American Journal of Medical Genetics 132A:391–394 (2005) Genomic and Transcription Studies as Diagnostic Tools for a Prenatal Detection of X-Linked Dilated Cardiomyopathy due to a Dystrophin Gene Mutation Paola Rimessi, 1 Francesca Gualandi, 1 Laurence Duprez, 2 Pietro Spitali, 1 Marcella Neri, 1 Luciano Merlini, 3 Elisa Calzolari, 1 Francesco Muntoni, 4 and Alessandra Ferlini 1 * 1 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Universita ` di Ferrara, Ferrara, Italy 2 Laboratoire de Cytoge ´ne ´tique, Service de Ge ´ne ´tique Me ´dicale, Ho ˆpital Erasme-ULB, Bruxelles, Belgique 3 Laboratorio di Miologia, Istituto Ortopedico Rizzoli, Bologna, Italy 4 Department of Paediatrics, Imperial College, Hammersmith Hospital Campus, London, United Kingdom X-linked dilated cardiomyopathy (XLDC) re- presents a form of dystrophinopathy with exclu- sive heart involvement. Here a prenatal diagnosis of this condition performed in a family with XLDC is described. In this family, the causative muta- tion was a pure intronic deletion, which induces the splicing of a novel, aberrant, and out-of-frame exon into the dystrophin transcript. The genetic test was performed by defining both the DNA (villous) and the RNA (amniocyte) configuration. The prenatal diagnosis determined that the fetus was female, and a carrier of the genomic deletion. RNA analysis on cultured amniocytes revealed the presence of an easily detectable dystrophin transcript, as well as the co-existence of both the wild-type and the abnormal splicing profile. Our analysis represents the first report of a prenatal diagnosis in XLDC and also indicates the feasi- bility of dystrophin mutation detection on RNA from amniocytes. This finding suggests that the dystrophin splicing pattern in amniocytes and skeletal muscle is similar, and that, therefore, this approach could be used in other prenatal dystro- phin mutation detection, where abnormal RNA splicing is thought to play a role, or for specific cases in which no mutations have been identified in the coding regions. ß 2005 Wiley-Liss, Inc. KEY WORDS: dystrophin; XLDC; RT-PCR; pre- natal; splicing INTRODUCTION X-linked dilated cardiomyopathy (XLDC) represents a rare allelic variant of Duchenne/Becker muscular dystrophies (DMD and BMD), significantly expanding the phenotypic spectrum of dystrophinopathies [Muntoni et al., 2003]. While cardiac involvement is invariably associated with DMD and BMD, in XLDC the mutations cause an almost exclusive cardiac involvement with isolated elevation of serum CK [Ferlini et al., 1999]. Two broad categories of mutations can be recognized as causes of XLDC: (a) mutations at the 5 0 end of the gene affecting specifically dystrophin transcription and/or splicing in the cardiac muscle and (b) mutations affecting the dystrophin gene regions corresponding to the rod-shaped domain of the protein. Therefore, the pathogenesis of XLDC seems to be based on the difference in expression of the dystro- phin mutations between cardiac and skeletal muscle. This difference may be mediated either by a tissue-specific splicing regulation effect of the mutations or by differences in functions of specific protein domains between skeletal and cardiac muscle [Muntoni et al., 2003]. We previously described an unusual XLDC mutation consist- ing of a purely intronic deletion within intron 11 [Ferlini et al., 1998; Gualandi et al., 2003]. This mutation resulted in the incorporation of part of dystrophin intron 11 as a novel exon into the dystrophin mRNA, abolishing the normal transcript in cardiac muscle but not in skeletal muscle. As this interrupts the reading frame, it resulted in the absence of dystrophin in the cardiac muscle. In this specific case, the RNA analysis was crucial for the molecular diagnosis of XLDC and it is worth noting that this mutation would have been missed following a purely genomic approach for standard mutation detection. Here, the prenatal diagnosis offered to the sister of the proband from this family who was a carrier of the dystrophin deletion [Ferlini et al., 1998] is described. The prenatal test performed on the chorionic villous tissue sample by using VNTR screening revealed that the fetus was a female. Mole- cular analysis on genomic DNA obtained from the villous sample confirmed the presence of the intron 11 deletion. The karyotype confirmed the sex and in addition showed an inversion of chromosome 9 (also detected in the fetus’ father). Transcription analysis performed on amniocyte RNA demon- strated the presence of abundant dystrophin transcript, together with the occurrence of the aberrant exon, which was shown to be spliced within the dystrophin messenger at low efficiency. The splicing characteristics of this dystrophin mutation, together with the possible diagnostic implications of this prenatal approach are discussed below. MATERIALS AND METHODS All the diagnostic procedures have been carried out following an informed consent from the parents. Cytogenetic Analysis Chorionic villous sampling was obtained at 10 weeks, 6/7. Short-term culture was set up with RPMI 1640. No long-term culture was set up (see Results). Grant sponsor: The Telethon-Italy; Grant number: GGP02311; Grant sponsor: European Union Finger; Grant number: QLG2- CT-1999-00920. *Correspondence to: Alessandra Ferlini, M.D., Ph.D., Diparti- mento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Universita ` di Ferrara, Via Fossato di Mortara, 74 44100 Ferrara, Italy. E-mail: fla@unife.it Received 12 May 2004; Accepted 5 October 2004 DOI 10.1002/ajmg.a.30513 ß 2005 Wiley-Liss, Inc.