Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles
Abstract
:1. Introduction
2. Results
2.1. ABCA4 Gene is Expressed in Human Hair Follicles and Skin
2.2. Hair Follicles and Skin Cells Express the Full-Length ABCA4 Transcript and Protein
2.3. Analysis of the ABCA4 Splice-Site Variants in Hair Follicles
2.4. Analysis of the ABCA4 Gene Expression in Hair Follicles of Patients with Stargardt Disease
3. Discussion
4. Materials and Methods
4.1. Patients and Controls
4.2. Culture of Primary Keratinocytes, Fibroblasts and Melanocytes
4.3. RNA Isolation and Quantitative mRNA Measurements
4.4. Sanger Sequencing of the ABCA4 Transcripts
4.5. Protein Extract Isolation and Western Blot Analysis
4.6. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
References
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Exon Number | 3–4 | 10–11 | 20–21 | 29–30 | 35–36 | 40–41 | 49–50 |
---|---|---|---|---|---|---|---|
Hair follicles (n = 8) | 33.72 | 28.18 | 28.53 | 27.65 | 27.22 | 27.05 | 27.28 |
Total skin samples (n = 4) | 37.77 | 32.51 | 33.17 | Not tested | 33.48 | Not tested | 31.92 |
Keratinocytes (n = 4) | 35.07 | 34.42 | 34.15 | Not tested | 33.02 | Not tested | 34.12 |
Fibroblasts (n = 4) | 33.67 | 32.75 | 32.54 | Not tested | 32.15 | Not tested | 31.48 |
Melanocytes (n = 4) | 37.87 | 37.07 | 37.01 | Not tested | 36.29 | Not tested | 38.46 |
TaqMan® Probes | Exon Boundary |
---|---|
Hs00979588_m1 | 3–4 |
Hs00979567_m1 | 10–11 |
Hs00979574_m1 | 20–21 |
Hs00979581_m1 | 29–30 |
Hs00979586_m1 | 35–36 |
Hs00979589_m1 | 40–41 |
Hs00979594_m1 | 49–50 |
ABCA4 Exons | Forward Primer (5′–3′) | Reverse Primer (5′–3′) | Amplicon Length | Ta |
---|---|---|---|---|
2–4 | GGAACTCGTGTGGCCTTTATCTTTATT | GTCCATGAATTGGGACAAGATGTGTAG | 331 | 58.2 |
4–8 | AGCTACACATCTTGTCCCAATTCATGG | TGTCTTCATACCAGTTGAAGGAGAGCA | 645 | 58.2 |
8–12 | CTCGGGTGCTCTCCTTCAACTGGTAT | TGGTAGAGAGCTGGTCCAGGGATACAT | 698 | 61.3 |
12–15 | GTGCCCTCTCTCTACTGGAGGAAAACA | AGACTGGCCTTGGAGAAGAAGGTGCT | 670 | 61.3 |
15–20 | CACCTTCTTCTCCAAGGCCAGTCTG | CAGGATGTTGTGCTGTGGACACATG | 787 | 61 |
20–25 | CACAGCACAACATCCTGTTCCAC | GAAATTCCAAAACTGCTGAGACCAA | 769 | 57.1 |
25–30 | GGCTGACCTTGGTCTCAGCAGT | CCTGCAGGATGGTGAAGGGTTG | 706 | 58.6 |
30–36 | CACAGGTCAACCCTTCACCATCCTG | TTGGATTTGTTCACCCGCTCCTGGATCA | 691 | 61 |
36–41 | ATCTGCGTGATTTTCTCCATGTCCTT | ATCATCTTCATCAACAATGGGCTCCT | 708 | 56.4 |
40–46 | GAAGGGGTGGTGTACTTCCTCCTGAC | CCTTCTCTGATGATGCTCACGATGAC | 707 | 62.7 |
44–49 | GTCCATCAAAATATGGGCTACTGTCCT | CTGGTCCAGTGTGGTCTGTGTGACT | 702 | 61 |
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Ścieżyńska, A.; Soszyńska, M.; Komorowski, M.; Podgórska, A.; Krześniak, N.; Nogowska, A.; Smolińska, M.; Szulborski, K.; Szaflik, J.P.; Noszczyk, B.; et al. Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles. Int. J. Mol. Sci. 2020, 21, 3430. https://doi.org/10.3390/ijms21103430
Ścieżyńska A, Soszyńska M, Komorowski M, Podgórska A, Krześniak N, Nogowska A, Smolińska M, Szulborski K, Szaflik JP, Noszczyk B, et al. Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles. International Journal of Molecular Sciences. 2020; 21(10):3430. https://doi.org/10.3390/ijms21103430
Chicago/Turabian StyleŚcieżyńska, Aneta, Marta Soszyńska, Michał Komorowski, Anna Podgórska, Natalia Krześniak, Aleksandra Nogowska, Martyna Smolińska, Kamil Szulborski, Jacek P. Szaflik, Bartłomiej Noszczyk, and et al. 2020. "Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles" International Journal of Molecular Sciences 21, no. 10: 3430. https://doi.org/10.3390/ijms21103430