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Splicing mutation in LAMP2 gene leading to exon skipping and cardiomyopathy development
Gene Reports 18 (2020) 100564
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Splicing mutation in LAMP2 gene leading to exon skipping and cardiomyopathy development
T
⁎
Larysa Sivitskayaa, , Tatiyana Vaikhanskayab, Nina Danilenkoa, Maryna Siniauskayaa, Aleh Liaudanskia, Tatsiyana Kurushkаb, Danat Yermakovicha, Oleg Davydenkoa a b
Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Akademicheskaya str. 27, 220072 Minsk, Belarus Republican Scientific and Practical Center of Cardiology, R. Luxembourg str. 110, 220036 Minsk, Belarus
A R T I C LE I N FO
A B S T R A C T
Keywords: Danon disease Dilated cardiomyopathy Exon skipping LAMP2 Splicing
Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease. It is an X-linked glycogen storage disease that includes cardiomyopathy (hypertrophic or dilated), skeletal myopathy and mental retardation. Here, we report a woman, carrying the mutation c.864 + 1G > A (IVS6 + 1G > A, rs727503119) in heterozygosity and affected with late-onset dilated cardiomyopathy. We demonstrated the effects of the mutation on the splicing LAMP2 in cardiac muscle. The variant c.864 + 1G > A is located in the canonical splice donor site in intron 6. Functional analysis reveals that mutation c.864 + 1G > A leads to the skipping of exon 6 in cardiac muscle. The protein loses 41 amino acids in the second luminal domain of LAMP2 without frameshift and premature termination of protein synthesis. Using qRT-PCR we revealed a significantly low level of wild-type LAMP2 mRNA in patient that can be explained by X-inactivation.
1. Introduction
2. Materials and methods
Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease (DD). It is an X-linked dominant metabolic disease selectively affecting cardiac muscle, while skeletal muscles involvement and mental retardation are quite variable. Men are severely affected earlier than women are, and men mostly die at an average age of 20 years due to heart failure (Boucek et al., 2011). The professional version of the Human Gene Mutation Database lists 93 mutations in the LAMP2 gene causing DD, of which truncating mutations are the most common. About one-quarter of them, represent splice site mutations with a variable and unpredictable expression. According to Bottillo et al. (2016), splicing mutations in LAMP2, like stop-gain variants, cause an earlier manifestation of DD than those not associated with protein truncation. We present a detailed clinical report of dilated cardiomyopathy (DCM) associated with a LAMP2 splice site mutation in a 59 years old female patient.
2.1. Editorial policies and ethical considerations Clinical surveillance and genetic investigations performed by the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments. Informed consent duly obtained from all participants. 2.2. Case report A 53 year old woman was referred for heart transplantation (New York Heart Association class IV). At the age of 51, she was diagnosed with DCM. Her family members had no heart disease. She had neither muscle weakness nor an intellectual disability. The electrocardiographic results showed normal sinus rhythm, wide QRS (158 ms), and left bundle branch block. Transthoracic echocardiographic findings showed left ventricular (LV) dilatation and systolic dysfunction, the LV end-diastolic diameter was 59 mm, the LV end-systolic dimension was
Abbreviations: B2M, beta-2-microglobulin; DCM, dilated cardiomyopathy; DD, Danon disease; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LAMP2, lysosomal-associated membrane protein 2; LV, left ventricular; LVH, left ventricular hypertrophy; MIF, macrophage migration inhibitory factor; MR, mental retardation; NGS, next-generation sequencing; qRT-PCR, quantitative RT-PCR; RT-PCR, reverse transcription PCR; VCF, variants call format; WT, wild type; XCI, X chromosome inactivation ⁎ Corresponding author. E-mail address: [email protected] (L. Sivitskaya). https://doi.org/10.1016/j.genrep.2019.100564 Received 27 June 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 Available online 13 November 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 18 (2020) 100564
L. Sivitskaya, et al.
Fig. 1. Images of the patient. A – Chest X-ray. Cardiac enlargement. B – A short-axis view of gadolinium-enhanced cardiac magnetic resonance imaging. Image shows marked left ventricular dilatation and late gadolinium enhancement in the sub endocardium and transmural in the interventricular septum, anterior and lateral walls.
analyzed by electrophoresis in 1% agarose gel. The band not corresponded to wild-type was extracted using PCR Clean-up Gel Extraction kit (Macherey-Nagel, Germany) and directly sequenced.
52 mm, and the LV ejection fraction (EF) was 30%. The systolic function decreased due to global hypokinesia with the akinetic apical segment. EF assessed by Simpson's method was 22%. Right ventricular function was impaired, with free wall hypokinesia (Tricuspid Annular Plane Systolic Excursion (TAPSE) - 9 mm). There were signs of pulmonary hypertension (tricuspid regurgitation gradient 50 mmHg) and moderate pericardial effusion (circumferential, maximum 8 mm laterally). Her chest X-ray revealed pulmonary congestion and cardiomegaly (Fig. 1). Magnetic resonance imaging also revealed cardiac enlargement with late gadolinium enhancement (Fig. 1). She underwent heart transplantation at the age of 54. At 59 years, her clinical status is stable without skeletal myopathy or intellectual problems.
2.4.2. Quantitative real-time RT-PCR Two TaqMan assays specific for exon 1 and 6 of the LAMP2 (NM_013995.2) using Beacon Designer software (Bio-Rad Inc., USA) were designed (Supplemental Appendix 1). The MIF and B2M were selected as endogenous reference genes for comparative analysis of gene expression (Miracco et al., 2006; Caracausi et al., 2017). We optimized the parameters for primers and probes as per the Real-time PCR Applications Guide (Bio-Rad). For relative quantification of LAMP2 mRNA expression, we chose three normal heart tissue samples as a control. They were obtained from three females (40, 42, and 64 years old), the tissue samples were taken during surgery for valve or septum correction. These women do not have DCM. We carried out the real-time PCR in triplicates in volume 40 μl each containing: 2XArtMix (ArtBioTech Ltd., Belarus), 7 μl cDNA, 600 nM primers and 400 nM TaqMan probe for each of the genes. The specificity of PCR products was confirmed by agarose gel electrophoresis. All data were analyzed in BioRad CFX Maestro (Bio-Rad Inc., USA). Relative quantification of mRNA level between patient and control group was calculated by the ΔΔCt method (Livak and Schmittgen, 2001). Student's t-test determined the statistical significance to have a value of p < .05, which was sufficiently significant.
2.3. DNA sequencing Genomic DNA was obtained from buccal epithelium by phenol/ chloroform extraction. We performed the targeted next-generation sequencing (NGS) using the TruSight Cardiomyopathy sequencing panel on the MiSeq System (Illumina Inc., San Diego, CA, USA). Confirmation of identified variant and family genotyping were carried out using Sanger sequencing with the Big Dye Terminator v3.1 cycle sequencing kit on 3500 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). We estimated the quality control of raw NGS data with FastQC, performed an allignment using BWA against the reference genome NCBIbuild37 (UCSC hg19), and generated the VCF files with a GATK4 HaplotypeCaller. Variants were annotated by ANNOVAR using dbSNP IDs, Exome Variant Server, the 1000 Genomes Browser, the Genome Aggregation Database, ClinVar, and REVEL.
3. Results A transition c.864 + 1G > A was detected in intron 6 of the LAMP2 gene (NM_002294). It is located in the canonical splice donor site and predicted to alter splicing according to the Human Splicing Finder. Sanger sequencing confirmed this variant. Segregation analysis in the family showed that the mutation appeared de novo and wasn't inherited by the patient's children. According to clinical examination, none of the family members had cardiac symptoms or abnormal cardiac studies. To prove the pathogenic role of the identified variant, we performed RT-PCR analysis. Patient's cardiac muscle samples obtained from the explanted heart were used for RNA extraction, reverse transcription, amplification, and sequencing of 5–7 exons region. The analysis revealed that the c.864 + 1G > A variant led to the skipping of the 6th exon (Fig. 2 A, B). Of note, the absence of exon 6 in the mature mRNA doesn't lead to a frameshift and a premature termination of protein synthesis. In our case, the mutant protein loses 41 amino acids in the second luminal domain of LAMP2 – p.Val248_Val288del. The protein spatial structure is predicted to change critically: two glycosylation sites and disulphide bond are lost because of skipping of exon 6 (Fig. 2C). To evaluate the total level of LAMP2 mRNA in our patient, we used TaqMan assay specific to exon 1 and control samples matched for age
2.4. Gene expression analysis 2.4.1. RNA isolation and RT-PCR We isolated total RNA from proband and control heart muscle using the innuSPEED Tissue RNA Kit (Analytik Jena, Germany). RNA quality was analyzed with electrophoresis in 1% agarose gel and spectrophotometry (A260/A280 > 2,0 and A260/A230 > 1,8). RNA was reverse transcribed using oligo-dT primers and ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs Inc., USA). In order to check splicing alteration in LAMP2, we designed primers specific to exon-exon boundaries in the mature mRNA and amplified the full length of 5–7 exons (Supplemental Appendix 1). The expected size of the RT-PCR fragment was 564 bp. Transcript variant NM_013995.2 was used for Primer-BLAST/BLAST and GenRunner version 6.5.48. The primers were tested for compatibility in OligoAnalyzer 3.1. The targeted LAMP2 fragment was amplified with FIREPol Master Mix (Solis BioDyne, Estonia), and then the RT-PCR products were 2
Gene Reports 18 (2020) 100564
L. Sivitskaya, et al.
Fig. 2. A – RT-PCR analysis of patient (P) and control (C) for c.864 + 1G > A in LAMP2. The smaller product (441 bp) in agarose gel electrophoresis corresponds to a transcript without exon 6. B – Results of smaller fragment sequencing. C – Topological model of LAMP2 protein and expected consequences of c.864 + 1G > A on its structure, showing loss of 41 amino acids with two glycosylation sites, and one disulfide bond. Reprinted from [Ruivo et al., 2009]. D – The mRNA level of wildtype LAMP2, measured by qRT-PCR in patient and control heart muscles. B2M and MIF were used as an internal control. Student's t-test, *P < .05.
4. Discussion
and gender. Neither in/dels nor SNPs were detected by Sanger sequencing in exon 1 of samples. The relative quantity calculated for LAMP2 was normalized with MIF and B2M as the endogenous control. PCR efficiency was assessed for each primer and probe from the slope of the standard curve. In most cases, the slope of the standard curve was close to −3.4, indicating maximal PCR efficiency. The normalized value of the control group was taken as the calibrator to calculate the relative expression level of target genes in the patient. We didn't find any differences in LAMP2 expression level between patient and control subjects (data not shown). It means, the mutation c.864 + 1G > A has no impact on the gene expression. The next step was to quantify the expression level of wild-type LAMP2 mRNA in our patient using TaqMan assay specific to exon 6. Fig. 2D shows the mRNA level for the wild-type form of LAMP2 measured by qRT-PCR. These results indicate that the expression level of normal LAMP2 is significantly lower in the heart of the patient with c.864 + 1G > A than in control subjects. Comparing with controls, the level of wild-type LAMP2 mRNA was ~50% lower in the patient (0.48 ± 0.04, P < .05). It means, only half of LAMP2 transcript can be translated into the native protein. Moreover, this data indicated random (50:50) X-chromosome inactivation (XCI) in the heart of our patient.
The variant c.864 + 1G > A (rs727503119) has already been registered in ClinVar as “pathogenic” based on its localization in the canonical splice donor site, but any detailed clinical reports associated with this mutation lack in literature. This is the first description of the DCM among women associated with c.864 + 1G > A. Her DD phenotype is presented with dilated cardiomyopathy that is typical for women. However, the manifestation is unusually late – 51 years old and is characterized with slow progression. Unfortunately, Danon disease often stays unrecognized in women due to the absence of specific signs. Frequency of DCM caused by LAMP2 mutations is not yet estimated. The genetic reason for DCM in our patient was the complete excision of exon 6 from mature mRNA. Thus, the effect of c.864 + 1G > A on the LAMP2 splicing was originally demonstrated by our research. To date, six different splice site mutations have been reported in this region, most of which result in skipping of the exon 6 (Katsumi et al., 1996; Nishino et al., 2000; Arad et al., 2005; Bui et al., 2008; Cottinet et al., 2011; Luo et al., 2014). Their clinical presentations are summarized in Table 1. According to the table, the disease manifests at different ages: the cardiomyopathy onset in women carrying mutations is evidently later. Of note, Arad et al. (2005) reported seven women with 3
Gene Reports 18 (2020) 100564
6 skipping
23 22 6 Puberty nr 51
Congenital 5 7
c.864 + 1_4delGTGA in LAMP2 in one family. Six of them were asymptomatic at the age of 14–49 years (no data in the table). Only one woman died from congestive heart failure at 44 years (Table 1). In contrast, the nucleotide substitution c.864 + 1G > T has been identified in a teenage female with severe HCM and left ventricular dilatation. Variability of DD onset and severity in women explained by XCI: a higher level of mutated X-chromosome inactivation in tissue leads to milder disease appearances (Deng et al., 2014). The XCI is indeed a major factor determining the severity of clinical manifestation in females carrying X-linked disease (Deng et al., 2014; Wu et al., 2014). We consider that the random XCI (50:50) in our patient determined the cardiomyopathy at her 50th. If the XCI were skewed and there were and extremely low level of normal LAMP2 mRNA, the disease would have manifested earlier. The phenotypic heterogeneity of DD in males is often reported: variability in the muscle pain and weakness, degree of mental retardation, age of disease onset, etc. (Table 1). Though not proved, probably it can be partly associated with LAMP2 expression level. Cottinet et al. (2011) demonstrated the mutation c.864 + 1delG had a severe impact on protein expression, which decreased dramatically in skeletal muscles. To date, it is unknown whether other mutations associated with the skipping of exon 6 have the same effect. Of note, splicing alterations due to splice site mutations can be tissue-specific. Cetin et al. (2016) showed that muscle tissue is more accurate for LAMP2 splicing investigations than leukocytes or fibroblasts. Using RNA-Seq analysis, they identified four different transcripts in a patient with c.65-2A > G, including the full-length mRNA. It was synthesized only in the muscle tissue of the patient. Its amount was too small to be detectable by RT-PCR and western blot analyses, but enough to protect the patient from the severe form of DD. Therefore, there are different ways for realization of splicing mutations to consider for functional analysis performing and variant interpretation. In summary, we described in detail the case of DCM in the woman with c.864 + 1G > A in LAMP2. We performed molecular experiments to confirm that the mutation results in exon skipping and to reveal a low level of wild-type LAMP2 mRNA in the patient. We classified the mutation c.864 + 1G > A as pathogenic based on splicing alteration, the strong association of LAMP2 mutations with Danon disease and typical phenotype for women with DD. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.genrep.2019.100564.
DD – Danon disease, CDM – cardiomyopathy, DCM – dilated cardiomyopathy, HCM – hypertrophic cardiomyopathy, LVH - left ventricular hypertrophy, MR - mental retardation, nr - not reported.
Died at the age of 34 years
High serum CK
Mild serum CK
Died from heart failure at 44 years. Sensoneural hearing loss, death at 15 years
Arad et al., 2005 Arad et al., 2005 Arad et al., 2005 Cottinet et al., 2011 Cottinet et al., 2011 Cottinet et al., 2011 Cottinet et al., 2011 Su-Shan Luo et al., 2014 Katsumi et al., 1996, Nishino et al., 2000 Bui et al., 2008 Sivitskaya et al., 2017 ClinVar rs727503119 This report Died at the age of 22 years
No No No Moderate Moderate Moderate No Moderate Moderate No Moderate nr No No No No No Moderate Mild No Severe Mild Severe Moderate nr No HCM HCM nr HCM HCM HCM Mild LVH HCM DCM HCM HCM HCM DCM 22 22 44 15 5 16 42 24 26 16 28 Teenager 59
M M F M M M F M M M M F F
c.864 + 1_4delGTGA c.864 + 1_4delGTGA c.864 + 1_4delGTGA c.864 + 1delG c.864 + 1delG c.864 + 1delG c.864 + 1delG c.864G > A c.864 + 5G > C c.864 + 3_6del c.864 + 3_6del c.864 + 1G > T c.864 + 1G > A
Exon Exon Exon Exon Exon Exon Exon Exon Exon nr nr nr Exon
6 6 6 6 6 6 6 6 6
skipping skipping skipping skipping skipping skipping skipping skipping skipping
16 22
MD CDM Onset DD Type of mutation Mutation Sex Age
Table 1 The phenotype of patients carrying donor splice site mutations of exon 6.
MR
Other
Ref
L. Sivitskaya, et al.
Acknowledgments The authors are thankful to the patient's family for cooperation. Scientific and Technical Program “DNA-identification” of National Academy of Sciences of Belarus, (ID: DNA/2017-6.6) supported this work. Author contribution L.S., N.D., and T.V. – study design, manuscript preparation; T.V. and T.K. – clinical investigations and sample collection; A.L. and D.Y. – NGS performing and raw data analysis; L.S. – NGS data and mutation analysis; M.S. – TaqMan assay design, O.D. – data interpretation and manuscript editing. Declaration of competing interest The authors declare they have no conflict of interests. References Arad, M., Maron, B.J., Gorham, J.M., Johnson Jr., W.H., Saul, J.P., Perez-Atayde, A.R., ... Seidman, J.G., 2005. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N. Engl. J. Med. 352 (4), 362–372. https://doi.org/10.1056/
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Katsumi, Y., Fukuyama, H., Ogawa, M., Matsui, M., Tokonami, F., Aii, H., ... Nonaka, I., 1996. Cerebral oxygen and glucose metabolism in glycogen storage disease with normal acid maltase: case report. J. Neurol. Sci. 140 (1–2), 46–52. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25 (4), 402–408. https://doi.org/10.1006/meth.2001.1262. Luo, S., Xi, J., Cai, S., Zhao, C., Lu, J., Zhu, W., ... Ye, Z., 2014. Novel LAMP2 mutations in Chinese patients with Danon disease cause varying degrees of clinical severity. Clin. Neuropathol. 33 (4), 284–291. https://doi.org/10.5414/NP300668. Miracco, C., De Nisi, M.C., Arcuri, F., Cosci, E., Pacenti, L., Toscano, M. … Tosi, P. (2006). Macrophage migration inhibitory factor protein and mRNA expression in cutaneous melanocytic tumours. Int. J. Oncol., 28, 345–352. doi.org/https://doi.org/10.3892/ ijo.28.2.345. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., ... Koga, Y., 2000. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406 (6798), 906–910. https://doi.org/10.1038/35022604. Ruivo, Raquel, Anne, Christine, Sagné, Corinne, Gasnier, Bruno, 2009. Molecular and cellular basis of lysosomal transmembrane protein dysfunction. Biochim. Biophys. Acta 1793 (4), 636–649. https://doi.org/10.1016/j.bbamcr.2008.12.008. Sivitskaya, L.N., Danilenko, N.G., Vaikhanskaya, T.G., Levdanskiy, O.D., Davydenko, O.G., 2017. Danon disease diagnosis by targeted next-generation sequencing: identification of LAMP2 mutations. Doklady Natsional'noi akademii nauk Belarusi [Doklady of the National Academy of Sciences of Belarus] 61 (3), 64–72 (in Russian). Wu, H., Luo, J., Yu, H., Rattner, A., Mo, A., Wang, Y., ... Nathans, J., 2014. Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease. Neuron 81 (1), 103–119. https://doi.org/10.1016/j.neuron. 2013.10.051.
NEJMoa033349. Bottillo, I., Giordano, C., Cerbelli, B., D'Angelantonio, D., Lipari, M., Polidori, T., ... Grammatico, P., 2016. A novel LAMP2 mutation associated with severe cardiac hypertrophy and microvascular remodeling in a female with Danon disease: a case report and literature review. Cardiovasc. Pathol. 25 (5), 423–431. https://doi.org/10. 1016/j.carpath.2016.07.005. Boucek, D., Jirikowic, J., Taylor, M., 2011. Natural history of Danon disease. Genet. Med 13 (6), 563–568. https://doi.org/10.1097/GIM.0b013e31820ad795. Bui, Y.K., Renella, P., Martinez-Agosto, J.A., Verity, A., Madikians, A., Alejos, J.C., 2008. Danon disease with typical early-onset cardiomyopathy in a male: focus on a novel LAMP-2 mutation. Pediatr. Transplant. 12, 246–250. https://doi.org/10.1111/j. 1399-3046.2007.00874.x. Caracausi, M., Piovesan, A., Antonaros, F., Strippoli, P., Vitale, L., Pelleri, M., 2017. Systematic identification of human housekeeping genes possibly useful as references in gene expression studies. Mol. Med. Rep. 16, 2397–2410. https://doi.org/10.3892/ mmr.2017.6944. Cetin, H., Wöhrer, A., Rittelmeyer, I., Gencik, M., Zulehner, G., Zimprich, F., Ströbel, T., Zimprich, A., 2016. The c.65-2A/G splice site mutation is associated with a mild phenotype in Danon disease due to the transcription of normal LAMP2 mRNA. Clin. Genet. 90 (4), 366–371. https://doi.org/10.1111/cge.12724. Cottinet, S.L., Bergemer-Fouquet, A.M., Toutain, A., Sabourdy, F., Maakaroun-Vermesse, Z., Levade, T., ... Labarthe, F., 2011. Danon disease: intrafamilial phenotypic variability related to a novel LAMP-2 mutation. J. Inherit. Metab. Dis. 34 (2), 515–522. https://doi.org/10.1007/s10545-010-9251-y. Deng, X., Berletch, J.B., Nguyen, Di K., Disteche, C.M., 2014. X chromosome regulation: diverse patterns in development, tissues and disease. Nat. Rev. Genet. 15, 367–378. https://doi.org/10.1038/nrg3687.
5
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Splicing mutation in LAMP2 gene leading to exon skipping and cardiomyopathy development
T
⁎
Larysa Sivitskayaa, , Tatiyana Vaikhanskayab, Nina Danilenkoa, Maryna Siniauskayaa, Aleh Liaudanskia, Tatsiyana Kurushkаb, Danat Yermakovicha, Oleg Davydenkoa a b
Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Akademicheskaya str. 27, 220072 Minsk, Belarus Republican Scientific and Practical Center of Cardiology, R. Luxembourg str. 110, 220036 Minsk, Belarus
A R T I C LE I N FO
A B S T R A C T
Keywords: Danon disease Dilated cardiomyopathy Exon skipping LAMP2 Splicing
Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease. It is an X-linked glycogen storage disease that includes cardiomyopathy (hypertrophic or dilated), skeletal myopathy and mental retardation. Here, we report a woman, carrying the mutation c.864 + 1G > A (IVS6 + 1G > A, rs727503119) in heterozygosity and affected with late-onset dilated cardiomyopathy. We demonstrated the effects of the mutation on the splicing LAMP2 in cardiac muscle. The variant c.864 + 1G > A is located in the canonical splice donor site in intron 6. Functional analysis reveals that mutation c.864 + 1G > A leads to the skipping of exon 6 in cardiac muscle. The protein loses 41 amino acids in the second luminal domain of LAMP2 without frameshift and premature termination of protein synthesis. Using qRT-PCR we revealed a significantly low level of wild-type LAMP2 mRNA in patient that can be explained by X-inactivation.
1. Introduction
2. Materials and methods
Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease (DD). It is an X-linked dominant metabolic disease selectively affecting cardiac muscle, while skeletal muscles involvement and mental retardation are quite variable. Men are severely affected earlier than women are, and men mostly die at an average age of 20 years due to heart failure (Boucek et al., 2011). The professional version of the Human Gene Mutation Database lists 93 mutations in the LAMP2 gene causing DD, of which truncating mutations are the most common. About one-quarter of them, represent splice site mutations with a variable and unpredictable expression. According to Bottillo et al. (2016), splicing mutations in LAMP2, like stop-gain variants, cause an earlier manifestation of DD than those not associated with protein truncation. We present a detailed clinical report of dilated cardiomyopathy (DCM) associated with a LAMP2 splice site mutation in a 59 years old female patient.
2.1. Editorial policies and ethical considerations Clinical surveillance and genetic investigations performed by the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments. Informed consent duly obtained from all participants. 2.2. Case report A 53 year old woman was referred for heart transplantation (New York Heart Association class IV). At the age of 51, she was diagnosed with DCM. Her family members had no heart disease. She had neither muscle weakness nor an intellectual disability. The electrocardiographic results showed normal sinus rhythm, wide QRS (158 ms), and left bundle branch block. Transthoracic echocardiographic findings showed left ventricular (LV) dilatation and systolic dysfunction, the LV end-diastolic diameter was 59 mm, the LV end-systolic dimension was
Abbreviations: B2M, beta-2-microglobulin; DCM, dilated cardiomyopathy; DD, Danon disease; EF, ejection fraction; HCM, hypertrophic cardiomyopathy; LAMP2, lysosomal-associated membrane protein 2; LV, left ventricular; LVH, left ventricular hypertrophy; MIF, macrophage migration inhibitory factor; MR, mental retardation; NGS, next-generation sequencing; qRT-PCR, quantitative RT-PCR; RT-PCR, reverse transcription PCR; VCF, variants call format; WT, wild type; XCI, X chromosome inactivation ⁎ Corresponding author. E-mail address: [email protected] (L. Sivitskaya). https://doi.org/10.1016/j.genrep.2019.100564 Received 27 June 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 Available online 13 November 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 18 (2020) 100564
L. Sivitskaya, et al.
Fig. 1. Images of the patient. A – Chest X-ray. Cardiac enlargement. B – A short-axis view of gadolinium-enhanced cardiac magnetic resonance imaging. Image shows marked left ventricular dilatation and late gadolinium enhancement in the sub endocardium and transmural in the interventricular septum, anterior and lateral walls.
analyzed by electrophoresis in 1% agarose gel. The band not corresponded to wild-type was extracted using PCR Clean-up Gel Extraction kit (Macherey-Nagel, Germany) and directly sequenced.
52 mm, and the LV ejection fraction (EF) was 30%. The systolic function decreased due to global hypokinesia with the akinetic apical segment. EF assessed by Simpson's method was 22%. Right ventricular function was impaired, with free wall hypokinesia (Tricuspid Annular Plane Systolic Excursion (TAPSE) - 9 mm). There were signs of pulmonary hypertension (tricuspid regurgitation gradient 50 mmHg) and moderate pericardial effusion (circumferential, maximum 8 mm laterally). Her chest X-ray revealed pulmonary congestion and cardiomegaly (Fig. 1). Magnetic resonance imaging also revealed cardiac enlargement with late gadolinium enhancement (Fig. 1). She underwent heart transplantation at the age of 54. At 59 years, her clinical status is stable without skeletal myopathy or intellectual problems.
2.4.2. Quantitative real-time RT-PCR Two TaqMan assays specific for exon 1 and 6 of the LAMP2 (NM_013995.2) using Beacon Designer software (Bio-Rad Inc., USA) were designed (Supplemental Appendix 1). The MIF and B2M were selected as endogenous reference genes for comparative analysis of gene expression (Miracco et al., 2006; Caracausi et al., 2017). We optimized the parameters for primers and probes as per the Real-time PCR Applications Guide (Bio-Rad). For relative quantification of LAMP2 mRNA expression, we chose three normal heart tissue samples as a control. They were obtained from three females (40, 42, and 64 years old), the tissue samples were taken during surgery for valve or septum correction. These women do not have DCM. We carried out the real-time PCR in triplicates in volume 40 μl each containing: 2XArtMix (ArtBioTech Ltd., Belarus), 7 μl cDNA, 600 nM primers and 400 nM TaqMan probe for each of the genes. The specificity of PCR products was confirmed by agarose gel electrophoresis. All data were analyzed in BioRad CFX Maestro (Bio-Rad Inc., USA). Relative quantification of mRNA level between patient and control group was calculated by the ΔΔCt method (Livak and Schmittgen, 2001). Student's t-test determined the statistical significance to have a value of p < .05, which was sufficiently significant.
2.3. DNA sequencing Genomic DNA was obtained from buccal epithelium by phenol/ chloroform extraction. We performed the targeted next-generation sequencing (NGS) using the TruSight Cardiomyopathy sequencing panel on the MiSeq System (Illumina Inc., San Diego, CA, USA). Confirmation of identified variant and family genotyping were carried out using Sanger sequencing with the Big Dye Terminator v3.1 cycle sequencing kit on 3500 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). We estimated the quality control of raw NGS data with FastQC, performed an allignment using BWA against the reference genome NCBIbuild37 (UCSC hg19), and generated the VCF files with a GATK4 HaplotypeCaller. Variants were annotated by ANNOVAR using dbSNP IDs, Exome Variant Server, the 1000 Genomes Browser, the Genome Aggregation Database, ClinVar, and REVEL.
3. Results A transition c.864 + 1G > A was detected in intron 6 of the LAMP2 gene (NM_002294). It is located in the canonical splice donor site and predicted to alter splicing according to the Human Splicing Finder. Sanger sequencing confirmed this variant. Segregation analysis in the family showed that the mutation appeared de novo and wasn't inherited by the patient's children. According to clinical examination, none of the family members had cardiac symptoms or abnormal cardiac studies. To prove the pathogenic role of the identified variant, we performed RT-PCR analysis. Patient's cardiac muscle samples obtained from the explanted heart were used for RNA extraction, reverse transcription, amplification, and sequencing of 5–7 exons region. The analysis revealed that the c.864 + 1G > A variant led to the skipping of the 6th exon (Fig. 2 A, B). Of note, the absence of exon 6 in the mature mRNA doesn't lead to a frameshift and a premature termination of protein synthesis. In our case, the mutant protein loses 41 amino acids in the second luminal domain of LAMP2 – p.Val248_Val288del. The protein spatial structure is predicted to change critically: two glycosylation sites and disulphide bond are lost because of skipping of exon 6 (Fig. 2C). To evaluate the total level of LAMP2 mRNA in our patient, we used TaqMan assay specific to exon 1 and control samples matched for age
2.4. Gene expression analysis 2.4.1. RNA isolation and RT-PCR We isolated total RNA from proband and control heart muscle using the innuSPEED Tissue RNA Kit (Analytik Jena, Germany). RNA quality was analyzed with electrophoresis in 1% agarose gel and spectrophotometry (A260/A280 > 2,0 and A260/A230 > 1,8). RNA was reverse transcribed using oligo-dT primers and ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs Inc., USA). In order to check splicing alteration in LAMP2, we designed primers specific to exon-exon boundaries in the mature mRNA and amplified the full length of 5–7 exons (Supplemental Appendix 1). The expected size of the RT-PCR fragment was 564 bp. Transcript variant NM_013995.2 was used for Primer-BLAST/BLAST and GenRunner version 6.5.48. The primers were tested for compatibility in OligoAnalyzer 3.1. The targeted LAMP2 fragment was amplified with FIREPol Master Mix (Solis BioDyne, Estonia), and then the RT-PCR products were 2
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Fig. 2. A – RT-PCR analysis of patient (P) and control (C) for c.864 + 1G > A in LAMP2. The smaller product (441 bp) in agarose gel electrophoresis corresponds to a transcript without exon 6. B – Results of smaller fragment sequencing. C – Topological model of LAMP2 protein and expected consequences of c.864 + 1G > A on its structure, showing loss of 41 amino acids with two glycosylation sites, and one disulfide bond. Reprinted from [Ruivo et al., 2009]. D – The mRNA level of wildtype LAMP2, measured by qRT-PCR in patient and control heart muscles. B2M and MIF were used as an internal control. Student's t-test, *P < .05.
4. Discussion
and gender. Neither in/dels nor SNPs were detected by Sanger sequencing in exon 1 of samples. The relative quantity calculated for LAMP2 was normalized with MIF and B2M as the endogenous control. PCR efficiency was assessed for each primer and probe from the slope of the standard curve. In most cases, the slope of the standard curve was close to −3.4, indicating maximal PCR efficiency. The normalized value of the control group was taken as the calibrator to calculate the relative expression level of target genes in the patient. We didn't find any differences in LAMP2 expression level between patient and control subjects (data not shown). It means, the mutation c.864 + 1G > A has no impact on the gene expression. The next step was to quantify the expression level of wild-type LAMP2 mRNA in our patient using TaqMan assay specific to exon 6. Fig. 2D shows the mRNA level for the wild-type form of LAMP2 measured by qRT-PCR. These results indicate that the expression level of normal LAMP2 is significantly lower in the heart of the patient with c.864 + 1G > A than in control subjects. Comparing with controls, the level of wild-type LAMP2 mRNA was ~50% lower in the patient (0.48 ± 0.04, P < .05). It means, only half of LAMP2 transcript can be translated into the native protein. Moreover, this data indicated random (50:50) X-chromosome inactivation (XCI) in the heart of our patient.
The variant c.864 + 1G > A (rs727503119) has already been registered in ClinVar as “pathogenic” based on its localization in the canonical splice donor site, but any detailed clinical reports associated with this mutation lack in literature. This is the first description of the DCM among women associated with c.864 + 1G > A. Her DD phenotype is presented with dilated cardiomyopathy that is typical for women. However, the manifestation is unusually late – 51 years old and is characterized with slow progression. Unfortunately, Danon disease often stays unrecognized in women due to the absence of specific signs. Frequency of DCM caused by LAMP2 mutations is not yet estimated. The genetic reason for DCM in our patient was the complete excision of exon 6 from mature mRNA. Thus, the effect of c.864 + 1G > A on the LAMP2 splicing was originally demonstrated by our research. To date, six different splice site mutations have been reported in this region, most of which result in skipping of the exon 6 (Katsumi et al., 1996; Nishino et al., 2000; Arad et al., 2005; Bui et al., 2008; Cottinet et al., 2011; Luo et al., 2014). Their clinical presentations are summarized in Table 1. According to the table, the disease manifests at different ages: the cardiomyopathy onset in women carrying mutations is evidently later. Of note, Arad et al. (2005) reported seven women with 3
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6 skipping
23 22 6 Puberty nr 51
Congenital 5 7
c.864 + 1_4delGTGA in LAMP2 in one family. Six of them were asymptomatic at the age of 14–49 years (no data in the table). Only one woman died from congestive heart failure at 44 years (Table 1). In contrast, the nucleotide substitution c.864 + 1G > T has been identified in a teenage female with severe HCM and left ventricular dilatation. Variability of DD onset and severity in women explained by XCI: a higher level of mutated X-chromosome inactivation in tissue leads to milder disease appearances (Deng et al., 2014). The XCI is indeed a major factor determining the severity of clinical manifestation in females carrying X-linked disease (Deng et al., 2014; Wu et al., 2014). We consider that the random XCI (50:50) in our patient determined the cardiomyopathy at her 50th. If the XCI were skewed and there were and extremely low level of normal LAMP2 mRNA, the disease would have manifested earlier. The phenotypic heterogeneity of DD in males is often reported: variability in the muscle pain and weakness, degree of mental retardation, age of disease onset, etc. (Table 1). Though not proved, probably it can be partly associated with LAMP2 expression level. Cottinet et al. (2011) demonstrated the mutation c.864 + 1delG had a severe impact on protein expression, which decreased dramatically in skeletal muscles. To date, it is unknown whether other mutations associated with the skipping of exon 6 have the same effect. Of note, splicing alterations due to splice site mutations can be tissue-specific. Cetin et al. (2016) showed that muscle tissue is more accurate for LAMP2 splicing investigations than leukocytes or fibroblasts. Using RNA-Seq analysis, they identified four different transcripts in a patient with c.65-2A > G, including the full-length mRNA. It was synthesized only in the muscle tissue of the patient. Its amount was too small to be detectable by RT-PCR and western blot analyses, but enough to protect the patient from the severe form of DD. Therefore, there are different ways for realization of splicing mutations to consider for functional analysis performing and variant interpretation. In summary, we described in detail the case of DCM in the woman with c.864 + 1G > A in LAMP2. We performed molecular experiments to confirm that the mutation results in exon skipping and to reveal a low level of wild-type LAMP2 mRNA in the patient. We classified the mutation c.864 + 1G > A as pathogenic based on splicing alteration, the strong association of LAMP2 mutations with Danon disease and typical phenotype for women with DD. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.genrep.2019.100564.
DD – Danon disease, CDM – cardiomyopathy, DCM – dilated cardiomyopathy, HCM – hypertrophic cardiomyopathy, LVH - left ventricular hypertrophy, MR - mental retardation, nr - not reported.
Died at the age of 34 years
High serum CK
Mild serum CK
Died from heart failure at 44 years. Sensoneural hearing loss, death at 15 years
Arad et al., 2005 Arad et al., 2005 Arad et al., 2005 Cottinet et al., 2011 Cottinet et al., 2011 Cottinet et al., 2011 Cottinet et al., 2011 Su-Shan Luo et al., 2014 Katsumi et al., 1996, Nishino et al., 2000 Bui et al., 2008 Sivitskaya et al., 2017 ClinVar rs727503119 This report Died at the age of 22 years
No No No Moderate Moderate Moderate No Moderate Moderate No Moderate nr No No No No No Moderate Mild No Severe Mild Severe Moderate nr No HCM HCM nr HCM HCM HCM Mild LVH HCM DCM HCM HCM HCM DCM 22 22 44 15 5 16 42 24 26 16 28 Teenager 59
M M F M M M F M M M M F F
c.864 + 1_4delGTGA c.864 + 1_4delGTGA c.864 + 1_4delGTGA c.864 + 1delG c.864 + 1delG c.864 + 1delG c.864 + 1delG c.864G > A c.864 + 5G > C c.864 + 3_6del c.864 + 3_6del c.864 + 1G > T c.864 + 1G > A
Exon Exon Exon Exon Exon Exon Exon Exon Exon nr nr nr Exon
6 6 6 6 6 6 6 6 6
skipping skipping skipping skipping skipping skipping skipping skipping skipping
16 22
MD CDM Onset DD Type of mutation Mutation Sex Age
Table 1 The phenotype of patients carrying donor splice site mutations of exon 6.
MR
Other
Ref
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Acknowledgments The authors are thankful to the patient's family for cooperation. Scientific and Technical Program “DNA-identification” of National Academy of Sciences of Belarus, (ID: DNA/2017-6.6) supported this work. Author contribution L.S., N.D., and T.V. – study design, manuscript preparation; T.V. and T.K. – clinical investigations and sample collection; A.L. and D.Y. – NGS performing and raw data analysis; L.S. – NGS data and mutation analysis; M.S. – TaqMan assay design, O.D. – data interpretation and manuscript editing. Declaration of competing interest The authors declare they have no conflict of interests. References Arad, M., Maron, B.J., Gorham, J.M., Johnson Jr., W.H., Saul, J.P., Perez-Atayde, A.R., ... Seidman, J.G., 2005. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N. Engl. J. Med. 352 (4), 362–372. https://doi.org/10.1056/
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