Creating a Disease-Causing Mutation at the Interface of Two

Opa1 Domains
Cr

Sara Backus and Dr. Andrew Kehr, PhD

Loras College, Division of Molecular and Life Sciences

Autosomal Dominant
Optic Atrophy and Opa1
Opa1 DNA with disease-causing Methods

Autosomal Dominant Optic mutation R445H was generated with 1. Site Directed Mutagenesis
Atrophy (ADOA) missense mutation
ADOA is…
o Leading cause of congenital optic
neuropathy Summary and Success of Methods Utilized
o Affects 1 in 30,000 people
o Vision loss varying from minor to Site Directed Enzymes Q5 Pfu
severe, or even blindness
Mutagenesis Create Opa1 DNA with R445H Mutation
o No yet curable  Annealing Temperatures (°C) 55.5, 54.8, 54, 53.7, 52.3, 51.1
(SDM)
 Primers 5’ and 3’ primers (1065,1066) 2. Bacterial Transformation
Above show all the methods utilized throughout the research. The conditions that produced the
R445H Opa1, but with mutation were Pfu polymerase, 54°C, and the 5’ and 3’ primers.

Above is the normal eye which shows the macula, retina, and Bacterial Transformation
optic nerve. In the patient, there is a prominent pallor color
showing the optic nerve degeneration
Using the process in step 2 of the methods, two
What is Opa1? colonies of bacteria grew on two of the
Kanamycin plates with the R445H Opa1 mutant. Insert R445H Mutated Opa1 into DH5αα
OPa1 is… Successful Conditions:
o A Dynamin Superfamily Protein • Pfu Polymerase
• Annealing Temperature of 54°C 3. PCR Screen and Sequencing
o Responsible for inner • Both 5’ and 3’ primers to insert the R445H
mitochondrial membrane fusion mutation
(IMM) 1 2 3
o A GTPase
o When mutated is contributes to PCR Screening
the development of ADOA
The definite structure of Opa1 protein is still Lane 1: ladder of base pairs (bp)
to be determined, but this is the predicted Lane 2 and 3: Successful R445H in Opa1 1000 bp
structure. Lane 4: Unsuccessful Clone Determine if Mutated Opa1 was
GTPase Domain
Transformed through PCR Screen and
Positive Screen indicates a band at 1000 bp
Middle/Stalk Further Sequencing
GTPase Effector
Sequencing of Opa1 Shows Cloned R445H Mutation within
the Opa1 Plasmid as well as a Missense Mutation Research Goal

References The overall goal of this research was

Both colonies (shown in to use different SDM techniques to
Kehr, 2019. Characterizing Opa1: A Protein Directly Linked to Blindness. lanes 2 and 3) contained successfully clone R445H mutation
mutated R445H Opa1 into the wildtype Opa1 plasmid.
Lenaers, G.; Hamel, C.; Delettre, C.; Amati-Bonneau, P.; Procaccio, V.; Bonneau, D.;
Reynier, P.; Milea, D. Dominant Optic Atrophy. Orphanet Journal of Rare Diseases 2012 Missense mutation that was found
Brewster, Madison. The Impact of Mutation on Lipid-Binding Interactions in Opa1 after sequencing the entire plasmid;
Protein. 2022. Loras College. DNA plasmids this mutation was a product of our
stock wildtype Opa1

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