Retinoblastoma (Rb) plays a significant role among tumors. It is the first neoplasm with the hereditary etiology that has been demonstrated. It helped in developing the "two-hits” hypothesis and the suppressor gene idea (4, 5, 15, 17).

The incidence of Rb is 1 in 25,000 of live-born infants. Despite its rarity, Rb is the most common intraocular neoplasm in children. In the majority of cases it is diagnosed before the age of 5 years. Rb scarcely occurs in adults. 60% of cases of Rb show a true sporadic character. These are the results of somatic mutations occurring in the retinal cells. The remaining 40% of patients are children with constitutive mutations. Among them 10-15% are familial cases and the remaining 25-30% are new in the family as the result of a de novo germinal constitutive mutation. The penetrance of the constitutive mutation reaches 90%. For the onset of neoplasm the inactivation of protein pRB is needed. It occurs after the function of both alleles has been lost or inactivated (17).

The probability of the appearance of two consecutive somatic mutations in the RB1gene in the same retina cell, as it happens in the sporadic form of Rb, is directly proportional to the duration of cell life. It explains the late onset and usually unilateral and unifocal development of such forms of Rb in comparison with Rb caused by constitutive mutation (fig. 1). According to the "two-hits” hypothesis the development of the tumors in patients with a constitutive mutation requires only a next simple somatic mutation in retina cell. This is more likely to happen earlier, compare to two accidental sporadic mutation, thus the average age of patients with hereditary form of Rb is lower. Furthermore, the second somatic mutation in a carrier of the constitutive mutation may appear in a greater number of cells, hence the multifocal and bilateral development of such tumors is common (4, 17).

Bilateral and unilateral multifocal cases of Rb are usually associated with the presence of a constitutive mutation which is either transmitted from ancestors or acquired de novo. In the majority of cases, they appear before the age of 3 years (fig. 2, 3). Constitutive mutations may also arise de novo in germ cells (fig. 2). However, tumors associated with such mutations have a sporadic family pedigree, they show different symptoms and an earlier onset, which make them more similar to familial cases. Certain patients, (20% in our own studies) despite the unilaterality of the Rb at the moment of diagnosis, carry a constitutive mutation. Compared to other unilateral cases of Rb, tumors in such patients occur earlier and are multifocal. What more, those patients are at a high risk of a new primary tumor growth in the same or other eye (10, 13, 14, 16, 17).

The RB1gene is of medium size (200 kb). It is located in the 13q14 chromosome region, and comprises a promoter and 27 exons. There is a single RNA transcript of 4.8 kb size known. Therefore, the alternative RNA splicing of the RB1gene presumably does not take place.

The RB1gene encodes a p105RB1 protein, which belongs to the pocket protein family and is a nuclear phosphoprotein of 105-110 kDa size. It participates in complex regulatory pathways, which control the pass from G1 into S phase in the cell cycle. Therefore, it is one of the key proliferation regulators. This process concerns all cells in the organism and the gene shows a high evolutionary conservation. The protein affects the functions of many other genes by inactivation (sequestration) of one of the main transcription regulators – E2F factor. This is performed by E2F binding to the pRB1 pocket domain. It disables the transactivation of dependent genes by E2F. The result is that cells remain in the G0 phase. Only the pRB1 protein with the hypophosphorylation on its serine residues has the ability for binding E2F; the hyperphosphorylation of the protein releases the transcription factor and enables the passing from G1 phase to S phase and the progression of the cell cycle. Some proteins of oncogenic viruses such as the E7 protein of the papilloma virus, the E1A protein of adenovirus, T-antigen of the SV40 virus and others bind competitively to the pocket domain of the pRB1 protein. This phenomenon allows for associating the virus ontogenesis processes with the appearance of genetically determined neoplasms. The pocket domain of pRB1 protein may bind many other proteins containing the -Leu-x-Cys-x-Glu- chain (4, 6, 7, 15, 26). Sequence mutations found in patients with Rb diagnosed are predominantly located in the protein pocket domain and its vicinity. They can affect the affinity for transcription factors. When such a blocking is not complete, mutations are of a low penetrance (see below).

The universal regulatory character of the pRB1 protein can explain its mutation involvement in arising from many others tumors, such as osteosarcoma, bladder cancer, small cell lung cancer etc., co-occurring in families with retinoblastoma history (1).

The activity of the RB1gene is regulated by the promoter and the associated transcription regulators. Few known promoter mutations downgrade this regulation and show clinical features of low penetrance (see below). One of them and the first de novo recognized mutation was identified in author's center (28, 29). Regular cell functioning requires the presence of only one normal allele. Constitutive mutation carriers, apart from having a predisposition to develop Rb, do not differ from healthy individuals having two normal alleles.

The peak incidence of retinoblastoma is at the age of about 42 months. More than 90% of cases are diagnosed before the age of 5 years. There are known cases where the tumor was diagnosed immediately after birth. Early symptoms indicating Rb are squinting, red eye and inflammation of the eyeball. However, the tumor is usually diagnosed by symptoms appearing later on. They are exophthalmos and a "cat's eye” noticed by parents – white pupil, which comes from the caseous surface of the tumor itself. Treatment at an early stage frequently allows preservation of the affected eyeball even with the vision field worsened. The diagnosis at a later stage, unfortunately the most common situation, when the "cat's eye” sign is present, usually requires the removal of the eyeball. It is often associated with radiotherapy and chemotherapy (6, 17).

Metastases of retinoblastoma are rare. The tumor spreads usually per continuitatem, mainly into adjacent tissues via the optic nerve. The prognosis depends mainly on the quality of diagnostics and health care. In developed countries with good medical care standards the mortality rate hardly exceeds 8% of the cases, and eyeball removal is necessary in about 10% of cases. In developing countries the mortality rate can reach 100%.

Currently RB1gene mutations have been noted in databases in ~1000 patients with constitutive retinoblastoma (8, 14, 25). They are classified with all generally known mutation categories: different types of point mutations, translocations, insertions, and deletions. Recently a relatively great occurrence of deletions among all mutations has been found. In previous years they were neglected. Particular classes of RB1gene alterations are point mutations in exonic and intronic sequences, mutations generating aberrant splicing, point mutations of promoter, and modifications of their methylation status (epigenetic changes) without alterations of sequence per se.

The majority of hereditary mutations, particularly in bilateral cases, are nonsenses and frameshifts. They generate premature stop codons and are localized in exons 1-25. No mutation has been identified until now in the last two exons 26 and 27. Although, they are potentially mutation prone sites due to two potential hot spots, CGA.

Hereditary predisposition to retinoblastoma is associated with the appearance of constitutive mutations of the RB1gene. There is a full service molecular RB1gene testing facility available in the author's institution (9, 27, 31). Gene sequence analysis is very expensive. However, it has been demonstrated that the expenses are lower after non-carriers have been excluded from the tests than the costs of extended prophylactic care for all children in the susceptible family (19). Full RB1gene sequencing identifies the mutation in about 80% of families, in which Rb is apparently hereditary. There are some preselective strategies (e.g. SSC, DGGE, PTT) that make this process easier to some extent but their sensitivity is lower than that of sequencing. Identification of the mutation in an affected individual allows for focusing the analysis on only the selected gene fragment in other members of the family (6, 8). Linkage analysis with intra-gene molecular probes can also be used especially in the diagnosis of numerous families (6, 12, 13, 29). Not only a classic PCR technique (exon-by-exon sequencing) but also multiplex PCR and quantitative multiplex PCR techniques (allowing simultaneous analysis of a number of exons), and new biochemical techniques for the detection of results of a mutation (e.g. HPLC) were introduced (21, 25, 30, 31).

Constitutive DNA extracted from a peripheral blood sample is analysed. Such samples contain encoding fragments (exons), as well as non-coding sections (introns) of the gene. The use of cDNA obtained in vitro from RNA and containing only the encoding sections of DNA improves the assessment technique and the following interpretation (8, 9). The diagnosis of the tumor DNA is not essential to identify the constitutive mutation. Nevertheless, it can verify the procedure and provide with unique information on somatic mutations ("second hit” and sporadic) (12, 13).

The findings of the mutations observed are stored in two free online databases. The Lohmann's Database holds fewer mutations and is rarely updated, but allows for simple discerning of constitutive mutations from somatic "second hits”. There is more new data (932 mutations) in the Valverde et al's Database, but the differentiation is not so simple. However, the analysis of the latter database has revolutionized the knowledge of constitutive mutations in the RB1gene (14, 25).

Until recently, it was considered that the mutations have random localization (8). It has been proven though that there are preferential sequences in the RB1gene where mutations accumulate. They correspond to the active sites of the pRB1 protein and especially to the pocket domain. 79% of the repetitive mutations are transitions C/T which appear in 11 triplets of the arginine codons (CGA); 40% of those are located in exons 8, 10, 11, 14, 15, 17, 18, and 23 (13, 25). These findings significantly change diagnostic sequencing technique, time and costs.

As expected, the immense majority of the mutations make the synthesis of the protein impossible or result in an inactive product. These are basically stop mutations (42% of cases), but also some types of deletion and splicing site mutations occur.

In contrast to the mutations that inactivate the product, the mutations that modify the activity of the molecule, yet not stopping the synthesis of the pRB protein completely, are randomly distributed.

This group consists of nonsense mutations and some of splicing site mutations. Stop codon producing mutations prevail. There is lower penetrance (60-70%) of promoter mutations and some splicing sites mutations (9, 12, 23, 28). Mutations skipping a generation are observed (obligatory carriers remain healthy) in susceptible families. Clinical presentation of the tumor is similar to that observed in Rb development as a result of somatic mutations (8, 22). The assessment of the risk of developing a tumor in such families may be difficult. However, mutations of low penetrance probably form only a small part of all mutations observed in the RB1gene (16, 18, 21, 22, 23, 24).