Increased incidence of mitochondrial cytochrome c-oxidase gene mutations in patients with...

Increased incidence of mitochondrial cytochrome c-oxidase

gene mutations in patients with myelodysplastic syndromes

Poluru L. Reddy, Vilasini T. Shetty, Diya Dutt, Aaron York, Saleem Dar, Suneel D. Mundle,

Krishnan Allampallam, Sairah Alvi, Naomi Galili, Gurveen Sethi Saberwal, Shalini Anthwal,

Malihi Shaikh, Samia Suleman, Shaista Y. Kamal and Azra Raza Rush Cancer Institute, Rush-

Presbyterian-St. Luke’s Medical Center, Chicago, IL, USA

Received 24 July 2001; accepted for publication 27 September 2001

Summary. Mitochondria (mt) play an important role in bothapoptosis and haem synthesis. The present study wasconducted to determine DNA mutations in mitochondrialencoded cytochrome c-oxidase I and II genes. Bone marrow(BM) biopsy and aspirate, peripheral blood (PB) and buccalsmear samples were collected from 20 myelodysplasticsyndrome (MDS) patients and 10 age-matched controls.Cytochrome c-oxidase I (CO I) and II (CO II) genes wereamplified using polymerase chain reaction and sequenced.CO I mutations were found in 13/20 MDS patients and theCO II gene in 2/10 normal and 12/20 MDS samples,irrespective of MDS subtype. Mutations were substitutional,deletional and insertional. CO I mutations were mostcommon at nucleotide positions 7264 (25%) and 7289(15%), and CO II mutations were most common at nuc-leotide positions 7595 (40%) and 7594 (30%), suggestingthe presence of potential Ôhot-spotsÕ. Mutations were not

found in buccal smears of MDS patients and were signifi-cantly higher in MDS samples compared with age-matched controls in all cell fractions (P < 0Æ05), with bonemarrow high-density fraction (BMHDF) showing a highermutation rate than other fractions (P < 0Æ05). MDS mar-rows showed higher levels of apoptosis than normal controls(P < 0Æ05), and apoptosis in BMHDF was directly related tocytochrome c-oxidase I gene mutations (P < 0Æ05). Electronmicroscopy revealed apoptosis affecting all haematopoieticlineages with highly abnormal, iron-laden mitochondria.These results suggest a role for mt-DNA mutations in theexcessive apoptosis and resulting cytopenias of MDS pa-tients.

Keywords: myelodysplastic syndromes, mitochondrial DNAmutations, cytochrome c-oxidase genes, apoptosis, iron-laden mitochondria.

Mitochondria (mt) were once independently living bacteriathat have become a permanent part of the eukaryotic cellthrough an ancient symbiotic contract (Margulis &Schwartz, 1999). They continue to exert some level ofautonomy, however, as they maintain 5–10 copies of theirown 16 500 base pair circular DNA (mt-DNA), replicateindependently of nuclear DNA, and code for 13 subunits ofthe respiratory chain multienzyme complex, 22 transferRNAs and 2 ribosomal RNAs (Anderson et al, 1981). Asevery cell has several mitochondria, and every mitochon-drion has multiple copies of mt-DNA, this amounts tothousands of copies of mt-DNA per cell. In addition tocoding for 13/80 subunits of the respiratory chain multi-enzyme complex essential for the electron transport systeminvolved in haem synthesis, a role for mitochondria has alsobeen identified in the initiation of cellular apoptosis (Petit

et al, 1995; Green & Reed, 1998; Hunault-Berger et al,1999). Given the appreciation of their significant role inmaintaining cellular homeostasis, it is no surprise thatmitochondrial mutations are associated with a number ofhuman diseases. These include both congenital diseasessuch as Pearson’s syndrome (Rotig et al, 1989) character-ized by a refractory sideroblastic anaemia and bone marrowdysplasia, as well as acquired ones such as various cancers(colorectal, head and neck, lung and primary bladdercancers) (Habano et al, 1999; Fliss et al, 2000) andacquired idiopathic sideroblastic anaemia (AISA) (Gatter-man et al, 1992, 1993, 1996, 1997; Gatterman, 1999;Matthes et al, 2000). Because of the contribution ofmitochondria in both haem synthesis and apoptosis, therehas been an increasing interest in investigating the role ofmt-DNA mutations in myelodysplastic syndromes (MDS) inwhich iron-laden, swollen and bizarre mitochondria areassociated with an acquired anaemia, variable cytopeniaand excessive apoptosis of haematopoietic cells (Jacobs,1986; Gatterman, 1999).

Correspondence: Azra Raza, M.D., Rush Cancer Institute, 2242W. Harrison Street, Suite 108, Chicago, IL 60612 – 3515, USA.

E-mail: [email protected]

British Journal of Haematology, 2002, 116, 564–575

564 Ó 2002 Blackwell Science Ltd

The MDS are a group of clonal, heterogeneous haema-topoietic disorders presenting with anaemia, a variablecytopenia and bone marrow dysplasia (Jacobs & Bowen,1992). Excessive intramedullary apoptosis of haematopoie-tic cells is postulated to account for the variable cytopeniasof MDS (Clark & Lampert, 1990; Mundle et al, 1995; Razaet al, 1995; Shetty et al, 1996). However, the precisemechanism by which haem synthesis is impaired leadingto anaemia and accumulation of iron in the mitochondria oferythroid precursors is not clear. Normally, iron is importedinto the mitochondria of erythropoietic cells, combines withprotoporphyrin IX to form haem and subsequently leavesthe mitochondria as haem iron (Jacobs, 1986; Gatterman,1999). In MDS, utilization of iron is obviously disturbed, sothat iron accumulates in the mitochondrial matrix andgives the appearance of ringed sideroblasts (Jacobs, 1986;Jacobs & Bowen, 1992). A possible explanation for thisdefect is that iron may not be in the right chemical form(Gatterman et al, 1992, 1993, 1997; Matthes et al, 2000).Iron deposits in the sideroblastic mitochondria are in theferric (Fe3+) state, while only ferrous iron can be used forincorporation into protoporphyrin IX by ferrochelatase(Porra & Jones, 1963). As ferrous iron is not stable underaerobic conditions, it is necessary for erythropoietic cells tohave an enzyme system that can maintain a supply of Fe2+

as substrate for ferrochelatase. It was shown that theelectrons for the conversion of ferric iron into ferrous ironare provided by the mitochondrial respiratory chains(Flatmark & Romslo, 1975), in particular complexes III(cytochrome b) and IV (cytochrome c-oxidase) (Gatterman,1999). It has been hypothesized that malfunction of one orof several of the enzyme complexes of the mitochondrialencoded respiratory chain genes may contribute to theinefficient reduction of iron in MDS and that this malfunc-tion could be caused by mutations of nuclear DNA or ofmitochondrial DNA, both of which contribute to theassembly of respiratory chain complexes (Jacobs, 1986;Jacobs & Bowen, 1992; Gatterman, 1999). Previous studieshave revealed the presence of mt-DNA mutations affectingcytochrome c-oxidase I gene in two patients with AISA(Gatterman et al, 1997), but the other varieties of MDS havenot been investigated. The current study was conducted tonot only detect mutations in mt-DNA encoded cytochromec-oxidase I and II genes, but also to explore the relevance ofthese mutations in the excessive apoptosis of haematopoieticcells in MDS. Our results indicate an unexpectedly high rateof mutations among 16/20 MDS patients examined.

An additional unique feature of this study is related todetailed morphological examination of mitochondria inbone marrow (BM) biopsy tissue of MDS patients. In thepast, electron microscopic (EM) studies could not beperformed on BM biopsies because the decalcificationprocess destroys ultrastructural detail (Cohen et al, 1997).A novel decortication technique was used to mechanicallytease apart the pieces of bone from the BM biopsy samplesprior to embedding the tissue for EM studies (Weiss, 1976).By eliminating the chemical decalcification step, it waspossible to examine the morphological details of iron-laden,swollen and bizarrely shaped mitochondria present in both

apoptotic and non-apoptotic dysplastic cells of MDS patients.In addition, both stromal and parenchymal MDS cells couldbe investigated precisely as they exist in vivo with well-preserved geographical relationships. These studies haveresulted in the recognition of novel, previously unrecog-nized ultrastructural details of mitochondria as well asapoptotic stromal and parenchymal cells in MDS bonemarrows. To our knowledge, the present study is the first toinvestigate simultaneously the morphological ultrastruc-tural details of mitochondria in MDS BM biopsies, and toexamine mitochondrial DNA for the presence of mutationsin cytochrome c-oxidase genes. Both aspects of the studyshed new light on the role of mitochondria in the pathologyand perhaps aetiology of cytopenias in patients withmyelodysplastic syndromes.

PATIENTS AND METHODS

The present study was conducted on peripheral blood (PB),bone marrow (BM) aspirates, BM biopsies and buccalsmears obtained from 20 patients with a confirmed diag-nosis of myelodysplastic syndromes and 10 normal, age-matched controls (mean ages were 64 years for MDSpatients and 62 years for controls). The normal controlsubjects were healthy volunteers. Informed consent wasobtained from every individual.

Samples. PB, BM aspirate, BM biopsy and buccal smearsamples were collected and transported on ice to thelaboratory. PB (10–15 ml) was collected in tubes contain-ing EDTA as an anticoagulant. BM aspirate (15–20 ml) wasaspirated directly into a syringe containing 3 ml of 2%sodium citrate. The low- and high-density mononuclear cellfractions (LDF, HDF) were separated from PB and BMaspirate samples using Ficoll–Hypaque 1077 density gradi-ent solution (Amersham Pharmacia Biotech, Piscataway,NJ, USA). Cells from high- and low-density fractions of PBand BM were used for detection of mitochondrial DNAmutations.

Detection of mitochondrial DNA (mt-DNA) mutations. Thetotal DNA was extracted from LDF and HDF mononuclearcells and buccal smear cells using the Capture Column Kitfrom Gentra Systems, Inc. (Minneapolis, MN, USA). Theprimers for cytochrome c-oxidase I and cytochrome c-oxid-ase II genes of mitochondria were designed using VECTORNTI (Infor Max, Inc. Bethesda, MD, USA) software programfrom the mitochondrial sequence (accession # NC 001807)and synthesized by Integrated DNA Technologies Inc.,Coralville, IA, USA. The primer sequences are provided inTable I. The mitochondrial DNA was amplified by thepolymerase chain reaction (PCR) using 35 cycles ofdenaturation (30 s at 94°C), annealing (45 s at 55°C) andextension (1 min at 72°C). The 100 ll PCR reactionscontained 10 mmol/l KCl, 20 mmol/l Tris-HCl (pH 8Æ8),400 lmol/l of each dNTP, 100 ng of each primer and5 units of Amplitaq DNA polymerase (Perkin-Elmer AppliedBiosystems, Foster City, CA, USA). The amplified PCRproducts for cytochrome c-oxidase I and cytochromec-oxidase II genes were visualized using ethidium bromidestaining of 2% agarose gels. The amplified PCR products

Mitochondrial DNA Mutations in Myelodysplasia 565

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 564–575

were sequenced using the automated DNA sequencer(MWG Biotech, High point, NC, USA). The sequencecomparison was made using BLAST SEARCH program(National Center for Biotechnology Information (NCBI),2001). The amino acid sequences for cytochrome c-oxidasegenes I and II were obtained using MITOMAP (2001).

Assessment of apoptosis using in situ end labelling (ISEL).Long-core BM biopsies were obtained from every individualand placed in saline. Under aseptic conditions, these biopsieswere cut into two segments. One half was placed in Bouinsfixative embedded in plastic using glycol methacrylate; 2- to3-lm thick sections were placed on Alcian blue-coatedcoverslips for detection of apoptosis by ISEL. The second halfwas used for electron microscopy (EM) as described below.Assessment of apoptosis was made using the ISEL techniqueas described by Wijsman et al (1993) and modified for use inplastic-embedded biopsy samples. Briefly, the cells werepretreated with sodium chloride and sodium citrate (SSC)solution at 80°C and 1% pronase (1 mg/ml in 0Æ15 mol/lphosphate-buffered saline; Calbiochem, La Jolla, CA, USA).The sections were then incubated at 18°C with a mixture of0Æ01 mol/l deoxyadenosine, deoxycytidine and deoxyguan-osine 5¢-triphophates (dATP, dCTP and dGTP) (Promega,Madison, WI, USA); 0Æ001 mol/l biotinylated uridine 5¢-tri-phosphate(bio-dUTP) (Sigma Chemical Co, St. Louis, MO,USA); and 20 U/ml DNA polymerase I (Promega). Incor-poration of bio-dUTP was finally visualized using an avidin–biotin peroxidase conjugate (Vectastatin Elite ABC kit;Vector, Burlingham, CA, USA) and diamino benzidinetetrachloride. Thus, cells labelled positively for ISEL showedbrown staining in their nuclei under the light microscope.The controls for these experiments were carried out asdescribed before (Mundle et al, 1995; Raza et al, 1995;Shetty et al, 1996). A subjective quantitative scale wasformulated to determine the degree of positivity as follows:negative, low, intermediate and high. Negative or absentindicates that there were less than 15% ISEL-positive cells;low, 1–3+ or 15–30%; intermediate, 4–5+ or 31–75%: andhigh 6–8+ or greater than 75% ISEL-positive cells.

Ultrastructural studies using electron microscopic studies ofdecorticated BM biopsy tissue. The second half of the BMbiopsy was processed for ultrastructural studies (Shettyet al, 2000). Briefly, the method involved perfusion of thebone with 3% glutaraldehyde, further immersion for15 min, and then careful teasing of the BM by decortica-tion. Processing for transmission EM was carried out usingstandard techniques. After fixation the cells were post fixed

in osmium tetroxide, treated with alcohol and propyleneoxide, and embedded in Araldite (EM sciences, Washington,PA, USA) at 58°C for 48 h. The biopsies were thenprocessed for semithin and ultrathin sections. Semithinsections were stained with toluidine blue and evaluated bylight microscopy. Ultrathin sections were contrasted withuranyl acetate and lead citrate and analysed using trans-mission electron microscope (TEM) (JEOL 200, Japan).

Statistical analysis. The non-parametric Mann–Whitney Uand chi-square tests were used for comparison between thetwo parameters.

RESULTS

The study included a total of 20 MDS patients and 10 normalhealthy subjects. Among the 20 MDS patients, seven patientshad refractory anaemia (RA), four had refractory anaemiawith ringed sideroblasts (RARS), five had refractory anaemiawith excess blasts (RAEB), two had refractory anaemia withexcess blasts in transformation (RAEB-t) and two had chronicmyelomonocytic leukaemia (CMMoL).

Mitochondrial DNA mutations in normal subjectsand MDS patientsThe primers used to amplify cytochrome c-oxidase I andcytochrome c-oxidase II genes (Table I) resulted in 375 and400 base pair PCR products respectively (Fig 1). The clinicalcharacteristics of MDS patients are presented in Table II.

Normal subjectsThe sequence comparison of these genes revealed that nonormal sample had any cytochrome c-oxidase I gene muta-tion (Table III) while 2/10 had cytochrome c-oxidase IImutations (Table III). In the normal subject (N2, Table III),PB LDF showed a deletional point mutation of A fromnucleotide position 7622 (Table III) in the cytochromec-oxidase II gene changing the amino acid sequence fromthreonine to leucine. BMLDF cells of this normal subjectalso showed an insertion of C at nucleotide position 7638which resulted in a change from glutamate to threonine(Table III). In the second normal subject (N7, Table III),PBLDF showed a substitution of A to G in nucleotideposition 7768, although this substitution did not changethe coding for the amino acid. The same substitution wasalso found at nucleotide position 7768 in the BMHDF cellsobtained from MDS patient #2 (Table III), and once againdid not result in an amino acid change.

Table I. The primers used to detect the mutations in mitochondrial encoded cytochrome c-oxidase I and

cytochrome c-oxidase II genes.

Gene name Primer sequence Size(bp) Primer binding position

(1) Cyt. c OXIF 5¢ACTACCCCGATGCATACACCACATG3¢ 375 7227Cyt. c OXIR 5¢TGCGCTGCATGTGCCATTAAGATAT3¢ 7601

(2) Cyt.c OX IIF 5¢ATGGCACATGCAGCGCAAGTAGG3¢ 400 7585

Cyt. c OXIIR 5¢GCAGGTCGCCTGGTTCTAGGAATAA3¢ 7984

566 P. L. Reddy et al


MDS patientsThe mitochondrial DNA mutations in MDS subjects des-cribed below are specific to the bone marrow and blood cells,as there were no mutations detected in matched buccalsmear cells. Sixteen of the 20 MDS cases studied showedmt-DNA mutations, four in cytochrome c-oxidase gene I only,three in cytochrome c-oxidase gene II only, and nine in bothgenes. Among the 13 cases with cytochrome c-oxidase geneI mutations, two had deletional, two had insertional, sevenhad substitutional mutations, while two patients (MDS #16and #18, Table II) had both deletional and insertionalmutations. Details of the specific mutations and theirconsequences in amino acid change are provided inTable III. This rate of mt-DNA mutations was significantlyhigher than age-matched normal subjects (P < 0Æ05).Cytochrome c-oxidase I mutations were most frequentlyseen at nucleotide positions 7264 (25%) and 7289 (15%),and cytochrome c-oxidase II gene mutations were mostfrequently seen at nucleotide positions 7594 (30%) and7595 (40%). The BMHDF cells showed a significantlyhigher percentage (45%) of cytochrome c-oxidase I muta-tions (Fig 2) than all other cell fractions tested (P < 0Æ05).The MDS subjects also showed a significantly higherpercentage of mutations in the cytochrome c-oxidase IIgene (P < 0Æ05) than their age-matched controls.

Fig 1. Ethidium bromide-stained agarose gel showing the poly-merase chain reaction (PCR) products of mitochondrial encoded

genes. Lane 1: DNA molecular weight marker, 100 bp ladder; Lane

2: 375 bp PCR product of cytochrome c-oxidase I gene. Lane 3:400 bp PCR product of cytochrome c-oxidase II gene.

Table II. Biological and clinical characteristics of myelodysplastic syndrome (MDS) subjects.

Subjects

Age

(years) Sex FAB type Cellularity ISEL Karyotype

Mutations in

Cyt.c oxidase I

Mutations in

Cyt.c oxidase II

MDS 1 50 Female RA hyper 5 46,XX[18] Yes Yes

MDS 2 71 Male RA hyper 7 46,XY[20] Yes Yes

MDS 3 47 Male RA hyper 4 46,XY,del (20)(q11.2q13.3)[19]/46,XY[1]

No Yes

MDS 4 71 Male RA hyper N/A 46,XY,del (20)(q11.2q13.1) No No

MDS 5 89 Female RA N/A N/A N/A No NoMDS 6 77 Male RA hyper 8 46,XY[20] Yes No

MDS 7 64 Female RA hyper 0 45,XX,)7[19]/46,XX[1] Yes Yes

MDS 8 28 Female RA hyper N/A 46,XX[20] Yes Yes

MDS 9 35 Female RARS normal 8 N/A Yes YesMDS 10 76 Male RARS hyper 6 46,XY,del(7)(q22q36)[13]/46,XY[6]/

NCA:

47,XY,del(7)(q22q36), +8

Yes Yes

MDS 11 62 Male RARS N/A 3 N/A Yes YesMDS 12 65 Male RARS N/A N/A N/A No Yes

MDS 13 70 Male RAEB hypo 3 46,XY,del(5)(q15q33)[10]/46,XY[10] No No

MDS 14 72 Male RAEB hyper 4 47,XY, +8[10]/45,XY,)7[6]/46,XY[4] No NoMDS 15 70 Male RAEB normal 5 46,XY[20] Yes Yes

MDS 16 72 Male RAEB hyper 1 46,XY[20] Yes No

MDS 17 79 Male RAEB-t hypo N/A 46,XY[20] Yes No

MDS 18 66 Female RAEB-t N/A 0 45,XX,)4,)5,add(17)(p11.2),mar[3]/45,XX,dup(1)(p21p32),

?del(4)(q21),)5,)6,)7,+12,add(12)

(q21),add(17)(p11.2),

)18,)19, +21, +2–3mar[16]/46,XX[1]

Yes Yes

MDS 19 64 Female CMMoL hyper 0 46,XX[20] Yes No

MDS 20 54 Male CMMoL hyper N/A 46,XY[20] No Yes

*N/A, data not available; FAB. French–American–British; ISEL, in situ end labelling.



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Types of mt-DNA mutations in cytochrome c-oxidase gene I(Table III)In cytochrome c-oxidase gene I, there were a total of 18mutations identified. Of these, nine were substitutions, fivewere deletions and four were insertions. Among the ninesubstitutions, three were C–A (np7264). The others wereC–G (np7502), A–G (np7519), G–C (np7444), T–G(np7549), T–C (np7572) and C–T (np7582). Four of the fivedeletions were a missing A at np7289 and the fifth was adeletion of C from np7264. The four insertions were one at T(np 7442), two were an insertion of G at np7463 and one wasan insertion of G at np7464. This makes the deletion of A (np7289) the most common mt-DNA mutation present in 4/18instances, followed by C–A substitution present in 3/18instances. The mutations at positions 7582 (MDS #6), 7519(MDS #10), 7572 (MDS #11) and 7549 (MDS #19) were inthe coding regions of t-RNA-aspartate and the mutations atpositions 7464 (MDS #8), 7463 (MDS #16 and 18) and 7502(MDS #17) were in the coding regions of t-RNA serine.

Types of mutations in cytochrome c-oxidase gene II (Table III)Thirty-four mutations were identified in gene II. Of these,25 were substitutions, five were insertions and four weredeletions. Eight patients had substitution of G–C(np7595), seven had a substitution between T–G(np7594). Four were substitutions of C–T at np7650,7868, 7891 and 7981. The others were substitution ofC–A in two instances, T–C in one, T–G in one, A–G inone and T–A in one. One had insertion of C at np7758,two had insertion of A at np7793, one had insertion of Aat np7814 and one had insertion of G at 7623. Amongthe four deletions, three had a deletion of A from np7622and one had a deletion of A from np7625. This makes G–C substitutions (present in eight instances), T–G substitu-tions (present in seven instances), C–T substitutions(present in four instances), and deletion of A (present infour cases) as the four most common types of mt-DNAmutations in gene II.

ÔHot spotsÕ for mitochondrial DNA mutations in cytochromec-oxidase gene I and II in MDSCertain Ôhot spotsÕ were identified in cytochrome c-oxidasegenes I and II that were frequently involved in a mutationalevent. The most common site of mt-DNA mutation wasnucleotide position 7595 in cytochrome c-oxidase gene IIwhere a substitutional change G–C affected a coding regionchanging the amino acid alanine to proline. This mutationwas found in eight MDS patients (40%), three belonging toRA, two to RARS, one RAEB-t and one CMMoL patient. Inonly two cases was this mutation detected in the BM MNC,both in the HDF (MDS # 8 and 18), while six cases had themutation in the PB, five of them in the LDF. The secondcommon mutation in cytochrome c-oxidase gene II was asubstitutional change in six MDS patients (30%) at nucleo-tide position 7594 from T–G resulting in a switch in aminoacid from histamine to glutamine. These seven patientswere the same ones who also had the substitutionalmutation at np7595 described above (Table III). Onceagain, 4/6 patients with this mutation had it in the PBM

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only, one in the BM only and one in both PB and BM. Infour (20%) cases, there was a mutation in cytochromec-oxidase gene I at np 7264, which was substitutional inthree (MDS # 1, 9 and 10), and in one (MDS # 15) it was adeletion. The substitutional mutation was from C to A in aterminating codon (Table III) in three MDS patients (MDS #1, 9 and 10), while in MDS # 15 a deletion of C at np 7264resulted in an amino acid change from serine to tyrosine.Cytochrome c-oxidase gene I np 7289 was mutated in 15%MDS patients (Table III).

Mitochondrial DNA mutations in PB versus BMand in mature versus immature cell compartmentsof individual patientsAn interesting observation was the detection of differentmitochondrial DNA mutations in the peripheral bloodversus bone marrows of the same patient as well asdifferences in different fractions of cells within the samecompartment. These differences are described in detail inTable III, affected both genes and were also present in anormal subject (N #2), as shown in Table III. In MDS #1,for example, there was a substitutional mutation (C–A)detected in the terminating codon of cytochrome c-oxidasegene I at np7264 in BMHDF, while a substitution betweenG–C was noted at np7595 in PBLDF resulting in an aminoacid change from alanine to proline. Table III shows thatthere were many variations present within the different cellcompartments in different patients. Patient MDS #7 showeda deletional mutation in gene I in BMHDF only, and none inBMLDF. In the same patient, PBLDF only showed threedifferent point mutations, one being deletional and twobeing substitutional, while none were detectable in PBHDF.One explanation for this seeming incongruity is that thereare several thousand mitochondria per cell and, given theirheteroplasmic transmission during mitosis, not all daughtercells receive the same share of mitochondria. Thus cellswhich are able to mature more (HDF) than others (LDF)may have different numbers of mitochondria containingmutated DNA. A similar explanation can be given for cells

that are able to exit the marrow (PB) versus those thatundergo intramedullary apoptosis (BM).

Mitochondrial DNA mutations and the FAB categoriesThere were eight patients with RA and six of them showedmutations, while all four RARS patients had mt-DNA muta-tions. Of the six RA cases with mutations, four showedmutations in both genes (MDS # 1, 2, 7 and 8, Table III),one in cytochrome c-oxidase I only (MDS # 6) and one(MDS # 3) in cytochrome c-oxidase gene II only (Table III).All three RARS patients had mt-DNA mutations affectingcytochrome c-oxidase gene I that were substitutional. MDS#9 and 12 had T–G substitution at nucleotide position7594. Both these mutations were present in a coding regionand resulted in an amino acid change from histamine toglutamine. Nucleotide position 7594 appears to be a ÔhotspotÕ for substitutional mutations (T–G) in cytochromec-oxidase gene II as it was affected in four other MDSpatients (MDS # 7, 8, 18 and 20), and resulted in each casein an amino acid change from histamine to glutamine(Table III). The mutation appeared to be independent ofFrench–American–British (FAB) subtype as the mutationswere seen in two RA (MDS # 7 and 8), two RARS (MDS #9and 12), one RAEB-t (MDS # 18) and one CMMoL (MDS #20). On the whole, 30% of MDS patients had mt-DNAmutations at this nucleotide position. Of the four RAEBpatients studied, two did not have any evidence of mt-DNAmutations (MDS # 13 and 14), while two had mutations(MDS # 15 and 16). Details regarding the type of mutationsand whether these affected a coding region or not and theirconsequences on amino acid change are described inTable III. There were two RAEB-t patients included in thestudy, and both showed mt-DNA mutations in cytochromec-oxidase gene I (MDS # 17 and 18), while one also had adeletional mutation in cytochrome c-oxidase gene II (MDS #18). Both CMMoL patients (MDS # 19 and 20) showedsubstitutional mutations, one in cytochrome c-oxidasegene I (MDS # 19) and the other in cytochrome c-oxidasegene II (MDS # 20).

Fig 2. The percentage distribution of myelo-dysplastic syndrome (MDS) patients and

normal healthy subjects showing the mitoch-

ondrial DNA encoded cytochrome c-oxidase I

and II gene mutations in low- and high-den-sity cell fractions of peripheral blood and bone

marrow.



Ultrastructural and apoptosis studies of BM biopsiesin MDS and normal subjectsMorphological details of the BM biopsies from normalsubjects and MDS patients were evaluated by EM. The ultrastructural details were well preserved in normal biopsies.MDS marrow showed an excessive degree of apoptosis incells belonging to all lineages (Fig 3). However, the apop-tosis was predominantly seen in the myeloid and erythroidlineages as reported earlier (Mundle et al, 1995; Raza et al,1995; Shetty et al, 1996). The stromal cells showedrelatively little apoptosis. Mitochondrial morphology isrepresented graphically in Fig 4. It was well preserved innormal marrow (Fig 4E); however, MDS bone marrowshowed grossly abnormal, iron-laden mitochondria in botherythroid (Fig 4A and C) and myeloid (Fig 4B and D)lineages. MDS bone marrow also contained large numbersof apoptotic cells with swollen and grotesquely distortedmitochondria.

Fig 3. The myelodysplastic syndrome (MDS) bone marrow showingoverall incidence of apoptosis in erythroid, myeloid and megak-

aryocytic lineages (uranyl acetate and lead citrate, original mag-

nification ·1600).

Fig 4. Ultrastructure studies of mitochondriafrom myelodysplastic syndrome (MDS)

patients (A–D) and normal (E) bone marrow.

(A) The ringed sideroblast showing the char-

acteristic iron-laden mitochondria (uranylacetate and lead citrate, original magnifica-

tion ·8300). (B) The myeloid cell showing

abnormal mitochondria (uranyl acetate and

lead citrate, original magnification ·6600).(C) Note the dense iron deposits in the mito-

chondria of a sideroblast (uranyl acetate and

lead citrate, original magnification ·33000).(D) Note the swollen cristae and dense deposits

in the mitochondria of a myeloid cell (uranyl

acetate and lead citrate, original magnifica-

tion ·33000). (E) A cell depicting a normalmitochondria with well-preserved cristae

(uranyl acetate and lead citrate, original

magnification ·20000).



Apoptosis studies in MDS and normal subjects using ISELtechniqueThe degree of apoptosis was measured in plastic-embeddedBM biopsies using in situ labelling of fragmented DNA(Fig 5). Apoptosis was seen not only in all the threehaematopoietic lineages ( myeloid, erythroid and mega-karyocytic) but also in stromal cells. MDS patients had asignificantly higher level of apoptosis than normals(P < 0Æ05). The level of apoptosis was significantly higherin patients with mutations in BM high-density fractions(P < 0Æ05).

DISCUSSION

We report an exceptionally high rate of DNA mutations inthe mitochondrial encoded cytochrome c-oxidase I and IIgenes in 16/20 MDS patients studied. Gatterman et al(1997) described mitochondrial DNA mutations in cyto-chrome c-oxidase I in acquired idiopathic sideroblasticanaemia, but to our knowledge this is the first report ofmt-DNA mutations affecting cytochrome c-oxidase gene IIin all subcategories of MDS patients. Several features of thisstudy were unique and are summarized below.

• mt-DNA mutations were only found in the haemato-poietic cells of MDS patients and were not found in matchedbuccal smear samples.

• Mutations were insertional, deletional, substitutionalor a combination.

• Cells obtained from the bone marrow high-densityfraction were the most likely to contain mt-DNA mutations.These cells represent the more mature fraction of thesample, and have already been shown to contain largenumbers of apoptotic cells in MDS patients. Cells containingmt-DNA mutations, albeit with a lesser frequency, were alsofound in low-density BM fraction and both low- and high-density fractions of peripheral blood compartments.

• mt-DNA mutations present in the bone marrow werenot necessarily the same as, or even present, in theperipheral blood and vice versa.

• Both cytochrome c-oxidase gene I (13/20 patients) andcytochrome c-oxidase gene II (12/20 patients) were affectedequally, and the majority of mutations were substitutional.

• Certain frequently involved Ôhot spotsÕ were identifiedfor cytochrome c-oxidase gene I mutations affecting nuc-leotide positions 7264 (25%) and 7289 (15%), and cyto-chrome c-oxidase gene II mutations affecting nucleotidepositions 7595 (40%) and 7594 (30%). The mutation atnp7595 resulted in a substitution of G to C changing theamino acid from alanine to proline, and at np7594,between T to G changing the amino acid from histamineto glutamine. Seven MDS patients had the substitutionalmutation at np7595 and six of these same patients also hadthe substitutional mutation at np7594. Although thesemutations were within the primer binding sites, they couldnot be artefactual because we sequenced both strands todetect these mutations.

• Patients belonging to all FAB categories of MDS werefound to have mt-DNA mutations, but they were mostconsistently present in RARS patients (4/4). No mt-DNAmutation was uniquely related to a specific FAB category.

• There was a significant association between mitoch-ondrial encoded cytochrome c-oxidase gene I mutations inthe high-density fraction of mononuclear cells and the levelof apoptosis in the BM biopsies (P < 0Æ05).

• None of the mitochondrial mutations described hereare polymorphic as they have not been recorded in thehuman mitochondrial genome database (MITOMAP, 2001).This does not exclude the presence of unknown polymor-phisms.

• Electron microscopic studies conducted on decorticatedbone marrow biopsy tissue enabled the first investigation ofultrastructural detail of mitochondria in both apoptotic andnon-apoptotic cells belonging to haematopoietic stromaland parenchymal lineages. Normal as well as bizarre,swollen and iron-laden mitochondria were clearly identified.

The first important point is that mt-DNA mutationsidentified in this study did not represent previously recordedpolymorphisms. Rather, they appeared to be acquiredmutations which were unique to the haematopoietic cellsof MDS patients as they were absent in buccal smearsobtained from the same patients. Mutations were morecommon in cytochrome c-oxidase gene I than in gene II. Ofthe 18 instances of mt-DNA mutations identified in gene I,there were nine substitutions, the most common being threeinstances of substitution between C–A. In cytochromec-oxidase gene II, the most common type of mutationswere substitutions present in 25 instances, followed by fiveinsertions. Certain nucleotide positions were frequentlyaffected in gene II and two of these at np7594 andnp7595 can definitely be considered as Ôhot spotsÕ formt-DNA mutations. These resulted in an amino acid changefrom alanine to proline when the G–C substitution occurredat np7595, and from histamine to glutamine when the T–Gsubstitution occurred at np7594.

Some of these mutations were only found in cellsobtained from either blood or the marrow, but not in bothsamples of the same patient. Even within a haematopoieticcompartment, mutations were frequently present only in a

Fig 5. High percentage of apoptosis in haematopoietic and stromalcells of myelodysplastic syndrome (MDS) bone marrow using the

in situ end labelling (ISEL) technique.



fraction of low- versus high-density cells. At first glance, thisappears counter intuitive. However, there are severalplausible explanations for this observation. First, this cannotrepresent a sampling error as no mutations were found innormal cells of the same patients (buccal smears). Further-more, many of these were recurrent rather than randommutations both across patients and across various sampleswithin the same patients, making it less likely to be amethodological issue. Second, it must be remembered thatthe polymerase chain reaction (PCR) method used for geneamplification followed by sequencing is likely to detect themost predominant cell population within a given sample.The marrow contains more immature cells than the blood,and if the mt-DNA predominated in the mature cells, then itwould only be detectable in the blood and vice versa. Thesame applies to various fractions of mononuclear cellsseparated on a density gradient, where the more mature aswell as apoptotic cells being heavier sink to the bottom andare recovered in the high-density fraction. Again, if themajority of mutated mitochondria are present in the moremature or dying cells, then only cells obtained from thehigh-density fraction of blood or marrow will show thepresence of the mutation.

Haematopoiesis in patients with myelodysplastic syn-dromes has repeatedly been shown to be monoclonal (Ananet al, 1995). An important question relates to the biologicalversus aetiological significance of mt-DNA mutations if theyare only present in a fraction of cells, be they mature orimmature, in an otherwise monoclonal population. Muta-tions could not possibly be the cause of the disease if theyare absent from a subpopulation of cells which haveotherwise descended from the transformed MDS parent cellas it would mean that the transformed MDS parent cell iscapable of generating both cells with mutated and wild-typemt-DNA. In other words, the presence of mt-DNA mutationsin a subset of cells would represent derivative populationsand evolving clones, and thereby reflect the biology and notthe aetiology. Given the unique multiplicity of mt-DNA andits peculiar independent replication, however, it is stillactually possible for such mutations to be the initiatingevent despite being manifested in a subset of cells in amonoclonal population. Further studies are needed tounderstand the role of mitochondrial DNA mutations inclonal selection by using the phenotypically well definedearly haematopoietic stem cells.

The increased level of apoptosis observed in MDS subjectsin the present study is in agreement with earlier reports(Mundle et al, 1995; Raza et al, 1995; Shetty et al, 1996).Recently, Shetty et al (2000) reported that the highestpercentage of apoptotic cells are recovered from the high-density fraction of MDS BM aspirates. The current observa-tion that the highest percentage of mitochondrial encodedcytochrome c-oxidase I gene mutations are present in thehigh-density fraction of MDS BM aspirates suggests thatthere may be a relationship between the mt-DNA mutationsand the level of apoptosis. However, the hypothesis that thetwo are related must remain speculative at best. Three mainmechanisms have been proposed to implicate the role ofmitochondria in apoptosis. The earliest mechanism involved

the disruption of the electron transport system (ETS). Forinstance, gamma irradiation induces apoptosis in thymo-cytes by disrupting the electron transport chain, probably atthe cytochrome b-c1/cytochrome c step (Scalfe, 1966). Theconsequence of the loss of ETS is a drop in ATP production.Such a drop in ATP production has been noted in apoptosis(Bossy-Wetzel et al, 1998). The second mechanism pro-posed was the activation of caspases by the cytochromeÔcÕ released from the mitochondria. Cytosolic cytochromeÔcÕ forms an essential part of the vertebrate ÔapoptosomeÕwhich is composed of cytochrome c, Apaf 1 and procaspase-9 (Li et al, 1997). The result is activation of caspase-9,which then processes and activates other caspases toorchestrate the biochemical execution of cells. In additionto cytochrome ÔcÕ, mitochondria are known to release otherpro-apoptotic mediators such as apoptosis-inducing factor(AIF) (Susan et al, 1999). The third mechanism proposedis the increased production of reactive oxygen speciesby mitochondria during apoptosis (Thress et al, 1999).Recently, Matthes et al (2000) showed an increased apop-tosis owing to decreased mitochondrial membrane potentialin acquired sideroblastic anaemia. Although it is not knownwhich of these mechanisms predominates in MDS, it is clearthat mitochondria constitute one of the key components inthe apoptotic process and the defect in complex III andcomplex IV of respiratory chain might contribute towardsan excessive apoptosis.

Another hallmark of the disease is that the bone marrowof MDS patients contain erythroblasts with a perinuclearring of iron-laden mitochondria as illustrated in Figs 4Aand C. Similar observations were made earlier (Cohen et al,1997). The intramitochondrial iron deposits contain iron inthe ferric (Fe3+) state and this ferric iron could beresponsible for an increased production of reactive oxygenspecies via the Fenton reaction (Halliwell & Gutteridge,1984). However, ferrochelatase, the enzyme that catalyseshaem synthesis, is known to use iron in the ferrous form.The electrons needed for the reduction of Fe3+ to Fe2+ arephysiologically generated through the electron transportsystem (Flatmark & Romslo, 1975). Mutations in cyto-chrome c-oxidase I and II genes might contribute towardsthe dysregulation of ETS such that the iron can no longer beconverted into the right chemical form for haem synthesis.The result is a deposition of the under-utilized iron in themitochondrial matrix of erythroid progenitors, giving rise tothe pathognomonic ringed sideroblasts.

The underlying cause for increased mutations in mito-chondrial encoded cytochrome c-oxidase I and II genesremains unknown. However, it has been reported thatexogenous factors such as toxins and viral infections cancause mutations in mitochondria. It has also been reportedthat one of the known human retroviruses, the humanimmunodeficiency virus (HIV), causes mitochondrial dys-function by selectively concentrating in mitochondria ofinfected cells (Somasundaran et al, 1994). Both toxins andviruses (Raza & Preisler, 2000; Reddy et al, 2000) havebeen proposed as being aetiological agents in the develop-ment of myelodysplastic syndromes, and may exert theirpathological effects through a disruption of mitochondrial



function. Further studies are needed to explore the possiblerole of mitochondria in the aetiology of MDS.

ACKNOWLEDGMENTS

This work was supported by a grant from the NationalCancer 1nstitute (PO1CA 75606), the Markey CharitableTrust (94–8) and the Dr. Roy Ringo Grant for basic researchin MDS. The authors wish to thank Ms. Lakshmi Venugopaland Ms. Sandra Howery for excellent administrative andsecretarial assistance. The authors also wish to thankSana Z. Raza, Zehra Raza, Seema Hussaini, Leena Joshi,William Townsend and Eileen Broderick for technicalassistance. We wish to thank Dr David Sidransky, Dr HimlaSoodyal and Dr Girish Modi for careful reading of themanuscript and helpful suggestions.

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