Influence of Growth Factors on Cell Division

Development, differentiation, and the maintenance of vital functions require exact regulation of the time and location of cell divisions. Rapidly multiplying cells in embryonic tissues must be controlled, just like those in stationary phases in adult tissues. Rapid response to injury or to foreign antigens requires controlled cell division. Multicellular organisms have an extensive repertoire of genetically regulated mechanisms at their disposal for controlling cell division and tissue proliferation. As a group, they are referred to as growth factors. Every growth factor has a specific cell surface receptor. Binding to the receptor initiates (or in some cases blocks) cell division. Most growth factors regulate only certain types of cells and tissues.

• Control of cell division by growth factors
  
  Basically, cell division (mitosis) can be controlled by stimulation or inhibition. In the absence of stimulation or with active inhibition, no mitosis occurs. Growth factors have an effect not only on specific types of cells, but also on defined phases of the cell cycle. The most frequently controlled phase of the cell cycle is the transition from G0 to G1. The growth factor group includes growth factors for epidermal cells (EGF), for nerve cells (NGF), for connective tissue or mesenchymal cells (fibroblasts, FGF), and for thrombus-forming cells in the inner lining (endothelium) of blood vessels (PDGF). Their stimulating effect may be opposed by an antagonistic effect (e.g., TGF!, transforming growth factor, or TNF, tumor necrosis factor). The function of each growth factor is mediated by a specific receptor.
  
• Activation of a growth factor receptor
  
  A growth factor receptor becomes activated by specific extracellular binding to the growth factor. The activated receptor in turn activates a substrate protein.
  
• PDGF-receptor kinases have an effect on numerous substrates
  
  A receptor such as the PDGF (platlet-derived growth factor) receptor can have an effect on numerous substrates. Substrates of the PDGF receptors include the Ras protein (see D), the Src protein (the name is derived fromthe tumor, a sarcoma, in which it was first found), phospholipase C (a signal transmitter), and others.
  
• Ras proteins as signal transmitters
  
  The Ras proteins play a central role as signal transmitters. They belong to the group of G proteins (guanosine-residue-binding proteins with signal-transmitting functions, see p. 266). The binding of growth factor, e.g., PDGF, activates the Ras protein by stimulating the conversion of associated GDP (guanosine diphosphate) to GTP (guanosine triphosphate) and triggering a short time-limited signal that initiates cell division. The signal is terminated by inactivation of Ras by a GTPase-activating protein (GAP), which converts GTP into GDP.Mutation of the Ras protein or of GAP can remove the time limit of the cell-stimulating signals and result in an active condition with uncontrolled cell division. This can lead to a tumor with uncontrolled growth (malignancy). Several mutations have been defined in the pertinent genes. (Diagrams adapted fromWatson et al., 1992.)
  
• References
  
  Lengauer, C., Kinzler, K.W., Vogelstein, B.:
  Genetic instabilities in human cancers. Nature
  396:643–649, 1998.
  Park, M.: Oncogenes: Genetic abnormalities of
  cell growth, pp. 589–611. In: C.R. Scriver, et
  al., eds., The Metabolic and Molecular Bases
  of Inherited Disease. 7th ed. McGraw-Hill,
  New York, 1995.
  Watson, J.D., et al.: Recombinant DNA. 2nd ed.
  Scientific American Books, W.H. Freeman,
  New York, 1992.

• Tumor suppressor gene
  
  In contrast to the cellular oncogenes, for which a change in one allele will alter normal function, both alleles of a tumor suppressor gene must lose their function before a tumor develops. The first event is usually a mutation by base exchange or deletion. The second event, affecting the other allele (allele 2), may also be a mutation, but the loss of function more often appears to be from loss of the chromosome after a faulty cell division (mitotic nondisjunction) or other mechanisms (e.g., mitotic recombination with gene conversion).
  
• Loss of heterozygosity in tumor cells
  
  Usually, in about half of the individuals who are heterozygous for DNA markers at the tumor suppressor gene locus of interest, the loss of one allele (event 2) can be demonstrated by Southern blot analysis. In contrast to normal somatic cells (blood), tumor cells contain only one allele (loss of heterozygosity, LOH). The remaining allele carries the mutation. Thus, the mutant allele can be identified by demonstration of LOH. LOH is useful in diagnosis as an indication of the existence of a tumor suppressor gene.
  
• Somatic and germinal mutation
  
  The first mutation in a suppressor gene can either be present in the zygote (germinal mutation, i.e., germ cell mutation due to transmission from an affected parent or due to new mutation) or occur in a single cell of the corresponding tissue (somatic mutation). Loss of function of one allele (corresponding to event 1 in A) predisposes the cell to tumor development. With a germinal mutation, all cells are predisposed. The tumor arises after loss of function of the second allele. When somatic mutation occurs in a single cell, loss of function of both alleles rarely affects the same cell. But with a germ cells mutation, loss of function of the second allele is frequent, since all cells carry the first mutation, i.e., are predisposed. With somatic mutation, the tumor occurs sporadically (is not hereditary) and arises unifocally from a single cell. In the hereditary form resulting from a germ cell mutation, several tumors may arise from different cells (multifocal tumor). The predisposition for the tumor in the hereditary form shows autosomal dominant inheritance.
  
• Examples of tumor suppressor genes
  
  Numerous types of tumors arise due to loss of function in both alleles of a tumor suppressor gene. Loss of heterozygosity (LOH) can be demonstrated in the tumor cells in about half of these patients.
  
  
• References
  
  Skuse, G.R., Ludlow, J.W.: Tumour suppressor
  genes in disease and therapy. Lancet
  345:902–906, 1995.
  Stanbridge, E.J.: Human tumor suppressor
  genes. Ann Rev Genet. 24:615–657, 1990.
  Weinberg, R.A.: Tumor suppressor genes.
  Science 254:1138–1146, 1991.
  

• Cellular and viral oncogenes
  
  A typical retrovirus contains an RNA genome that codes for three genes or groups of genes: gag (group-specific antigen), pol (polymerase), and env (coat protein, envelope). As with all genes of higher organisms, a cellular oncogene (c-onc) consists of exons and introns with defined structure and sequence, as in the gene src (the name is derived from sarcoma, a tumor that is induced by a change in this gene). The virus may contain parts of the cellular oncogene (c-scr). This is designated viral oncogene (v-src) (Rous sarcoma virus). In chickens, it induces a malignant tumor (a sarcoma), first observed by Peyton Rous in 1911. Since many cellular oncogenes are also known in an altered, viral form, it is assumed that the viruses have integrated parts of the respective cellular oncogenes into their own genomes. Virus-induced tumors are known especially in chickens, rodents, and cats. In man, they do not play a general role in the induction of tumors. Important exceptions are papilloma virus-induced carcinoma of the cervix and carcinoma of the liver secondary to hepatitis virus infection.
  
• Mechanisms of oncogene activation
  
  A cellular oncogene controls cell division. It controls the time and location of the orderly proliferation of cells and tissues (normal growth). Genetic changes can lead to disorders of the regulation of cell divison, increased proliferation of cells, and formation of a tumor. This can be traced back to relatively few mechanisms. A point mutation in a critical region of the gene can lead to disturbances in the regulation of cell division. Examples are mutations in codon 12 or 63 or the H-ras gene. An inactive cellular oncogene may become activated when it is moved by chromosomal translocation to the vicinity of an active gene. In Burkitt lymphoma, an inactive gene is moved to the region of an active gene for the H or L chain of an immunoglobulin. In other cases, the breakpoint of the chromosome translocation may lie within a cellular oncogene and thereby affect its expression. An example is the Philadelphia translocation (see p. 332). Multiplication (amplification) of a gene is a futher mechanism that can lead to altered (usually increased) expression.
  
  
• Examples of cellular oncogenes and their proteins
  
  The table shows examples of the about 60 known cellular oncogenes, their basic functions, a fewtumors induced inman by mutation of the cellular oncogene (c-onc), and tumors induced in vertebrates by the homologous viral onogene (v-onc). (Data from Lodish et al., 2000; Cannon-Albright et al., 1992.)
  
• References
  
  Lodish, H., et al.: Molecular Cell Biology. 4th ed.
  2000.
  Cannon-Albright, L.A., et al.: Assignment of a locusfor
  familial melanoma, MLM, to chromosome
  9p13-p22. Science 258:1148, 1992.
  Levine, A.J., Broach, J.R., eds.: Oncogenes and
  cell proliferation. Current Opin Genet and
  Development 5:1–150, 1995.
  Park, M.: Oncogenes, p. 205—228, In: Vogelstein,
  B., Kinzler, K.W., eds., The Genetic
  Basis of Human Cancer. McGraw-Hill, New
  York, 1998.
  Park, M.: Oncogenes, p. 589–611. In: Scriver,
  C.R. et al., eds.: TheMetabolic and Molecular
  Bases of Inherited Disease. 7th ed.McGraw-
  Hill, New York, 1995.

• The human p53 protein
  
  The active form of the human p53 protein is a tetramer of four identical subunits. Each subunit has 393 amino acids and five highly conserved regions, I–V. Region I is part of the transcription- activation domain; regions II–V belong to sequence-specific DNA-binding domains. The carboxyl end beyond amino acid 300 consists of a nonspecific DNA interaction domain and the tetramerization domain. Proteins encoded by DNA tumor viruses bind to p53 and inhibit its activity.Mutations in the p53 gene on human chromosome 17 at p13 (spanning 20 kb of DNA and yielding a 2.8 kb mRNA transcript from 11 exons) have the greatest effect when they occur in the conserved regions II–V in codons 129–146 (exon 4), 171–179 (exon 5), 234–260 (exon 7), and 270–287 (exon 8). Particularly vulnerable are the conserved amino acids arginine (R) in positions 175, 248, 249, 273, and 282 and glycine (G) in position 245. Mutations occur mainly as missense, resulting from base-pair substitutions, but some are insertions and deletions and exert a dominant negative effect. Knockout mice develop normally, but develop tumors at a high rate. Activated benzopyrene induces mutations at codons 175, 248, and 275 in cultured bronchial epithelial cells.
  
• Germline mutations of p53
  
  In 1969, Li and Fraumeni identified families in whom other members were affected with diverse types of tumors, mainly soft-tissue sarcomas, early-onset breast cancer, brain cancers, cancer of the bone (osteosarcoma) and bone marrow(leukemias), and carcinoma of the lung, pancreas, and adrenal cortex. Similar observations had been reported as “cancer family syndrome” by Lynch. This autosomal dominant cancer syndrome is called the Li–Fraumeni syndrome (McKusick 114480). In the pedigree shown in panel 1, four individuals (II-2, II-3, III- 1, III-2) are affected by different types of tumors. A mutation in codon 248 of the p53 gene (CGG arginine, to TGG, tryptophan) is present in these patients. The mutation is also present in individuals I-1 and III-5. This places these individuals at increased risk for one of the types of cancer mentioned above and shown in panel 2. In contrast, absence of the mutation in individuals III-3 and III-4 indicates that they do not have an increased risk of cancer (Data of D. Malkin). A subset of patients with Li–Fraumeni syndrome does not show p53 mutations.
  
• Model of function of the p53 gene
  
  Normally, the p53 gene is inactive (1). p53 plays an important role in regulating growth in damaged cells (2). DNA damage in cells leads to increased expression of p53 and interruption of the cell cycle in G1. If DNA repair is successful, the cell can continue its cycle. If repair is not successful, the cell dies (cell death, apoptosis). Damaged cells with p53 protein that is mutant are not arrested in G1. (After Lane, D.P.: Nature 358:15–15, 1992.)
  
• References
  
  Bell, D.W., et al.: Heterozygous germline hCHK2
  mutations in Li-Fraumeni syndrome.
  Science 286:2828–2831, 1999.
  Hanahan, D., Weinberg, R.A.: The hallmarks of
  cancer. Cell 100:57–70, 2000.
  Lodish, H., et al.: Molecular Cell Biology (with an
  animated CD-ROM). 4th ed.W.H. Freeman &
  Co., New York, 2000.
  Malkin, D.: The Li-Fraumeni syndrome, pp. 353–
  407. In: Vogelstein, B., Kinzler, K.W., eds.,
  The Genetic Basis of Human Cancer.
  McGraw-Hill, New York, 1998.

• The main signs of NF1
  
  NF1 is very variable. Lisch nodules of the iris (1) in more than 90% of patients, café-au-lait spots (2) (more than five spots of more than 2 cm diameter are considered diagnostic) in more than 95%, and multiple neurofibromas (3) in more than 90% of patients are the most important signs.
  
• Neurofibromatosis gene NF1 on human chromosome 17 at q11.2
  
  The localization of the NF1 gene revealed the gene on a 600 kb NruI restriction fragment. A CpG island (CpG-1) and two translocation breakpoints at t(17;22) and t(1;17) served as important anchor points for gene identification. The NF1 gene has 79 exons, which span about 335 kb of genomic DNA. Three unrelated genes, OMGP, EVI2B, and EVI2A, are embedded within the NF1 gene in intron 35 on the opposite DNA strand. Mutation analysis of the NF1 gene shows deletions, insertions, base substitutions, and splice mutations leading to truncated and presumably nonfunctional gene products. Currently mutations are found in about 60–70% of patients. (Figure adapted from Claudio and Rouleau, 1998).
  
• NF1 gene product (neurofibromin)
  
  The NF1 gene encodes a gene product with 2810 amino acids, called neurofibromin. Between amino acids 840 and 1200, this large protein contains a domain that corresponds to a GTPase-activating protein. The homology includes a gene product in yeast (S. cerevisiae), IRA1 (inhibitor of ras mutants). Mutations at the NF1 locus interrupt a signal pathway to the ras genes. (After Xu et al., 1990).
  
• Neurofibromatosis gene NF2 on human chromosome 22 at q12.1
  
  The NF2 genewas identified in 1993 by Rouleau et al. and Trofatter et al. within a cosmid contig contained in YAC clones (yeast artificial chromosomes). Two deletions observed in unrelated patients aided in finding the almost 100 kb gene with 17 exons. Mutations can be detected in more than 50% of patients (large deletions including the entire gene or several exons and small deletions are frequent). The gene product, called schwannomin, is related to a family of cytoskeleton-membrane proteins (erythrocyte protein 4.1, see p. 374, and the ERM family ezrin, radixin, and moesin) and a family of protein tyrosine phosphatases. The basic function of these proteins is to maintain cellular integrity. (Figure adapted from Claudio and Rouleau, 1998).
  
• References
  
  Carey, J.C., Viskochil, D.H.: Neurofibromatosis
  Type 1: a model condition for the study of
  the molecular basis of variable expressivity
  in human disorders. Am. J. Med. Genet.
  (Semin. Med. Genet.) 89:7–13, 1999.
  Claudio, J.O., Rouleau, G.A.: Neurofibromatosis
  type 1 and type 2, pp. 963–970. In: Principles
  of Molecular Medicine, J.L. Jameson,
  ed. Humana Press, Totowa, NJ, 1998.
  Huson, S.M.: What level of care for the neurofibromatoses?
  Lancet 353:1114–1116, 1999.
  Messiaen, L.M., et al.: Exhaustive mutation
  analysis of the NF1 gene allows identification
  of 95% of mutations and reveals a high
  frequency of unusual splicing defects. Hum.
  Mutat. 15:541–555, 2000.
  Riccardi, V.M., Eichner, J.E.: Neurofibromatosis.
  Phenotype, Natural History and Pathogenesis.
  2nd ed., Johns Hopkins University Press,
  Baltimore, 1992.
  Rouleau, G.A., et al.: Alteration in a new gene
  encoding a putative membrane-organizing
  protein causes neurofibromatosis type 2.
  Nature 363:515–521, 1993.
  Trofatter, J.A., et al.: A novel Moesin-, Ezrin-,
  Radixin-like gene is a candidate for the neurofibromatosis
  2 tumor suppressor. Cell
  72:791–800, 1993.
  Xu, G., et al.: The neurofibromatosis type 1 gene
  encodes a protein related to GAP. Cell
  62:599–608, 1990.

• Polyposis coli and colon carcinoma
  
  Familial polyposis (FAP) is an autosomal dominant hereditary disease. In late childhood and early adulthood, up to 1000 and more polyps develop in the mucous membrane of the large intestine (colon) (1). Each polyp can develop into a carcinoma (2). In about 85% of affected persons, small hypertrophic areas that do not affect vision are present in the retina (3). Hereditary non-polyposis colorectal cancer (HNPCC) affects about one in 200–1000 individuals (3% of all colorectal cancers). It results from germline mutation in one of the DNAmismatch repair genes hMSH1, hMLH2, hPMS1, and hPMS2 or related genes. Microsatellite instability is an important feature of HNPCC. (Photos 1 and 2 from U. Pfeifer, Institut für Pathologie der Universität Bonn; photo 3 from W. Friedl et al., 1991.)
  
• Mutations at different gene loci in polyposis coli and carcinoma of the colon
  
  At least six gene loci are involved in the development of carcinoma of the colon associated with polyposis coli. Somatic mutations may occur in two recessive oncogenes (Ras genes KRAS1 and KRAS2) and in four dominant tumor suppressor genes. Most forms of carcinoma of the colon are not associated with polyposis coli.
  
• The APC gene and distributions of mutations
  
  The APC gene (adenoma polyposis coli) consists of 8538 bp in 15 exons encoding a 2843-aminoacid protein (not 8535 bp and 2844 amino acids as shown in C). Exon 15 is very large, 6579 base pairs. Over 95% of mutations result in a nonfunctional truncated protein due to nonsense mutations (40%), deletions (41%), insertions (12%), and splice site mutations (7%). The APC gene is also involved in sporadic colorectal cancer.
  
• Indirect DNA diagnosis in FAP
  
  Linked DNAmarkers (RFLPs) near the APC locus (1) can be used for indirect DNA diagnosis. The alleles of three flanking marker pairs (K,k and E,e on the centromere side and A,a on the distal side) form the haplotypes, e.g., e–K–a and E–k–a in individual I-1 in the pedigree (2). The mutation-carrying haplotype must be e–K–a. Since individual III-2 has inherited this haplotype, he is at risk for the disease, whereas individual III-1 is not.
  
• Several mutations in the production of colon carcinoma
  
  Tumor formation goes through several stages. It starts with a somatic or germinal mutation in the APC gene. After loss of the other allele (LOH), an adenoma develops with less-differentiated cells and polyp formation. Mutations in other genes lead to malignant transformation and eventually to tumor development. (Diagram after Fearon and Vogelstein, 1990.)
  
• References
  
  Bronner, C.E., et al.: Mutation in the DNA mismatch
  repair gene homologue hMLH1 is associated
  with hereditary nonpolyposis
  colon cancer. Nature 368:258–261, 1994.
  de la Chapelle, A., Peltomäki, P.: The genetics of
  hereditary common cancers. Curr. Opin.
  Genet. Develop. 8:298–303, 1998.
  Fearon, E.R., Vogelstein, B.: A genetic model for
  colorectal tumorigenesis. Cell 61:759–767,
  1990.
  Fearon, E.R., Cho, K.R.: The molecular biology of
  cancer, pp. 405–438, In: D.L. Rimoin, J.M.
  Connor, R.E. Pyeritz, eds., Emery and
  Rimoin’s Principles and Practice of Medical
  Genetics. 3rd ed. Churchill-Livingstone,
  Edinburgh, 1996.
  Groden, J., et al.: Identification and characterization
  of the familial adenomatous polyposis
  coli gene. Cell 66:589–600, 1991.
  Kinzler, K.W., Vogelstein, B.: Colorectal tumors,
  pp. 565–587. In: B. Vogelstein, K.W. Kinzler,
  eds., The Genetic Basis of Human Cancer.
  McGraw-Hill, New York, 1998.
  

• The breast cancer susceptibility gene BRCA1
  
  The BRCA1 gene on chromosome 17 at q21.1 consists of 24 exons spanning 80 kb of genomic DNA that encode a 7.8 kb mRNA transcript. The protein has 1863 amino acids. Exon 11 is quite large (3.4 kb). About 55% of all mutations occur in exon 11. Although some mutations occur relatively frequently in other exons, they tend to be evenly distributed throughout the gene (only some mutations are shown). The deletion of an adenine (A) and a guanine (G) in nucleotide position 185 (185delAG) and the insertion of a cytosine in position 5382 (5382insC) account for about 10% of mutations each. These mutations are particularly frequent in the Ashkenazi Jewish population.
  
  The protein has five main functional domains. The RING finger region near the N-terminus at amino acids 1–112 defines a zinc-binding domain of conserved cysteine and histidine residues that mediate protein—protein or protein- DNA interactions. This region is also the site of heterodimerization of BRCA1 and BARD1 (BRCA1-associated RING domain 1). Other functional domains define the central part of the BRCA1 protein. These are two nuclear localization signals (NLS) and two protein-binding domains, one for p53 protein, retinoblastoma (RB) protein, and RAD50 and RAD51. RAD50 and 51 are proteins involved in recombination during mitosis and meiosis, and in recombinational repair of double-stranded DNA breaks. The C-terminus contains a region involved in transcriptional activation and DNA repair.
  
• The breast cancer susceptibility gene BRCA2
  
  The BRCA2 gene on 13q12 comprises 27 exons spanning 80 kb of genomic DNA that encode a 10.4 kb mRNA transcript. Its protein has 3418 amino acids. Exon 11 is large (11.5 kb), as in BRCA1. Mutations occur throughout the gene (only some are shown). A deletion of thymine at nucleotide position 6174 (6174delT) is relatively (1%) frequent in the Ashkenazi Jewish population.
  
  The BRCA2 protein has a transcriptional activation domain near the N-terminus and a nuclear location signal (NLS) near the C-terminus. A large central domain consists of eight copies of a 30–80-amino-acid repeat, which are conserved in all mammalian BRCA2 proteins (BRC repeats). Four of these interact with the RAD51 protein.
  
  The BRCA1 and BRCA2 genes are expressed ubiquitously with the highest levels of expression in thymus and testis. The spatial and temporal expression patterns of Brca1 and Brca2 in the mouse fetal and adult tissues are essentially identical, with highest expression of both in rapidly dividing tissues during differentiation, especially in mammary epithelium. In the mammary gland both genes are expressed during puberty and pregnancy, and their expression is reduced during lacation. (Figures redrawn fromWelcsh et al., 2000.)
  
• References
  
  Miki, Y., et al.: A strong candidate for the breast
  and ovarian cancer susceptibility gene
  BRCA1. Science 266:66–71, 1994.
  Welcsh, P.L., Schubert, E.L., King, M.C.: Inherited
  breast cancer: an emerging picture. Clin.
  Genet. 54:447–458, 1998.
  Welcsh, P.L., Owens, K.N., King, M.C.: Insights
  into the functions of BRCA1 and
  BRCA2. Trends Genet:16:69–74, 2000.
  Wooster, R., et al.: Identification of the breast
  cancer susceptibility gene BRCA2. Nature
  378:789–792, 1995.

• Phenotype
  
  Retinoblastoma occurs in one eye or both eyes. An important early sign is the so-called “cat’s eye,” awhite shimmer out of the affected eye (1) or the development of strabismus. One or several tumors originate from the retina (2). The tumor progresses rapidly (3). The relative proportions of the genetic types of retinoblastoma are about 60% somatic mutations (nonhereditary form) and 40% germline mutations, transmitted as an autosomal dominant trait (hereditary form, in about 10–15%, due to transmission from a parent; the remainder due to a new mutation). New mutations usually affect a paternal allele (about 10: 1). In about 10% of carriers of a germline mutation no tumor develops (nonpenetrance).
  
• Retinoblastoma locus on chromosome 13
  
  The RB1 locus at 13q14.2 was first defined with cytogenetically visible interstitial deletions.
  
• Retinoblastoma gene BR-1 and the pRB protein
  
  The RB1 gene is organized into 27 exons spanning 183 kb of genomic DNA (1). The RB1 gene is ubiquitously expressed and transcribed into a 4.7 kb mRNA (2). The gene product (pRB protein) has 928 amino acids (3). It is a 100 kD phosphoprotein with important functions in the regulation of the cell cycle. It is activated by phosphorylation (P) during cell cycle progression from G0 to G1 (p. 112) at about 12 distinct serine and threonine residues. Three functional domains, A, B, and C, and a nuclear localization signal (NLS) can be distinguished.
  
• Diagnostic principle
  
  Molecular diagnosis of retinoblastoma greatly contributes to its early recognition and to the correct assessment of individual risks within families. In about 3–5% of patients an interstitial deletion 13q14 or a larger deletion is visible by chromosomal analysis (1). In familial retinoblastoma indirect DNA diagnosis can be achieved by segregation analysis using DNA markers at the RB1 locus (2). In the example shown, the affected girl (II-1) has inherited haplotype a from the unaffected father and haplotype c from the unaffected mother. In tumor cells, obtained after one eye had to be removed, haplotype a only is present (loss of heterozygosity, LOH, see p. 318). This reveals that haplotype a represents the mutation-carrying RB1 allele. In the family shown (3), I-2 and II-2 are affected (3). Sequence analysis reveals a C-to-T transversion in codon 575 in the two affected individuals (CAA glutamine to TAA stop codon). The mutational spectrum in hereditary retinoblastoma involves deletions (~26%), insertions (~9%), and point mutations (~65%), including splice-site mutations. (Illustrations courtesy ofW. Höpping (A) and D. Lohmann (C and D).)
  
  
• References
  
  Lohmann, D.R.: RB1 gene mutations in retinoblastoma.
  Hum. Mutat. 14:283–288, 1999.
  Lohmann, D.R., et al.: Spectrum of RB1 germline
  mutations in hereditary retinoblastoma.
  Am. J. Hum. Genet. 58:940–949,
  1996.
  Newsham, I.F., Hadjistilianou, T., Cavenee,W.K.:
  Retinoblastoma. pp. 363–392. In: B. Vogelstein,
  K.W. Kinzler, eds. The Genetic Basis
  of Human Cancer. McGraw-Hill, New York,
  1998.

• The Philadelphia chromosome (Ph1) in different forms of leukemia
  
  A Philadelphia chromosome is present in the bone marrow cells of most patients with the chronic form of the disease (CML). If it is not present, the illness progresses more rapidly than usual and has a poorer prognosis. In addition, the Philadelphia chromosome may be found in some acute leukemias (acute lymphocytic leukemia, ALL; acutemyelocytic leukemia, AML) in adults and in children. Here, Ph1 indicates a poor prognosis, whereas its absence is favorable.
  
• The Ph1 translocation [t(9;22)(q34;q11)]
  
  The Philadelphia chromosome arises by reciprocal translocation between a chromosome 22 and a chromosome 9. The breakpoints are in 9q34 and 22q11. A good half of the long arm of a chromosome 22 is translocated to the long arm of a chromosome 9. A very small segment of the distal long arm of a chromosome 9 (9q34), not visible under the light microscope, is translocated to a chromosome 22. The Philadelphia chromosome (22q–) consists of the short arm and the proximal one-third of the long arm of a chromosome 22 and the small distal segment from the long arm of a chromosome 9. For demonstration of the Philadelphia translocation by in-situ hybridization, see p. 192).
  
• The Ph1 translocation leads to the fusion of two genes
  
  The breakpoints of the Ph1 translocation are located in the BCR gene of chromosome 22 and in the ABL gene of chromosome 9. The translocation leads to the fusion of these genes. The exact locations of the breakpoints differ from patient to patient, but in the BCR gene they are limited to a small region of just 6 kb (thus the designation BCR, or breakpoint cluster region). In CML, the breakpoints lie in exons 10–12 of the BCR gene; in acute Ph1-positive leukemias (e.g., ALL) they lie further in the 5! direction in exon 1 or 2. The breakpoint region in the ABL gene extends over 180 kb between exons 1a and 1b, which are separated by an intron.
  
• The gene fusion leads to changes in transcription and gene products
  
  The ABL gene codes formRNA transcripts of 7 kb (exon 1b, 2–11) and 6 kb (exon 1a, 2–11) by differential splicing; these in turn code for a protein of about 145000 Da (p145abl). From the fusion of the two genes in CML, an 8.5 kb mRNA transcript results, which codes for a fusion protein of 210000 Da (p210bcr/abl). In the acute form of leukemia (ALL), a transcript results that codes for a fusion protein of 190000 Da (p190bcr/abl). In contrast to the normal protein, it has high tyrosinase activity. This results in uncontrolled cell division in the affected cells and tumor growth.
  
• References
  
  Bartram, C.R. et al.: Translocation of c-abl oncogene
  correlates with the presence of a
  Philadelphia chromosome in chronic myelocytic
  leukaemia. Nature 306:277–280,
  1983.
  Cline, M.J.: The molecular basis of leukemia.
  New Eng. J. Med. 330:328–336, 1994.
  Faderl, S. , et al.: The biology of chronic myeloid
  leukemia. New Eng. J. Med. 341:164–172,
  1999.
  Hentze, B.M., Kulozik, A.E., Bartram, C.R.:
  Einführung in die medizinische Molekularbiologie.
  Grundlagen, Klinik, Perspektiven.
  Springer, Berlin, 1990.
  Kurzrok, R., Gutterman, J.U., Talpaz, M.: The
  molecular genetics of Philadelphia-positive
  leukemias. New Eng. J. Med. 319:990, 1988.
  Sawyers, C.L.: Chronic myeloid leukemia. New
  Eng. J. Med. 340:1330–1340, 1999.

• Bloom syndrome (BS)
  
  In Bloom syndrome (McKusick 210900) (1), prenatal and postnatal growth deficiency is pronounced (birth weight 2000 g, birth length !40 cm, adult height around 150 cm). The phenotype (2) includes a narrow face. Usually, but not always, a sunlight-induced erythema develops on the cheeks, eyelids, mouth, ears, and back of the hands (a and b). The photograph on the right (c) shows a boy with Bloom syndrome and acute leukemia. Metaphase cells show about a tenfold increase in the rate of sister chromatid exchanges (SCE), !60 instead of about 6 per metaphase in normal cells (3). (Sister chromatid exchanges are explained in the glossary, p. 423). Metaphases of patients contain increased breaks in one or both chromatids and exchanges between homologous chromosomes in about 1–2% of cells. In Bloom syndrome patients, different types of malignancies occur in a distribution comparable to that of the general population, but at a much earlier age (mean age 24.7, range 2–48 years). Some patients have multiple primary tumors, which underlines the striking susceptibility to cancer in Bloom syndrome. Chemotherapy is very poorly tolerated.
  
  Homozygosity for mutations in the Bloom syndrome gene (BLM) results in an increased rate of somatic mutations, a manifestation of genomic instability. The BLM gene on chromosome 15 at q16.1 encodes a member of the RecQ family of DNA helicases. The 1417-amino-acid protein shows homology to the yeast SGS1 gene product (slow growth suppressor) and the human WRN gene product (Werner syndrome, McKusick 277700). Allozygous nonsense mutations (two different mutations in the two alleles of the same gene) are frequent in the BLM gene. A characteristic homozygous 6 bp deletion/7 bp insertion at nucleotide 2281 occurs in Ashkenazi Jewish individuals as a result of a founder effect.
  
• Fanconi anemia (FA)
  
  Fanconi anemia (hereditary pancytopenia) (McKusick 227650) is a malformation syndrome (1) with variable clinical expression. Growth deficiency (2), hypoplastic or absent thumbs (3), and short or absent radii are characteristic physical signs. FA cells are hypersensitive to DNA-crosslinking agents, such as diepoxybutane (DEB). Several complementation groups can be distinguished. Three FA genes have been identified, at chromosome 16q24.3 (FAA), 9q22.3 (FAC), and 3p22– 26 (FAD). FAA is the most prevalent group in 60–65% of patients.
  
• Ataxia telangiectasia
  
  Ataxia telangiectasia (McKusick 208900) is a pleiotropic, variable disease due to mutations in the ATM gene on chromosome 11q23. Preferential reciprocal translocations between chromosomes 7 and 14 with breakpoints at 7p14, 7q14, 14q11, and 14q32 occur in a small proportion of metaphases. The ATM gene has 66 exons spanning more than 150 kb of genomic DNA. From its 13-kb transcript (and smaller alternatively spliced products), a 3056-amino-acid protein kinase ATM(350 kDA) is translated. ATM is activated in response to double-strand DNA breaks. It is part of a network of proteins that regulate cellular responses to DNA damage (p. 80). Clinically different disorders are related at the cellular level (Nijmegen breakage syndrome, McKusick 251260, and others).
  
• References
  
  Auerbach, A.D., Buchwald, M., Joenje, H.: Fanconi
  anemia, pp. 317–332. In: B. Vogelstein,
  K.W. Kinzler, eds., The Genetic Basis of
  Human Cancer. McGraw-Hill, New York,
  1998.
  Gatti, R.: Ataxia telangiectasia, pp. 275–300. In:
  B. Vogelstein, K.W. Kinzler, eds., The Genetic
  Basis of Human Cancer. McGraw-Hill, New
  York, 1998.
  German, J., Ellis, N.A.: Bloom syndrome, pp.
  301–315. In: B. Vogelstein, K.W. Kinzler,
  eds., The Genetic Basis of Human Cancer.
  McGraw-Hill, New York, 1998.
  Zhao, S. , et al.: Functional link between ataxiatelangiectasia
  and Nijmegen breakage syndrome
  gene products. Nature 405:473–477,
  2000.