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We thank Dr Elena Morandi, Dr Alessandra Vucetich, and Dr Stefano Garbo with whom we first discussed the possibilities offered by HIV-infected

manipulation; Dr Giulia Rognoni, Dr Simonetta Giuntelli, Dr Guglielmo Ragusa, and Dr Enninia Lubelli who have greatly helped in the semen

djnia) screening of the couples, in the sonographic monitoring, and with the insemination procedure; and Mr Gabriel R. Bouygue, for his critical advice on

the script.

REFERENCES 1. Istituto Superiore di Sanitá: Report on AIDS cases. March 31, 1992. 2. Zagury D, Bernard J, Leibovitch J, et al. HTLV-III in cells cultured from semen of two patients with AIDS. Science 1984; 226: 449. 3. Borzy MS, Connell RS, Kiessling AA. Detection of human immunodeficiency virus in cell-free seminal fluid. J Acquir Immune Defic Syndr 1988; 1: 419-24. 4. Ashida ER, Scofield VL. Lymphocyte major histocompatibility complex-encoded Class II structures may act as sperm receptors. Proc Natl Acad Sci USA 1987; 84: 3395-99. 5. Wolff HK, Zhang W, Anderson DJ. The CD4 antigen (HIV receptor) is not detectable on human testicular germ cells or spermatozoa. Abstract 2547. IV International Conference on AIDS, Stockholm, June 1988. 6. Semprini AE, Vucetich A, Morandi E, Parravicini CL, Pardi G, Beer AE. Removal of p18 immunoreactive cells from the semen of HTLV-III/ LAV seropositive men. Colloque INSERM 1987; 154: 462. 7. Semprini AE, Vucetich A, Garbo S, et al. Attempts to obtain spermatozoa concentrates with low-infectivity from ejaculates of HIV + subjects. VI International Conference on AIDS, San Francisco, June 1990: Abstr SC.713 p 267. 8. Semprini AE, Morandi E, Vucetich A, et al. Successful removal of HIV infected immunocompetent cells from semem of HIV-seropositive men. Society for Gynecological Investigation: 1991, 534: Abstr. 9. Anderson DJ, Politch JA, Oneta M, Tucker L, Semprini AE. Efficacy of conventional semen processing techniques in separation of motile sperm from HIV-1 and HIV-1 host cells. 48th Annual Meeting American Fertility Society. New Orleans, 1992 (abstr). 10. Saracco A, Musicco M, Nicolosi A, Costigliola P, Angarano G, Lazzarin A. Italian Partner Study. Man to woman sexual transmission of HIV: a prospective study of 343 steady partners of infected men. J Acquir Immune Defic Syndr (in press).

ADDRESS: Department of Obstetrics and Gynecology, San Paolo Biomedical Institute, University of Milan Medical School, Via di Rudini’ 8, 21042, Milan, Italy (A. E. Semprini, MD, P. Levi-Setti, MD, M. Bozzo, MD, M. Ravizza, MD, A. Taglioretti, MD, P. Sulpizio, MD, E. Albani, PhD, M. Oneta, PhD, Prof G. Pardi, MD).

Correspondence to

Dr A. E.

Semprini.

Inherited idiopathic dilated cardiomyopathy with multiple deletions of mitochondrial DNA

Idiopathic dilated cardiomyopathy (DCM) is often familial, but the pathogenetic mechanisms of DCM unknown. We report a woman and her son who both died of DCM. The son’s cardiac and skeletal muscles showed a high proportion of mitochondrial DNA (mtDNA) with multiple large deletions by Southern-blot hybridisation and polymerase chain reaction analyses. Amplification of the mother’s cardiac mtDNA from 20-year-old paraffinembedded sections showed that she also had deletions of mtDNA. These data suggest that a subgroup of inherited DCMs is associated with mtDNA mutations. are

Fig 1-Southern-blot hybridisation analysis of patient 2.

DNA from

Lane 1, control skeletal muscle; lane 2, heart muscle; lane 3, skeletal muscle. Several hybridising signals were detected in lanes 2 and 3 with the probe K3. In addition to the normal 166 kb mtDNA, faster migrating mtDNA populations were found in the patient’s samples, ranging from 155 kb to 70 kb, corresponding to deletions of 1 kb to 95 kb. Arrows indicate migration of the DNA standard (bacteriophage lambda DNA digested with Hind III: 23kb, 9-4 kb, 67 kb).

Dilated cardiomyopathy (DCM) is the most common of severe cardiomyopathy in young and middle-aged people, with an estimated prevalence of 3-65/10 000.1 It is characterised by dilatation and dysfunction of one or both ventricles. Over 20% of cases are familial,l and some of the familial cases show X-chromosomal inheritance.2 The genes involved and the pathogenesis of the disease are not known. Mitochondria provide cellular energy by oxidative phosphorylation in the respiratory chain, and thirteen subunits of the enzyme complexes involved in this process are encoded in the mitochondria by mitochondrial DNA (mtDNA). Mutations of mtDNA have been shown to cause cardiomyopathies. A disease-specific point mutation was reported in a fatal infantile cardiomyopathy3 and in a maternally inherited myopathy-cardiomyopathy.4 We have studied mtDNA of affected family members with DCM. cause

Patient 1

was the only child of healthy parents. She developed dyspnoea at the age of 35. Chest radiographs showed an enlarged heart with interstitial pulmonary oedema. Right heart catheterisation revealed increased pressure in the right atrium (mean 13 mm Hg) and pulmonary artery (46/30, mean 36 mm Hg). The mean pulmonary artery wedge pressure was also raised (27 mm Hg). The cardiac index was 10 1/min per m2. The patient died 2 years later of progressive heart failure. Necropsy showed dilatation of the left atrium and ventricle with endocardial thickening. Histological examination revealed myocardial fibre-size variation and degeneration, with pleomorphic nuclei and endocardial severe

fibrosis. Patient 2 was the youngest of three children of patient 1. His two sisters are healthy. He was successfully treated with digoxin at the age of 6 months due to an enlarged and hypokinetic left ventricle. At 17 years of age severe pulmonary oedema, enlarged liver, and ankle oedema developed. Echocardiography showed striking biventricular dilation with secondary mitral and tricuspid regurgitation. The left ventricle was diffusely hypokinetic with an ejection fraction of 25%. Therapy with diuretics and convertingenzyme inhibitor was started. Severe muscle fatigue and weakness became evident, but no ptosis or ophthahnoplegia were detected. A muscle-biopsy specimen showed type-1-fibre predominance and type-2 hypertrophy, but no ragged-red fibres. Mitochondria were ultrastructurally normal but subsarcolemmally their numbers were increased. During the subsequent 5 years the patient’s clinical condition steadily deteriorated. Due to pulmonary hypertension

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Fig 2-PCR analysis. Amplification of heart (lane 2) and skeletal muscle (lane 3) DNA samples from patient 2 with primers 3 and 4 resulted in more than ten fragments from4 Okb to01 kb, corresponding to deletions from 1 8kb to 57 kb (primers are 58 kb apart). No amplification is seen in the control (lane 1) because the fragment of mtDNA of normal size is too long for efficient amplification. Amplification of DNA from paraffinembedded heart-muscle samples from patient 1 (lane 5) and patient 2 (lane 6) with primers 1 and 2 resulted in one amplifying fragment (arrow) of about 500 bp, corresponding to a deletion of 5,0 kb (normal primer distance 5kb). Primers4 and 5 gave two amplifying fragments of about 250 bp and 350 bp (arrows), corresponding to deletions of 26 kb and 25 kb (primer distance 2 85 kb) in samples from patient 1 (lane 8) and patient 2 (lane 9). No amplification is seen in the control paraffin-embedded sample (lanes4 and 7). Lane VI = molecularweight marker VI (Boehringer Mannheim, Germany).

(pulmonary vascular resistance 9-10 Wood units), the patient was not considered a candidate for cardiac transplantation. He died suddenly at the age of 22 years. At necropsy, macroscopic findings were consistent with the diagnosis of DCM. Endocardial fibrosis and the myocardial changes were consistent with the findings seen in the mother. Ultrastructurally, intermyofibrillar mitochondria seemed numerous. The extraction, Southern-blot hybridisation analyses, and the densitometry, polymerase chain reaction (PCR) amplification of total DNA from the skeletal muscle and heart samples of patient 2 were done as described previously. The probe K3 (a fragment of human mtDNA cloned in pTZ 19, mitochondrial map position at 1-740, not likely to be deleted) was used in the preliminary Southern-blot analysis. Deletions were localised with probes K7, K9, K10, K12, and K14 as describedpreviously.5 DNA from the paraffin-embedded heart samples of patients 1 and 2 was amplified by PCR as described by Shibata et al.6 The oligonucleotide primers (with corresponding nucleotides in mtDNA) were: 1 (nt 8239-8263), 2 (nt 13692-13712), 3 (nt 9174-9193), 4 (nt 14944-14967), and 5 (nt 12105-12128). As controls for DNA analyses we used muscle samples from patients who had orthopaedic surgery and sections of an old paraffinembedded heart sample comparable to that of patient 1. Control subjects did not have symptoms or signs of muscle or heart disease.

Southern-blot hybridisation and PCR analyses of the son’s heart and muscle DNA detected several mtDNA populations with large deletions, in addition to normal mtDNA (figs 1 and 2). The proportion of normal mtDNA was quantified from autoradiographs by densitometry and was found to be 50% of total mtDNA in heart and 65% of total mtDNA in muscle samples. PCR amplification of the mother’s paraffin-embedded samples showed products identical to those found in the son’s sample but not found in controls (fig 2). From this we concluded that the mother also had deletions. Deletions were localised to the area that includes genes for cytochrome c oxidase subunits 1, 2, and 3, ATPase 8 and 6, and NADH-dehydrogenase (ND) subunits 3, 4, 5, and 6. Most downstream breakpoints were localised to the ND5 gene or to the 3’ direction from the gene.

In previous studies/.8 PCR showed deletions in the heart mtDNA of three individuals with dilated or hypertrophic cardiomyopathy, but Southern-blot hybridisation analysis did not reveal these deletions. The importance of these minor populations of mutated mtDNA is unknown, since a small proportion of deleted mtDNA can be found by PCR in healthy aged individuals.9 By Southern-blot hybridisation analysis we have shown multiple deletions of mtDNA in the heart of a patient with familial DCM. The mtDNA populations with deletions of different size made up about 50% of the total mtDNA. These deletions resembled in number and location those found previously in familial progressive external ophthalmoplegia" a condition that shows autospmal dominant transmission. This fmding suggests that the deletions may not be inherited as such, but rather that an unknown nuclear gene defect leads to multiple mtDNA deletions. In our family, DCM was found in mother and son and both showed deletions, thus making both autosomal and maternal inheritance patterns possible. Multiple deletions of mtDNA in the heart and muscle of patients with familial DCM offer one clue to the cause of this large and, most likely, heterogeneous group of cardiomyopathies. We thank Dr T. Paavonen for undertaking the necropsy of patient 2. This study was supported by the Emil Aaltonen Foundation, the Finnish Research Foundation of Neuromuscular Diseases, the Arvo and Lea Ylppö Foundation, the Foundation of Finnish Medicine, and the Oskar Oflund Foundation.

REFERENCES 1. Michels VV, Moll PP, Miller FA, et al. The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated

cardiomyopathy. N Engl J Med 1992; 326: 77-82. BA, Swift M. X-linked dilated cardiomyopathy. N Engl J

2. Berko

Med

1987; 316: 1186-91. 3. Tanaka M, Ino H, Ohno K,

et al. Mitochondrial mutation in fatal infantile cardiomyopathy. Lancet 1990; 336: 1452. 4. Zeviani M, Gellera C, Antozzi C, et al. Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNALeu(UUR). Lancet 1991; 338: 143-47. 5. Suomalainen A, Majander A, Haltia M, et al. Multiple deletions of mitochondrial DNA in several tissues ofa patient with severe retarded depression and familial progressive external ophthalmoplegia. J Clin Invest 1992; 90: 61-66. 6. Shibata DK, Arnheim N, Martin WJ. Detection of human papillomavirus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 1988; 167: 225-30. 7. Ozawa T, Tanaka M, Sugiyama S, et al. Multiple mitochondrial DNA deletions exist in cardiomyocytes of patients with hypertrophic or dilated cardiomyopathy. Biochem Biophys Res Commun 1990; 170:

830-36. 8. Hattori K, Ogawa T, Kondo T, et al. Cardiomyopathy with mitochondrial DNA mutations. Am Heart J 1991; 122: 866-69. 9. Cortopassi GA, Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res 1990; 18: 6927-33. 10. Zeviani M, Sevidei S, Gellera C, Bertini E, DiMauro S, DiDonato S. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 1989; 339: 309-11.

ADDRESSES:

Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland (A. Suomalainen, MD, L. Peltonen, MD); and Department of Pathology (A. Paetau, MD), First Department of Medicine (H. Leinonen, MD), Department of Medical Chemistry (A. Majander, MD), and Department of Neurology (H. Somer, MD, A. Suomalainen), University of Helsinki, Helsinki. Correspondence to Dr Anu Suomalainen.