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Inheritance and Mutations in a Single-Gene Disorder

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INTRODUCTION Some diseases are caused by the environment. For example, exposure to chemicals or extremely bright lights can cause certain forms of blindness. But other forms of blindness are inherited, meaning that they are passed on from one generation to another. In this activity, you will learn about a young woman, Molly Troxel, who has an inherited form of blindness called Leber congenital amaurosis (LCA). The activity explores how LCA is inherited, the mutations that cause it, and how the disease can be treated to help Molly and other patients. PART 1: Determining LCA’s Pattern of Inheritance Watch the short film Genes as Medicine until time 1:42. You will meet Molly and learn about what causes her blindness. Use what you learn from the film to answer the following questions. 1. Imagine you’re a doctor treating a patient with severe vision issues. What questions might you ask to determine whether these issues are more likely to be inherited or caused by environmental factors?

I will ask if any of his/her parents, maternal/paternal relatives or grand parents had this disease or not, I will also enquire about his/her siblings and cousins if they have this condition or not. If someone has this disease in his ancestory then I will create a pedigree chart and try to find patterns if it could be genetical environmental. Some inherited diseases, including LCA, may be caused by mutations in a single gene. Pedigrees can be used to determine the patterns of inheritance for these diseases. In a 1998 study, scientists analyzed a series of pedigrees showing the family histories of LCA patients. Some of their pedigrees are shown in the following figures. Squares represent males, circles represent females, and shading indicates that an individual has LCA.

Figure 1. A pedigree of a family that has individuals with LCA. Adapted from Morimura et al. (1998).

Figure 2. Two pedigrees of families that have individuals with LCA. Adapted from Morimura et al. (1998).

2. Based only on the pedigree in Figure 1, can you determine whether LCA is or isn’t inherited according to an autosomal dominant, autosomal recessive, or X-linked recessive pattern? Explain your answer.

In pedigree in figure 1 it is not sufficient data to conclude if LCA is autosomal dominant or recessive or X linked recessive. Yes up to some extent we can conclude that it is genetic but it is not guaranteed until more individuals are provided in the chart. 3. Based on the pedigrees in Figure 2, can you determine whether LCA is or isn’t inherited according to an autosomal dominant, autosomal recessive, or X-linked recessive pattern? Explain your answer.

Pedigree in figure 2 exactly fits for X- linked recessive pattern but it cannot be autosomal dominant, though there is a very slight chance that the pattern is autosomal recessive but we can say that the pattern is X- linked recessive.

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Inheritance and Mutations in a Single-Gene Disorder

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Figure 3 shows the pedigrees of seven families in the 1998 study. The scientists used these pedigrees to determine the inheritance pattern for LCA.

Figure 3. Seven pedigrees of families that have individuals with LCA. Adapted from Morimura et al. (1998).

4. Based on the pedigrees in Figure 3, is LCA inherited according to an autosomal dominant, autosomal recessive, or X-linked recessive pattern? Use evidence from the pedigrees to support your claim, making sure to explain the evidence that rules out the inheritance patterns you didn’t choose.

In figure number 3, X linked condition would have caused the disease to all males. Autosomal dominant would cause disease to the majority of the people and won't just appear in F1 without parents having it. The pedigree explains it to be autosomal recessive.

5.

Recall that a person typically has two copies, called alleles, of every gene. One or both of these alleles may be mutated in certain individuals. Use what you know about LCA’s inheritance pattern to write the genotypes for Family #4 in Figure 3, using L to represent an unmutated allele and l to represent a mutant, disease-causing allele for the LCA-related gene. a. What are the possible genotypes of both parents in this family? Explain your answer.

The possible genotype of both the parents would be Ll because a cross between Ll and Ll only can result in a diseased child. b. What are the possible genotypes of the female child without LCA? Explain your answer.

The possible genotype of the female child without LCA can be Ll or LL because a cross between Ll and Ll can result in either LL or Ll or ll and ll is disease trait.

6. Although Molly has LCA, her parents do not. Draw a pedigree for Molly and her parents using the same style as in the previous pedigrees. Write the genotypes for each person using L and l as before.

Molly has LCA represented by ll. Parents are not diseased must have Ll.

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Inheritance and Mutations in a Single-Gene Disorder

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PART 2: Examining Mutations That Cause LCA Many genes contain instructions for making proteins, which have important functions in the body. Mutations, changes in a gene’s DNA sequence, can affect these proteins and cause inherited diseases, such as LCA. Continue watching the Genes as Medicine film until time 2:44. Figure 4 shows two illustrations from the film.

Figure 4. Illustrations comparing unmutated and mutated, disease-causing copies of a gene, and their resulting proteins.

7. Based on the film, what is the difference between the two proteins shown in Figure 4? The gene or the specific sequences of nucleotides in the deoxyribonucleic acid has the information for the protein molecules. Mutation is the change in the nucleotide sequence of these genes. Mutation can result in a modified or dysfunctional protein that may no longer perform its regular function in the cell.

8. Consider what mutations Molly and her parents may have in the gene related to Molly’s LCA. Using Figure 4 as a guide, draw the gene’s two alleles and their resulting proteins for both of Molly’s parents and Molly. Leber congenital amaurosis is an inherited eye disorder that causes vision loss in children. It is the most common cause of the inherited blindness. LCA affects both photoreceptors, the rod cell (allow to see in dark) and the central cone cells in the retina of eye. Due to mutation, the light detecting cells or the photoreceptors progressively malfunction and die. In Molly, there are two copies of the mutated RPE65 genes and her parent have one normal copy and other mutated copy of RPE65 as shown in the figure.

9. Use the diagram you drew to explain why Molly’s parents do not have LCA, but Molly does. Since humans are diploid, each gene can have two same or two different alleles (different version of the same gene) in the cell. Molly’ s parents are heterogenous for the given gene that mean in both parents, one allele has normal gene sequence and the other allele has mutation. However, this do not affect them because at least one gene is functional providing functional protein. But in Molly, there gene is homogenous for the mutated allele, that means, the two mutated alleles from each of the parents have been transferred (inherited) to Mo s genome. Since Molly has both alleles mutated, there is no production of functional protein necessary for the vison, and therefore lose vision.

Continue watching the Genes as Medicine film to time 8:47. 10. Look at the diagram shown at time 8:26 of the film. What are the similarities and differences between this diagram and the one you drew in Question 8? The similarity in both the figure is that Molly’ s parent genes encode one functional and one dis-functional protein, while, Molly genes encodes two dysfunctional genes. The difference is that Molly and Molly’ s parents’ gene is showing mutations in different locus. Different locus can state different genes which is not correct. Both the normal and mutated allele should be shown in the same locus as alleles are nothing but different version of the same gen

11. Is Molly homozygous or heterozygous for the RPE65 gene? Are her alleles for RPE65 both dominant, both recessive, or does she have one of each? Explain your answers. The mutation that causes LCA is a recessive mutation and is not expressed when resent with a dominant allel. However, the diagram shows that the mutation is expressed in Molly, meaning that it is present in recessive homozygous condition. Both alleles for RPE65 in Molly are recessive as the mutation is itself recessive and would not have manifested in the presence of a dominant allele.

Scientists can use gene sequencing to identify mutations that cause disease. Molly’s LCA, for example, was found to be due to mutations in the gene RPE65. This gene codes for a protein, RPE65, that plays an important role in the eye. Tables 1 and 2 compare parts of the unmutated RPE65 amino acid sequence with those of two LCA patients: a 9year-old and an 11-year-old. The tables show sequences for the proteins produced by each RPE65 allele in both patients. The “…” indicate amino acids in the full protein sequences that are not included in the tables. Genetic Disease www.BioInteractive.org

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Inheritance and Mutations in a Single-Gene Disorder

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Table 1: Partial RPE65 protein sequence (amino acids 41–60) for the 9-year-old LCA patient. Unmutated Protein Sequence

START…Ser-Leu-Leu-Arg-Cyc-Gly-Pro-Gly-Leu-Phe-Glu-Val-Gly-Ser-Glu-Pro-Phe-Tyr-His-Gly…STOP

Patient’s Allele 1 Protein Sequence

START…Ser-Leu-Leu-Gln-Cyc-Gly-Pro-Gly-Leu-Phe-Glu-Val-Gly-Ser-Glu-Pro-Phe-Tyr-His-Gly…STOP

Patient’s Allele 2 Protein Sequence

START…Ser-Leu-Leu-Gln-Cyc-Gly-Pro-Gly-Leu-Phe-Glu-Val-Gly-Ser-Glu-Pro-Phe-Tyr-His-Gly…STOP

Table 2. Partial RPE65 protein sequence (amino acids 61–70 and 291–300) for the 11-year-old LCA patient. Unmutated Protein Sequence

START…Phe-Asp-Gly-Gln-Ala-Leu-Leu-His-Lys-Phe…Ile-Ala-Asp-Lys-Lys-Arg-Lys-Lys-Tyr-Leu…STOP

Patient’s Allele 1 Protein Sequence

START…Phe-Asp-Gly-Gln-Ala-Leu-Leu-Tyr-Lys-Phe…Ile-Ala-Asp-Lys-Lys-Arg-Lys-Lys-Tyr-Leu…STOP

Patient’s Allele 2 Protein Sequence

START…Phe-Asp-Gly-Gln-Ala-Leu-Leu-His-Lys-Phe…Ile-Ala-Asp-Lys-STOP

Source: Data from Russell et al. (2017).

Use Tables 1 and 2 to answer the questions below. 12. Circle the mutations in each RPE65 allele for both patients. 13. Does each patient have the same mutation in both of their alleles? Explain your reasoning.

For number 12. the highighted should be Gln in table 1 parent allele 1 and 2. Also Tyr in table 2 patients allele 1. His ---> Tyr 5'-CAC-3' ---> 5'-TAC-3' It is a transition and nonsynonymous mutation 14. Based on your answers to Questions 11 and 13, which patient’s genotype for RPE65 is more similar to Molly’s? Explain your answer.

Lys ---> Stop 5'-AAA-3' ---> 5'-UAA-3' It is a transversion and nonsense mutation. 15. Assume that the parents of both patients do not have LCA. a. Based on Table 1, predict the RPE65 genotypes of the parents of the 9-year-old patient. From the sequence we can see that the 9 year old has mutation in the same region for both the alleles indicating that the patient is homozygous (Arg to Gln substitution in 4th position). As the parents doesn't have LCA indicating that they can be either homozygous dominant or heterozygous. Since the pateint is homozygous and the parents is normal we can conclude that the parents are heterozygous. Thus the patient will be homogzygous recessive. If R is the dominant allele and r recessive allele for RPE65. Then the genotype of both the parent will be Rr and the genotype of the patient will be rr.

b. Based on Table 2, predict the RPE65 genotypes of the parents of the 11-year-old patient. From the sequence we can see that the 11 year old has different mutation in both the alleles in the same gene. We know that one allele is obtained from each parent, and as the parents doesn't have LCA indicating them to be heterozygous but the mutation in recessive allele will be different for each parents. That is the mutation of recessive allele which is Tyr to His substitution will be in one parent and stop codon replacing end protein will be in another parent . As the mutation is different we can say that the patient is compound heterozygous. If R is the dominant allele and r' for recessive allele in one parent and and r" for recessive allele in another parent. Then the Genotype of one parent will be Rr' and other parent will be Rr" and that of patient will be r'r" . Genetic Disease www.BioInteractive.org

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Inheritance and Mutations in a Single-Gene Disorder

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PART 3: Using Gene Therapy to Treat LCA Continue watching the Genes as Medicine film to the end. As shown in the film, scientists treated Molly’s blindness with gene therapy. To do this, they used a modified virus to deliver copies of the unmutated RPE65 gene into cells in Molly’s eye. 16. Like Molly, the LCA patients in Tables 1 and 2 have nonfunctioning RPE65 proteins. However, their mutations in the RPE65 gene may be different from Molly’s. If so, could the gene therapy that helped Molly work for either of these patients? Justify your answer.

Yes, As the mutation is occured in the same gene ( and same protein) as that of Molly, same gene therapy could be used. Gene therapy introduced gene produce functional RPE65 protein. This protein will serve the function which is otherwise not done by the mutated protein produced from inherited (mutated) RPE65 gene.

RPE65 is not the only gene that, when mutated, can cause LCA. Mutations in other genes that code for proteins involved in vision can also cause this type of blindness. 17. Do you think that the same gene therapy that helped Molly could also help LCA patients with mutations in genes other than RPE65? Explain your reasoning.

No, As mentioned in the question LCA can be caused by mutation in different genes other than RPE65. Hence patients with mutation in genes other than RPE65 cannot be treated with the same gene therapy. However if the mutation is in RPE64 alone Molly's gene therapy can be used. Hence different types of gene therapy should be used for the patients having mutation in the genes other than RPE65.

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