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M l l K g t r i g m ORIGINALRESEARCH ARTICLEpublished: 30 December 2013

doi: 10.3339/fphys.2013.00393

First description of phosphofructokinase deficiency in

Spain: identification of a novel homozygous missense

mutation in the PFKM gene

Joan-Lluis Vives-Corrons1 * Pavia Korafkova

2, Josep fA

Richard Van Wijk4Grau

3, Maria dei Mar Manu Pereira

1 and

' Red Cell Pathology Unit, Biomedical Dianostic Centre. University Hospital Clinic ds Barcelona, Barcelona. Spain2Faculty of Medicine and Dentistry, Department of Biology, Paiacky University, Ciomouc, Czech Republic

3Service of Interna! Medicine and Muscle, University Hospital Clinic de Barcelona. Barcelona, Spain

4 Department of Clinical Chemistry and HaematoiogxUniversity Medical Canter 'Jfecht, Utrecht. Netherlands

Edited by:

Anna Bogdsnova. University of

Zurich, Switzerland

Reviewed by:

Michael White. Drexei University

College of Medicine, Canada

Lillian DeBnjin, Wilfrid Laurier

University, Canada

'Correspondence:Joan-Lluis Vives-Corrons, Red Cell

Pathology Unit. Biomedical

Dianostic Centre, University

Hospital Clinic de Barcelona,

c/VUIarroel 17Q, 08036-Barcelona,

Spain

e-maii: jivives@clinic. ub. ss

Phosphofructokinase deficiency is a very rare autosomal recessive disorder, which belongs

io group of rare inborn errors of metabolism called glycogen storage disease. Here we

report on a new mutation in the phosphofructokinase (PFK) gene PFKM identified in a

65-vears-oid woman who suffered from lifelong intermittent muscle weakness and painful

spasms of random occurrence, episodic dark urines, and slight haemolytic anemia. After

ruling out the most common causes of chronic haemolytic anemia, the study of a panel of

24 enzyme activities showed a markedly decreased PFK activity in red blood cells (RBCs)

from the patient. DNA sequence analysis of the PFKMgene subsequently revealed a novel

homozygous mutation: c.926A>G; p.Asp309Giy. This mutation is predicted to severely

affect enzyme catalysis thereby accounting for the observed enzyme deficiency. This case

represents a prime example of classical PFK deficiency and is the first reported case of

this very rare red blood cell disorder in Spain.

Keywords: phosphofructokinase deficiency, glycogen storage disease, PFKM gene, missense mutation, enzymecatalysis

been charac terized. Th e gene enco di ng th e M su bu ni t {PFKM)

(ATP: D- fruct ose-6 -pho sphat e-I- has been assigned to chrom osome 12ql3.3 and spans 30k b. ItINTRODUCTION

Phosphofructokinase

ph os ph ot ra ns fe ra se ; EC 2.7 .1. 11; PFK) is a key re gu la to ryenz yme o f the glycolytic, cycle and catalyse s the conversio n offhictose-6-p hosphate to fructose- 1,6-dip hosphate (Figure I).

Human PFK is composed of three isoenzymes, muscle (M), liver(L), and platelet (P) (Vora, lv*3; Nakajima et a)., 2002). The Ptype is also known as Fibroblast type (F). Mammalian PFK isa tetrameric enzyme that is subjected to allosteric regulation.Tissue isozymes randomly aggregate to form homotetramersor heterotetramers depending on the relative abundance of thesubunits in a particular tissue. PFK-M is the sole subunit inmuscle cells whereas red blood cells (RBCs) contain both LandM subunits and form their hybrids (M4, L4, M3L, M2L2,and ML3).

Phosphofructokinase deficiency (OMIM 171 850) is a veryrare autosomal recessive condition with heterogeneous clinicalsymptoms, mainly characterized by myopathy and/or haemolysis(Hirano and Di Mauro. H-^v). Myopathy is caused by the accu-

. mu lat ion of glycogen in muscle tissue due to the metabol ic defectand is also known as glycogenosis type VII or Tarui disease. It ischaracterized by muscle pain, exercise-induced fatigue, cramps,and myoglobinuria. The observed clinical symptoms reflect lackof muscle PFK activity and partial reduction of enzymatic activ-ity in erythrocytes. The latter usually is associated with raildhaemolysis.

Up to now, only about 100 patients with PFK deficiency havebe en -r ep or te d worl dw id e an d 22 PFK- defici en tPFKM allele.; have

contains 24 exons and at least 3 promoter regions (Elson et aL,1)90; Yam ad a et aL, 2004). Amon g the d etected m utat ion s aremosdy missense mutations and splicing defects.

We now describe here a Spanish patient with a clinical his-tory of anemia, haemolysis, and intermittent muscle weakness,who was found to be homozygous for a novel mutation in thePFKMgene (c.926A>G). This mutation encodes the substitutionof aspartic acid by glycine at residue 309 (p.Asp309Gly). This isthe first descripti on of PFK deficiency in Spain.

CASE REPORT

A 65-years-old woma n with long standing hypertension and type2 diabetes was referred to our Unit because of intolerance to exer-cise and chronic fatigue. From youth she suffered from spasmsof random occurrence associated with muscle weakness, painfulintolerance to small efforts, and intermittent dark urines, espe-cially after exercise due to myoglobinuria, as revealed by urinaly-

sis. Physical examination showed no weakness of muscle atrophyand only a moderate splenomegaly without hepatomegaly or lym-ph ad en op at hy . Parent s were unavail able fo r stud y an d ther e wasno known consanguinity. The same clinical picture, however,was present in a non-smoker brother whereas the three patient'sdaughters were normal. Patient's clinical condition has remainedstable in follow-up.

Complete blood count (CBC) show red moderate anemia(Hb : 115 g/L), wit h slight macro cyto sis (105 1) a nd incre ased

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Vives-Corrons et si. Anaemia, haemolysis, myopathy, phosphofruc tokinase, muta tion

? -

Embden-ft/leyerh of Pathway

glucose

' _ I iHexokmase :A D P H 1 1

giucose-6-phosphaia

A

IGlucosephosphate I. isom'eras e )

fructose-6-phosphale

ATP

A D Pi Phosphofructokinase I

fructose-1.S-diphosphate

l Aldolase !

dihydroxyacetone *

phosphate r

g!ycera!dshyde-3-phosphate

! Triosephosphate j

isomerase ;

N A D J j Glyceraidehyde-3 -phosphale ,

N A D H -A ! dehydroge nase

{Rapoport-Luebering Shunt") j

j bi sphosphoglycerate

j mu tase .

-- 1.3-diphosphoglycerale

2,3-biphosphoglycerate

} bisphosphoglycerate !

| phosphatase !

A D P

ATP} Phosphoglycerate kinase.

-phosphoglycerate

4

jMonophosphogiycerate mutase

2-phospftogiycerate

A

| Enolase ;

phospoenolpyruvate

A D PATP '

kinasej

pyruvate

NADH

NAD '! Lactate dehydrogenase

*tectate

FIGURE 1 j Etnbden Meyerhof Pathway of R3C metabolism. Phosphofructokinase (PFK) catalyzes the transformation of fructose 6-phospnate into fructose

1,'6 diphosphate. [Reproduced wi th permission from Van WijK and van ; ."L J

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Vives-Corrons et ai.

number of circulating reticulocytes (110 x 109/L). Leukocyteand platelet counts, as well as general serum biochemical anal-ysis, were within normal range, except for a moderate risein non-conjugated bilirubin, lactate dehydrogenase (LDH) anduric acid (hyperuricemia). Biological signs of diabetes melli-tus, type 2 were also present. The studies performed to ruleout the origin of the anemia, discarded nutritional deficiencies(serum iron tests, cobalamin and serum folate were all nor-mal), haemoglobinopathies (HPLC and thermal stability), and

paroxysmal nocturna l haemogloh inuria (norma l flow cyt ome-try measurement of CD45 and CD49 in leukocytes and RBCs).Hereditary REC membrane defects were ruled out by morpholog-ical observation of May-Grunwald-Giemas stained blood smears,and a normal osmotic fragility test. Extensive study of RBCenzyme activity measurements demonstrated a marked decrease(< 30 % of normal) in PFK activity (Table 1). DNA sequence anal-ysis of individual exons ofPFKM, including flanking splice sites,revealed that the patient was homozygous for a rnissense muta-tion in exon 11: c.929A>G. This mutation, that has not beenpreviously reported in the litera ture, encodes the substi tut ion of

aspartic acid by glycine at residue 309 (p.Asp309Gly). Mutationpredic tion pro grams PolyPhen-2 (Adzhubei et a!., 2010) and SIFT(Kumar et at, 2009) predict this mutation to be pathogenic (i.e.,disease causing).

The complete lack of PFK activity in muscle was confirmedon both histological preparations and muscle extracts. Muscleabnormality was also confirmed by electromyography (EMG),that showed mild myopathic changes and by the forearm test,characterized by a plane lactate curve with normal increase ofammonium. As usual in patients with glycogenosis, a painfulspasm occurred at the end of the forearm test. Muscle biopsyshowed slight amounts of polysaccharide (PAS) not digested bydiastase and abnormalities in NADH-TR reaction.

S i S C U S S J G ^In this report, we describe, for the first time, the occurrence ofPFK deficiency in Spain in an adult woman of 65 years of age. Hermoderate haemolysis was associated with the accumulation ofmuscle glycogen comparable to that of Tarui disease. Muscle painand exercise-induced fatigue and weakness associated with darkurines were the most relevant clinical manifestations. She wasfou nd to be homozygous for a novel misscnse muta tion inPFKM.,the gene that encodes the PFK-M subunit. On the amino acidlevel, this mutation (c.929A>G) causes the substitution of aspar-tic acid by glycine at residue 309. Asp309 is part of an a-helix, and

Table 1 } Ret blood cel i enzyme act ivi ty measurements .

Patient Reference value

G6PD activit y (lU/g Hb) 7.0 6.2-9.9

HK activity (lU/g Hb) 1.2 0.6-1.3

PGI activity (lU/g Hb) SI.9 '46.0-66.0

PK activity

PFK activit y (lU/g Hb) 3.07 6.4-13.9

Giucose-6-phosphste dehydrogenase; HK. Hexokinase; PGI, Phosphcglucose

isomerase; PK, Pyruvate kinase; PFK. Phosphofructokinase.

replacement with glycine could disrupt this a-helix. Furthermore,structural analysis using the 3D molecular model of rabbit mus-cle type PFK (PDB entry 308L) showed that the p.Asp309Glysubstitution is located in direct vicinity of the nucleotide (ADP)bin ding site in the center of the PFKM subun it (Figure 2A). PFKis allosterically activated by ADP, and the ADP binding site has

been recently identi fied (Bar.aszak et a I., 2011). Asp309 does not

directly interact with ADP, however, substitution of Asp309 byglycine is likely to disrupt multiple hydrogep bonds with Glyl77,GM79, Ser 180, and Ser306 (Figure 2B). 'This loss of hydrogen

bo nd s an d the change of polari ty and electrostat ic interactio nsupon mutation of Asp309 will likely affect correct positioning ofthe side-chains of amino acids directly involved in ADP bind-ing (Figure2B), in particular neighboring residue Phe308, whichmakes stacking interaction with the adenine ring of ADP. Thishypothesis is further supported by recent functional studies ofthe Asp543Ala change in PFKM. This mu tati on is associated withTarui disease, an d substituti on of Asp543 by Ala was shown to dis-rup t hydrogen bond s with ADP (Bri et a L 2012). We therefo repos tulate that decreased binding of the alloster ic act ivator ADPwill inhibit PFK enzymatic activity, in particu lar u nder low energy

levels. These findings support the physiological importance ofAsp390 for the recently identified ADP binding site.

The rnissense mutation is thus predicted to lead to a less func-tional PFK-M subunit. In accordance with this, the complete lackof PFK activity in muscle was confirmed on both histological

preparat ion s and muscle extracts . The muscle abnormality wasalso confirmed by electromyography, ischemic exercise testing,histochemistry and electron microscopy. Furthermore, partialred blood cell PFK deficiency was reflected by the moderatelydecreased enzymatic activity in red blood cells, leading to mildhaemolytic anemia ,

PFK is a key regulatory enzyme for glycolysis (Van Wiik andvan Solingf. 2005) and catalyzes the irreversible transfer of pnos-

phoryl from ATP to fructos e-6-phosp hate, and conver ts it tofructose-1,6-bisphosphate. Thus, tissues deficient in PFK cannotuse free or glycogen-derived glucose as a fuel source and accu-mulate glycogen (glycogenosis). PFK deficiency (Tarui disease)was the first disorder recognized to directly affect glycolysis (Taruiet 1., 1963). Since th is first desc ription of the disease, a wide rangeof biochemical, physiological and molecular studies have greatlycontributed to our knowledge concerning not only PFK functionin normal muscle, but also on the general control of glycolysisand glycogen metabolism. So far, more than one 100 patientshave been described with prominent clinical symptoms charac-terized by muscle cramps, exercise intolerance, rhabdomyolysisand myoglobinuria, often associated with haemolytic anemia andhyperuricemia. In classic Tarui disease, the genetic defect involves

the M isoform, resulting in the absence of enzymatic activity inthe muscle. Erythrocytes lack the M4 and hybrid isozymes andonly express the L4 homotetramers, resulting in about 50% ofnormal PFK activity (Figure 3). Thu s, haemolysis is a result of

par tia l ery throcy te PFK deficiency .

Despite PFK deficiency is a very rare autosomal recessive dis-ease; its t rue inciden ce may be highe r due to lack of recognition, assymptoms may be quite mild. In fact, our case highlights the lat-ter, because the mild clinical present ation led her to be diagnosed,

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Vives-Corrons et a!. * u , , .Anaemia, haemolysis, myopathy, phosphotructokinase, mutati on

; HGUEE 2 j 3D crystal str uctur e of PRC subunit from rabbit sksie taimuscie and 3D model of normei/mutaisa PFKM jPDB308Lprotein

i databank). {A}Allosteric nucleotide (ADP) binding site residues (purple) in

| the center of the rabbit subunit of PFKM. and the allosteric activator ADP are

' shown (atom coloring: carbongreen , nitrogenbi ue, oxygenred,

j phosphororange). (B) Close-up of the c.Asp309G ly mu tat ion in PFKM.

j Asp309 is in close proxi mit y of the ADP binding site . Asp309 does not

directly interact with ADP However, substitution of Asp309 by glycine is likely

tc disrupt multiple hydrogen bonds with Giy177, Gly179, Ser 180 and Ser306

(black dotted lines). This loss of hydrogen bonds, and the change of polarity

ar i electrostatic interactions upon mutation of Asp309 will likely affect

cur: act positioning of the side-chains of amino acids directly involved in ADP

biruiing; in particular neighboring residue Phe308, which makes stacking

in section with the adenine ring of ADR

for many years, as chr onic fatigue, until the consideration of dark

urines ultimately led to the study of chronic haemolysis. In ourcase the combination of RBC enzyme activity measurements andmuscle biopsy analysis allowed for the correct diagnosis even attliis late stage of life.

Generally, PFK deficiency presents in childhood. Clinical his-tory however, defines 4 main subtypes: (1) classic, (2) infan-tile onset, (3) late onset, and (4) haemolytic. Most of therepo rted cases belong to t he classic form, cha racterized by exercii.;intolerance, fatigue, muscle cramps with pain and myoglobin-uria (Hu'ano and Di Mauro, 1999). A compensated haemolysiswith jaundice, increased serum creatine kinase (CK) and hype-ruricemia is also commonly present Sometimes, nausea andvomiting appear after intense physical, efforts Patients with theinfantile onset may manifest as "floppy babies" that die within

the first year of life. Symptoms include myopathy, psychomo-tor retardation, cataracts, joint contractures, and death duringearly childhood. They can also show evidence of arthrogrypo-sis and mental retardation. Patients with the late-onset formmay present in adulthood with progressive muscle weakness,cramps and myalgias in later life. Exercise ability, however, islow already in childhood, and a mild muscle weakness mayappear in the 5th decade leading to severe disability. Diagnosisdepends on patient history, physical examination and the findings

from muscle biopsy, electromyography, ischemic forearm test-

ing, CK testing. E.BC enzyme activity measurement is, however,die easiest way to establish the definitive diagnosis. Fatal infan-tik type and late-onset forms of PFK clinical expression arevery rare, with only several reported cases. The haemolytic formpresents with heredita ry non-sph erocyt ic haemolyt ic anemiawith out muscle man ifestat ions (Fuji! and Mivva, 2000). Due to themolecular genetic heterogeneity, a clear-cut genotype-phenotypecorrelation has not been recognized in patients with PFK defi-ciency (Toscano and Musumeci, 2007). Unfortunately, no spe-cific treatment or cure of enzyme deficiency exists. Althoughdiet therapy may be highly effective at reducing clinical man-ifestations, Tarui disease resolves with rest. Fortunately, theco dition does not progress to severe disability. Because theliver and kidneys express only the L isoform, these organs are

spored.About 100 cases of PFK deficiency have been reported in

pa rent s with Taru i disease fr om Europe, USA,and Japan withso; . pred omin ance in Ashkenazi Jews (Fujii and Miwa, 2000).Up lo know, 22 different mutations of PFK have been described,r.lii,: ense and splicing mutations are the most frequendy occur-ring mutations in PFKIM. Intriguingly, PFK-deficient AshkenaziJews share 2 common mutations in the gene. A splicing defectcau:..:d by the G>A base change at the first nucleotide in intron

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Vives-Corrons et ai. Anaemia, haemolysis, myopathy, phosphofructokinase, mutation

Musc le

M

L iver

L

P l a t e l e t s

Pc.12 c.21

T

c.10

PFKP

Norma Situation

M U S C L E RSC LIVER

Mu sc e

M

Liver

LP l a t e l e t s

p

fc12;Tl 7/f f "

c.21 i f c.10

PFK-M Deficiency(Tarui Disease)

MUSCLE LIVER

T

GLYCOGENOSIS associated with HAEMOLYSIS

\ RGURE 3 | tVloiecular basis of PFK defi ciency. In classic Tarui disease, the homotet ramers (M4 and L4) and three hybrid isozymes (M3L, M2L2 and

| genetic defect involves the M, isoform of PFK enzyme, resulting a severe ML3), lack the M4 isofo rm and the hybrid isozymes, and only express the L4

j enzyme def iciency in muscle. Erythrocytes that normally have two homote tramers , resulting in about 50% of normal PFK activ ity

5 (c.237+lG>A) accounts for 68% of mutant Ashkenazi alleles, case described to be affected by this very rare disease. The iden-and a single base deletion in exon 22 (c.2Q03deiC) accounts for tificat ion of a novel homozygous missense muta tion furtherabout 27% of mutant Ashkenazi alleles (Raben and Sherman, extends the repertoire of PFK deficiency-associated muta tions in1995). The here described homozygous patient is the first Spanish PFKM.

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ACKNOWLEDGMENTS

This study has been partially supported by a Research Grantof die Spanish M inistry of Hea lth (HS Ref PI 10/01460).'We are indebted to the European Network for Rare andCongenital Anaemias (ENERCA) that has facilitated theconnection and information exchange between the centersparti cip ati ng in thi s study. Pavla Koralkova is suppor ted by

the grants NT11208 (Ministry of Health, Czech Republic)and LF_2013_010 (Internal Grant Agency of PalackyUniversity).

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Raben, N., and Sherman,J. B. (1995). Mutations in muscle phosphofructokinasegene. Hum. Mutat. 6, 1-6. doi: 10.1002/humu.l380060102

Tarui, S., Okuno, G., Ikura, Y., Tanaka, T., Suda, M., and Nisbikawa, M.(1965). Phosphofructokinase deficiency in skeletal muscle: a new type ofglycogenosis. Biochem. Biophys. Res. Commnn. 19, 517-523. doi: 10.1016/0006-291X(65)90156-7

Toscano, A., and Musumeci, O. (2007). Tarui disease and distal glycogenoses:clinical and genetic update. Acta Myol. 26, 1 0 5 - 1 0 7 . ,

Van Wijk, R., and van Solinge, W. W. (2005). The energy-less red blood cell islost: erythrocyte enzyme abnormalities of glycolysis.Blood.106,4034-4042. doi:lG.ll82/blood-2005-04-1622

Vera, S. (1983). Isozymes of huma n phosphofruc tokinase: biochemical and genetic

aspects.Isozymes Curr. Top. Biol. Med. Res. 11,3-23.Yamada, S., Nakajima, H., and Kuehn, M. R. (2004). Novel testis- and embryo-

specific isoforms of the phosphofructokinase-1 muscle type gene. Biochem.Biophys. Res. Commun. 316,580 -587. doi: 10.1016/j.bbrc.2004.02.089.

i

Conflict of Interest Statement: The authors declare that the research was con-

ducted in the absence of any commercial or financial relationships that could be

construed as a potential conflict of interest.

Received: 02 November 2013; accepted: 13 December 2013; published online: 30December 2013.

Citation: Vives-Corrons J-L, Koralkova P, Grau JM, Manit Pereira MdM and Van Wijk

R 12013) First description of phosphofructokinase deficiency in Spain: identification of

il novel homozygous missense mutation in the PFKM gene. Front. Physiol. 4:393. doi:

10J389/fphys.2013.00393

This article was submitted to Membrane Physiology and Membrane Biophysics, a

section of the journal Frontiers in Physiology.

Copyright 2013 Vives-Corrons, Koralkova, Grau, Manu Pereira and Van Wijk.

This is an open-access article distributed under the terms of the Creative Commons

Attribution License (CC BY). The use, distribution or reproduction in other forums is

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