both eyes, while in some rarer cases retinoblastoma would develop later in life and only be unilateral. This unique development pattern allowed Knudson and several other scientific groups in 1971 to correctly hypothesize that the early development of retinoblastoma was caused by inheritance of one loss of function mutation to an RB germ-line gene followed by a later de novo mutation on its functional Rb gene allele. The more sporadic occurrence of unilateral development of retinoblastoma was hypothesized to develop much later in life due to two de novo mutations that were needed to fully lose tumor suppressor properties. This finding formed the basis of the two-hit hypothesis. In order to verify that the loss of function of tumor suppressor genes causes increased tumorigenicity, interstitial deletion experiments on chromosome 13q14 were conducted to observe the effect of deleting the loci for the Rb gene. This deletion caused increased tumor growth in retinoblastoma, suggesting that loss or inactivation of a tumor suppressor gene can increase tumorigenicity. Two-hit hypothesis Unlike oncogenes, tumor suppressor genes generally follow the two-hit hypothesis, which states both alleles that code for a particular protein must be affected before an effect is manifested. If only one allele for the gene is damaged, the other can still produce enough of the correct protein to retain the appropriate function. In other words, mutant tumor suppressor alleles are usually recessive, whereas mutant oncogene alleles are typically dominant. Proposed by A.G. Knudson for cases of retinoblastoma. He observed that 40% of U.S cases were caused by a mutation in the germ-line. However, affected parents could have children without the disease, but the unaffected children became parents of children with retinoblastoma. This indicates that one could inherit a mutated germ-line but not display the disease. Knudson observed that the age of onset of retinoblastoma followed 2nd order kinetics, implying that two independent genetic events were necessary. He recognized that this was consistent with a recessive mutation involving a single gene, but requiring bi-allelic mutation. Hereditary cases involve an inherited mutation and a single mutation in the normal allele. Non-hereditary retinoblastoma involves two mutations, one on each allele. Knudson also noted that hereditary cases often developed bilateral tumors and would develop them earlier in life, compared to non-hereditary cases where individuals were only affected by a single tumor. There are exceptions to the two-hit rule for tumor suppressors, such as certain mutations in the