A novel noncontiguous duplication in the DMD gene escapes the reading-frame rule

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1 c Indian Academy of Sciences RESEARCH NOTE A novel noncontiguous duplication in the DMD gene escapes the reading-frame rule LUZ BERENICE LÓPEZ-HERNÁNDEZ 1, BENJAMÍN GÓMEZ-DÍAZ 2, ELIGANTY BAHENA-MARTÍNEZ 1, TERESA NERI-GÓMEZ 3, ALEJANDRA CAMACHO-MOLINA 4, LUIS A. RUANO-CALDERÓN 5, SILVIA GARCÍA 1 and RAMÓN M. CORAL-VÁZQUEZ 3 1 Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Subdirección de Enseñanza e Investigación, Centro Médico Nacional 20 de Noviembre, México, C. P , D.F., México 2 Instituto Nacional de Rehabilitación, Secretaría de Salud, México, C. P , D.F., México 3 Instituto Politécnico Nacional, Escuela Superior de Medicina, México, C. P , D.F., México 4 Programa de Medicina Genómica, Dirección Médica, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, México, C. P , D.F., México 5 Hospital General de Durango, C. P , Durango, México [López-Hernández L. B., Gómez-Díaz B., Bahena-Martínez E., Neri-Gómez T., Camacho-Molina A., Ruano-Calderón L. A., García S. and Coral-Vázquez R. M A novel noncontiguous duplication in the DMD gene escapes the reading-frame rule. J. Genet. 93, ] Introduction Most mutations in the DMD gene disrupting the reading frame result in Duchenne muscular dystrophy (DMD) (OMIM: ), whereas in-frame mutations producing small but functional dystrophin result in a milder form, Becker muscular dystrophy (BMD) (OMIM: ). This reading frame rule holds true for more than 90% of cases (Tuffery-Giraud et al. 2009). Besides DMD/BMD, intermediate phenotypes (IMD) have also been identified (Prior and Bridgeman 2005) but the molecular mechanisms involved are not yet clear. A novel noncontiguous duplication of exons 1, 42 and 43 was detected using multiplex ligation-dependent probe amplification (MLPA) in a patient with an attenuated phenotype; not as severe as classic DMD but not as subtle as in BMD. His ambulation decline began at 12 years, without steroid treatment, and he presented learning difficulties. His mother was carrier of the rearrangement. Immunodetection analysis showed dystrophin expression in the muscle biopsy, with a slight reduction in the carboxy-terminal region. Complex rearrangements may underlie uncommon subphenotypes in dystrophinopathies. For correspondence. lblhmedgen@gmail.com. Clinical description Materials and methods The 14-year-old proband is the first of four children, born when the father was 23 years old and the mother 21. Both parents are healthy and the marriage is nonconsanguineous. Family history was negative for neuromuscular disorders. The propositus was born at 35 weeks of gestation; his weight was 1.85 kg and height 43 cm. He stayed in hospital for 12 days for immaturity; he had difficulty in suckling and swallowing, as well as motor developmental delay. He could sit at one year and walk at 18 months but frequent falls were noticed and he was not able to run or jump. He is on a wheelchair, has scoliosis, and has girdle weakness, areflexia, hypotonia and deformity of lower limbs and weakness of neck muscles. He did not attend school; learning difficulties were reported by the mother although formal evaluation on this matter was not performed. Although the mother did not show any signs or symptoms of neuromuscular disease, once the mutation was found genetic testing was offered to establish carrier status; sisters of the proband were not tested because they were too young to request genetic counseling; one of them had subtle mental retardation. Both the patient and his mother signed informed-consent letters prior to the analysis, according to institutional ethical guidelines. Some authors have recognized subphenotypes different from DMD/BMD. Desguerre et al. defined classic DMD Keywords. noncontiguous duplications; DMD gene; reading frame. Journal of Genetics, Vol. 93, No. 1, April

2 Luz Berenice López-Hernández et al. phenotype with a loss of ambulation at ± 15.6 months (Desguerre et al. 2009) whereas others, based on age at wheelchair dependency, defined DMD < 13 y, BMD 16 y, and 13 y IMD < 16 y (Tuffery-Giraud et al. 2009). Ambulation decline began at 12 years because of progressive weakness girdle associated to calf hypertrophy and Gower s sign, at 13 years old he was only able to stand and walk with difficulty (wheelchair dependency). He did not receive steroids or physiotherapy, even though his age at wheelchair dependency (156 months) was higher than the mean of our patients with definitive/probable diagnosis according to MD STARNET criteria (Mathews et al. 2010) and without steroid treatment (117.6 ± 21.7 months, n = 33, Student s t test P < 0.05). Therefore, we considered him an outlier or intermediate phenotype (Lopez-Hernandez et al. 2014). Dystrophin analysis A biopsy of muscle and skin was obtained from deltoids frozen into liquid-nitrogen-chilled isopentane and kept at 70 C until analysis. Frozen sections were subjected to hematoxylin eosin staining and immunodetection analysis. Indirect immunofluorescence was carried out in muscle, for DYS1 (rod domain), DYS2 (carboxy-terminal) and DYS3 (amino-terminal) antibodies (Novocastra, Newcastle upon Tyne, UK) and Western blot was performed for VPD-505 (carboxy-terminal) and VP-D508 (rod domain) antibodies (Vector Laboratories, Burlingame, USA as described previously (Gomez-Diaz et al. 2012). Additionally, skin biopsy was analysed for dystrophin expression because muscle is often damaged when the specimen is taken in advanced stages of the disease (Tanveer et al. 2009). Mutation analysis Peripheral blood samples were obtained from the propositus and his mother, and genomic DNA was isolated by the CTAB DTAB method (Gustincich et al. 1991). Mutation detection was performed at first by multiplex PCR as described previously (Coral-Vazquez et al. 1997). Due to the absence of deletions in the analysed exons and the possible presence of duplications, samples were additionally analysed by MLPA according to the manufacturer s instructions (MRC-Holland, Amsterdam, The Netherlands). MLPA, unlike multiplex PCR, detects gross deletions and duplications in all 79 exons of the DMD gene by direct hybridization and ligation of exon-specific half probes to DNA. After that, ligated probes are amplified by fluorescence multiplex PCR which reveals copy number alterations in the gene of interest. The PCR products were visualized by capillary electrophoresis using an ABI 310 sequence detection system (Applied Biosystems, Foster City, USA). Data analysis was done using R software (Softgenetics, Pennsylvania, USA). GeneMarker Results Analysis of the patient biopsy showed dystrophic changes such as central nuclei and muscle fibres of different size (data not shown). Immunofluorescence and immunohistochemistry analysis of the muscle and skin biopsies of the Figure 1. Immunodetection analysis. (a) Immunofluorescence showing dystrophin expression in muscle (C, negative control; C+, normal control; P, patient). (b) Western blot confirms expression of dystrophin and shows reduced staining of carboxy-terminal region. 226 Journal of Genetics, Vol. 93, No. 1, April 2014

3 Complex rearrangement in the DMD gene patient showed a reduction in the staining for the carboxyl terminal domain of dystrophin (figure 1, panel a). This decrease (62.36%) was also observed by Western blot assay; in contrast, the staining for the rod domain was almost normal when compared with the control (figure 1, panel b). After the multiplex PCR screening, mutation analysis was negative (data not shown). The MLPA analysis showed a novel noncontiguous duplication involving exons 1, 42 and 43. The complex rearrangement was found in both the patient and his mother (figure 2). This rearrangement c.[-244-?_31dup; 5923-?_6290+?dup] [NM_ ] was not previously reported in the Leiden database ( thus the genotype phenotype correlation was unknown. Two mutation hotspots have been described for duplications in the DMD gene, one in the 5 region (duplication of exon 2 and exons nearby) and the other in the distal region (exons 45 53) (Gualandi et al. 2009); both duplications are located near those commonly duplicated sites. To gain insight into the splicing process involved in this phenomenon, RNA was isolated from muscle biopsy of the patient, but it was insufficient to complete the analysis. Discussion In agreement with conventional notions of the disease, the presence of a mutation in the DMD gene is a criterion for a definite Duchenne case (Mathews et al. 2010); of particular interest would be how the pathogenicity of a genetic variant or variants is determined. Schwartz et al. (2007) reported a complete deletion of exon 16 in the DMD gene in a healthy control, whereas in the present case, we show a novel noncontiguous duplication of exons 1, 42 and 43 in a patient with an intermediate phenotype not as severe as classic DMD, but not that subtle to be considered as Becker type. By itself duplication of exon 1 might not modify protein translation and/or structure, because it is known that exon 1 of the skeletal muscle isoform is largely noncoding (Muntoni et al. 2003). In addition, an alternative translation initiation site exists (Gurvich et al. 2009). A duplication including the transcription initiation site could produce transcripts including or excluding the duplicated segment or a combination of both. A duplication of noncoding regions like exon 1 and 5 untranslated region may not abolish dystrophin expression Figure 2. Mutation analysis. (a) MLPA dosage diagram shows the noncontiguous duplication of exons 1, 42 and 43 (red); deleted probes are Y chromosome control probes. (b) Peak height differences show duplicated exons. Journal of Genetics, Vol. 93, No. 1, April

4 Luz Berenice López-Hernández et al. but could affect RNA stability ( nevertheless in this case, the probes for DP427c are not duplicated, thus although the breakpoints of duplications were not determined duplication may not encompass the 5 of the muscle promoter. Duplication of exons 42 and 43 solely predicts disruption of the reading frame, nonetheless in the case presented here dystrophin is present. Therefore, we speculate that due to the presence of this noncontiguous duplication, skipping of exon 44 may occur owing to alternative splicing events, simultaneously with the exclusion of exons 42 and 43 from mrna; this could restore the reading frame in this case. The proportion of in-frame and out-of-frame transcripts would determine the expression of dystrophin in muscle and as a consequence the patient s phenotype. Complex rearrangements such as noncontiguous duplications, deletions and triplications account for 1% of mutational events (Lalic et al. 2005). Interestingly, IMD phenotypes are also present in 1.1% of cases (Tuffery-Giraud et al. 2009). Immunodetection analyses showed only a reduction in the expression of the carboxy-terminal domain. A possible explanation is that as a consequence of the duplication of exons 42 and 43, alternative splicing may be induced (Gualandi et al. 2009) producing a protein nearly the original size but with a different conformation, which may cause a failure in antibody recognition. Despite the production of dystrophin in this case, the phenotype is not that slight to be considered as Becker type. Interestingly mutations affecting the spectrin-like repeats 16 and 17 (R16/17) of dystrophin (encoded by exons 42 45) that disrupt anchoring of neuronal nitric oxide synthase (nnos) are associated with severe phenotype in Becker patients (Gentil et al. 2012), therefore nnos binding site could be altered in our patient; leading to the observed phenotype. We have previously reported another genotype phenotype discordance involving dystrophin rod domain in a severe DMD patient with an in-frame deletion of exons 24 41(Lopez-Hernandez et al. 2011). Thus, although internally truncated dystrophins are thought to be functional, certain sites within the rod domain are not dispensable. Although complex rearrangements are rare events, other authors have reported noncontiguous duplications and triplications in the DMD gene (Lalic et al. 2005; Fenollar-Cortes et al. 2008; White et al. 2006; Oshimaet al. 2009; Zhang et al. 2008); in this regard, mechanisms causing complex rearrangements remain unrevealed. Helmrich et al. (2011) suggested that large genes such as the DMD gene are prone to collisions between transcription and replication machineries, whenever the duration of the former is large and of the latter short. This may potentially explain why some mutations are likely to occur during meiosis and present as inherited in the family, and others occur during mitosis in the first divisions of the zygote, which are apparently de novo mutations. Therefore, reporting genetic variants and their parental origin is important to elucidate mechanisms involved in mutational events. Now it is possible to estimate recurrence risks for germ-line mosaicism depending on mutation type and location within the DMD gene (Helderman-van den Enden et al. 2009). Some of the implications of the case presented here are: (i) a complete genetic screening of the DMD gene is crucial, because the presence of double mutations could modify the diagnosis as well as genetic counselling. (ii) The molecular effect of genetic variants acting together may change the predicted effect on gene expression. (iii) As a consequence of this, the predicted phenotype according to the detected mutation does not always follow the reading-frame rule (Monaco et al. 1988) and dystrophin expression does not assure phenotype rescue. This should be taken into account when establishing genotype phenotype correlations or designing mini-dystrophins for therapeutic purposes. Acknowledgements We thank financial support from Administración del Patrimonio de la Beneficencia Pública-Secretaría de Salud, and Consejo Nacional de Ciencia y Tecnología (CONACYT) becas mixtas , CONACYT Salud We thank Emmelien Aten for MLPA training, Lucila Sandoval and Bladimir Roque for help in sample processing, and Graciela Romero for useful suggestions and comments. 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