1. X-linked chronic granulomatous disease (CGD) is an uncommon disorder characterized by a defect in host defense that leads to severe, recurrent, and often fatal pyogenic infections beginning in early childhood. The X-linked CGD locus encodes the heavy chain of cytochrome b, a component of the oxidase that generates superoxide in phagocytes. Because interferon-γ (IFN-γ) is known to enhance the oxidase activity of normal phagocytes, IFN-γ was administered to boys with X-linked CGD to see whether their oxidase activity increased. Before treatment, the phagocytes of some less severely affected patients had small but detectable bursts of oxidase activity (unlike those of severely affected patients), suggesting that increased activity in these less severely affected subjects is the result of greater production of cytochrome b from the affected locus. In these less severe cases, IFN-γ increased the cytochrome b content, superoxide production, and killing of Staphylococcus aureus in the granulocytes. The IFN-γ effect was associated with a definite increase in the abundance of the cytochrome b chain. Presumably, the cytochrome b polypeptide of these patients is partially functional, and increased expression of the residual function improved the physiological defect. Describe the genetic differences that might account for the fact that the phagocytes of some patients with X-linked CGD respond to IFN-γ in vitro and others do not.

2. Identify some of the limitations on the types of proteins that can be considered for extracellular replacement therapy, as exemplified by polyethylene glycol–adenosine deaminase (PEG-ADA). What makes this approach inappropriate for phenylalanine hydroxylase deficiency? If Tay-Sachs disease caused only liver disease, would this strategy succeed? If not, why?

3. A 3-year-old girl, Rhonda, has familial hypercholesterolemia due to a deletion of the 5′ end of each of her low-density lipoprotein (LDL) receptor genes that removed the promoter and the first two exons. (Rhonda’s parents are second cousins.) You explain to the parents that she will require plasmapheresis every 1 to 2 weeks for years. At the clinic, however, they meet another family with a 5-year-old boy with the same disease. The boy has been treated with drugs with some success. Rhonda’s parents want to know why she has not been offered similar pharmacological therapy. Explain.

4. What classes of mutations are likely to be found in homocystinuric patients who are not responsive to the administration of large doses of pyridoxine (vitamin B₆)? How might you explain the fact that Tom is completely responsive, whereas his first cousin Allan has only a partial reduction in plasma homocystine levels when he is given the same amount of vitamin B₆?

5. You have isolated the gene for phenylalanine hydroxylase (PAH) and wish ultimately to introduce it into patients with PKU. Your approach will be to culture cells from the patient, introduce a functional version of the gene into the cells, and reintroduce the cells into the patient.

a. What DNA components do you need to make a functional PAH protein in a gene transfer experiment?

b. Which tissues would you choose in which to express the enzyme, and why? How does this choice affect your gene construct in (a)?

c. You introduce your version of the gene into fibroblasts cultured from a skin biopsy specimen from the patient. Northern (RNA) blot analysis shows that the messenger RNA (mRNA) is present in normal amounts and is the correct size. However, no PAH protein can be detected in the cells. What kinds of abnormalities in the transferred gene would explain this finding?

d. You have corrected all the problems identified in (c). On introducing the new version of the gene into the cultured cells, you now find that the PAH protein is present in great abundance, and when you harvest the cells and assay the enzyme (in the presence of all the required components), normal activity is obtained. However, when you add ³H-labeled phenylalanine to the cells in culture, no ³H-labeled tyrosine is formed (in contrast, some cultured liver cells produce a large quantity of ³H-labeled tyrosine in this situation). What are the most likely explanations for the failure to form ³H-tyrosine? How does this result affect your gene therapy approach to patients?

e. You have developed a method to introduce your functional version of the gene directly into a large proportion of the hepatocytes of patients with PAH deficiency. Unexpectedly, you find that much lower levels of PAH enzymatic activity are obtained in patients in whom significant amounts of the inactive PAH homodimer were detectable in hepatocytes before treatment than in patients who had no detectable PAH polypeptide before treatment. How can you explain this result? How might you overcome the problem?

6. Both alleles of an autosomal gene that is mutant in your patient produce a protein that is decreased in abundance but has residual function. What therapeutic strategies might you consider in such a situation?

7. A Phase III clinical trial is undertaken to evaluate the effectiveness of a small molecule drug that facilitates skipping over nonsense mutation codons. The drug had been shown in earlier trials to have a modest but significant clinical effect in patients with cystic fibrosis with at least one CFTR nonsense mutation. Two cystic fibrosis (CF) patients each have a nonsense mutation in one CFTR allele, but at different locations in the reading frame. One patient responds to the drug, whereas the other does not. Discuss how the location of the nonsense mutation in the predicted reading frame of the protein could account for this differential response.