Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction
Matteo Vatta, PhD*,
Bhagyalaxmi Mohapatra, PhD*,
Shinawe Jimenez, MD*,
Ximena Sanchez, PhD*,
Georgine Faulkner, PhD ,
Zeev Perles, MD*,
Gianfranco Sinagra, MD ,
Jiuann-Huey Lin, MD*,
Thuy M. Vu, BS*,
Qiang Zhou, PhD||,
Karla R. Bowles, PhD*,
Andrea Di Lenarda, MD,
Lisa Schimmenti, MD¶,
Michelle Fox, MS||,
Michelle A. Chrisco, BS*,
Ross T. Murphy, MD#,
William McKenna, MD#,
Perry Elliott, MD#,
Neil E. Bowles, PhD*,
Ju Chen, PhD||,
Giorgio Valle, PhD** and
Jeffrey A. Towbin, MD, FACC* ,*
* Department of Pediatrics (Cardiology), Baylor College of Medicine, Houston, Texas, USA;
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
Department of Cardiology, Ospedale Maggiore, Trieste, Italy
|| Institute of Molecular Medicine and Department of Medicine, University of California at San Diego, School of Medicine, La Jolla, California, USA
¶ Department of Pediatrics (Genetics), University of California at Los Angeles, Los Angeles, California, USA
# Department of Cardiological Sciences, St. George's Hospital Medical School, London, United Kingdom
** CRIBI Biotechnology Centre, Universita degli Studi di Padova, Padova, Italy
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Figure 1 Cypher/ZASP genomic structure. (A) Representation of the Cypher/ZASP genomic structure (top), and six messenger ribonucleic acid isoforms, termed Cypher/ZASP-1, -2, -3, -4, -5, and -6. The PDZ domain is encoded by exons 1, 2, and 3; the three LIM domains are encoded by exons 12-16. (B) The location of mutations identified in this study. Note that the mutation in C/Z1 will also be present in the C/Z2 and 3 isoform (not shown), and the mutation in C/Z4 will also be present in C/Z5 and 6 isoform (not shown).
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Figure 2 Mutation detection in FDCM 066. (A) Denaturing high performance liquid chromatography (DHPLC) of exon 10 identifies an abnormal DHPLC pattern in the proband (top panel) that is absent in controls (bottom panel). (B) The deoxyribonucleic acid (DNA) sequence analysis of genomic DNA identifies a C to G base substitution at position 1056. (C) Pedigree of family FDCM 066 showing the nucleotides identified at 1056. The arrow identifies the proband.
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Figure 3 Mutation detection in FDCM/INLVM 065. (A) Denaturing high performance liquid chromatography (DHPLC) analysis of exon 4 identifies an abnormal DHPLC pattern in the proband (top panel) that is absent in controls (bottom panel). (B) The deoxyribonucleic acid (DNA) sequence analysis of genomic DNA identifies a C to T base substitution at position 587. (C) Pedigree of family FDCM/INLVM 065. The arrow identifies the proband. (D) Blast homology analysis of Cypher/ZASP amino acid sequence for residue S196.
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Figure 4 Mutation detection in DCM 035. (A) Denaturing high performance liquid chromatography (DHPLC) analysis of exon 4 identifies an abnormal DHPLC pattern (top panel), which is absent in controls (bottom panel). (B) The deoxyribonucleic acid (DNA) sequence analysis of genomic DNA identifies a C to T base substitution at position 638. (C) Blast homology analysis of Cypher/ZASP amino acid sequence for residue T213.
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Figure 5 Mutations identified in INLVM-11, INLVM-17, and DCM-31. (A) The deoxyribonucleic acid (DNA) sequence analysis of exon 6 identifies a G349A substitution in Patient INLVM-11. Note that the reverse sequence is shown. (B) The DNA sequence analysis of genomic DNA from Patient DCM-31 identifies an A407T transversion. (C) Blast homology analysis of Cypher/ZASP amino acid sequence for residues D117 and K136.
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Figure 6 Analysis of the expression of Cypher/ZASP isoforms in human heart. The Cypher/ZASP exons were detected in human cardiac ribonucleic aid by reverse transcription-polymerase chain reaction (A) and Northern blot analysis (B). The exons identified are shown at the bottom of each panel. M = 100 bp deoxyribonucleic acid ladder (A). Positions of ribonucleic acid size markers are indicated (B).
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Figure 7 Immunohistochemical analysis of Cypher/ZASP expression. Immunohistochemical analysis of C2C12 cells transfected with the pcDNA3.1/NT-GFP-TOPO vector (A to C), or with constructs expressing wild type Cypher/ZASP-1-GFP (D-F) or D117N-Cypher/ZASP-1-GFP (G to I). Actin staining (red) is shown in the left panels, GFP (Cypher/ZASP ) staining (green) is shown in the middle panels, while the right panels are the overlay of the ZASP, actin and DAPI (nuclei) images. All images were obtained using 40x magnification.
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Figure 8 Western blot analysis of ZASP expression. Western blot analysis of protein isolated from C2C12 cells transfected with wild type (lanes 1 and 3), D117N (lanes 2 and 4) Cypher/ZASP, separated into insoluble (IF) and soluble (SF) fractions.
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