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Modern Genetic Analysis Chapter 15. Regulation of Cell Number: Normal and Cancer Cells
Solved Problem

1.

In the inherited form of retinoblastoma, an affected child is heterozygous for an rb mutation, which is either passed on from a parent or has newly arisen in the sperm or the oocyte nucleus that gave rise to the child. The heterozygous RB / rb cells are nonmalignant, however. The RB allele of the heterozygote must also be knocked out in the developing retinal tissue to create a tumorous cell. Such a knockout can occur through an independent mutation of the RB allele or by mitotic crossing-over such that the original rb mutation would now be homozygous.
a. If retinoblastoma is passed on to other siblings as well, could we determine if the original mutation was derived from the mother or the father? How?
b. Could we determine if the rb mutation was maternally or paternally derived if it arose de novo in a germ cell of one parent?

See answer

Solution
a. If the trait is inherited, we can determine from which parent it came. The most straightforward approach is to identify DNA polymorphisms, such as restriction fragment length polymorphisms (RFLPs), that map within or near the RB gene. RB has the curious property of being inherited as an autosomal dominant, even though it is recessive on a cellular basis. Given the dominant pattern of inheritance, by finding DNA differences in the parental genomes that map near each of the four parental alleles, we should be able to determine which allele is passed on to all affected offspring. That allele is the mutant one.
b. If the trait arose de novo in the sperm or the oocyte, we can still possibly determine from which parent it came, but with considerable difficulty. One way to do so would be to use recombinant DNA cloning techniques to isolate each of the two copies of the RB gene from normal cells. Only one of these alleles should be mutant. When the gene has been cloned, by DNA sequencing of the two alleles we may be able to identify the mutation that inactivates RB. If it arose de novo in the sperm or the oocyte, this mutation would not be present in the somatic cells of the parents. If sequencing also reveals some polymorphisms (for example, in restriction-enzyme recognition sites) that distinguish the alleles, we should then be able to go back to the parents' DNA and find out if the mother or the father carries the polymorphisms in the cloned mutant allele. (Whether this approach will work depends on the exact nature of the parental alleles and the mutation.)

If the mutation arose by mitotic crossing-over, we have additional tools available to us. In this case, the entire region around the rb gene will be homozygous for the mutant chromosome. By examining DNA polymorphisms known to map in this region, we should be able to determine if this chromosome derived from the mother or the father. This becomes a standard exercise in DNA fingerprinting similar to that described in part a.

See question

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