Case 29
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 RangeSerum calcium, mg/dL / 5.1 / 8.7-10.1
Serum phosphorous, mg/dL / 4.2 / 2.4-4.3
Serum 1α, 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 1α,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
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 / MutationJustin / 319 / G → A
Patient A / 374 / G → A
Patient B / 1004 / G → C
Patient C / 1144 / C → T
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 4 would lead to non-functional enzymes.
Figure 29.1: Vitamin D metabolism.
Table 29.2: The nucleotide sequence of the defective enzyme in pseudovitamin D deficiency.
ATGACCCAGA CCCTCAAGTA CGCCTCCAGA GTGTTCCATC GCGTCCGCTG GGCGCCCGAG 60TTGGGCGCCT CCCTAGGCTA CCGAGAGTAC CACTCAGCAC GCCGGAGCTT GGCAGACATC 120
CCAGGCCCCT CTACGCCCAG CTTTCTGGCC GAACTTTTCT GCAAGGGGGG GCTGTCGAGG 180
CTACACGAGC TGCAGGTGCA GGGCGCCGCG CACTTCGGGC CGGTGTGGCT AGCCAGCTTT 240
GGGACAGTGC GCACCGTGTA CGTGGCTGCC CCTGCACTCG TCGAGGAGCT GCTGCGACAG 300
GAGGGACCCC GGCCCGAGCG CTGCAGCTTC TCGCCCTGGA CGGAGCACCG CCGCTGCCGC 360
CAGCGGGCTT GCGGACTGCT CACTGCTGAA GGCGAAGAAT GGCAAAGGCT CCGCAGTCTC 420
CTGGCCCCGC TCCTCCTCCG GCCTCAAGCG GCCGCCCGCT ACGCCGGAAC CCTGAACAAC 480
GTAGTCTGCG ACCTTGTGCG GCGTCTGAGG CGCCAGCGGG GACGTGGCAC GGGGCCGCCC 540
GCCCTGGTTC GGGACGTGGC GGGGGAATTT TACAAGTTCG GACTGGAAGG CATCGCCGCG 600
GTTCTGCTCG GCTCGCGCTT GGGCTGCCTG GAGGCTCAAG TGCCACCCGA CACGGAGACC 660
TTCATCCGCG CTGTGGGCTC GGTGTTTGTG TCCACGCTGT TGACCATGGC GATGCCCCAC 720
TGGCTGCGCC ACCTTGTGCC TGGGCCCTGG GGCCGCCTCT GCCGAGACTG GGACCAGATG 780
TTTGCATTTG CTCAGAGGCA CGTGGAGCGG CGAGAGGCAG AGGCAGCCAT GAGGAACGGA 840
GGACAGCCCG AGAAGGACCT GGAGTCTGGG GCGCACCTGA CCCACTTCCT GTTCCGGGAA 900
GAGTTGCCTG CCCAGTCCAT CCTGGGAAAT GTGACAGAGT TGCTATTGGC GGGAGTGGAC 960
ACGGTGTCCA ACACGCTCTC TTGGGCTCTG TATGAGCTCT CCCGGCACCC CGAAGTCCAG 1020
ACAGCACTCC ACTCAGAGAT CACAGCTGCC CTGAGCCCTG GCTCCAGTGC CTACCCCTCA 1080
GCCACTGTTC TGTCCCAGCT GCCCCTGCTG AAGGCGGTGG TCAAGGAAGT GCTAAGACTG 1140
TACCCTGTGG TACCTGGAAA TTCTCGTGTC CCAGACAAAG ACATTCATGT GGGTGACTAT 1200
ATTATCCCCA AAAATACGCT GGTCACTCTG TGTCACTATG CCACTTCAAG GGACCCTGCC 1260
CAGTTCCCAG AGCCAAATTC TTTTCGTCCA GCTCGCTGGC TGGGGGAGGG TCCCACCCCC 1320
CACCCATTTG CATCTCTTCC CTTTGGCTTT GGCAAGCGCA GCTGTATGGG GAGACGCCTG 1380
GCAGAGCTTG AATTGCAAAT GGCTTTGGCC CAGATCCTAA CACATTTTGA GGTGCAGCCT 1440
GAGCCAGGTG CGGCCCCAGT TAGACCCAAG ACCCGGACTG TCCTGGTACC TGAAAGGAGC 1500
ATCAACCTAC AGTTTTTGGA CAGATAG 1527
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.
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