PMC3959660_01
Diagram of the patient's mutation in exon 4 of ATP7A. The normal sequence contains two consecutive TG dinucleotides in this region (nucleotides 1282 to 1285), one of which was deleted in this patient. Whichever TG was deleted results in the same translational frameshift, predicting addition of three novel amino acids and then a stop codon (all in red font).
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F2
Western blot of fibroblast proteins from a well-characterized normal male fibroblast cell line (CRL2076), the patient, and a Menkes disease patient with deletion of exons 20-23. Red arrow denotes normal ATP7A protein (178 kD). Various smaller sized bands are detected in these samples by the C-terminal antibody that do not however correspond to functional versions of ATP7A, and which we consider to denote non-specific binding. The blot was re-probed with an anti-beta-actin antibody as a control for loading (lower panel).. Reverse-transcription PCR to assess presence of full-length ATP7A transcripts shows that only the normal control cell line produces a stable transcript containing ATP7A exons 21 and 22 (94 bp RT-PCR fragment). Partial length beta-actin transcripts (57 bp RT-PCR fragment) were amplified in the patient as well as controls.. Confocal microscopy of cultured fibroblasts after staining with an anti-ATP7A antibody shows the typical condensed perinuclear pattern in the normal cell line, and only faint background staining in the patient. Scale bars=10 microns.. Characterization of ATP7A protein and transcripts in the patient's fibroblasts.
nihms-553540-f0003.jpg
PMC3959660_01_nihms-553540-f0003.jpg
F3
Western blot analysis of fibroblast protein from the patient did not detect ATP7A protein (Figure 3a). Reverse transcription-polymerase chain reaction (PCR) to assess the potential presence of ATP7A mRNA showed that the patient's fibroblast cell line did not produce transcripts harboring ATP7A exons 21 and 22 (94 bp RT-PCR fragment) (Figure 3b). Confocal microscopy of the patient's cultured fibroblasts after staining with an anti-ATP7A antibody also showed no ATP7A signal (Figure 3c).