British Journal of Haematology ,2001, 113,43±48 Molecularcharacterizationofthe PK-LR geneinsixteen pyruvatekinase-deficientpatients Alberto Zanella, 1 Paola Bianchi, 1 Elisa Fermo, 1 Alessandra Iurlo, 1 Manuela Zappa, 1 Cristina Vercellati, 1 Carla Boschetti, 1 Luciano Baronciani 1 and Frederic Cotton 2 1 Divisione di Ematologia, IRCCS Ospedale Maggiore of Milan, Italy, and 2 Department de Biologie Clinique, Hopital Erasme, Brussels, Belgium Received 8 August 2000; accepted for publication 19 December 2000 Summary. We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated witherythrocytepyruvatekinasedeficiency.Fifteendifferent mutations were detected among the 28 mutated alleles identified:twodeletionsdel1010G,del1042±1044);one four nucleotide duplication nt 1515±1518, GGTC); one splice site [IVS622)t]; nine missense 991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T,1483A, 1529A); andtwononsense721T,1675T)mutations.Eightofthem [del1010G,del1042±1044,dupl1515±1518,IVS622)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042±1044causesthelossofLys348.Deletion1010Gand duplication 1515±1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS622)t leads to an alteration of the 5 0 and 3 0 splice site consensus sequence; the cDNA analysisshowsa67-bpdeletioninthefirstpartofexon11 del 1437±1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics oftheenzymeandclinicalcourseofthedisease. Keywords: pyruvatekinasedeficiency, PK-LR gene,chronic haemolyticanaemia,erythrocytemetabolism,mutations. Pyruvate kinase PK; ATP: pyruvate 2-o-phosphotransfer- ase, EC 2´7´1´40) catalyses the conversion of phosphoenol pyruvatePEP)topyruvate,coupledtothesynthesisofone ATPmolecule.Thereactionisthelaststepoftheglycolytic pathwayandisirreversibleunderphysiologicalconditions. FourisozymesarepresentinmammaliantissuesImamura & Tanaka, 1972; Ibsen, 1977): L-type hepatic) and R-type erythrocytic),encodedbythe PK-LR geneunderthecontrol of two tissue-specific promoters Noguchi et al,1987);and M1- muscle and brain) and M2- fetal and most adult tissues) types encoded by the PK-M gene by alternative mRNAsplicingNoguchi et al,1986). PK deficiency is the most common cause of hereditary non-spherocytic haemolytic anaemia. Although the defect has a worldwide geographical distribution, it has been recognized most commonly in Northern European popula- tions Dacie, 1985). Since its first detection in 1961 Valentine et al,1961),over400caseshavebeendescribed, but many more remain unreported in the absence of unusual clinical or molecular features Zanella & Bianchi, 2000). PK deficiency is transmitted as an autosomal recessive trait. Clinical symptoms, ranging from neonatal jaundice requiring exchange transfusions to a fully compensated haemolyticanaemiaMentzer&Glader,1989),occuronly in homozygotes and in compound heterozygotes for two mutant alleles. Heterozygotes are haematologically normal with rare exceptions Valentine et al, 1961; Noguchi et al, 1986;Mentzer&Glader,1989;Tanaka&Paglia,1995). The PK-LR gene has been localized on the long arm of chromosome1Satoh et al,1988),andthecDNAofthe R- typehasbeenclonedandsequenced;itis2060bplongand codesfor574aminoacids.Thecodifyingregionissplitinto 12 exons, 10 of which are common to the two isoforms, whereasexons1and2arespecificfortheerythrocyticand thehepaticisoenzymerespectivelyTani et al,1988;Kanno et al,1991). Sofar,134differentmutationshavebeenidentifiedinPK deficiency Bianchi & Zanella, 2000; Zarza et al, 2000). They are spread all over the coding region, with no preference for the active site as determined by crystal- lographicanalysisofthecatmuscleenzymeMuirhead etal, 1986; Allen & Muirhead, 1996). So far, only three mutationshavebeenreportedinthepromoterregionvan Solinge et al, 1997; Kugler et al, 1999). The mutations identified are mostly missense, splicing and stop codon, q 2001BlackwellScienceLtd 43 Correspondence: Alberto Zanella, MD, Divisione di Ematologia, IRCCS Ospedale Maggiore, Via F. Sforza, 35, 20122 Milano, Italy. E-mail:div_emat@polic.cilea.it