Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

Mitochondrial iron overload in acquired idiopathic sideroof species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial geblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respinomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood ratory chain dysfunction, thereby impairing reduction of ferric iron (Fe) to ferrous iron (Fe). The reduced form of iron samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenelectrophoresis. One of the mutations involves a T r C transition in nucleotide position 6742, causing an amino acid esis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial change from methionine to threonine. The other mutation is a T r C transition at nt 6721, changing isoleucine to threomembrane. q 1997 by The American Society of Hematology. nine. Both amino acids are highly conserved in a wide range


S
one or several of the enzyme complexes of the respiratory IDEROBLASTIC ANEMIA is characterized by inadequate formation of heme and excessive accumulation of chain are disturbed in such a way that efficient reduction iron in erythroblast mitochondria.Whereas recent molecular of iron no longer occurs. 14Under these circumstances, the biology approaches identified abnormalities of erythroid d- imported iron cannot be used for heme synthesis and will aminolevulinic acid synthase in the hereditary, X-linked type accumulate in the mitochondrial matrix.The proposed malof sideroblastic anemia, 1-7 the pathogenesis of the most prevfunction could be due to mutations of nuclear DNA or mitoalent form of sideroblastic anemia, acquired idiopathic sidchondrial DNA (mtDNA), because both genomes contribute eroblastic anemia (AISA), has remained an enigma.For to the assembly of respiratory chain complexes.Human many years, investigators tried to clarify the disorder by mtDNA is a 16,569-bp circular molecule that codes for the searching for enzyme defects of heme biosynthesis (Fig 1).
genes of 13 essential polypeptide subunits of the respiratory A major shortcoming of this approach was its inability to chain, together with the 12S and 16S ribosomal RNAs and explain why the penultimate product of the heme synthetic the 22 mitochondrial transfer RNAs that are required for the pathway, protoporphyrin IX, is elevated rather than deintramitochondrial translation of the protein-coding units.creased in most AISA patients. 8In our opinion, this finding In recent years, a variety of disorders with predominantly provides a strong argument for a possible defect in the last neurologic symptoms have been linked to mutations in step of heme synthesis, namely insertion of iron into proto-mtDNA. 15,16orphyrin IX.This step is catalysed by ferrochelatase.However, this enzyme is unlikely to be responsible, because increased red blood cell (RBC) protoporphyrin concentrations Our focus on mtDNA is partly explained by the fact that were found.Until 1996, the course of the disease was uneventful, Pearson's syndrome, a rare congenital disorder, points to a with Hb values fluctuating between 8.5 and 10 g/dL.In May 1996,   relation between mtDNA mutations and sideroblastic anethe patient received her first blood transfusion because her Hb level mia.This condition presents in infancy with a variety of had decreased to 8 g/dL.Meanwhile, transfusion of 2 U of packed symptoms, including refractory sideroblastic anemia, throm-RBCs is required every 3 to 4 weeks.In the bone marrow, there has bocytopenia, neutropenia, pancreatic exocrine dysfunction, been no evidence of progression toward a more advanced type of and lactic acidosis. 17The bone marrow shows dysplastic myelodysplastic syndrome (MDS).
changes including ring sideroblasts and prominent vacuol-Patient no. 2. A 68-year-old man presented with exertional dysization of precursor cells. 18,19The cause of Pearson's synpnea and fatigue in December 1989.The Hb level was 7.8 g/dL, the MCV was 77.7 fL, the MCH was 32.2 pg, and the serum ferritin drome was discovered in 1989, when Ro ¨tig et al 20 found an level was 664 mg/mL.The leukocyte count was 3.8 1 10 3 mL, and mtDNA deletion in a patient with the disorder.Investigation the WBC differential was 27% neutrophils, 1% bands, 3% eosinoof further patients confirmed that large deletions of mtDNA phils, 3% monocytes, and 66% lymphocytes.The platelet count was are a constant feature of this disease. 21However, the relation 338,000/mL.Bone marrow biopsy showed hyperplasia and dysplasia between mtDNA deletions and sideroblastic anemia was not of the erythroid series, with megaloblastoid changes and nuclear understood.According to our pathogenetic model, 14 deficient lobulation and fragmentation.Iron staining showed 70% to 80% ring iron reduction, attributable to respiratory chain dysfunction, sideroblasts.Multinucleated megakaryocytes were found.Granuloprovides the missing link.cytopoiesis appeared inconspicuous, and there was no excess of Having excluded large deletions of mtDNA as a cause of blasts.A diagnosis of RARS was made.In addition, the patient was AISA, 22 we started looking for short deletions and point found to have a multinodular goiter and to be hyperthyroid.He was successfully treated with radioactive iodine.In March 1990, a control mutations.Recently, we reported a patient who had a heterobone marrow biopsy showed no significant change, but the patient plasmic point mutation of mitochondrial tRNA Leu(CUN) in his became transfusion dependent.In September 1991, anemia bone marrow cells. 23However, this mutation did not help to worsened, bone marrow biopsy showed greater than 90% ring siderolocalize the crucial site of iron reduction and transport blasts, and cytogenetic examination was abnormal for the first time through the mitochondrial inner membrane, because mito-[in 4 of 20 mitoses, a 5q0 abnormality was found, ie, del(5) (q13chondrial tRNA mutations cause a general impairment of 14)].

mitochondrial protein synthesis and can thus affect all respi-
The patient was entered into a pilot study of all-trans retinoic ratory chain complexes possessing components encoded by acid 24 and responded with an increase in Hb values up to 11.8 g/ mtDNA.More informative in this respect are the two point dL.Ring sideroblasts in the bone marrow decreased to 45% (July mutations presented here.1992).The ferritin level was 385 ng/mL.In January, 1993, anemia worsened again, a bone marrow biopsy showed 70% ring sidero-MATERIALS AND METHODS blasts, and karyotype analysis confirmed the 5q0 anomaly.The patient received a second trial of ATRA until June 1993.He re-

Case Reports
sponded again, became independent of transfusions, and showed an Patient no. 1.A 58-year-old woman was first evaluated for mod-Hb level of 10.2 g/dL.The disease remained stable from 1993 to erate macrocytic anemia in 1992.The hemoglobin (Hb) level was 1995, when the patient turned up for a follow-up examination: Hb 11 g/dL, an RBC count of 2.94 1 10 6 /mL, a hematocrit level of level of 11 g/dL, RBC count of 4.52 1 10 6 /mL, hematocrit level of 33%, a mean corpuscular volume (MCV) of 112 fL, a mean cor-34.6%,reticulocytes level of 13.4%, and MCV of 76.7 fL.The puscular Hb (MCH) level of 37.8 pg, and a mean corpuscular Hb platelet count was 227,000/mL.Bone marrow biopsy showed dyscontent (MCHC) of 33.7 g/dL.The leukocyte count was 8.5 1 10 3 / plastic changes in all three cell lines, including some megakaryocytes mL, and the white blood cell (WBC) differential was 50% neutrowith nonlobulated nuclei.Ring sideroblasts were 90%.Karyotype phils, 2% bands, 2% eosinophils, 1% basophils, 5% monocytes, and analysis showed 5q0.Despite the high proportion of ring sidero-40% lymphocytes.The platelet count was 596,000/mL.Bone marrow blasts, Hb levels have remained stable.biopsy showed a hypercellular marrow with dysplastic changes in all three myeloid cell lines.On iron staining, 40% of erythroblasts

Preparation of Cell Fractions
were ring sideroblasts.There was no excess of myeloblasts.A diagnosis of refractory anemia with ring sideroblasts was made (RARS; The patients were part of a series of 20 patients with MDS whose bone marrow samples were subjected to restriction fragment length according to the French-American-British [FAB] classification).Cytogenetic analysis showed a normal karyotype; in particular, there polymorphism (RFLP) screening for mtDNA mutations.Among those 20 patients, 12 had sideroblastic anemia.Fifty hematologically was no evidence of a 5q0 abnormality.The patient had psoriatic arthritis since 1979, but was never treated with methotrexate or normal individuals (cardiac surgery patients) were specifically screened for the mutations of cytochrome c oxidase (COX) at nucleo-cyclophosphamide. On follow-up examinations, bone marrow biopsies showed that ring sideroblasts were greater than 50%; there were tide positions 6721 and 6742 that we identified in AISA patients; AID Blood 0002 / 5h42$$$$21 11-10-97 12:01:52 blda WBS: Blood For personal use only.on April 19, 2017.by guest www.bloodjournal.orgFrom bone marrow samples from the controls had been obtained for a mtDNA Amplification previous study on mtDNA deletions. 25In the two patients under mtDNA was amplified in 17 overlapping segments using the PCR.discussion, mtDNA was extracted from an unfractionated bone mar-The exact position of segments according to the standard Cambridge row sample and from peripheral blood cells of different lineages.
sequence 29 and the respective primer sequences were as previously Platelet-rich plasma was obtained from citrated blood samples by reported. 23DNA was amplified using 35 cycles of denaturation (1 centrifugation at 100g for 15 minutes at room temperature.Contamiminute at 94ЊC), annealing (30 seconds at 55ЊC to 71ЊC, depending nating blood cells were removed by centrifugation at 200g for 10 on the pair of primers used), and extension (60 seconds at 72ЊC).minutes.Platelets were pelleted from the supernatant by centrifuga- The 100 mL PCR reactions contained 10 mmol/L KCl, 20 mmol/L tion at 1,000g for 10 minutes and resuspended in phosphate-buffered Tris-HCl (pH 8.8), 10 mmol/L (NH 4 ) 2 SO 4 , 4 mmol/L MgS0 4 , 400 saline.Granulocytes were enriched by depletion of peripheral blood mmol/L of each dNTP, 50 pmol of each primer, and 1 U of Vent DNA mononuclear cells through density gradient centrifugation with polymerase (New England Biolabs, Dreieich, Germany).Double-Lymphoprep 1.077 (Nycomed, Oslo, Norway).A 200 mL sample stranded PCR products were purified with a commercial DNA purifrom the granulocyte/erythrocyte pellet was used for DNA extracfication system (Wizard PCR Preps; Promega, Heidelberg, Gertion.Erythrocytes do not contain mtDNA, 26 because mitochondria many), separated in 1% agarose gels, and visualized by ethidium are eliminated during the reticulocyte stage. 27Therefore, mtDNA bromide staining.isolated from the granulocyte/erythrocyte pellet mainly derives from mature peripheral blood granulocytes, apart from a small contamination by reticulocyte mtDNA.For isolation of B and T lymphocytes All restriction enzyme reactions were performed according to manubeads were removed with a magnet.Buccal mucosa cells were pelfacturer's recommendations, using approximately 100 to 200 ng of leted from a vigorous mouthwash performed with 20 mL normal PCR-generated DNA for digestion with 5 U of enzyme.Restriction saline and depleted of contaminating granulocytes by density gradifragments were separated by electrophoresis in 3.5% Visigel matrix ent centrifugation with Nycoprep 1.150 (Nycomed).A skin biopsy (Stratagene, Heidelberg, Germany) and visualized by ethidium bro-(right buttock) was obtained from patient no. 2, and fibroblast culmide staining and UV illumination.tures were established by outgrowth from this biopsy according to a standard method. 28Cells were grown to confluency in Dulbecco's modified Eagle's Medium (Flow, Meckenheim, Germany) supple-

Electrophoresis (TGGE)
penicillin (400 mg/mL), streptomycin (50 mg/mL), and 10% fetal Point mutations in double-stranded DNA can be detected by denacalf serum (FCS) on tissue culture dishes in a humidified atmosphere turing-gradient gel electrophoresis (DGGE) 30 or TGGE, 31,32 because of 5% CO 2 and 95% air at 37ЊC.mutations alter the denaturation pattern of double-stranded DNA at the site of the mutation and in its neighborhood.The sensitivity of TGGE analysis is greatly enhanced through heteroduplex generation.

Colony Assay of Hematopoietic Cells
During a denaturation/renaturation cycle, hybrids are formed be-Mononuclear cells were obtained from peripheral blood (PBtween wild-type DNA fragments and homologous fragments car-MNC) through density gradient centrifugation with Lymphoprep rying the mutation.The mismatch in the double helix at the site of 1.077 (Nycomed), washed three times in RPMI 1640 medium, and the mutation leads to significant lowering of the midpoint melting plated in tissue culture-grade 35-mm Petri dishes (2 1 10 5 cells per temperature of the heteroduplex fragments.When exposed to indish) in a volume of 1 mL MethoCult H4433 (StemCell Technolocreasing temperatures during electrophoresis (the TGGE procedure), gies, Vancouver, British Columbia, Canada), containing 0.9% methheteroduplex hybrids show partial denaturation earlier than homoduylcellulose, 5% serum (containing phytohemagglutinin-leukocyte plexes do.Because early partial denaturation causes early retardation conditioned medium [PHA-LCM]), 30% FCS, 10% bovine serum in the gel, heteroduplexes can be resolved from homoduplex molealbumin, 13% Iscove's medium, erythropoietin (150 U/mL), 2-mercules by TGGE.captoethanol (0.1 mol/L), and L-glutamine (200 mmol/L).After 14  In the two patients reported, intraindividual heteroduplex analysis days of incubation at 37ЊC and 5% CO 2 , colonies and clusters were was performed, ie, TGGE was used to examine whether heteroexamined using an inverted microscope.For polymerase chain reacduplexes formed after PCR amplification of a mtDNA fragment of tion (PCR) amplification of a particular COX I segment of mtDNA, interest.This only happens when there is coexistence of wild-type individual colonies were picked with a micropipette and entered and mutated mtDNA in the patient's sample, which is also called directly into PCR reaction mixtures (before adding specific primers heteroplasmy.Among other segments of mtDNA, a 420-bp segment and polymerase).

DNA Isolation
lowed by quantitative denaturation of the PCR product (heating in Total DNA was isolated from the various cell samples using the 4 mol/L urea at 95ЊC for 5 minutes) and subsequent renaturation QIAamp Kit (Qiagen, Hilden, Germany), which is based on cell (incubation at 50ЊC for 20 minutes).Samples were then loaded onto lysis in the presence of guanidine hydrochloride and subsequent thin polyacrylamide gels (5%) covalently bound to polyethylene binding of DNA to an anion-exchange resin in a spin-column.plained fragments of 594 and 497 bp and a very short fragment of 14 bp that was not visualized in the gel.The relative intensities of bands were estimated by gel photography with an electronic camera and data analysis with suitable software (Enhanced analysis system, EASY plus; Herolab GmbH, Wiesloch, Germany).The system sums up the light intensities of all pixels belonging to a specific band (with automatic background subtraction).The intensities were 182, 113, and 66, respectively, for the 497-bp band representing normal molecules and the 281-bp and 216-bp bands representing cleavage products of mutated mtDNA molecules.After correcting for molecular weight, this translated into 49% mutated mtDNA in the patient's bone marrow.
The same proportion (50% mutated mtDNA) appeared when the calculation was based on data of another experi- and the absence of the mutation in the patient's mother and selected on Luria broth (LB) medium containing 150 mg/L ampicildaughters, excluded an inherited mutation.We suggest that lin and X-Gal. 34Plasmid DNA from transformants was prepared the mutation arose in a myeloid stem cell of the colonyusing Tip20 columns (Qiagen) and double-stranded DNA was seforming unit granulocyte, erythroid, monocyte, megakaryoquenced with the Sequenase kit (US Biochemicals, Cleveland, OH) cyte (CFU-GEMM) type, although we cannot exclude with using 35 S-dATP, according to the method of Sanger et al. 35 certainty the involvement of a pluripotent hematopoietic stem cell.The aberrant pattern was not detectable in 10 other

RESULTS
patients with sideroblastic anemia, 8 patients with other types In two patients with AISA we identified point mutations of MDS, and 50 hematologically normal controls (data not of mtDNA in the region encoding subunit I of cytochrome shown).c oxidase (nt 5904-7444).These mutations, in the following An mtDNA fragment containing the mutation was cloned referred to as COX I mutations, are located at nt 6721 and in E coli.Five clones were sequenced; three yielded the nt 6742, affecting amino acids M273 and I280, respectively, wild-type sequence and two showed a point mutation characof the human sequence.
terized by a T r C transition at nt 6721 (  this patient, too. Figure 10 clearly shows that skin fibroblasts and buccal mucosa cells were not affected by the mutation, 1 and her two daughters.This inherited homoplasmic genetic marker stands in contrast to the acquired heteroplasmic mu-whereas myeloid cells showed the abnormal restriction pattern.Analysis of band intensities showed a proportion of tation at nt 6721, which occurred in the patient's hematopoietic system and was detectable neither in cells of extramedul-52% mutated mtDNA in the bone marrow, 59% in a peripheral blood sample, and 71% in platelets.Faint pathologic lary origin nor in the patient's relatives.
bands in B and T lymphocytes could again be due to contam-Patient No. 2 ination with phagocytic cells during immunomagnetic bead isolation of lymphocytes or represent a low percentage of When the mutation at nt 6721 had been detected in patient mutated mtDNA in lymphocytes.As in patient no. 1, the data no. 1, the other patients in our series were screened for suggest that the mutation was restricted to the hematopoietic mutations in the COX I region by TGGE analysis.As a system.result, a further COX I mutation was found in patient no. 2 that had not been detected by RFLP analysis.Figure 7 shows a typical heteroduplex finding in perpendicular TGGE, and

Interspecies Comparison Fig 8 shows a parallel TGGE with heteroduplex bands of
The positions of both mutations are in close proximity to patients no. 1 and 2 in comparison with normal findings.
each other within subunit I of cytochrome oxidase and affect DNA sequencing showed that the mutation in patient no. 2 amino acids M273 and I280 of the human sequence.During was a T r C transition at nt 6742 (Fig 9).This mutation evolution, methionine and isoleucine in these positions have changes an amino acid from isoleucine (ATT) to threonine been strongly conserved, as shown by interspecies comparison (Table 1).Furthermore, high resolution screening of human populations involving hundreds of individuals has never shown restriction site polymorphisms at nt 6721 or showed that all clusters and colonies that we picked (erythro-6742. 36oietic, granulocytic, and monocytic) showed the same pathologic restriction pattern as the mtDNA derived from uncultured blood or bone marrow cells of the respective mtDNA From Clonal Cultures patient (data not shown).The percentage of mutated mtDNA Whereas erythropoietic cell growth in culture was comvaried between different colonies, but the average percentage pletely lacking in patient no. 1, erythropoietic clusters and was similar to that observed in whole blood or bone marrow some smaller colonies were observed in patient no. 2.
samples.In patient no. 2, there was no significant difference Growth of colony-forming units-granulocyte (CFU-G) and in the percentage of mutated mtDNA between erythropoietic colony-forming units-monocyte (CFU-M) appeared unimclusters and granulocytic colonies.paired in both patients.PCR amplification and RFLP analysis of the mtDNA segment containing the COX I mutation

DISCUSSION
The finding of mtDNA mutations affecting cytochrome c oxidase supports our hypothesis that AISA is a disorder involving mitochondrial respiratory chain dysfunction. 14Cytochrome c oxidase (COX) is a good candidate for playing a major role in mitochondrial iron metabolism.A strong hint in this direction comes from the copper connection, 37 which refers to the close relation between copper and iron in hematopoiesis, including the role of copper in mitochondrial iron metabolism.COX is the only respiratory chain complex that contains copper, namely Cu A1 and Cu A2 in subunit II and Cu B in subunit I. 38,39 Copper is an essential part of the metal redox centers of this enzyme complex.1][42][43][44][45][46][47] Williams et al 48 found that mitochondria isolated from copper-deficient animals were deficient in COX activity and failed to synthesize heme from Fe 3/ and protoporphyrin at the normal rate.The rate of heme synthesis correlated with the COX activity.Through experiments with respiratory chain inhibitors, these investi- For personal use only.on April 19, 2017.by guest www.bloodjournal.orgFrom as heteroplasmy in postmitotic tissues such as muscle or nerve.The matter is more in the bone marrow because the dynamics of hematopoietic cell proliferation must be taken into account.Thus, the degree of heteroplasmy that we observed with the two COX mutations may reflect a balance between (1) a putative replicative advantage of mutated mtDNA over wild-type mtDNA, (2) the proliferative advantage of the cells belonging to the pathologic clone of sideroblastic anemia, and (3) a proliferative disadvantage of cells containing a particularly high proportion of mutated mtDNA.
A very interesting aspect of heteroplasmy is its potential to explain a peculiar feature of AISA, namely the heterogeneity in the degree of mitochondrial iron accumulation.][67] Whereas such intraclonal heterogeneity is hard to explain in terms of mutations involving nuclear genes, mtDNA mutaboth COX I mutations that we identified are located in transtions could easily account for the phenomenon.Each cell membrane helix no.VII in the immediate vicinity of the contains hundreds of mitochondria and thousands of copies heme a 3 -Cu B center, 38 it is tempting to speculate that this of the mitochondrial genome.Therefore, cells can harbor metal redox center is involved in supplying electrons to Fe 3/ .mixtures of mutant and normal mtDNAs (heteroplasmy), and The copper connection has recently been strengthened by each time a heteroplasmic cell divides, the mutant and northe finding that high-affinity transmembrane uptake of iron mal mtDNAs are randomly segregated into the daughter in Saccharomyces cerevisiae requires copper.This is excells. 15In each cell, the severity of the resulting respiratory plained by the involvement of a copper-containing oxidorechain defect depends on the proportion of mutated mtDNA.ductase, FET3, in iron uptake. 49,50Yeast mutants defective A heterogeneous population consisting of normal and grossly in the fet3 gene are deficient in high-affinity Fe 2/ transport, abnormal as well as intermediate cells can thus be the conseand copper depletion of wild-type S cerevisiae also results quence of an mtDNA mutation.in a selective reversible decrease in Fe 2/ transporter activity.
It is conceivable that the COX mutations that we identified The FET3 protein exhibits extensive similarity to the familiy mainly disturb the process of mitochondrial iron reduction, of blue multicopper oxidoreductases, an enzyme superfamily without producing significant impairment of other respirathat also includes mitochondrial cytochrome c oxidase.tory chain functions.Although such a defect would interfere Both COX I mutations fulfill the criteria that are generally applied to exclude nonpathogenic polymorphisms of mtDNA, namely (1) the observed nucleotide change is absent in unaffected individuals; (2) it occurs in an evolutionary conserved region; and (3) the mutation displays heteroplasmy.As far as the first criterion is concerned, data are available from extensive population genetic studies involving more than 1,800 individuals from various ethnic backgrounds. 36,51-61To our knowledge, these studies never identified nucleotide changes in positions 6721 or 6742 of the mitochondrial genome.The second criterion is not reliable, because mutations of conserved nucleotides can occur without having a direct effect on the clinical phenotype. 62The third criterion, heteroplasmy, requires special consideration.When an mtDNA mutation arises, it creates an intracellular mixture of mutant and normal mtDNA molecules called heteroplasmy.Heteroplasmy is considered an indicator for pathogenic mutations because it is typical of diseases associ- The amino acids mutated in patients no. 1 and 2, Met 273 and Ile 280 according to the human sequence, have been strongly conserved during evolution.Sequence data are from earlier studies. 29, withe major metabolic activity of erythroblasts (ie, heme cap rather than a growth advantage for proliferating cells.25,68,69 A hematopoietic stem cell affected with a pathosynthesis), causing considerable damage through secondary genic mtDNA mutation must therefore acquire an additional effects of iron loading, its consequences may be minimal for nuclear mutation conferring growth advantage to become other cell lineages.In accordance with a defect primarily capable of establishing a clonal bone marrow disease such disturbing erythropoiesis, we found that erythropoietic colas sideroblastic anemia.In patient no. 2, the 5q0 anomaly ony growth (burst-forming unit-erythroid [BFU-E] and colmay have been the transforming event.ony-forming unit-erythroid [CFU-E]) was virtually absent The mtDNA mutations reported here fit into the mitochonor severely impaired, whereas growth of CFU-GM was not drial-DNA mutation hypothesis of cell ageing, 70,71 which is significantly reduced, despite the fact that cells in granulobased on (1) an approximately 10 to 20 times higher frecyte and macrophage colonies carried the same mtDNA muquency of mutations in mitochondrial as compared with nutation.
clear DNA; (2) the compactness of the mitochondrial ge-In general, mtDNA mutations associated with mitochonnome (no introns), increasing the probability that mutations drial diseases arise in the female germline and are present will affect functionally important regions; (3) the lack of in various tissues, albeit at widely differing percentages.To sufficient repair mechanisms; and (4) the increased producour knowledge, the two COX mutations described (and a tion of oxygen radicals in mitochondria with increasing age, mitochondrial tRNA mutation that we recently reported 23 ) promoting chemical modification of mitochondrial proteins are the first examples of disease-associated mtDNA mutaand mtDNA.Apparently, hematopoietic stem cells must be tions that are specifically related to a particular organ, ie, included among cells suffering age-related changes to their the hematopoietic system.As a prerequisite for this unusual mitochondrial genome. 25constellation, a mtDNA mutation must arise in a hematopoietic stem cell, which in turn must populate the bone marrow

Fig 1 .
Fig 1.Schematic representation of the heme biosynthetic pathway and its connection with the electron transport chain of the inner mitochondrial membrane.(1) 5-Aminolevulinic acid synthase; (2) 5-aminolevulinic acid dehydratase; (3) porphobilinogen deaminase; (4) uroporphyrinogen III synthase; (5) uroporphyrinogen III decarboxylase; (6) coproporphyrinogen III oxidase; (7) protoporphyrinogen III oxidase.Complex I, NADH dehydrogenase; complex II, succinate dehydrogenase; complex III, b/c1-complex !ubiquinol cytochrome c reductase; complex IV, cytochrome c oxidase; Q, coenzyme Q !ubiquinone; c, cytochrome c.The broken line (grey) represents the outer mitochondrial membrane.In accordance with the point mutations identified in mtDNA-encoded subunit I of cytochrome c oxidase, the reduction of Fe 3" to Fe 2" has been placed into complex IV of the respiratory chain.R from EDTA-anticoagulated blood, immunomagnetic beads (Dyna-Each amplified mtDNA segment was entered into 20 different beads M-450 Pan-B [CD19] and Dynabeads M-450 Pan-T [CD2], restriction enzyme digestions using Alu I, BamHI, Cfo I, Dde I, respectively) were used according to the recommendations of the EcoRI, Hae II, Hae III, HincII, HindIII, HinfI, Hpa I, Msp I, Pvu manufacturer (Dynal, Oslo, Norway).With antibody-coated beads II, Rsa I, Sac I, Sca I, Stu I, Taq I, Tru9I, and Xba I. Endonucleases still bound to the cell surface, lymphocytes were entered into the were purchased from Boehringer Mannheim (Mannheim, Germany).cell lysis step of the DNA extraction procedure.After cell lysis,

Fig 2 .
Fig 2. Detection of a heteroplasmic mtDNA mutation in a bone marrow aspirate from patient no. 1. RFLP analysis of mtDNA segment

Fig 5 )
. The mutation changes an ATG codon to ACG, thus causing an amino Patient No. 1 acid change from methionine to threonine.Complementary strands of mtDNA were also sequenced and showed the A heteroplasmic mutation was detected by RFLP analysis expected A r G transition at nt 6721 (data not shown).of unfractionated bone marrow cells when Rsa I digestion On RFLP analysis in patient no. 1, we also detected a of mtDNA segment no. 7 (nt 6504-7608) showed an aberrant hitherto unknown homoplasmic Taq I polymorphism at nupattern with two unexpected bands representing fragments cleotide position 8958 (Fig 6).Cloning and sequencing (data of 281 and 216 bp length (Fig 2, lane 9).Their appearance not shown) showed a C r T transition, changing the Taq I implied the presence of a new Rsa I site, because the two regular sites in this segment (nt 6999 and 7013) only ex-recognition sequence TCGA to TTGA.This polymorphism,

Fig 3 .
Fig 3. Cell lineage involvement of the COX I mutation in patient no. 1.Total DNA was extracted from different types of cells: Bm, bone marrow; Bl, peripheral blood; Plt, platelets; G, erythrocyte/granulocyte pellet; T, T lymphocytes; B, B lymphocytes; Mu, buccal mucosa cells; lane 8, DNA size marker; M, peripheral blood from the mother of patient no.1; D1 and D2, peripheral blood from the first and second daughter of patient no. 1. See text for explanation of the restriction pattern.

Fig 4 .Fig 8 .
Fig 4. Cell lineage involvement of the COX I mutation in patient no. 1. Immunomagnetic bead isolation of T and B lymphocytes was repeated with another peripheral blood sample, and a normal Rsa I Fig 5. Sequencing of the COX I mutation in patient no. 1. mtDNA light strand; normal sequence on the left, mutated sequence on the restriction pattern of mtDNA segment 7 (nt 6504-7608) was obtained for T and B lymphocytes.For abbreviations, see Fig 3. right.The pathologic clone showed a T r C transition at nt 6721.

Fig 9 .
Fig 9. Sequencing of the COX I mutation in patient no. 2. mtDNA

Fig 10 .
Fig 10. Cell lineage involvement of the COX I mutation in patient