British3'ournal ofOphthalmology 1993; 77: 84-88

Retinitis pigmentosa, ataxia, and mental retardationassociated with mitochondrial DNA mutation in anItalian family

Piero Puddu, Piero Barboni, Vilma Mantovani, Pasquale Montagna, Angelina Cerullo, MichelaBragliani, Carla Molinotti, Roberto Caramazza

AbstractAn Italian pedigree including two sisters andtheir mother affected by a neuro-ophthalmicdisease characterised by retinitis pigmentosa,ataxia, and psychomotor retardation isreported. Molecular analysis of mitochondrialDNA showed the presence of heteroplasmic8993 point mutation in the subunit 6 of theATPase gene. The clinical features and geneticfindings in this family were comparable withthose recently described in an English family.The mitochondrial DNA analysis of the familyshowed a correlation between the amount ofmutated DNA and the disease severity in theprobands, and indicated the presence of athreshold amount ofmutated genome inducingophthalmic defects. Moreover, the comparativeanalysis of blood, hairs, muscle, and urinarytract epithelia of two probands revealed anessentially similar distribution of mutated andwild type mitochondrial genomes. Our resultssuggest that the 8993 mitochondrial DNAmutation characterises a disease with similarclinical features in different populations.(BrJ Ophthalmol 1993; 77: 84-88)

Institute ofOphthalmology,University of Bologna,ItalyP PudduP BarboniC MolinottiR Caramazza

Department ofPathology, MalpighiHospital, Bologna, ItalyV MantovaniM Bragliani

Institute of ClinicalNeurology, University ofBologna, ItalyP MontagnaA CerulloCorrespondence to:Professor Piero Puddu,Istituto di Clinica Oculistica,Policlinico S Orsola-Malpighi,Via Massarenti, 9 1-40138Bologna, Italy.Accepted for publication17 September 1992

A variety of ophthalmic findings such as retinitispigmentosa, progressive external ophthalmo-plegia, and optic atrophy may occur in differentassociations with neurological diseasescharacterised by disorders of mitochondrialfunctions.'' Mitochondria are cytoplasmicorganelles essential to cellular bioenergy,producing ATP by means of oxidativephosphorylation. Each mitochondrion containsseveral copies of a circular double strandedmolecule ofDNA (mtDNA) of 16 569 nucleotidepairs (np) coding for two rRNAs, 22 tRNAs, and13 of the 61 protein subunits of mitochondrialrespiratory complexes: NADH dehydrogenasesubunits (complex I), cytochrome b subunit(complex III), cytochrome c oxidase subunits(complex IV), and ATP synthase subunits(complex V).5-7 The mtDNA is transmittedthrough the maternal lineage and the mito-chondria of the zygote are provided only by theovum.8

Genetic studies of some neuro-ophthalmicdiseases have revealed various defects inmtDNA. Point mutations of mtDNA were

reported as the genetic defect in Leber'shereditary optic neuropathies,'-'2 myoclonus

epilepsy with ragged red fibres (MERRF),'3mitochondrial myopathy, encephalopathy, lacticacidosis and stroke-like episodes syndrome(MELAS),'4 and in other mitrochondrial myo-

pathy,'5-'8 Other genetic defects of mtDNA assingle or multiple deletions and tandem dupli-cations were described in chronic progressiveexternal ophthalmoplegia, Kearns-Sayresyndrome, and encephalomyopathies. 19-24 Avariable proportion of mutated and wild type mtgenomes may be present in different cells andtissues; this condition goes by the name ofheteroplasmy and is a typical feature of mito-chondrial diseases.'1125 The variable degrees ofheteroplasmy in tissues with different energyrequirements may explain the widely differentclinical features often encountered in mito-chondrial diseases.

Holt et al26 recently reported a point mutationof np 8993 of mtDNA in a family with a newneuro-ophthalmic disease characterised byretinitis pigmentosa, dementia, seizures, ataxia,proximal neurogenic muscle weakness, sensoryneuropathy, and maternal inheritance.We report an identical association between

clinical and genetical features in an Italianfamily. We found the presence of the sameheteroplasmic mutation in three maternallyrelated family members; moreover we evaluatedthe segregation of mutated and wild typemtDNA in different tissues.

Patients and methods

PATIENTSThe pedigree of this Italian family included threeaffected female (two sisters and their mother)and other three asymptomatic relatives (Fig LA).The family history had no records of otherrelatives with neuro-ophthalmic disorders.

Patient II-1, a 41-year-old woman, was themother oftwo daughters. She complained of life-long muscle fatigue, headache, and, in the lastyears, some memory loss and mild paraesthesialocalised in her toes. Examination showed diffusehypotonia, slight proximal leg weakness, loss ofknee and ankle jerks, and minimal Rombergsign. Ophthalmoscopic examination was normal;electroretinography showed normality ofphotopic cone and scotopic rod responses (Fig2). Creatinine kinase (CK), lactate at rest andafter a standardised effort, electrocardiogram(ECG), electroencephalogram (EEG), braincomputed tomography (CT), quantitativeelectromyelogram (EMG), and motor andsensory conduction velocities (CV) were allnormal. Muscle biopsy was normal, in particularragged-red fibres were absent. Spectrophoto-metric assay of respiratory chain enzymes wasalso normal.

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Retinitis pigmentosa, ataxia, and mental retardation associated with mitochondnialDNA mutation in an Italianfamily

Figure I (A) The pedigreeofthe family: filled symbolsindicate affectedindividuals. (B) The gelelectrophoresis shows theAva I digestion ofamplifiedmtDNA with primersEMIIMT19. Fragment 872indicates the wild typemntDNA andfragments 656znd 216 indicate the mutatedmntDNA. (C) shows thesubsequent Southern blotting2nd hybridisation withMTI7 probe. M=sizemarker; bp=base pair.

A I

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Patient III-1, her 13-year-old daughte-r, hadhad neonatal jaundice and psychomotorretardation since birth. During infancy she hadseveral episodes of marked worsening of musclestrength, ataxia, vomiting, and confusion upontrivial fevers. On examination, dysarthria,diffuse hypotonia, and muscle wasting withweakness, dysmetria, incoordination, gait ataxiawith a Romberg sign, and weak tendon jerkswere found. Ophthalmoscopic examinationshowed a bilateral pigmentary pattern of retinitispigmentosa, with pigmentary bone spicule-likedeposition in the midperiphery (Fig 3). Electro-retinography disclosed subnormal photopicresponses: amplitudes of photopic a and b-waveswere reduced and b-wave implicit time wasprolonged (Fig 2). CK lactate at rest and aftereffort, ECG were normal. Brain CT showedcerebellar and brainstem atrophy (Fig 4); EEGslow background activity and paroxysmal(spikes, spike slow waves) diffuse activity; EMGshowed neurogenic atrophy and decreasedamplitude of sensory evoked potentials (SEP) inthe legs. Muscle biopsy was normal withoutragged-red fibres. Respiratory chain enzymeswere normal on spectrophotometric assay.Patient III-2, 19-year-old daughter, had neonatalicterus and febrile convulsions at 6 months ofage. Severe psychomotor retardation andhypotonia were present since birth. Markedworsening of strength, gait, and confusion

Figure 3 Fundusphotographfrom right eye ofpatient III-Idemonstrating vessel tortuosity, wrinkling ofinner limitingmembrane and bone spicule pigmentforms in all quadrants ofthe midperipheiy.

occurred upon even trivial fevers. Visual lossbecame apparent since the age of 16 years. Shepresented fundus changes in both eyes, typical ofadvanced retinitis pigmentosa (pigmentary bonespicule-like depositions, arteriolar narrowing,pallor of the disc); an ERG recording showed areduction in photopic a and b-wave amplitudeswith a prolonged implicit time (Fig 2). Neuro-logical signs comprised besides the severe mentalretardation, dysarthria, muscle hypotonia with-out any muscle weakness or atrophy, dystonicposturing of trunk and limbs, and gait ataxia.CK, ECG, EEG, and resting lactate were normal.Brain CT showed cerebellar brainstem, andslight cerebral cortical atrophy; EMG showedneurogenic atrophy and decreased amplitude ofsensory potentials.

Ophthalmoscopic examination performed alsoin subjects I-1, I-2 and II-2, who were completelyasymptomatic; was normal; electroretinographictesting of grandmother (1-2) showed normalphotopic cone and scotopic rod responses.

MethodsMolecular analysis ofmtDNA was performed inthe six family members (Fig 1). Since the 8993mtDNA mutation creates an Ava I26 and removesa Bst NI restriction site, we performed anendonuclease digestion with these enzymes, afterpolymerase chain reaction (PCR) amplificationof mtDNA.27 Total DNA was extracted fromleucocytes, epithelial cells of the urinary tract

Patient 111-1

Figure 2Electroretinographicfindings ofpatients II-I,III-I, and II-2. In the twosisters cone a and b-waveamplitudes were reduced andb-wave implicit time wasprolonged. In the motherphotopic cone and scotopicrod responses were normal.RE=right eye; LE=lefteye; SE=skin electrodes;GA =general anaesthesia.

RE * RE

LE LE

SE1o0 l15-33 ms

GA62-5 ,uj

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RE

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Figure 4 Brain computedtomography ofpatient III-Ishowing marked cerebellarand brainstem atrophy.

and fr(muscleisoamysolubilipH 8.2formedsodiumRNAsebeforepairs(5'-AT8335 toTGCA(MT39from 8amplif)respectsites.5were c(volumeTris-Higelatin,and drTaq-pcUSA).for 90 :and 72Cof the Iovernig

FigureS (A) Gel (aboutelectrophoresis showing the restrictBst NI digestion of Beverlyamplified mtDNA withprimers MT391MT19. electroFragment 421 indicates themutated mtDNA andfragments 214 and 207indicate the wild typemtDNA. M=size marker;bp=base pair. (B) Analysisofmuscle, hair, and unrnarytract epithelia mtDNA. Thepanel shows the Ava Idigestion ofamplifiedmtDNA with primers 421-MT391MT19. Fragment421 indicates the wildtypemtDNA andfragments 216 214and 205 indicate the mutatedmtDNA. M=size marker; 207 -m=muscle; b=blood;h=hair; u=urinary tractepithelia.

om fractionated homogenates of frozenwith phenol and chloroform-

rlalcohol, precipitated with ethanol andised in 10 mM Tris-HC1/l mM EDTA!8DNA preparation from hairs was per-I by adding 0 15 mg proteinase K, 50 tldi dodecyl sulphate (SDS) 20%, 0-05 mgz, 40 iim dithiothreitol for 70 hours,the conventional DNA extraction. Theof oligonucleotide primers EM1TAAGAGAACCAACACCTC-3' fromD 8354 np/MT19 (5'TATGTGTTGTCG-GGTAGA-3' from 9206 to 9185 np), and(5'-TCCTGCCTCACTCATTTACA-3'

3786 TO 8805 np)/MT19 were used toy 872 and 421 np regions of mtDNAtively, including the Ava I and Bst NITwenty five cycles of PCR amplificationompleted with 1 ,ug of DNA in a 100 1tlreaction containing 50 mM KCI, 10 mM

[Cl (pH 8 3), 1 5 mM MgCl2, 100 tLg/mlI 200 FtM each of dATP, dCTP, dGTP,PTP, 1 FtM of each primer and 2 5 unitsilymerase (Perkin Elmer-Cetus, Norwalk,Each cycle consisted of 94°C denaturationseconds, 55°C annealing for 150 seconds°C extension for 4 minutes.29 The presence8993 mtDNA mutation was detected byght digestion of the total amplified DNA2 ,tg) with 10 units of Ava I and Bst NItion enzymes (New England Biolabs,y, MA, USA). The samples were thenphoresed on 3% Nusieve plus 1% agarose

gel stained with 1 [ig/ml ethidium bromide andphotographed under ultraviolet light.

After staining, the gel was denatured in 0 4 NNaOH/0-6 M NaCl for 30 minutes at roomtemperature and neutralised in 15 M NaCl/0 5M Tris-HCl pH 7 5 for 30 minutes at roomtemperature. The DNA was then transferredovernight to a nylon membrane by the Southernmethod.30 The oligonucleotide probe MT175'CTATTGGTTGAATGAGTAGGCTGA-3'from 8989 to 8966 np), 5'end-labelled with32P-dATP, was used for hybridisation. The filterwas prehybridised for 1 hour at 37°C in 5 xDenhardt's (0 -1 Ficoll/0 1% polyvinylpyrroli-done/0 1% bovine serum albumin), 5xSSPE(750 mM NaCl/50 mM NaH2PO4/5 mMEDTA), 0-5% SDS and 200 mg/ml of denaturedherring sperm, and then hybridised for 1 hour at50°C in a solution consisting of the prehybridisa-tion solution plus the radiolabelled probe(2 x 106 cpm/ml). After hybridisation the filterwas washed in O1xSSPE/01% SDS for 10minutes at room temperature and for 10 minutesat 40°C. The filter was then exposed to KodakXAR-5 at -800C with intensifying screen for 2hours and/or overnight. In order to test theabsence of 8993 mtDNA mutation in the Italianpopulation, we included 51 unrelated healthycontrols in the study.

ResultsThe Ava I and Bst NI restriction analyses wereperformed in mtDNA from leucocytes of thewhole pedigree. Both daughters and their motherpresented the Ava I restriction site, producingthe 656 and 216 np fragments, because of the8993 mutation (Fig iB). Likewise, all threeprobands lacked the Bst NI site owing to thesame mutation (Fig SA).

In restriction analyses with both Ava I and BstNI enzymes, the presence of heteroplasmy inleucocytes was revealed by the presence of allthree fragments. A residual amount of wild typemtDNA was found in variable proportions in thethree probands: about 25% in both sisters andabout 90% in their mothers. These results wereconfirmed by the Southern blotting andhybridisation of the digestion (Fig IC).

Additional molecular analysis was performedin mtDNA obtained from epithelial cells of theurinary tract of subjects I-2, II-1, and III-1, frommuscle of patients II-1 and III-1, and from hairsof patient III-1 (Fig 5B). The degree of hetero-plasmy in all three probands detected in muscle,

111-1

111-1 111-2 11-1 1-2 11-2 M bp

421 -

216 %205--

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Retinitis pigmentosa, ataxia, and mental retardation associated with mitochondrialDNA mutation in an Italianfamily

urinary tract epithelia and hair was the same asthat found in blood.The Ava I and Bst NI restriction analyses were

also performed in both grandmothers (I-1 and I-2) and in the aunt of the sisters (II-2). The threesubjects did not show the mutation either inblood (I-1, I-2 and II-2) or in urinary cells (I-2).All the 51 unrelated healthy controls studied didnot present the mutation.

DiscussionRetinitis pigmentosa, ataxia, and mentalretardation were the clinical features presentboth in our Italian family and in the pedigreepreviously described by Holt et al. 6 Therefore,these clinical signs seem to be typical of this newneuro-ophthalmic disease. However, the samefindings were found by Tatuch et al in a familywhich presented, in addition, the clinical pheno-type of Leigh syndrome.3'

Molecular analysis showed the presence of the8993 point mutation of mtDNA in all probandsof this family. This mutation causes a trans-version from thymine to guanine, converting ahighly conserved leucine into arginine in ATPasesubunit 6. A variable proportion of mutated andwildtype mtDNA (heteroplasmy) was present inthe probands and the percentage of wildtypedecreased in the second affected generation.These findings are in line with the hypothesisthat mutated mitochondria shift toward a homo-plasmic condition during the meiotic segregation,possibly due to a positive selection of defectivemitochondria."2-35As in the other two pedigrees, we found a

correlation between the amount of mutatedmtDNA and the disease severity in the probands;the two sisters showed higher levels of mutatedmtDNA (about 75%) than their mother (about10%) (Fig 1). In our family the mtDNA analysisseems to indicate the presence of a thresholdamount of mutated mtDNA inducing the retinaldegeneration. More than one type ofpigmentaryretinopathy of different severity can be associ-ated with mitochondrial diseases.' 36 The findingof defective mitochondria in the retinal pigmentepithelium suggests a correlation betweendegenerative processes of the retina and defectsin oxidative phosphorylation.37

Moreover, we evaluated the mitotic segre-gation of the two mtDNA types in differenttissues. The comparative analysis of blood,muscle, hairs, and urinary tract epithelia of twoprobands revealed an essentially similarheteroplasmy (Fig SB). However, we are unableto rule out the hypothesis of a higher number ofmutated mt genomes in brain and retina.We did not find traces of mutation in the

grandmothers and in the aunt of the sisters,though we used a more sensitive radioisotopicmethod (Fig 1). Furthermore, the analysis ofurinary tract epithelia of the grandmother (I-2)excluded a different segregation of mtDNA inthis tissue. This result suggested a recent originof the mutation in the family. Our geneticfindings confirmed the mitochondrial origin ofthe disease.Our results indicate that the 8993 mtDNA

mutation characterises a new neuro-ophthalmic

disease, with typical clinical and genetic features,which remains unchanged even in differentpopulations.

We are particularly indebted to the family members, withoutwhose interest and cooperation no work would have been possible.The genetical assistance of A Zacchini, P Malpassi, P Selva,D Bastia, and the electrophysiological assistance of P Fiorl'ni,L Fulco, and S Bontempi are gratefully acknowledged. We wish tothank F Barboni for his valued contribution and assistance in thiswork.

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