Download Identifying CYP4v2 mutation in Bietti`s crystalline dystrophy patient

Identifying CYP4V2 mutation in Bietti’s crystalline dystrophy patient
Malini
1
Naidu ,
1University
of
Cate Lockhart,
2
PharmD ,
2
Washington, Department
Edward Kelly,
2
PhD
of Pharmaceutics, Seattle, Washington
Abstract: A patient with Bietti’s crystalline dystrophy had their DNA extracted to be screened for a mutation in CYP4V2. The extraction of the DNA from the patient
allowed the lab to locate the point mutation in the gene. Using PCR (polymerase chain reaction), exons 1 through 11 were amplified and then genotyped to see exactly
where the point mutation occurs. After sequencing the patient’s DNA, a mutation on exon 9 (a combined sequence since both exons are short) was found. The
mutation is expected to be a point mutation with C-T (cytosine – thymine) on nucleotide c1198.
Introduction
Methods & Materials
o Bietti's crystalline dystrophy (BCD) is a disease that
is found in the retina and/or cornea of the eye.
o BCD is characterized be yellow/white crystallization
in the retina/cornea, and is thought to be caused by
complex lipids localized in these regions.
o BCD is an autosomal recessive disease.
o The end stage of BCD is blindness.
o The early symptoms include : decline in central
vison, night blindness, and gradual constriction of
the visual field.
o The negative effects caused by BCD are associated
with gene mutations in CYP4V2.
o CYP4V2 is closely studied when researching BCD
because of mutations in the gene that causes the
enzyme to not be functional.
1. PCR (Polymerase chain reaction)
Conclusion
3. DNA purification
PCR amplifies a strand of DNA and
creates many copies of that sequence.
The patient analyzed by the lab has a mutation that is
consistent with a diagnosis of BCD. The results from
the sequence of exon 9 have shown that in fact the
patient does have BCD because of a mutation at
CYP4V2. There have been 60 other mutations found
on the other exons (1 through 11), and the
identification of this mutation has shown that there
are other point mutations that can cause the illness.
4. Genotyping
2. Gel electrophoresis
Figure 1.
Autosomal
recessive
genetics in
a family.
Figure 8. Mutations found from exon 1 – 11 on CYP4V2.
Figure 5. PCR
run through a
gel of the BCD
patient’s DNA.
Results
Bietti’s crystalline dystrophy (BCD) is caused by mutations in
CYP4V2, which causes the illness. On exon 9 a C>T homozygous point
mutation was found. The amino acid change is arginine to cysteine at
nucleotide 400 (pR400C).
Acknowledgements
This research project was supported by the
University of Washington GenOM Project (NIH
5R25HG007153-03), and all the help given by the
coordinators, teachers, and counselors. Special
thanks to Drs. Anne Dinning and Michael Wolf for
the generous gift. Acknowledgements also go to Dr.
Ed Kelly and his lab.
References
Figure 6. Gene sequence (forward) of the patient on exon 9. The red box signifies the point mutation.
Figure 4.
Anatomy of the
human eye
(Nakano, 2014)
Figure 7. Gene sequence (reverse) of the patient on exon 9.
University of Washington GenOM Project: ALVA 2014
Bacterial Genomic DNA Isolation Kit." - For the Rapid Preparation of Genomic DNA from Bacteria. Norgen Biotek
Corporation. Accessed 12 Aug. 2014. <https://norgenbiotek.com/display-product.php?ID=35>
Bietti, G (1937). "Ueber familiaeres Vorkommen von 'Retinitis punctata albescens' (verbunden mit
'Dystrophia marginalis cristallinea corneae'), Glitzern des Glaskoerpers und anderen degenerativen
Augenveraenderungen.“ Klinische Monatsblätter für Augenheilkunde. 99 (1937): 737–757.
Hunt, Margaret. Real Time PCR Tutorial. 2010. Microbiology and Immunology On-Line. University of South
Carolina School of Medicine. Accessed 12 Aug. 2014.
Kelly, EJ, M Nakano, P Rohagti, V Yarov-Yarovoy, and AE Rettie. "Finding Homes for Orphan Cytochrome
P450s." Molecular Interventions. 11.2 (2011): 124-32.
Nakano, M, CM Lockhart, EJ Kelly, and AE Rettie. "Ocular Cytochrome P450s and Transporters: Roles in
Disease and Endobiotic and Xenobiotic Disposition." Drug Metabolism Reviews. 46.3 (2014): 247-260.