Propionic acidemia: mutation update and functional and structural effects of the variant alleles

Molecular Genetics and Metabolism 83 (2004) 28–37

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Propionic acidemia: mutation update and functional and structural eVects of the variant alleles

L.R. Desviat, B. Pérez, C. Pérez-Cerdá, P. Rodríguez-Pombo, S. Clavero, M. Ugarte¤

Centro de Biología Molecular “Severo Ochoa” CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 22 June 2004; received in revised form 27 July 2004; accepted 2 August 2004Available online 11 September 2004

Abstract

Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, alife-threatening inborn error of metabolism with autosomal recessive inheritance. To date, 41 mutations in the PCCA gene and 54 inthe PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of smallinsertions and deletions and splicing defects. A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with noprevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied. For thePCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Orientalpopulations. These two populations show a diVerent mutational spectrum, only sharing some involving CpG dinucleotides probablyas recurrent mutational events. Functional characterization of the mutant missense alleles has been accomplished using diVerent pro-karyotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models. For the PCCAgene, the main molecular eVect of the expressed mutations is related to protein instability, except two mutations in the active site pre-dictably aVecting ATP binding. In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site confor-mation resulting in diminished activity. A few carboxy-terminal PCCB mutations aVect the interaction between subunits and theassembly with PCCA to form a functional PCC oligomer. The amount of normal transcripts resulting from some PCCA and PCCBsplicing mutations has also been analyzed. Overall, the data generated from the expression analysis reveal potential genotype–pheno-type correlations for this clinically heterogeneous disorder. 2004 Elsevier Inc. All rights reserved.

Keywords: Propionic acidemia; Mutations; PCCA; PCCB; Propionyl-CoA carboxylase; Expression analysis; Structural models; Genotype–phenotype

Introduction

Propionic acidemia (PA; MIM #606054) is an auto-somal recessive disorder of organic acid metabolismcaused by a defect of propionyl-CoA carboxylase (PCC,EC 6.4.1.3) [1]. This biotin-dependent mitochondrialenzyme consists of two non-identical subunits, �- and�-encoded by PCCA (MIM 232000) and PCCB (MIM232050) genes, respectively. The �-subunit contains thebiotin attachment site at the C-terminus and a biotin

* Corresponding author. Fax: +3491 734 7797.E-mail address: [email protected] (M. Ugarte).

1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ymgme.2004.08.001

carboxylase domain. An enzyme deWciency can rise frommutations in either the PCCA or PCCB genes. PCCcatalyses the carboxylation of propionyl-CoA to D-meth-ylmalonil-CoA in the catabolic pathway of odd-num-bered carbon fatty acids and aminoacids, namelyisoleucine, valine, threonine, and methionine. Thebiochemical features of PA include mild to severeketoacidosis, hyperammonemia, hyperglycinemia-uria,mild to moderate elevation of lactate, low levels of freeand total carnitine, high concentration of plasma odd-chain fatty acids and a diagnostic urine organic acid pro-Wle (3-hydroxypropionate, methylcitrate, propionylglycine,and tiglylglycine) [2]. The common clinical presentationinclude frequent vomiting, lethargy, refusal to feed, and

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L.R. Desviat et al. / Molecular Genetics and Metabolism 83 (2004) 28–37 29

hypotonia. In most of the patients there is a neonatalclinical onset associated with development delay andearly death, but late-onset patients are also describedwith a milder course [3,4]. Conventional treatment of PAattempts to limit Xux through protein degradation. Thisrequires the dietary restriction of propionicogenic aminoacids, increase of caloric intake, avoidance of long-fast-ing periods and carnitine supplementation. On thewhole, PA is a severe disorder with a poor survival ratefor the early-onset patients [5]. Survivors may havenutritional problems and various degrees of develop-mental delay and neurological impairment.

The worldwide incidence of PA is estimated as verylow (»1:50,000), although in speciWc populations, suchas the Greenland Inuit, the incidence is much higher(1:1000) due to a founder eVect and genetic drift [6].However, recent neonatal screening employing tandemmass spectrometry has revealed a highly variable fre-quency of PA, ranging from 1:17,400 in Japan [7] or1:27,264 in Saudi Arabia [8], to 1:129,000 in USA [9] or1:250,000 in Germany [10]. In Japan, a recent study ana-lyzes the molecular basis of the mild form of PA identi-Wed at high frequency by tandem mass screening [7]. Theclinical signiWcance of this Wnding remains uncertain asmost of the neonatally detected patients exhibit a mildform of the disease, some of them remaining asymptom-atic up to date. In addition, this correlates with a muta-tional spectrum quite diVerent from that of patientsdiagnosed based on clinical symptoms, as also describedfor medium-chain acyl-CoA dehydrogenase deWciency,recently reported to appear with high frequency in neo-natal screening programs [11].

The diagnosis of PA is based on clinical symptomsand biochemical analysis, and the enzyme deWciency isconWrmed by assaying PCC activity in lymphocytes and/or skin Wbroblasts. The defective gene, PCCA or PCCB,can be determined by complementation studies in whichpatient Wbroblast cell lines are fused with tester strains ofthe known complementation groups followed by assayfor restoration of propionate metabolism [12,13]. Analternative method involves transfection of normalPCCA and PCCB cDNAs in the patient’s cells and mea-suring the recovery of enzyme activity when the defectivegene is expressed [14].

Molecular basis of PA

Complementation analysis of cultured skin Wbro-blasts from patients with PA has revealed two maincomplementation groups, pccA and pccBC [12]. Mutantsof the pccA group have a primary defect in the PCCAgene (MIM#232000), encoding the � subunit of propio-nyl-CoA carboxylase. Mutants of the pccBC group andsubgroups have defects in the PCCB gene(MIM#232050), encoding the � subunit.

The cDNAs for the � and � subunits have beencloned (GenBank Accession Nos. AF385926 andNM_00532, respectively) and the corresponding nucleargenes PCCA and PCCB have been mapped to chromo-somes 13q32 and 3q13.3-q22, respectively. The structureof the nuclear genes PCCA and PCCB has been eluci-dated. The PCCA gene comprises 24 exons (GenBankAccession Nos. AY035786–AY035808) and the transla-tion initiation codon has been redeWned 75 nucleotidesupstream of the originally reported, which accounts for25 extra amino acids in the amino terminal region of theprotein. Although the mature protein is predicted to bethe same, this new initiation codon has resulted in therenumbering of nucleotides and amino acids [15]. ThePCCB gene contains 15 exons (GenBank Accession Nos.AJ006499–AJ006487) [16]. Mutations in either gene cancause propionic acidemia. A website with informationon propionyl-CoA carboxylase and its mutations ismaintained at the University of Colorado(www.uchsc.edu/sm/cbs/pcc/pccmain.htm).

Variant alleles

Up to date, 44 variant alleles (41 mutations and 3SNPs) have been reported in the PCCA gene and 55 (54mutations and one SNP) in the PCCB gene (Tables 1and 2). In both genes, missense mutations are the mostfrequent defects (39 and 46%, for PCCA and PCCB,respectively), followed by small insertions/deletions andsplicing mutations (24–29% each in either gene), most ofthem predictably resulting in a truncated protein. Themutations are distributed all along the gene sequences,although some clustering is observed in exon 13 in thePCCA gene and in exons 12 and 15 in the PCCB genesuggesting that they could be hot spots for mutations orpart of important functional domains.

In the PCCA gene none of the mutations detected ispredominant in Caucasians, while in Japanese, threemutations, 922-923insT, 1644-6C > G, and R399Q,account for 56% of the total mutant alleles [17]. Themutational spectrum is clearly population speciWc (Table1), the only exception is mutation R77W, detected inJapanese and in Spanish patients, probably as the resultof recurrence as it aVects a CpG dinucleotide.

In the PCCB gene a few mutations account for mostof the patients in diVerent ethnic groups, although theoverall prevalence of each mutation is unknown. AmongCaucasians, 1218del14ins12 is the most common muta-tion [16,18], although 1172_1173insT and E168K arealso frequent speciWcally in the Spanish and Latin Amer-ican populations [16], while among Oriental populationsT428I and R410W are prevalent [17,19,20]. Once again,the presence of mutations R165W, R410W, and R512Cin Orientals and Caucasians could be explained by arecurrence mechanism, as they are present on CpG

http://www.uchsc.edu/sm/cbs/pcc/pccmain.htm














30 L.R. Desviat et al. / Molecular Genetics and Metabolism 83 (2004) 28–37

dinucleotides. In the Inuit population in Greenland,mutation A513_R514insP is prevalent, with a carrier fre-quency of 5% and a high degree of linkage disequilib-rium with an adjacent STR marker allele, suggesting it isa founder mutation in this region [6].

Functional and structural analysis of the variant alleles

To conWrm the pathogenic eVect of the identiWed mis-sense mutations and to analyze their functional conse-quences, diVerent expression systems have been used:

Table 1Mutations and polymorphisms in the PCCA gene

Nucleotide numbering starts at the ATG translation initiation codon using the new nomenclature described by [15]. Designation corresponds to thatdescribed by [43]. In the case of insertions the nucleotide change also includes (in brackets) the latest recommended designation, i.e., as duplicationsdesigning the most 3� nucleotide (http://www.hgvs.org/).

Nucleotide change Exon/intron Predicted eVect Origin References

Missense mutationsc.223G > C E3 A75P USA [32]c.229C > T E3 R77W Spain, Japan [17,38,39]c.412G > A E5 A138T Spain [38]c.491T > C E7 I164T Spain [38]c.590G > A E7 G197E Japan [17]c.686T > A E9 M229K USA [32]c.890A > G E11 Q297R Japan [17,39]c.1103A > G E13 D368G France [32]c.1118T > A E13 M373K Brazil, Austria [38]c.1136G > T E13 G379V USA [32]c.1192T > C E13 C398R Japan [17]c.1196G > A E13 R399Q Japan [39]c.1268C > T E14 P423L Japan [39]c.1676G > T E19 W559L Japan [39]c.1891G > C E21 G631R Spain [38]c.2002G > A E22 G668R USA [32]

Nonsense mutationsc.937C > T E12 R313X Canada, USA, Spain [38,40]c.1685C > G E19 S562X Spain [38]

Deletions/insertionsc.419delA E6 H140fs USA [41]c.440delC E6 S147X Spain [15]c.721_722delG E10 G241fs USA [41]c.775del5 E10 L259fs USA [40]c.922_923insT (c.923dupT) E12 L308fs Japan [17,41]c.1096_1097insG (c.1097dupG) E13 L367fs Croatia [41]c.1135_1136delG E13 G379fs Spain [41]c.1190del4 E13 E397fs France [40]c.1593delATT E18 L532del USA [41]c.2103delT E23 T704fs Spain [15]c.2133del3 E24 C712del Unknown [32]

Splicing mutationsc.184-17delTG I2 T62_S100del39 (�exons 3_4) Italy [41]c.231+44del4 I3 T62_S100del39 (�exons 3_4) Italy [41]c.1284+1G > A I14 V356_G428del73 (�exons 13_14) USA [15]c.1540+1G > A I17 V478_G514del37 (�exon 17) Japan [17,39]c.1643+1G > A I18 G514f (�exon 18) Japan [17]c.1644-6C > G I18 R548f (�exon 19) Japan [17]c.1746G > A E19 R548f (�exon 19) Japan [17]c.1746+5G > C I19 R548f (�exon 19) USA [40]c.1846-2del9 I20 C616_V633del18 (�exon 21) Spain [42]c.1899+3del4 I21 C616_V633del18 (�exon 21) Spain [42]c.1899+3insCT I21 C616_V633del18 (�exon 21) Spain [42]c.2041-2A > G I22 V681_T706del26 (�exon 23) USA [41]

Polymorphismsc.627A > G E8 A209A Spain [38]c.1423A > G E16 I475V Spain [38]c.1651G > T E19 V551F Arab [41]

http://www.hgvs.org/











b Multiple splicing abnormalities reported.

Table 2Mutations and polymorphisms in the PCCB gene

Nucleotide numbering starts at the ATG translation initiation codon. Designation corresponds to that described by [43]. In the case of insertions thenucleotide change also includes (in brackets) the latest recommended designation, i.e., as duplications designing the most 3� nucleotide (http://www.hgvs.org/).

a Activated cryptic splice site.

Nucleotide change Exon/intron Predicted eVect Origin References

Missense mutationsc.49C>A E1 L17M Spain [44]c.131G>C E1 R44P Spain [16]c.201G>C E2 R67S Spain [41]c.318C>A E3 S106R Spain [16]c.319G>A E3 V107M Croatia [41]c.335G>A E3 G112D Italy, USA [41]c.391G>C E4 G131R Spain [16]c.457G>C E5 A153P Japan [17]c.493C>T E5 R165W Japan, Ecuador [16,17,20]c.494G>A E5 R165Q Spain [41]c.502G>A E5 E168K Spain, Chile [16]c.562G>A E6 G188R Italy, USA [41]c.593G>A E6 G198D Spain [16]c.614T>A E6 V205D Austria [44]c.683C>T E7 P228L USA [45]c.737G>T E7 G246V Spain [41]c.1228C>T E12 R410W Japan, Spain, Brazil, Greece [16,17,19,45]c.1283C>T E12 T428I Japan, Korea [17,20,46]c.1304A>G E13 Y435C Japan [7]c.1316TA>G E13 Y439C Japan, Korea [7,46]c.1325T>C E13 M442T Spain [44]c.1402G>A E14 A468T Japan [7]c.1534C>T E15 R512C Spain, Japan [16,17]c.1556T>C E15 L519P Spain [16]c.1606A>G E15 N536D USA [45]

Nonsense mutationsc.280G>T E2 G94X Spain [41]c.1495C>T E14 R499X Japan [7,17,20]c.1593G>A E15 W531X Spain [16]

Insertions/deletionsc.31del10 E1 G11fs Korea [46]c.416_427ins12 (c.416_427dup) E4 dupKICK140 USA [45]c.790insG (c.790dupG) E8 D264fs Spain [44]c. 989_990insT (c.990dupT) E10 E331X Brazil [41]c.1018_1020delATC E10 I341del Spain [41]c.1172_1173insT (c.1173dupT) E11 V392fs Spain, Chile, USA [16,41,47]c. 1204delG E12 A402fs USA [41]c.1218del14ins12 E12 G407fs USA, Spain, Chile, Ecuador [16,18,48]c.1222del3 E12 I408del USA [18,48]c.1297_1298insA (c.1298dupA) E12 A434fs Chile [16]c.1357insT (c.1357dupT) E13 Y453fs Korea [46]c.1527del3 E15 S510del Korea [46]c.1539_1540insCCC (c.1538_1540dup) E15 A513_R514insP Greenland [6]

Splicing mutationsc.183+3G>C I1 not determined Spain [16]c.429+3del4 I4 D125_K143del19 (�exon 4) Japan, USA [17,41,49]c.543+1G>A I5 I144_L181del38 (�exon 5) Arab [41]c.653A>G E6 V217_K218del2a Spain [41]c.764-2delA I7 G255fsb �exon 8) USA [41]c.966+1G>T I9 S295fs (�exon 9) Japan [17]c.1091-11del6 I10 C365_G400del36 (�exon 11) Spain [16]c.1199-2A>G I11 G400_T401del2 or T401fsa,b Austria [41]c.1288A>C E12 T401_A434del34 (�exon 12)b Japan [7]c.1299+3del8 I12 T401_A434del34 (�exon 12) Japan [17,50]c.1300-8T>A I12 T401_A434del34 (�exon 12) Korea [46]c.1398+1G>T I13 A434_K466del33 (�exon 13) USA [45]c.1499-1G>C I14 G500fsa USA [41]

Polymorphismsc.1490C>T E14 A497V Spain [16]













chaperonin mediated assembly in Escherichia coli [21–23],a more physiological approach in human deWcient Wbro-blasts [24,25], cell-free coupled transcription-translation,and a mammalian two-hybrid system for the analysis ofheteromeric (�/� subunits) and homomeric (�/� subunits)interactions in the PCC oligomer [26]. The structuraleVects of the changes have also been analyzed in somecases using the available crystal structures of homologousbacterial proteins. The biotin carboxylase subunit of E.coli acetyl-CoA carboxylase (PDB coordinates 1DV2) [27]has been used as template for the biotin carboxylasedomain of PCCA and both the E. coli biotinyl carboxylcarrier protein (BCCP) (PDB coordinates 1BDO) [28] andthe 1.3S subunit of the Propionibacterium shermanii trans-carboxylase complex (PDB coordinates 1DCZ) [29] aretemplates for the biotinylation domain. For the PCCBprotein a homology model based on the crystal structureof the transcarboxylase 12S from P. shermanii (PDB coor-dinates 1ON3 and 1ON) has been recently described [30].

For the PCCA gene, 11 mutations (10 missense andan in-frame deletion caused by diVerent splicing muta-tions) have been expressed in deWcient Wbroblasts and ina cell-free in vitro system [24]. Most mutant proteinsshow an increased turnover in rat liver mitochondriaafter in vitro synthesis, and all are functionally deWcient,with residual activities ranging from 1 to 27% of wild-type value (Table 3). All the mutated residues are wellconserved in homologous proteins, and those in the bio-tin carboxylase domain were all found to clustertogether in the E. coli 3-D structure clearly deWning aregion probably important for function and/or stabilityof the � subunit (Fig. 1). One exception is mutationM229K, in the active site and presumably involved inthe interaction with the adenine moiety of ATP [24]. Thehomologous mutation in E. coli biotin carboxylase wasrecently analyzed for its kinetic behaviour along with

other active site residues including an Arg mutated inPA patients (R399Q mutation) [31]. Both mutants haveelevated apparent Km values for ATP, impaired Vmax,and exhibit negative cooperativity for bicarbonate. Theirdisease causing nature is clearly related to theirdecreased activity and there is no intrinsic instability andsubsequent degradation of the enzyme due to the muta-tions [31]. This was also observed after expression inhuman Wbroblasts of the M229K mutation, having simi-lar turnover rate to wild-type protein (Table 3) and alsocorrelates with the Wnding in patient’s cells of stable bio-tinylated PCCA protein [32].

The only mutation directly aVecting biotinylation ofthe PCCA protein is G668R, which along with G631Rand the in-frame C615_V633del18, map to the biotinyla-tion domain. The main molecular eVect in patients’ cellsof these mutations is probably related to protein insta-bility [24].

For the PCCB gene, the results of in vitro expressionanalysis of disease causing mutations (18 missense, oneamino acid deletion, one insertion, and three carboxy-terminal truncations) are shown in Table 4. These analy-ses have conWrmed the pathogenicity of the changesstudied, although in the prokaryotic system, some muta-tions exhibit wild-type activity, albeit associated withreduced stability. For one mutant (R410W) similar sta-bility and activity to wild-type was reported. This couldbe explained by the previously discussed fact that invitro expression analysis may overestimate enzymeactivity [33], specially in the prokaryotic systememployed where there could be a stabilizing inXuence ofthe co-expressed GroESL necessary in this system topermit the functional assembly of the PCC oligomer [21].This may also explain the diVerences in stabilityobserved for some mutants in the prokaryotic andeukaryotic systems (Table 4).

Table 3Expression data for PCCA mutations and associated phenotypes in homozygous and functionally hemizygous patients

n.d., not done.a Data from [24,31,38].b Referred to relative intramitochondrial stability after in vitro synthesis [24].c Mild: patient alive with near normal development. Moderate: Patient alive with moderate psychomotor delay. Severe: patient dead or severely

retarded.

Mutation In vitro expressiona Clinical presentation in homozygous and functional hemizygous patientsc

Patients’ reference

% PCC activity Biotinylation Protein stability b

A75P 26.7 Normal Reduced — —R77W 3.6 Normal Reduced Mild [5]A138T 9.4 Normal Reduced Mild [5]I164T 15.8 Normal Reduced Mild [5]M229K 2.8 Normal Normal Mild [32]D368G 4.3 Normal Reduced Severe [32]M373K 3.5 Normal Reduced Severe [38]G379V 5.4 Normal Reduced — —G631R 3.3 Normal Reduced Severe [5]G668R 1.4 DeWcient n.d. Severe [32]C616_V633del18 (�exon 21) 1.0 Normal Reduced Moderate [5]


Fig. 1. Localization of residues involved in missense mutations in the 3-D PCCA homology model of the biotin carboxylase domain, generated usingthe alignment of its sequence with biotin carboxylase of E. coli acetyl-CoA carboxylase (PDB Accession No. 1DV2) and the Swiss-Model server [54].The model was displayed using WebLab Viewer software (Molecular Simulations). Shown inside the box are mutations found to cluster together, ina region of unknown function opposite the active site.

Fig. 2. Localization of homologous residues aVected by PCCB mutations in the 3-D structure of transcarboxylase 12S from P. shermanii. (A) 12Shexamer (PDB Accession code 1ON3) bound to its substrate methylmalonyl-CoA (backbone in red). (B) Closer view of the region with residues pre-dictably aVecting hexamer formation [30]. (C) Closer view of the active site region. Mutant residues were identiWed by alignment using CLUSTALWand shown as CPK representation, with the same colours as the parental subunit.


In the eukaryotic system only two of the mutantsstudied, R410W and N536D retain partial activity, therest are deleterious mutations, resulting in close to nullenzyme activity [25]. From the results of the analysis invitro and in patients’ cells, where in most cases somePCCB protein is detected by Western blot analysis [5], ingeneral the analyzed mutations do not have a profoundimpact on the overall stability of the protein. Consistentwith this is the prediction that few of the missense muta-tions are incompatible with monomer folding when ana-lyzed in the PCCB homology model constructed using astemplate the crystal structure of the 12S transcarboxy-lase from P. shermanii [30]. This model consists of anhexameric core, resulting from the stacking of two tri-mers related by a 2-fold symmetry, with the substratebinding-sites located at the trimer–trimer interface.Many of the mutations (G131R, R165Q, G246V,R165W, E168K, V205D, R410W, and M442T) arelocated at the inter–trimer interface and are predicted toalter the active site conformation by producing a localdistortion (Fig. 2C). The C-terminal mutations R512C,L519P, and N536D are predicted to interfere with hexa-mer formation, aVecting intra-trimer interactions and/ormonomer folding [30] (Fig. 2B). The interactionsbetween � subunits and between these and � subunitshave also been explored using a mammalian two-hybrid

system [26]. Consistent with the structural approach, thecarboxy-terminal mutations R512C, L519P, W531X,and N536D exhibited a defective �/� and/or �/� interac-tion. In the prokaryotic system, some PCCB mutationshave also been reported to aVect the interaction with�-subunits and thus the assembly to form a functionalPCC oligomer [22].

Clearly, each expression system may provide comple-mentary information on the structural and functionalconsequences of disease causing alleles, although dis-crepancies arise due to the diVerent intracellular milieuin each system and to the experimental approachesemployed. However, all systems are useful as the demon-stration that a mutation has analogous alterations ofenzyme properties in diVerent systems provides evidenceof a genuine eVect.

Finally, the eVect of speciWc splicing mutations hasbeen analyzed in detail using minigenes and real-timePCR methods [34]. Two mutations in the PCCA gene(c.1899+3del4 and c.2041-2A > G) and one in the PCCBgene (c.1091-11del6) were shown to produce some nor-mal splicing in patients’ cells, at very low levels, albeitsuYcient to moderate the phenotype, as the patients pre-sented a mild course of the disease [34]. Accurate charac-terization of the functional eVect of splicing mutations(very frequent in PA, see Tables 1 and 2) is necessary

Table 4In vitro expression data of pathogenic PCCB mutations and associated phenotypes in homozygous and hemizygous patients

a Data from [6,21–23,25].b Determined by Western blot analysis, in bacterial extracts (prokaryotic system) or in transfected Wbroblast cell extracts (eukaryotic system).c Mild: patient alive with near normal development. Moderate: Patient alive with moderate psychomotor delay. Severe: patient dead or severely

retarded.

Mutation In vitro expressiona Clinical presentation in homozygous and functional hemizygous patientsc

Patients’ reference

% PCC activity Protein stabilityb Prokaryotic/eukaryoticProkaryotic system Eukaryotic system

R44P 0 <2 Normal/normal Moderate [5]R67S nd <2 nd/normal Moderate [41]S106R 0 <2 Normal/normal Severe [5]G112D nd <2 nd/reduced Severe [51]G131R 0 <2 Normal/normal Severe [5]R165W 106 <2 Reduced/normal Severe [25]R165Q nd <2 Normal/normal Mild [25]E168K 106 <2 Reduced/normal Mild/moderate/severe [5]D178H 120 nd Reduced/nd — —G198D 0 0 Normal/normal Severe [52]V205D 0 <2 Reduced/normal Mild/asymptomatic [53]P228L 78 nd reduced/nd — —Del408 0 nd Normal/nd — —R410W 92 11.5 Normal/normal Moderate [4,17,19]T428I 0 nd Normal/nd — —M442T 0 nd Normal/nd Mild [5]R499X 0 nd Reduced/nd — —R512C 3.9 <2 Reduced/reduced Severe [5]A513_R514insP 0 nd Reduced/nd Severe [6]R514X 0 nd Reduced/nd — —L519P 0 <2 Reduced/reduced Mild [5]W531X 10 <2 Normal/normal Severe [5]N536D 0 80 Reduced/normal Mild [25]


using adequate cellular-based model splicing systems(minigenes).

Genotype–phenotype correlations

One of the driving forces behind the expression analy-sis of mutant alleles in PA is the idea that it may providea basis for the prediction of the phenotype from thegenotype. This would enable assessment of the prognosisand implementation of individually tailored treatmentsaimed at speciWc functional defects. Several studies haveexplored the genotype–phenotype correlations in PAbased on the Wndings in a collection of patients and onthe expression analysis of mutant alleles [5,22–25].Clearly, some correlations have been found. Mutationscategorized as null alleles, which include those predictedfrom the nature of the DNA change (nonsense muta-tions, out-of-frame deletions and insertions, splicingmutations presumably resulting in frameshifts, ƒ) andthose deWned experimentally (mutations with no detect-able enzyme activity or protein in expression analysis),are associated with the most severe phenotypes. Mis-sense mutations retaining partial activity in vitro (e.g.,A138T, I164T in the PCCA gene, N536D in the PCCBgene) are associated with a mild phenotype (Tables 3and 4). However, this can only be asserted for homozy-gous and functional hemizygous (with a second nullallele) patients. Many patients are compound heterozy-gous or carry rare missense mutations not analyzedfunctionally, which precludes an accurate prediction ofthe phenotype. In addition, some inconsistencies havebeen found. For example, mutation E168K results in abroad spectrum of phenotypic manifestations rangingfrom mild to severe, which was suggested to depend onthe modulation by genetic and epigenetic factors aVect-ing folding, given the temperature-sensitive behaviour ofthe mutant protein in the mammalian two-hybrid system[26].

Some mutations are found associated with a milder,up to date asymptomatic form of propionic acidemiadiagnosed in Japan through neonatal screening pro-grams using tandem mass spectrometry [7]. This is thecase of mutation Y453C in the PCCB gene, found inhomozygous fashion in nearly half of the patients identi-Wed this way (frequency for the homozygotes 1/29,929).The patients’ residual activity of PCC in lymphocyteswas also high [7], which suggests a mild impairment ofPCC function.

The results described above for PCCA and PCCBsplicing mutations also provide relevant information onthe complexity of genotype–phenotype correlations andsuggest that, in some cases, the regulation of gene splicingcould also inXuence phenotypic parameters in PA, similarto what has been described in other diseases and opens analternative possibility for therapeutic intervention [35].

Summary and future prospects

Since the last review on propionic acidemia mutations[36], a much clearer understanding of the relationbetween structure and function of PCC and the relation-ship of mutations to disease pathology has beenobtained, based on the results of the characterization ofdiVerent variant alleles in several expression systems. Inthis scenario, the elucidation of the crystal structure ofPCC will provide further insight into the disease causingmechanisms of each mutation.

The expression analysis of PCCA and PCCB muta-tions has revealed potential genotype–phenotype corre-lations although the considerable genetic heterogeneitydeWes any simplistic conclusions to be drawn, and, inother cases, modulation of the eVect of particular muta-tions by genetic and epigenetic factors contribute to phe-notypic variability.

As in other human genetic diseases, animal modelsmay represent useful tools to investigate the pathogene-sis of PA and to test novel therapeutic strategies. APCCA knock-out mouse model of PA was recently cre-ated [37]. The fatal outcome in the knock-out mice canbe avoided with the introduction of a liver-speciWc trans-gene, albeit with very low expression levels (accountingfor 10–20% of endogenous wild-type PCC activity),which provides relevant information on the suitability ofgene therapy for this disease. Other potential applica-tions of the knock-out mice include the evaluation of theimpact of particular mutations and subsequent develop-ment of therapies aimed at speciWc functional defects.

Acknowledgments

This work was supported by grants from Fondo deInvestigaciones Sanitarias (REDEMETH G03/054 andRECGEN C03/07). S.C. is supported by ComunidadAutónoma de Madrid. The Wnancial support of Funda-ción Ramón Areces to the Centro de Biologia Molecular“Severo Ochoa” is gratefully acknowledged.

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Propionic acidemia: mutation update and functional and structural effects of the variant alleles

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