Pseudovitamin D Deficiency Focus concept An apparent Vitamin D deficiency is act

Question

Pseudovitamin D Deficiency

Focus concept

An apparent Vitamin D deficiency is actually caused by a mutation in an enzyme leading to the vitamin’s synthesis.

Prerequisites

· Vitamins and coenzymes.

· The genetic code.

Background

Your patient is a 2-year-old male infant named Justin N. He is suffering from hypotonia, weakness and growth failure, and is unable to walk. His mother has just brought him into the emergency room from the family beach house, where they have been spending the summer, because he has had a seizure. X-rays indicate that the infant is suffering from rickets, which is a result of a nutritional deficiency of Vitamin D. But the infant’s mother insists that her son’s diet is not Vitamin D-deficient. He drinks three glasses of milk a day, and his diet also includes meat and eggs.

You decide to carry out further analysis and take a sample of the infant’s blood. The laboratory results are shown in Table 29.1.

Table 29.1: Laboratory results from a patient with a suspected Vitamin D deficiency.

Patient

Normal Range

Serum calcium, mg/dL

5.1

8.7-10.1

Serum phosphorous, mg/dL

4.2

2.4-4.3

Serum 1, 25-dihydroxycholecalciferol, pg/mL

13

20-76

Serum 25-hydroxycholecalciferol, ng/mL

48

10-55

A simplified scheme of Vitamin D metabolism is shown in Figure 29.1. The chemical name of active Vitamin D is 1,25-dihydroxycholecalciferol, and it is synthesized via the pathway shown. Catalysts, both in the form of enzymes and ultraviolet light, are required for Vitamin D synthesis. The two main sources of active Vitamin D are diet and sunlight. Food supplemented with “Vitamin D” usually contains cholecalciferol (Vitamin D3), or possibly a biologically equivalent analog. In the liver, dietary cholecalciferol is converted to 25-hydroxycholecalciferol. Next, in the kidney, the 25-hydroxycholecalciferol is converted to the active Vitamin D. Sunlight is also responsible for producing Vitamin D3. The skin contains a precursor, 7-dehydrocholesterol. In the presence of ultraviolet light, which acts as a catalyst, a ring-opening reaction occurs which is followed by the spontaneous conversion of this intermediate to Vitamin D3. Vitamin D3 is then converted to active Vitamin D via the pathway just described.

Active Vitamin D is a steroid-like compound that acts in combination with other hormones to increase the concentration of Ca2+ via a variety of mechanisms, one of which includes increasing the intestinal absorption of dietary calcium (intestinal absorption of dietary phosphate, a calcium counter- ion, also increases as a result). Calcium ions are required to form hydroxyapatite, Ca5(PO4)3OH, the main mineral constituent of bone.

Questions: (Please answer questions 2 & 4)

1) After obtaining the results from the laboratory, you suspect that your patient might have a defective enzyme in the Vitamin D synthetic pathway. Which enzyme do you think is defective, and why?

2) Next, you and your colleagues attempt to isolate the gene coding for the defective enzyme. The gene sequence is shown in Table 29.2. You compare the sequence of the gene from your patient with three other patients you have had with the same symptoms. What is the amino acid change in the enzyme from your patient? What amino acid changes are associated with the enzymes from the other three patients?

Patient

Base Pair Location

Mutation

Justin

320

GA

Patient A

374

GA

Patient B

1004

GC

Patient C

1144

CT

3) Once you have isolated the mutated gene from your patient, you wish to demonstrate that the gene does in fact code for a nonfunctional protein. You introduce the cloned gene into an expression vector, and these cells produce the protein of interest. Design an experiment in which you test the enzymatic activity of your gene product. Assume that you have cultured cells expressing the cloned mutated gene that you can use for this assay. You also have a culture of control cells. Describe the expected results.

4) Explain why you think that the amino acid changes listed in Question 2 would lead to non-functional enzymes.

Figure 29.1: Vitamin D metabolism (above)

Table 29.2: The nucleotide sequence of the normal enzyme in pseudovitamin D deficiency. (below)

Reference
Kitanaka, S., Takeyam, K, Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi,
H., Yanagisawa, J., Tanaka, T., and Kato, S. (1997) N. Eng. Jour. Med., 338, pp. 653-661.

Patient

Normal Range

Serum calcium, mg/dL

5.1

8.7-10.1

Serum phosphorous, mg/dL

4.2

2.4-4.3

Serum 1, 25-dihydroxycholecalciferol, pg/mL

13

20-76

Serum 25-hydroxycholecalciferol, ng/mL

48

10-55

HO HO HOT CH CH 7-dehydrocholesterol uv radlatlon skin) CH CH CHs spontaneous CH CH CH3 CH3 5-hydroxylase CH CHS cholecalciferol IVitamin D3) HOT 1 hydroxylase kidney) CH CH. Ca intestine) (blood) Cas(PO, (bone) CHs CHs OH 25-hydroxy- choiecalcMerol CH CH 3 OH 1a, .25 dlhydrox- cholecalciferol (Active Vitamin D

Explanation / Answer

Que 2

In this case Arginine is replaced by Histidine.

Que 4

If any single nucleotide is replaced in codon region that may alter the amino acid coded for it. Moreover, this amino acid is constituent of an active site of an enzyme, the product enzyme shall be able to conduct its function.

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