pcrmo ag ootof cohort large a of spectrum clinical 1243-1255. and 91(5): genetic 2017, child International, The Chinese Kidney al. acidosis[J]. a et tubular in 364. renal F, mutations 19(1): distal Becherucci ATP6V1B1 2018, with A, heterozygous report[J]. patients Provenzano compound case V, Novel a Palazzo acidosis: al. [17] tubular et renal Y, 567-579. distal novel Gao primary 97(3): a J, with 2020, as patient ATP6V1C2 International, Lu tryptophan- identified Kidney X, by sequencing acidosis[J]. Zhao caused exome tubular [16] Whole renal acidosis distal al. tubular recessive et renal for M, 409-418. Tarsio gene Distal V, candidate 94(5): Klambt 2018, al. T, Genetics, Jobst-Schwan Clinical et [15] mutations[J]. N, Mutations (WDR72) Sawasdee Recessive 72 with domain C, Patients repeat Nettuwakul in aspartate N, Deafness and Rungroj Acidosis [14] al. 1041-1048. et 29(3): N, 2018, Edwards FOXI1[J]. D, in Nilsson American S, the Enerback of [13] Journal vacuolar 3027-3038. acidosis[J]. (a4) ATP6V0A4 14(12): tubular the renal 2003, of distal Nephrology, regulation of of and form Society Localization inherited an al. in et defective KE, subunit distal 1858-1866. H+-ATPase Finberg inherited N, cells[J]. 17(7): cause Schulz duct PA, 2006, that collecting Stehberger Nephrology, mutations medullary [12] of subunit inner vacuolar Society in B1 -ATPase trafficking American kidney and H+ the Vacuolar assembly new of pump al. Journal a proton et affect encoding SK, acidosis Singh tubular ATP6N1B, renal G, in Nature Li hearing[J]. Q, Mutations preserved Yang with [11] acidosis al. tubular 71-75. renal et 26(1): distal Molecular KA, recessive 2000, (review)[J]. cause Genetics, Choate subunit, developments J, 116-kD recent pump Skaug proton (AE1): AN, 3 Smith band [10] of 155-165. function 14(4): and Renal 1997, Distal structure Biology, with 513-521. Membrane The Patients 43(2): Pediatric MJ. 2018, in Tanner Research, Analysis [9] Genotype-Phenotype Pressure Blood al. et and HS, Kidney Hyun Acidosis[J]. Tubular MH, Cho E, Biochemical Park in Trends [8] splicing[J]. pre-mRNA regulating 169-178. hereditary in structure 35(3): of RNA cause 2010, of frequent Sciences, Role most JA. the Berglund mutations MB, affecting Warf splicing [7] 1900-1903. variants Are 579(9): DNA al. 2005, human et 39-90. Letters, C, Understanding FEBS Ouzounis 103: B, disease?[J]. 2019, Audit al. Genetics, N, in et Lopez-Bigas Advances C, [6] era[J]. Masotti NGS RT, the in Interdiscip Carmo splicing Wiley do splicing[J]. pre-mRNA pre-mRNA LG, Biology, in Dufner-Almeida definition Cell intron [5] Molecular and 49-60. Exon Reviews: 4(1): E. Nature 2013, Buratti spliceosome[J]. RNA, M, Rev Baralle the L, of Conti life De [4] the in minigene day a 108-121. A using 15(2): variations Z. nucleotide 2014, Wang F8 AG, 26 Matera of analysis [3] Splicing 1126: 306-315. al. 2014, 25(2): et Biology, 2019, C, Haemophilia, Molecular Nougier assay[J]. in M, Fretigny Methods Y, Jourdy minigenes[J]. [2] of splicing Cell-based KW. Lynch 243-255. SA, Smith [1] Reference applicable. Not Acknowledgements 9 Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n ed oarltvl idrtnlpeoyeJ.IvsiaieOhhlooyadVsa cec,2010, skipping Science, exon Visual induces and CEP290 Ophthalmology in Investigative mutation phenotype[J]. nonsense novel retinal is A mild Cl-/HCO3- 3646-3652. acidosis relatively al. 3) 51(7): a tubular et (band RW, to renal AE1 Collin leads JW, distal the and Pott in dominant KW, mutation 6380-6388. Autosomal Littink R589H 273(11): [31] the 1998, Chemistry, al. for Biological heterozygosity et of with D, Journal AE1 families exchanger[J]. Prabakaran 6337-6342. gene three the 95(11): C, exchanger in of 1998, Shayakul chloride-bicarbonate associated Proceedings America, P, of the acidosis[J]. States Jarolim tubular in United renal [30] Mutations the distal of recessive al. 1 Sciences autosomal of exchanger et Academy not anion AZ, National but of Gyory dominant mutation FJ, autosomal R589C Gainza novo 644-648. cause FE, de 18(7): A Karet 2003, OCRL Nephrology, [29] Pediatric al. Exonic acidosis[J]. et of tubular S, renal Analysis Vasuvattakul distal S, Splicing causing branch Kirdpon 9(1). 7 S, al. 2018, intron Sritippayawan Disease[J]. et the [28] Dent-2 including E, or gene Ramos-Trujillo Syndrome SLC5A2 Lowe A, the 33920. Causing Perdomo-Ramirez in 6: Mutations deletion L, 2016, recurrent Reports, Suarez-Artiles A Scientific [27] glucosuria[J]. al. renal et familial Y, 188. for Lang 9: responsible L, Spliceogenic 2018, site Cui Eight Genet, X, Front of Assays[J]. Zhao Identification of functional Minigene [26] Journal al. by and 16 et European Exon predictions B, genes[J]. BRCA2 Diez-Gomez bioinformatics BRCA in A, Variants of Valenzuela-Palomo the E, Contribution of Fraile-Bethencourt variants [25] unclassified al. 1052-1058. of 19(10): et MSH2 2011, interpretation P, and Genetics, the Gaildrat MLH1 Human to of S, 145-154. effect assays Krieger 27(2): the splicing 2006, for JC, Mutation, analysis Thery Human mRNA splicing[J]. Systematic [24] aberrant al. on et mutations mutations C, silent Navarro exonic and MP, missense nonsense: Busine 285-298. understanding J, 3(4): and Auclair 2002, silence [23] Genet, to Rev Listening Nat AR. 2018, splicing[J]. Krainer report[J]. affect c.3610G SL, case that Chew TSC2 a L, novel complex: Cartegni a sclerosis [22] of tuberous a classical Identification in with Skipping patient al. 173. Exon a et Cause in 19(1): Q, splicing Gene Wang aberrant SLC5A2 J, to the Wang contribute in Variants R, Exonic Zhang 585064. Six [21] 11: al. Renal 2020, et Distal Genet, J, Primary Front tubular Wang with Assay[J]. renal Y, Children Minigene Wang Chinese hereditary 599-606. S, in a Wang 22(10): Mutations Novel 2018, [20] of Five Biomarkers, discussion Mol al. Test function et Genet Y, and Acidosis[J]. Lang Screening Tubular C, Wang R, al. 159. Zhang [19] et 11(3): YB, 2020, gene[J]. Zhu pathogenic HL, family Wang acidosis L, Chen [18] 10 > ,pG24 mutation p.G1204R A, Posted on Authorea 7 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161273789.95036962/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. by-minigene-assay eight-exonic-variants-in-the-slc4a1-atp6v1b1-and-atp6v0a4-gene-that-alter-rna-splicing- 1.pdf Table file Hosted vial at available https://authorea.com/users/394318/articles/507714-identification-of- 11