Clinical Genetics

1. Clinical Genetics Lecture 4

2. What drives cancer cells to grow and divide uncontrollably and to escape cell death? • Tumor suppressor genes often function to restrain inappropriate cell growth and division, • Also stimulate cell death to keep our cells in proper balance. • Some of these genes are involved in DNA repair processes, which help prevent the accumulation of mutations in cancer-related genes. • In this way, tumor suppressor genes act as "brakes" to stop cells in their tracks before they can take the road to cancer. • Loss of tumor suppressor gene function can be disastrous, and it often puts once-normal cells on the fast track to disease.

3. The Two-Hit Hypothesis: Knudson and RB1 • Like all genes, tumor suppressor genes may undergo a variety of mutations • Mutations that occur in tumor suppressor genes are recessive in nature. T • Thus, in order for a particular cell to become cancerous, both of the cell's tumor suppressor genes must be mutated known as the "two-hit" hypothesis • it was first proposed by geneticist Alfred Knudson in 1971. • Today, this hypothesis serves as the basis for researchers' understanding of how mutations in tumor suppressor genes drive cancer.

4. Retinoblastoma • The two-hit hypothesis arose of out Knudson's interest in the genetic mechanisms underlying retinoblastoma. • Under normal circumstances, a population of cells in the developing eye (retinoblasts) stops growing and dividing during embryogenesis and differentiates into retinal photoreceptor (light-capturing) cells and nerve cells. • Typically, these differentiated cells do not divide very often, if ever. • In the case of retinoblastoma, however, the retinoblasts fail to differentiate; thus, these cells continue to divide, forming tumors in the retina. If left untreated, the retinal tumor cells will eventually metastasize (spread) to other parts of the body.

5. Knudson study on retinoblastoma • Between 1944 and 1969, Knudson studied 48 patients with retinoblastoma who had been admitted to the hospital (Knudson, 1971). • For each patient, Knudson tabulated the age at diagnosis, sex, family history, whether the tumor occurred in one eye (unilaterally) or in both eyes (bilaterally), and an estimation of the number of tumors present in each eye. • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC389051/pdf/pnas00079-0129.pdf

6. At the time of Knudson's study, researchers believed that retinoblastoma could be caused by either somatic or germ-line mutations. • Knudson's combined data itself showed that retinoblastoma was caused by a germ-line mutation in approximately 40% of U.S. cases. • However, he was puzzled by the observation that some children with an affected parent were disease-free, but these unaffected individuals later bore children with retinoblastoma; this finding suggested that an individual could inherit a germ-line mutation but not have the disease. • He also noted that while the majority of children with an affected parent had bilateral tumors (25%–30%), some had only unilateral tumors (10%–15%). • Furthermore, he determined that approximately 60% of retinoblastoma cases in the U.S. were unilateral and were not associated with a family history of the disease.

7. These semilog plots of the fraction of 23 bilateral (heritable) cases and 25 unilateral (most expected to be non-heritable) cases that were still not diagnosed at plotted ages (data were analyzed retrospectively) show that the bilateral cases match the expected shape of a one-hit curve, whereas the unilateral cases match the shape of a two-hit curve. As the bilateral cases inherit one genetic hit, both heritable and spontaneous retinoblastoma are due to two hits.

8. Knudson knew that if retinoblastoma were caused by a recessive mutation in a single gene, both copies of the gene would need to be mutated in order for retinoblastoma to occur. • and the mutation rate would be the same for each allele of the gene. • Therefore, individuals who inherited a mutation in one allele of the gene would only need to accumulate a single mutation in the remaining normal allele of any retinoblast in order for cancer to occur. • However, without an inherited mutation, the same cell would need to accumulate two mutations—one in each allele of the gene—and this process would be much slower.

9. Based on calculations using this clinical data, Knudson concluded that retinoblastoma was caused by two mutations: • one in each copy of a single tumor suppressor gene (now called RB1). • He also estimated that each of the two mutations would occur at a rate of 2 x 10-7 per year. • Patients who inherited an RB1 mutation would develop tumors earlier, and they would often develop more than one tumor. • In contrast, individuals who did not inherit a mutation would almost always be affected by a single tumor. • This statement, which Knudson called the two-mutation hypothesis, is now known as the two-hit hypothesis (Knudson, 1971).

10. Two-hit tumor formation in both hereditary and nonhereditary retinoblastoma

11. Based on the predicted mutation rate, Knudson expected that many individuals in the general population would acquire a single somatic mutation in theRB1 gene over their lifetime, and that the retinas of most people would therefore likely contain small groups of retinoblasts that had received one "hit" in the RB1 gene. • In order to become cancerous, each retinoblast with one mutant copy of the RB1 gene would need to acquire a mutation in the remainingwild-type copy of the gene. • Most individuals who had one hit did not develop retinoblastoma, however, because most of their mutated cells had already differentiated and quit dividing before they could receive a second hit (Knudson, 2001).

12. Loss of Heterozygosity and Mechanisms of Tumor Suppressor Gene Inactivation • "Loss of heterozygosity" describes the process that leads to the inactivation of the second copy of a tumor suppressor gene. • During this process, a heterozygous cell receives a second hit in its remaining functional copy of the tumor suppressor gene, thereby becoming homozygous for the mutated gene. • Mutations that inactivate tumor suppressor genes, called loss-of-function mutations, are often point mutations or small deletions that disrupt the function of the protein that is encoded by the gene; chromosomal deletions or breaks that delete the tumor suppressor gene; or instances of somatic recombination during which the normal gene copy is replaced with a mutant copy.

13. Mechanisms of RB inactivation • Retinoblastoma (RB) contains multiple protein binding sites and functions as a molecular scaffold to promote the assembly of transcription complexes. • Disassembly of these complexes is mediated by RB inactivation by means of four known mechanisms. • The RB gene is mutated (dashed line), causing release of its associated factors. • RB mutations have been detected in retinoblastoma and a small fraction of sporadic tumors. • RB is sequestered by viral oncoproteins, such as E1A, which prevent it from binding other factors. Phosphorylation (P) of RB by CDK-cyclin complexes during cell-cycle progression disrupts its ability to assemble transcriptional complexes. • RB is degraded by a caspase-dependent proteolytic pathway during apoptosis. Except for RB phosphorylation, the other three mechanisms of RB inactivation have been associated with sensitization to apoptosis.

14. Mechanisms of RB inactivation