UvA-DARE (Digital Academic Repository) The x-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies d' Adamo, P.; Fassone, L.; Gedeon, A.; Janssen, E.A.M.; Bione, S.; Bolhuis, P.A.; Barth, P.G.; Wilson, M.; Haan, E.; Orstavik, K.H.; Patton, M.A.; Green, A.J.; Zammarchi, E.; Donati, M.A.; Toniolo, D. Published in: American Journal of Human Genetics DOI: 10.1086/514886 Link to publication Citation for published version (APA): d' Adamo, P., Fassone, L., Gedeon, A., Janssen, E. A. M., Bione, S., Bolhuis, P. A., ... Toniolo, D. (1997). The xlinked gene G4.5 is responsible for different infantile dilated cardiomyopathies. 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UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) Download date: 20 May 2020 Am. J. Hum. Genet. 61:862–867, 1997 The X-Linked Gene G4.5 Is Responsible for Different Infantile Dilated Cardiomyopathies Patrizia D’Adamo,1 Lucia Fassone,1 Agi Gedeon,2 Emiel A. M. Janssen,3 Silvia Bione,1 Pieter A. Bolhuis,3 Peter G. Barth,3 Meredith Wilson,4 Eric Haan,5 Karen Helen Örstavik,6 Michael A. Patton,7 Andrew J. Green,8 Enrico Zammarchi,9 Maria Alice Donati,9 and Daniela Toniolo1 1 Institute of Genetics, Biochemistry and Evolution-CNR, Pavia, Italy; Departments of 2Cytogenetics and Molecular Genetics and 5Medical Genetics and Epidemiology, Women’s and Children’s Hospital, North Adelaide, Australia; 3Department of Neurology, Academic Medical Centre, Amsterdam; 4Department of Clinical Genetics, The New Children’s Hospital, Westmead, Australia; 6Department of Medical Genetics, Ulleval Hospital, Oslo; 7Department of Medical Genetics, St. Georges Hospital Medical School, London; 8Department of Medical Genetics, University of Cambridge, Cambridge; and 9Department of Pediatrics, University of Florence, Florence Summary Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia. The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life. X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic. We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5. In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies. Mutations in the G4.5 gene were found in nine of the patients analyzed. The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069). Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a ‘‘less severe’’ phenotype. Introduction The dilated cardiomyopathies are a very heterogeneous group of heart disorders of largely unknown ethiology. Genetic causes have become increasingly evident with mapping of loci and identification of genes responsible Received March 28, 1997; accepted for publication July 11, 1997. Address for correspondence and reprints: Dr. Daniela Toniolo, IGBE-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy. E-mail: [email protected] q 1997 by The American Society of Human Genetics. All rights reserved. 0002-9297/97/6104-0013$02.00 862 for different forms of the disorder (Keating and Sanguinetti 1996). The dilated cardiomyopathies are often idiopathic, and in adults they represent a primary indication for heart transplantation. Less frequent and more severe are the infantile forms, which often cause cardiac failure during the first months of life. One of these severe forms is Barth syndrome (BTHS), an X-linked inherited disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia (Barth et al. 1983). The disease has infantile onset of symptoms and is often fatal in childhood, because of cardiac failure or sepsis: the clinical manifestations of the disease are, in general, quite variable also within families, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding. Which of the other manifestations of the disease may also be diagnostic is an open question. Xlinked infantile cardiomyopathies with cardiac dilatation but apparently lacking the other symptoms of BTHS have been reported, and it has been proposed that the BTHS may be allelic with both X-linked endocardial fibroelastosis (EFE1; OMIM 305300) and other infantile cardiomyopathies (Gedeon et al. 1995; OMIM 300069). BTHS has been localized to Xq28 (Bolhuis et al. 1991). We have recently reported the identification of a novel gene, G4.5, which we have proposed as the genetic locus responsible for the disease (Bione et al. 1996). The G4.5 gene is a small single-copy gene with a complex pattern of expression. As many as 10 mRNAs differing either in the region encoded in the central exons (5–7) or in the 5* end can be synthesized. Two forms, containing all three exons or lacking exon 5 only, were consistently more abundant and ubiquitously expressed. Since the differently spliced mRNAs maintain the same open reading frame (ORF), the tafazzins, the putative proteins encoded by the gene, can differ in the N-terminus and in a central portion. All the mutations found in the four familial cases of BTHS studied so far have introduced a stop codon in the ORF, but in all instances D’Adamo et al.: X-Linked Dilated Cardiomyopathies they would not interfere with the synthesis of all the possible tafazzins. We now report the study of a larger group, of patients affected with BTHS and of patients affected with severe cardiac disorders compatible with X-linked inheritance. We show that mutations are to be found also in this group of patients and that the G4.5 gene is responsible for most of the X-linked infantile dilated cardiomyopathies. Material and Methods Mutation Detection Genomic DNA preparation, amplification, and direct sequencing of the BTHS gene were as described elsewhere (Bione et al. 1996). Sequences were from the primers described, and they were designed to sequence all exons and exon-intron junctions from both strands. As a control for PCR errors, at least two independent PCR products were sequenced in the region of each mutation. The sequence of each patient was always compared with that of at least one normal individual, in the same gel. As a control for polymorphism, all the mutations were searched in DNA from 100 normal chromosomes randomly selected among different populations available to the laboratory and containing both males and females. Sequence Analysis Sequence reactions were run in a Perkin Elmer 373A Automated Sequencer, and they were analyzed by use of SeqEd and Sequence Navigator software. Sequences were compared with the GenBank sequences by use of BLAST, and they were aligned with CLUSTAL. Results Mutation Analysis of BTHS Patients In our previous work we studied patients from four families affected with BTHS. To gain more information on the mutations causing this disorder, we collected eight additional cases. Some were definitely familial. In other families relatives were reported as possibly affected, but the disease could not be well defined before death occurred. Some of the clinical manifestations of the propositi of each family are schematically listed in table 1. Other affected family members were usually studied less thoroughly and are not reported in the table: some of the families were previously published, and more data are available from the literature. In all families, cardiac dilatation and cardiac failure within the 1st year of life were the common feature. Neutropenia (usually cyclic or episodic), myopathy, growth retardation, and alterations in urinary 3-methylglutaconic acid (Kelley et al. 1991) were consistently reported. Life expectancy was very variable, possibly depending on medical treatment. Cardiac transplantation was reported in 863 two of the patients (BS and GW) and seems to have increased life expectancy. In the search for mutations, PCR products were prepared from genomic DNA of either one patient or an obligate carrier from each family and were sequenced directly, as described elsewhere (Bione et al. 1996). Mutations were found in six of the familial cases, and they are listed in table 1; their localizations with respect to the G4.5 gene are shown in figure 1. Some of the corresponding sequences are shown, aligned with the normal sequence, in figure 2. Two new mutations were found in exon 2, one was in exon 6, and the remaining three were in exon 8. One of the mutations in exon 2 (family 1) was a deletion causing a frameshift and early stop; all the remaining mutations were missense. As a control for polymorphism, 100 chromosomes from normal individuals were sequenced in the region of the mutations, and they were all normal. On the other hand, additional affected individuals and obligate carriers, when available, were sequenced, and the mutations were all confirmed. In family 1 we sequenced the mother’s DNA; in family 6 the mother’s and the grandmother’s DNAs; in family 8 the mother’s and the sister’s DNAs; and in family 9 the mother’s, the aunt’s, and a normal brother’s DNAs. In the remaining two families, published as putative BTHS (families 11 and 12; Örstavik et al. 1993), no mutations were found. Since only the coding region and the splice junctions were studied, and since only the DNAs of obligate carriers were available, we cannot absolutely exclude an involvement of the G4.5 gene. Four sporadic patients were also studied, and no mutations were found. X-Linked Infantile Cardiomyopathies: Mutations in the BTHS Gene Gedeon et al. (1995) reported a large family presenting with X-linked inheritance of a fatal infantile cardiomyopathy. The gene was mapped to Xq28. The cardiomyopathy in this family is consistently of congenital onset and is fatal in infancy. The clinical features were insufficient to permit a definite diagnosis, but the possibility that this disease is allelic with BTHS was discussed. We sequenced the G4.5 gene in two affected individuals of the family, and we found deletion of a C at position 919 in exon 8, causing frameshift and a stop codon after 18 nucleotides (patient MH in fig. 1). The mutation may cause the production of a truncated protein or, possibly, a reduced amount of the corresponding mRNAs and therefore no protein at all (Maquat 1995). We have also studied patients from two families with several affected male relatives (brothers and cousins) who died very early of heart failure. The clinical data were very limited, but after autopsy these patients’ hearts showed left-ventricular dilatation and hypertrophy, and they were diagnosed as affected with EFE1. 864 Table 1 Data on BTHS Patients Family 1 2 3 4 5 6 7 8 9 10 11 12 Patient Source Author Affected Relative(s) BM V.22 BS K GW FW OAT MF FT BV II-1 II-2 R.M. P.A.B. M.A.P. P.A.B. A.G. K.H.O. A.G. M.A.D. P.A.B. P.A.B. K.H.O. K.H.O. Brother Several (18) Two brothers Not proved Nephew Brother Several Not proved Not proved Not proved Brother Brother Referencea Year of Birth Age (years) ... 1 2 ... ... 3 4 ... ... ... 3 3 1986 1984 1992 1989 1978 1984 1989 1994 1991 1983 1987 1977 10 5 19 5 16 Age at Death (mo) Dilated Cardiomyopathyb Neutropeniab Living 10 Living 13 Living 9 ú21 14 Living Living 1 20 / / /c / /d / / / / / / / Cyclic / //0 0 Cyclic / / / Cyclic / 0 0 Myopathyb 3Methylglutaconic Aciduriab Growth Retardationb Abnormal Mitochondriab Mutation Exon Referencea / //0 //0 / 0 ND / ND / / //0 0 / / / //0 / ND ND / / / ND ND / / / / / / ND ND / / 0 0 ND / ND ND / ND ND ND ND ND /f /f 428del13 Y51X H69Q 527-1 GrA 527-1 GrC F178Ie 868insT G197R G216R G216R Nonee Nonee 2 2 2 ... 5 ... 5 5 ... 5 ... ... ... ... ... 6 7 8 8 8 a 1 Å Barth et al. (1983); 2 Å Patton et al. (1994); 3 Å Örstavik et al. (1993); 4 Å Àdes et al. (1993); and 5 Å Bione et al. (1996). / Å Present; 0 Å absent; //0 Å uncertain status; and ND Å not done. c Cardiac transplant at age 4 mo. d Cardiac transplant at age 14 mo. e Sequence was determined on the basis of the mother’s DNA. f Only in myocardium. b Am. J. Hum. Genet. 61:862–867, 1997 865 D’Adamo et al.: X-Linked Dilated Cardiomyopathies Figure 1 Mutations in G4.5 gene, and their localization in a schematic representation of the gene. Blackened boxes are invariant exons; and diagonally striped boxes are alternatively spliced exons. One family was published by Lindenbaum et al. in 1973 (Lindenbaum et al. 1973). In the second large and yetunpublished family (fig. 3), four affected males were described: three (IV-1, IV-16, and V-5) died within the first 3–4 mo of life of cardiac failure. The fourth, SWH (IV-4), had cardiac failure at age 5 wk, survived, and now, at age 25 years, is normal. In both families we found the same mutation (a GrA change at nucleotide 1006) in exon 10, causing a GrR change in the sequence of the protein (G240R). The mutation was not found in 100 normal chromosomes. In the family studied by Lindenbaum et al., the mutation was found in three obligate carriers. In the family of SWH, all the patients and the mother (II-1) of SWH were sequenced and shown to carry the mutation. Missense Mutations: Change of Conserved Amino Acids The G4.5 gene is conserved in evolution. A BLAST search of GenBank showed that ORFs encoding very similar protein sequences exist in Caenorhabditis elegans and Saccharomyces cerevisiae genomes. The alignment of the tafazzins from the three organisms is shown in figure 4. The region corresponding to the alternative exon 5 in human is missing in the C. elegans gene; in S. cerevisiae the corresponding region is present, but it is not conserved. Striking conservation was observed in most of the rest of the protein. The four regions boxed in figure 4 are the most conserved: ú50% of the amino acids are identical between the three species, and ú80% are conservative substitutions. The missense mutations found in the patients are indicated in figure 4, and in all instances they correspond to residues conserved between two or all three organisms. No function is known for the C. elegans or S. cerevisiae tafazzins, since they correspond to ORFs identified by genomic sequencing. Portions of chromatograms showing mutations in some Figure 2 patients (BM, BS, and MF [bottom sections of panels]) or in an obligate carrier (HM [bottom section of panel]), compared with the normal sequence (top sections of panels). Lowercase letters denote intron sequences. Numbers correspond to nucleotide positions in the cDNA (Bione et al. 1996). Arrowheads point to mutations. A 13-base deletion is boxed. Discussion In this paper we have presented the results of a study of mutations in the BTHS gene, G4.5. We show that mutations in this gene are found in BTHS and in other 866 Figure 3 Pedigree of family SWH. The asterisk (*) indicates the ‘‘normal’’ male (II-4) carrying the mutation. Blackene boxes denote affected males; and diagonally striped boxes denote males who died of heart disorder different from BTHS. X-linked dilated cardiomyopathies, previously considered different conditions and listed with different OMIM numbers (305300 and 300069). The patients diagnosed as affected with BTHS were often well characterized, and at least one patient in each family was thoroughly studied. We have tried to summarize the most common features of the disease, and the data in table 1 demonstrate that, in addition to cardiac failure in the 1st year of life, growth arrest, cyclic neutropenia, and methylglutaconic aciduria appear to be reliable diagnostic signs of BTHS. Patients Figure 4 Am. J. Hum. Genet. 61:862–867, 1997 affected with either EFE1 or severe cardiomyopathy have not been as thoroughly studied, and they were just described as affected with dilated cardiomyopathy. Whether the other symptoms of BTHS were present cannot be established with the available clinical data, but our findings suggest that mutations in the G4.5 gene have to be considered as a possible cause of infantile dilated cardiomyopathies affecting males, even in the absence of the typical BTHS signs. Seven different mutations have been reported in this work. They were searched and not found in 100 normal chromosomes, and, when DNA of family members was available, they were shown to segregate with the disease. The 11 mutations described thus far in the BTHS gene are null or missense mutations localized in alternative, as well as in invariant, parts of the gene. Both missense and null mutations seem to be responsible for a similar disease, since the clinical characteristics and life expectancy of the patients in each BTHS family do not appear to profoundly differ. This is unlike the situation in many other disorders, where missense often causes a phenotype less severe than that caused by null mutations. However, since comparison with similar sequences in distant species has indicated that the amino acids changed by the mutations are highly conserved residues, the very severe effect of missense mutations could be ascribed to a drastic structural modification of the pro- Alignment of amino acid sequence of tafazzins with C. elegans and S. cerevisiae homologues, done by use of CLUSTAL. Asterisks (*) indicate identical amino acids; and dots indicate conserved amino acids. Highly conserved regions are boxed; and amino acids changed in the patients indicated are in boldface. 867 D’Adamo et al.: X-Linked Dilated Cardiomyopathies teins, as a consequence of their substitution with different amino acid. In the family affected with X-linked fatal infantile cardiomyopathy (Gedeon et al. 1995), the mutation is a 1-base deletion in exon 8, causing a frameshift and eventually a stop codon after 18 nucleotides (patient MH), and it is unique among the 13 patients studied, since it causes knockout of all the putative tafazzins. The very severe clinical manifestations of the disorder in this family (described in six affected males and in eight males suspected of being affected) could thus be related to the severity of the mutation. More patients should be studied to confirm this observation, but the results suggest that null mutations in the invariant part of the tafazzins, near the C-terminus of the protein, may be rare and may have a more severe phenotypic effect. The direct study of the tafazzins, by determination of which are the proteins present in each affected cell type, will help to further clarify this point. The function of the G4.5 gene is presently unknown, nor did the gene product show similarity to known proteins. The sequence of the tafazzins is very conserved in evolution: ORFs encoding proteins highly homologous to the tafazzins have been found in S. cerevisiae and C. elegans. The conservation, together with the very severe phenotype associated with mutations in the gene, suggests that the role of the tafazzins must be of great importance for the correct function of the heart and other organs during fetal and neonatal life. In this paper, we have presented a family in which at least one individual (SWH) carrying a mutation causing a severe phenotype had a very severe heart failure but survived to live a normal life. In the same family, another male (II-4) also must have carried the mutation: one of his sisters (II-5) and his daughter (III-15) are obligate carriers, and his grandson was affected. Despite the mutation, he was able to reproduce and to transmit the disease. We do not know much about and cannot study II-4, but the finding of the SWH patient and the characteristics of his family suggest that some ‘‘protecting’’ factor(s) could act in fetal life and early after birth. Accordingly, some other patients seem to be able to survive to age ú10 years (see table 1 and one patient reported by Christodoulou et al. [1994] who is suspected to have BTHS). These findings suggest that the role of the tafazzin(s) may be very important early in life and for a limited time after birth and that in later life it may be substituted by other functions. Such function(s) may be responsible also for the very large phenotypic heterogeneity of the symptoms within families. Acknowledgments We thank Dr. Mueller and Dr. R. Savrirayan for providing patient DNA and information. We also thank F. liBergolis for technical assistance. This work was supported by Progetto Finalizzato CNR Ingegneria Genetica and Telethon Italy (support to D.T.). S.B. is a fellow of the Ph.D. program of the U. of Pavia. References Adès LC, Gedeon AK, Wilson MJ, Latham M, Partington MW, Mulley JC, Nelson J, et al (1993) Barth syndrome: clinical features and confirmation of gene localization to distal Xq28. Am J Med Genet 45:327–334 Barth PG, Scholte HR, Berden JA, Van der Klei-Van Moorsel JM, Luyt-Houwen IEM, Van’t Veer-Korthof ETH, Van der Harten JJ, et al (1983) An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leukocytes. J Neurol Sci 62:327–355 Bione S, D’Adamo P, Maestrini E, Gedeon AK, Bolhuis PA, Toniolo D (1996) A novel X-linked gene, G4.5, is responsible for Barth syndrome. Nat Genet 12:385–389 Bolhuis PA, Hensels GW, Hulsebos TJM, Baas F, Barth PG (1991) Mapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28. Am J Hum Genet 48:481–485 Christodoulou J, McInnes RR, Jay V, Wilson G, Becker LE, Lehotay DC, Platt BA, et al (1994) Barth syndrome: clinical observation and genetic linkage studies. Am J Med Genet 50:255–264 Gedeon AK, Wilson MJ, Colley AC, Sillence DO, Mulley IC (1995) X linked fatal infantile cardiomyopathy maps to Xq28 and is possibly allelic to Barth syndrome. J Med Genet 32:383–388 Keating MT, Sanguinetti MC (1996) Molecular genetic insights into cardiovascular disease. Science 272:681–685 Kelley RI, Cheatham JP, Clark BJ, Nigro MA, Powell BR, Sherwood GW, Sladky JT, et al (1991) X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3-methylglutaconic aciduria. J Pediatr 119:738–747 Lindenbaum RH, Andrews PS, Khan ASSI (1973) Two cases of endocardial fibroelastosis possible X-linked determination. Br Heart J 35:38–40 Maquat LE (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1:453–465 Örstavik KH, Skjörten F, Hellebostad M, Hågå P, Langslet A (1993) Possible X linked congenital mitochondrial cardiomyopathy in three families. J Med Genet 30:269–272 Patton MA, Taylor R, Jeffrey S, Jeffrey I, Burn J (1994) Prenatal diagnosis of X-linked cardiomyopathy with neutropenia (Barth syndrome) using DXS15. J Med Genet 31:169–170