Leber Congenital Amaurosis (LCA) causes progressive vision loss due to defects in the gene that encodes RPE65 isomerase. Affected individuals are homozygous recessive for mutant alleles of the RPE65 gene. You are trying to determine the molecular nature of the mutations in three individuals with LCA. For ease of analysis, you may assume that each individual is homozygous for the same mutant allele (though the three individuals have different mutations than each other). You use the polymerase chain reaction to amplify DNA from each patient and you determine the sequence of the DNA and compare it to unaffected individuals. You identify the following differences. Note that the non-template strand of DNA is given and the changes are highlighted using red boldface. You can assume that the sequences are in the first reading frame (eg. the first three nucleotides of each sequence is a codon). The coding region of the gene is 1602 bp and the position of the sequences shown below is indicated. Individual 1 has the following change in the sequence. An NdeI restriction enzyme site is underlined in both the ‘normal’ and ‘individual 1’ sequence . ‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311 Individual 1 sequence: 1288 5’CCT TAC ACA TAT GCG TAT TGA CTT 3’ 1311 Individual 2 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘individual 2’ sequence. ‘Normal’ sequence: 859 5’ GTT TGG CTT CAT ATT GCT GAC AAA 3’ 882 Individual 2 sequence: 859 5’ GTT TGG CTT CAT ATG GCT GAC AAA 3’ 882 Individual 3 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘normal’ sequence. ‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311 Individual 3 sequence: 1288 5’CCT TAC ACA TCC ATG CGT ATG GAC TT 3’ 1313 For each individual, indicate whether the change is an example of a missense, nonsense, silent or frameshift mutation. Describe exactly how the protein would be changed by the mutation. For example: ‘A glycine amino acid at position 300 (remember to convert nucleotide numbers to amino acid numbers) in the protein would be changed to a leucine amino acid.’ BONUS: For any missense mutation, indicate whether it is conservative or non-conservative. A second mutation was identified in individual 3 that changed a 5’ GGG 3’ codon to 5’ GGC 3’. Why was this not considered to be the cause of LCA for that individual? A fourth individual is tested and no mutations are identified in the coding region of the gene. However, antibody tests show that the individual makes no RPE65 isomerase protein. What type of mutation might cause this? You design PCR primers that hybridize to the beginning and end of the RPE65 isomerase gene, and use them to amplify DNA from a normal control (C) individual as well as individuals 1-3. You then digest the PCR product with NdeI restriction enzyme. Draw in the fragments you would generate in the image of the agarose gel below. The first lane contains molecular weight markers (M) to show the sizes of the DNA. One of the ways that we study human disease is by developing ‘mouse models’ of human diseases. Review this video on the CRISPR-Cas9 genome editing technology (https://www.youtube.com/watch?v=2pp17E4E-O8). Which method would you use if you wanted to make a mouse model of LCA? Explain why you chose that method and, briefly, how you would make the transgenic mice. Note in particular whether the Cas9 induced break in the DNA will be repaired with non-homologous end joining (NHEJ) or homology dependent repair (HDR).

Leber Congenital Amaurosis (LCA) causes progressive vision loss due to defects in the gene that encodes RPE65 isomerase. Affected individuals are homozygous recessive for mutant alleles of the RPE65 gene. You are trying to determine the molecular nature of the mutations in three individuals with LCA. For ease of analysis, you may assume that each individual is homozygous for the same mutant allele (though the three individuals have different mutations than each other). You use the polymerase chain reaction to amplify DNA from each patient and you determine the sequence of the DNA and compare it to unaffected individuals. You identify the following differences. Note that the non-template strand of DNA is given and the changes are highlighted using red boldface. You can assume that the sequences are in the first reading frame (eg. the first three nucleotides of each sequence is a codon). The coding region of the gene is 1602 bp and the position of the sequences shown below is indicated. Individual 1 has the following change in the sequence. An NdeI restriction enzyme site is underlined in both the ‘normal’ and ‘individual 1’ sequence . ‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311 Individual 1 sequence: 1288 5’CCT TAC ACA TAT GCG TAT TGA CTT 3’ 1311 Individual 2 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘individual 2’ sequence. ‘Normal’ sequence: 859 5’ GTT TGG CTT CAT ATT GCT GAC AAA 3’ 882 Individual 2 sequence: 859 5’ GTT TGG CTT CAT ATG GCT GAC AAA 3’ 882 Individual 3 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘normal’ sequence. ‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311 Individual 3 sequence: 1288 5’CCT TAC ACA TCC ATG CGT ATG GAC TT 3’ 1313 For each individual, indicate whether the change is an example of a missense, nonsense, silent or frameshift mutation. Describe exactly how the protein would be changed by the mutation. For example: ‘A glycine amino acid at position 300 (remember to convert nucleotide numbers to amino acid numbers) in the protein would be changed to a leucine amino acid.’ BONUS: For any missense mutation, indicate whether it is conservative or non-conservative. A second mutation was identified in individual 3 that changed a 5’ GGG 3’ codon to 5’ GGC 3’. Why was this not considered to be the cause of LCA for that individual? A fourth individual is tested and no mutations are identified in the coding region of the gene. However, antibody tests show that the individual makes no RPE65 isomerase protein. What type of mutation might cause this? You design PCR primers that hybridize to the beginning and end of the RPE65 isomerase gene, and use them to amplify DNA from a normal control (C) individual as well as individuals 1-3. You then digest the PCR product with NdeI restriction enzyme. Draw in the fragments you would generate in the image of the agarose gel below. The first lane contains molecular weight markers (M) to show the sizes of the DNA. One of the ways that we study human disease is by developing ‘mouse models’ of human diseases. Review this video on the CRISPR-Cas9 genome editing technology (https://www.youtube.com/watch?v=2pp17E4E-O8). Which method would you use if you wanted to make a mouse model of LCA? Explain why you chose that method and, briefly, how you would make the transgenic mice. Note in particular whether the Cas9 induced break in the DNA will be repaired with non-homologous end joining (NHEJ) or homology dependent repair (HDR).

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Description: Leber Congenital Amaurosis (LCA) causes progressive vision loss due to defects in the gene that encodes RPE65 isomerase. Affected individuals are homozygous recessive for mutant alleles of the RPE65 gene. You are trying to determine the molecular n

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Individual 1 has the following change in the sequence. An NdeI restriction enzyme site is underlined in both the ‘normal’ and ‘individual 1’ sequence .

‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311

Individual 1 sequence: 1288 5’CCT TAC ACA TAT GCG TAT TGA CTT 3’ 1311

Individual 2 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘individual 2’ sequence.

‘Normal’ sequence: 859 5’ GTT TGG CTT CAT ATT GCT GAC AAA 3’ 882

Individual 2 sequence: 859 5’ GTT TGG CTT CAT ATG GCT GAC AAA 3’ 882

Individual 3 has the following change in the sequence. An NdeI restriction enzyme site is underlined in the ‘normal’ sequence.

‘Normal’ sequence: 1288 5’CCT TAC ACA TAT GCG TAT GGA CTT 3’ 1311

Individual 3 sequence: 1288 5’CCT TAC ACA TCC ATG CGT ATG GAC TT 3’ 1313

For each individual, indicate whether the change is an example of a missense, nonsense, silent or frameshift mutation. Describe exactly how the protein would be changed by the mutation. For example: ‘A glycine amino acid at position 300 (remember to convert nucleotide numbers to amino acid numbers) in the protein would be changed to a leucine amino acid.’

BONUS: For any missense mutation, indicate whether it is conservative or non-conservative.

A second mutation was identified in individual 3 that changed a 5’ GGG 3’ codon to 5’ GGC 3’. Why was this not considered to be the cause of LCA for that individual?

A fourth individual is tested and no mutations are identified in the coding region of the gene. However, antibody tests show that the individual makes no RPE65 isomerase protein. What type of mutation might cause this?

You design PCR primers that hybridize to the beginning and end of the RPE65 isomerase gene, and use them to amplify DNA from a normal control (C) individual as well as individuals 1-3. You then digest the PCR product with NdeI restriction enzyme. Draw in the fragments you would generate in the image of the agarose gel below. The first lane contains molecular weight markers (M) to show the sizes of the DNA.

One of the ways that we study human disease is by developing ‘mouse models’ of human diseases. Review this video on the CRISPR-Cas9 genome editing technology (https://www.youtube.com/watch?v=2pp17E4E-O8). Which method would you use if you wanted to make a mouse model of LCA? Explain why you chose that method and, briefly, how you would make the transgenic mice. Note in particular whether the Cas9 induced break in the DNA will be repaired with non-homologous end joining (NHEJ) or homology dependent repair (HDR).

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