-Hereditary disease and X -linked dis.pptx

mutant allele resides in X-chromosome-females are carriers but not affected-e.g:Hemophilia,Duchenne muscle dystrophy and fragile x syndrome.other hereditary diseases linked to autosomes e.g hereditary breast cancer,thrombophilia..and inherited colon cancer

1. INHERETED DISEASES by Prof Dr. Ekbal Abo Hashem Professor of clinical pathology

2. In X-linked diseases, the mutant allele resides on the X chro­mosome. In X-linked recessive diseases, females are carriers of the disease with one normal and one mutant allele but typically are not affected. C. X-LINKED DISEASES

3. Males receiving the mutant allele from their mothers and having only one X chromosome have no normal allele and thus are affected. All daughters of affected males are carriers of a mutant allele. C. X-LINKED DISEASES

4. Carrier females have a 25% chance of transmitting their normal allele to a son, a 25% chance of having an affected son, a 25% chance of having a daughter who carries the mutant allele, and a 25% chance of having a daughter who receives her normal allele. In the absence of a family history, an affected male can have a mutant allele that rose de novo as a new mutation during the formation of the egg. C. X-LINKED DISEASES

5. 1. Hemophilia A

6. 1. Hemophilia A

7. 1. Hemophilia A

8. Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) and affects approximately 1 in 10,000 males worldwide. The disease is characterized by prolonged bleeding after injuries or surgery, renewed bleeding after the initial bleeding has ceased, and in severe cases, spontaneous bleeding into the joints. a. Phenotype 1. Hemophilia A

9. 1. Hemophilia A

10. The severity of the disease is determined by the amount of FVIII coagulant activity present in the plasma; mild, moderate, and severe disease have corresponding FVIII activity levels of 5% to 30%, 1 % to 5%, and <1 % of control, respectively. 1. Hemophilia A a. Phenotype

11. In the patient with severe or moderate hemo­philia A disease, the activated partial thromboplastin time ( aPTT ) will be prolonged while all other routine coagulation test results are normal. 1. Hemophilia A a. Phenotype

12. In patients with mild hemophilia A disease, however, aPTT is often normal. The age of diagno­sis is typically earlier in cases with severe disease and a family history. Although most patients with severe disease are diagnosed in the first year of life, patients with mild disease may not be diagnosed until several years. 1. Hemophilia A a. Phenotype

13. The FVIII gene is positioned on Xq28 (long arm of the X chromosome band 28) and was cloned in 1984. The gene spans more than 186,000 base pairs and includes 26 exons. A plethora of nucleotide substitutions, gene dele­tions, insertions, and rearrangements throughout the FVIII gene have been reported in patients with hemophilia. 1. Hemophilia A b. FVIII Gene

14. The variability accounts for much of the clinical heterogeneity that is observed with this disease. Interestingly, although routine screening of the coding, splice junctions, promoter region, and polyadenylation site of the gene could detect the mutation in the majority of patients with mild to moderate disease, the disease-causing mutation in about half of the patients with severe disease remained elusive for several years. 1. Hemophilia A b. FVIII Gene

15. A common inversion mutation was identified in patients with severe disease. The inversion mutation arose from genetic recombination between a small intronless gene within intron 22, gene A, and one of two additional copies of the gene A located approxi­mately 500 kb upstream from the FVIII gene. 1. Hemophilia A c. Inversion Mutation

16. The mecha­nism for the inversion involves flipping of the tip of the X chromosome allowing pairing between homologous sequences and genetic recombination between one of the upstream copies of gene A and the copy of gene A within intron 22. 1. Hemophilia A c. Inversion Mutation

17. FVIII is synthesized as a single polypeptide chain of 2351 amino acid residues and an approximate weight of 280 kD . The encoded FVIII protein is predominantly produced in the liver, circulates in the plasma, and is stabilized through non-covalent binding to the complex multimeric glycopro­tein von Willebrand's factor ( vWF ). 1. Hemophilia A d. Protein Structure and Function

18. In the intrinsic coagu­lation pathway, proteolytic activation of FVIII by small amounts of thrombin frees it from vWf , where it then participates as a cofactor with activated factor IX to catalyze the conversion of factor X to factor Xa . Factor Xa hydrolyzes and activates prothrombin to thrombin. 1. Hemophilia A d. Protein Structure and Function

19. The treatment for bleeding episodes is intravenous infusions of FVIII concentrate as quickly as possible to prevent pain, disability, and chronic joint disease. However, management of 15% to 33% of severe and mod­erately affected patients is complicated by antibody forma­tion to exogenous FVIII caused by repeated infusions. The antibodies can rapidly neutralize infused FVIII. 1. Hemophilia A d. Protein Structure and Function

20. i ) DNA testing for hemophilia A primarily involved the use of ­linkage studies and restriction fragment length polymorphisms to determine the carrier status of at-risk females in the family and to perform prenatal testing. PCR and the analysis of poly­morphic microsatellite repeats within the FVIII gene increased the number of families in which linkage studies could be performed and significantly improved turnaround times . 1. Hemophilia A e. DNA Testing

21. ii) The most significant improvement in DNA testing was the identification of the inversion mutation in families with severe disease and the ability to offer direct mutation analy­sis as opposed to linkage studies. 1. Hemophilia A e. DNA Testing

22. For clinical samples, if the patient has a negative test result for a common inversion mutation, DNA sequence analysis of the FVIII gene is typically per­formed, and the turnaround time is about 4 to 8 weeks. Once the mutation segregating in the family has been identified, direct mutation testing for prenatal studies or the carrier status for other at-risk females in the family is available with a usual turnaround time of 2 weeks. 1. Hemophilia A e. DNA Testing

23. 2. Duchenne's Muscular Dystrophy

24. Duchenne's muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive skeletal muscle wasting. The incidence of DMD is about 1 in 3500 male births, making it the most common severe neuromus­cular disease in man. 2. Duchenne's Muscular Dystrophy e. DNA Testing

25. The onset of DMD is typically before 3 years of age with gait difficulty, progressive myopathic weakness with pseudohypertrophy of calves, and grossly ele­vated serum creatine kinase (CK) as a result of degenerating fibers. 2. Duchenne's Muscular Dystrophy e. DNA Testing

26. Carrier females can be asympto­matic or have varying degrees of clinical symptoms depending on the degree of inactivation of the X chromo­some harboring the mutant DMD gene in the various tissues where the DMD protein is expressed. Females with severe disease most often result from a carrier female with skewed lyonization or an X-autosome translocation involving the DMD gene. 2. Duchenne's Muscular Dystrophy e. DNA Testing

27. Electromyography and muscle biopsy are used to confirm the diagnosis. Most DMD patients are wheelchair bound between 10 and 15 years of age. Continual degeneration and regenera­tion of muscle eventually lead to the replacement of muscle tissue by adipose and connective tissue, causing progressive disease; death usually occurs before the age of 30 from res­piratory or cardiac failure. 2. Duchenne's Muscular Dystrophy e. DNA Testing

28. Gene expression studies of diseased and normal muscle have revealed the overexpression of genes involved in muscle structure and regeneration processes Although not yet reality, technological advances in many areas of medicine suggest that gene therapy for DMD lies in the not so distant future. 2. Duchenne's Muscular Dystrophy b. Gene and Protein

29. The DMD gene is the largest gene in the human genome known to date, spans 2.2 mega bases (Mb), contains 79 exons, and has multiple promoter regions. The protein product, dystrophin with 3685 amino acids, has a molecular weight of 427 kD and represents approximately 0.002% of total striated muscle protein. 2. Duchenne's Muscular Dystrophy b. Gene and Protein

30. Dystrophin is a cytoskeletal protein associated with a protein complex, dystrophin ­associated protein complex (DAPC), which in skeletal muscle plays a structural role connecting the actin cytoskel­eton to the extracellular matrix, stabilizing the sarcolemma during repeated cycles of contraction and relaxation, and transmitting force generated in the muscle sarcomeres to the extracellular matrix. 2. Duchenne's Muscular Dystrophy b. Gene and Protein

31. Without dystrophin , an integral component of DAPC, sarcolemmal integrity is compro­mised. a result, there is an influx of extracellular calcium triggering calcium-activated proteases and fiber necrosis 2. Duchenne's Muscular Dystrophy b. Gene and Protein

32. Duplications of gene sequences are observed in about 5% of patients. Both deletions and duplications can be rapidly detected by use of a combination PCR reactions. Electrophoretic gels of the amplified DNA will show the loss of a band or bands or an increased intensity of one or more bands 2. Duchenne's Muscular Dystrophy c. Gene Mutation and Detection

33. Because of the tremendous size (2.2 Mb), complexity (8promoters), and diversity of mutations within the DMD gene, DNA testing for DMD presents a challenge for clinical laboratories. Although DNA testing is often not required for diagnosis, identification of the mutation causing the disease in a family is required for carrier detection of at-risk females and for prenatal testing. 2. Duchenne's Muscular Dystrophy c. Gene Mutation and Detection

34. Becker muscular dystrophy (BMD), a milder and less common form of muscular dystrophy with an estimated incidence of 1 in 18,500 births, is an allelic variation of DMD caused by different mutations within the DMD gene. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

35. About 55% of BMD patients have deletions within the dystrophin locus, with those having deletions around the distal rod domain of dystrophin (exons 45 to 60) showing a more classic BMD phenotype and in some cases even remaining free of symptoms until their 50s. However, BMD patients with deletions involving the amino-terminal domain of dystrophin (exons 1 to 9) have a more severe phenotype with an earlier age of onset and a more rapid progression of disease. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

36. i ) For DMD or BMD families in which no deletion or dupli­cation is detected, carrier and prenatal testing can be offered by clinical laboratories using DNA linkage studies. These studies require the use of both multiple intragenic and 5' and 3' flanking markers for an accurate carrier or prenatal result. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

37. ii) Germline mosaicism is a phenomenon in which no DMD gene mutation is present in lymphocyte DNA, but DMD gene mutations are present in the germline tissue. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

38. iii) In mutation-negative families, carrier assessment involves measurement of serum CK activity and linkage studies. Serum CK is, by definition, above the reference interval (defined as the 95th percentile of a healthy reference group of age-matched women) in 1of 20 noncarrier women. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

39. iii) Moreover, serum CK decreases in DMD carrier women as they age. Thus for DMD and/or BMD families, identification of the mutation in the family best enables rapid and accurate direct mutation analysis for carrier and prenatal testing in that family. 2. Duchenne's Muscular Dystrophy d. Becker Dystrophy, Mutation and Phenotype

40. 3. Fragile X Syndrome

41. 3. Fragile X Syndrome

42. 3. Fragile X Syndrome

43. Fragile X syndrome is one of the most commonly inherited forms of mental retardation, with an estimated incidence of 1 in 3500 males and 1 in 9000 females. The name of the condition reflects the cytogenetic abnormality of a break­point or fragile site in the X chromosome. 3. Fragile X Syndrome a. Cytogenetic Abnormality

44. The disease is characterized by the presence of a marker X chromosome in the leukocytes of some mentally retarded males following incubation of cells in cell culture media depleted of folate and thymidine; the marker segregated with mental retardation in the family. The chromosomal locus for this fragile site would later be localized to Xq27.3 (the long arm of the X chromosome band 27.3). 3. Fragile X Syndrome a. Cytogenetic Abnormality

45. Common clinical features associated with fragile X syndrome are mental retardation, delayed motor and speech development, macroorchidism , long face, prominent forehead and jaw, large ears, flat feet, and abnormal behavioral characteristics that include hyperactivity, hand flapping, temper tantrums, persevering speech patterns, poor eye contact, and occa­sionally autism. 3. Fragile X Syndrome b. Clinical Features

46. Common clinical features associated with fragile X syndrome are mental retardation, delayed motor and speech development, macroorchidism , long face, prominent forehead and jaw, large ears, flat feet, and abnormal behavioral characteristics that include hyperactivity, hand flapping, temper tantrums, persevering speech patterns, poor eye contact, and occa­sionally autism. 3. Fragile X Syndrome b. Clinical Features

47. As a sex-linked disease, fragile X syndrome has a compli­cated inheritance pattern. Affected females are heterozygous for the mutation, and unaffected males can transmit the mutation through the family. For this reason, fragile X syndrome is an X-linked dominant disorder with reduced penetrance (79% for males and 35% for females), but the penetrance of the disease appeared to increase in subsequent generations within a family. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

48. The mechanism of this "paradox" was resolved when the gene causing fragile X syndrome, FMRl (fragile X M ental Retardation) was cloned. FMRl was the first gene discovered to cause disease through an expansion of an unstable trinucleotide repeat sequence. The unstable CGG repeat is located in the 5'-untranslated region of the FMRl gene in exon 1. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

49. The gene spans 38 kb, contains 17 exons, and encodes a 4.4 kb transcript. Alleles contain blocks of CGG repeats usually 7 to 13 repeats in length, which can be interspersed with single AGG repeats. Allelic diversity results from the variable number and lengths of these CGG-repeat blocks. There are no distinct bound­aries separating the repeat number categories. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

50. As a sex-linked disease, fragile X syndrome has a compli­cated inheritance pattern. Affected females are heterozygous for the mutation, and unaffected males can transmit the mutation through the family. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

51. For this reason, fragile X syndrome is an X-linked dominant disorder with reduced penetrance (79% for males and 35% for females), but the penetrance of the disease appeared to increase in subsequent generations within a family. The mechanism of this "paradox" was resolved when the gene causing fragile X syndrome, FMRl (fragile X M ental Retardation) was cloned. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

52. FMRl was the first gene discovered to cause disease through an expansion of an unstable trinucleotide repeat sequence. The unstable CGG repeat is located in the 5'-untranslated region of the FMRl gene in exon 1. The gene spans 38 kb, contains 17 exons, and encodes a 4.4 kb transcript. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

53. Alleles contain blocks of CGG repeats usually 7 to 13 repeats in length, which can be interspersed with single AGG repeats. Allelic diversity results from the variable number and lengths of these CGG-repeat blocks. There are no distinct bound­aries separating the repeat number categories. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

54. Normal alleles have 5 to 45 repeats; gray zone alleles have 46 to 54; pre mutation alleles have 55 to 200; and full mutation expansion alleles contain >200 repeats. Indi­viduals with a normal number of CGG repeats do not have fragile X syndrome nor are they at risk of having an affected child. Individuals with 46 to 54 repeats represent alleles in the upper range of normal or a smaller than average pre­mutation allele. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

55. These individuals do not have fragile X syn­drome, yet may have a slightly increased risk of repeat instability and expansion to a full mutation in their offspring in some families, Premutation alleles are unstable and can expand to a larger allele in the premutation range when transmitted or can expand to a full mutation allele and an offspring with fragile X syndrome. 3. Fragile X Syndrome c. Inheritance and Trinucleotide Expansion

56. The fragile X phenotype occurs following expansion of the CGG repeat, hypermethylation , and histone deacetyla­tion of the adjacent CpG island in the promoter region of the FMRI gene, transcriptional silencing of the gene, and ultimately no production of FMRP. 3. Fragile X Syndrome e. FMRI Gene Mutation

57. DNA testing for fragile X syn­drome using Southern blot analysis of peripheral blood enables detection of all possible genotypes. PCR analysis with capillary electrophoresis is used to com­plement the testing by providing the precise CGG-repeat number. Chorionic villus samples or amniotic fluid can be tested, but the methylation pattern expected in adult tissue may be absent. 3. Fragile X Syndrome f. DNA Testing

58. As expected, as the CGG-repeat length increases, the risk of expansion from a premutation to a full mutation in pre­mutation carrier females increases. This information is most useful for determining the risk of an affected offspring during genetic counseling of a premutation carrier female. 3. Fragile X Syndrome f. DNA Testing

59. 1. Thrombophilia

60. 1. Thrombophilia

61. 1. Thrombophilia

62. 1. Thrombophilia

63. 1. Thrombophilia

64. 2. Inherited Breast Cancer

65. Mutations in the two major breast cancer genes, BRCA 1 and BRCA2, predispose patients to breast and ovarian cancer and to prostate and colon cancer (BRCA1) or pancreatic cancer (BRCA2). 2. Inherited Breast Cancer a. BRCA1 and BRCA2 Mutation Predisposes to and Accelerates the Progression of Caner

66. 2. Inherited Breast Cancer

67. 2. Inherited Breast Cancer

68. 2. Inherited Breast Cancer

69. 2. Inherited Breast Cancer

70. 2. Inherited Breast Cancer

71. The progression rate of breast neoplasia is accel­erated in women who carry BRCA1 or BRCA2 mutations compared with other patients who have breast carcinoma with or without a family history. In families in which breast cancer is segregating, but no BRCA1 or BRCA2 mutation has been detected, additional genes that predispose to breast cancer are likely but have yet to be identified. 2. Inherited Breast Cancer a. BRCA1 and BRCA2 Mutation Predisposes to and Accelerates the Progression of Caner

72. The inabil­ity to identify breast cancer susceptibility genes in these fam­ilies may reflect: (1) genetic heterogeneity in the family with mutations in several genes, (2) low penetrance of these mutations, making it difficult to distinguish family members without mutation from asymptomatic carriers in the studies, (3) an autosomal recessive mode of inheritance, or (4) breast cancer acting as a complex disease that results from the inter­action of both several genes and environmental factors, thereby making it difficult to tease out the genetic compo­nent of the disease. 2. Inherited Breast Cancer b. Causes of Absence of Susceptibility Genes Detection

73. Recently developed gene-expression assays of tumor tissue may be instrumental for classification of these families into subsets, aiding studies aimed at deter­mining the molecular origin of these cancers. Mutations in tumor-suppressor genes TP53 or ATM are also associated with "familial" cancer, including breast cancer in mutation ­positive females. 2. Inherited Breast Cancer b. Causes of Absence of Susceptibility Genes Detection

74. Mutations in BRCA1 and BRCA2 are inherited in an auto­somal dominant fashion, with offspring of known carriers or of affected patients possessing a 50% chance of inherit­ing the predisposing cancer gene mutation. Inheritance of the mutation does not convey a certainty of developing cancer nor indicate the type of cancer or the age of onset. 2. Inherited Breast Cancer c. Mode of Inheritance and Cumulative Risk

75. The average cumulative risk of breast cancer for mutations in either BRCAI or BRCA2 is about 27% to age 50 and 64% to age 70. Both environmental and genetic factors play role in the development of breast or other cancers in muta­tion-positive patients as does the type of DNA mutation in BRCA1 or BRCA2. 2. Inherited Breast Cancer c. Mode of Inheritance and Cumulative Risk

76. Using DNA linkage studies, the gene was mapped for early onset familial breast cancer to 17 q21 (long arm of chromosome 17 banding region 21). BRCA1 was cloned and later confirmed as the susceptibility gene in breast and ovarian cancer kindreds . 2. Inherited Breast Cancer d. BRCA1 and BRCA2 Tumor-Suppressor Genes

77. 2. Inherited Breast Cancer d. BRCA1 and BRCA2 Tumor-Suppressor Genes

78. The BRCA2 gene, unrelated in sequence to BRCA1, spans 70kb, contains 26 exons, encodes an 11.5-kb mRNA, and is translated into a protein of 3418 amino acids. BRCA1 and BRCA2 are considered tumor-suppressor genes requiring inactivation of both alleles for progression to neoplasm. 2. Inherited Breast Cancer d. BRCA1 and BRCA2 Tumor-Suppressor Genes

79. In a patient with famil­ial breast cancer, a mutant allele is inherited, and the second allele-the patient's wild-type allele-is inactivated through somatic mutation. BRCAI and BRCA2 proteins are multi­functional, interacting with numerous other proteins in complex and separate systems involved in response to DNA damage, regulation of transcription, remodeling of chro­matin, and regulation of cell growth. 2. Inherited Breast Cancer d. BRCA1 and BRCA2 Tumor-Suppressor Genes

80. Testing for disease-associated mutations is made difficult by the heterogeneity of disease-causing mutations and the complexity of the BRCA1 and BRCA2 genes. 2. Inherited Breast Cancer e. Heterogeneity of Mutation

81. Interestingly the majority of BRCAI and BRCA2 disease-associated muta­tions result in premature truncation of the protein and thus a loss of function. For this reason, the protein truncation test (PTT) is often employed as a screening method for muta­tion detection. 2. Inherited Breast Cancer f. Protein Premature Truncation and Protein Truncation Test

82. In this methodology, multiple PCR primer pairs are designed to span the gene with each primer pair, including one primer that includes an RNA polymerase pro­moter and a translation initiation sequence. in vitro translation system, and the synthesized proteins are subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis for detection of truncated proteins. 2. Inherited Breast Cancer f. Protein Premature Truncation and Protein Truncation Test

83. Common screening assays include microarrays, dena­turing high-performance liquid chromatography (DHPLC), single-strand conformation polymorphism (SSCP), dena­turing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), and fluorescence assisted mismatch analysis (FAMA). 2. Inherited Breast Cancer g. DNA Screening Assays

84. More recently, other unique methods have been described as well, including allele-specific gene expres­sion analysis (AGE), multiplex ligation-dependent probe amplification (MLPA), and restriction endonuclease fingerprinting, single-strand conformation polymorphism coupled with capillary electrophoresis. 2. Inherited Breast Cancer g. DNA Screening Assays

85. Although screening assays can detect DNA perturbations, DNA sequence (DS) analysis is required for precise identifi­cation of the base or bases involved. 2. Inherited Breast Cancer h. Importance of DNA Sequencing

86. In many instances, a combination of screening methods is used, thereby reducing the region of the gene that requires DNA sequencing ­analysis. An alternative approach is to eliminate screening of the gene and rather perform direct DS analysis of the BRCA1 and BRCA2 genes on each specimen. 2. Inherited Breast Cancer h. Importance of DNA Sequencing

87. BRCA1 or BRCA2 mutation in a patient is increased to 10% or greater if: Breast cancer was diagnosed in two women in the family before the age of 50. Breast cancer was diag­nosed in women in the family before age 50, and ovarian cancer was detected in one or more. women in the family. 2. Inherited Breast Cancer The likelihood of mutation

88. BRCA1 or BRCA2 mutation in a patient is increased to 10% or greater if: Breast cancer was diagnosed after the age of 50 in one woman, and ovarian cancer was detected in two or more women in the family. Ovarian cancer is present in two or more family members. Male breast cancer is present, and breast or ovarian cancer is present in the family 2. Inherited Breast Cancer The likelihood of mutation

89. 2. Inherited Breast Cancer

90. 3. Inherited Colon Cancer

91. 3. Inherited Colon Cancer

92. 3. Inherited Colon Cancer

93. 3. Inherited Colon Cancer

94. The molecular basis of sporadic and inherited CRC involves two distinct pathways, one of chromosomal instability and one associated with microsatellite instability. The original model of chromosome instability has been further characterized to reveal a complex chain of events whereby normal colon lining (mucosa) is transformed into adenomatous and then into malignant mucosa via the inactivation of tumor-suppressor genes and the activation of genes involved in tumor cell proliferation. 3. Inherited Colon Cancer

95. 3. Inherited Colon Cancer

96. The cascade of events proceeds with continued activation of the KRAS (Kirsten rat sarcoma virus) proto­oncogene on 12p 12.1 (short arm of chromosome 12 banding region 12.1) through somatic gene mutations (most frequently occurring in codons 12, 13, or 61), which in the pres­ence of APC inactivation increases growth and proliferation of the cell. 3. Inherited Colon Cancer a. Chromosomal Instability Pathway

97. 3. Inherited Colon Cancer

98. Subsequent inactivation of the tumor-suppres­sor gene DCC (deleted in colon cancer) and frequent loss of adjacent tumor-suppressor genes on 18q (long arm of chromosome 18) including SMAD4 and SMAD and the inactivation of tumor-suppressor gene TP53 on 17p (short arm of chromosome 17) are identified in late adenoma and carcinoma. 3. Inherited Colon Cancer a. Chromosomal Instability Pathway

99. The chromosomal instability (CIN) pathway begins with the loss of function of the adenomatous polyposis coli (APC) tumor-suppressor gene product, most often because of a somatic inactivating gene mutation on one allele followed by a chromosomal deletion encompassing the second APC allele and adjacent flanking DNA on chromosome 5q. Since APC is involved early in the tumorigenic process, it has been referred to as the "gate­keeper". 3. Inherited Colon Cancer a. Chromosomal Instability Pathway

100. The microsatellite instability (MSI) pathway in sporadic CRC arises from mutations or altered expression of genes involved in DNA mismatch repair (MMR). As a result of an altered and thus dysfunctional MMR system, DNA replication errors, pri­marily within microsatellite repeats or repetitive sequences, remain uncorrected and accumulate 3. Inherited Colon Cancer b. microsatellite instability pathway

101. Inherited CRC syndromes initiate as a result of an inher­ited mutation in one of the genes involved in the CIN or MSI pathway. Although several CRC syndromes exist, the two most common are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). 3. Inherited Colon Cancer c. Familial Adenomatous Polyposis and Hereditary Non- plyposis Colorectal Cancer

102. The gene responsible for FAP, the APC gene, was cloned following linkage to chromosome 5q21 (long arm of chromosome band 21) in FAP kindreds . 3. Inherited Colon Cancer d. APC Gene and Protein

103. The APC gene spans 8535 base pairs, contains 15 exons, and encodes a protein of 2843 amino acids with a molecular weight of about 312kD, The APC protein is a multidomain , multifunctional protein, participating in several cellular processes, including cell adhesion and migration, signal transduction, microtubule assembly, and chromosome segregation. Best understood is the regu­lation of  -catenin levels through interaction with APC. 3. Inherited Colon Cancer d. APC Gene and Protein

104.  -catenin is required for binding with E-cadherin, a member of the calcium-dependent cell adhesion molecules, and is also involved in signal transduction pathways. Mutations altering the association of  -catenin, with APC minimize degradation and increase cytoplasmic levels of  -catenin; this can affect cell-cell adhesion and the transcription of genes, promoting cell proliferation (specifically CMYC) or inhibition of cell death. 3. Inherited Colon Cancer d. APC Gene and Protein

105. Studies on FAP families indicate that a wide variety of germline mutations exist; >95% result in truncated proteins, either because of a nonsense mutation (30%) or by a frameshift mutation, and most are contained within the 5`-half of the gene. 3. Inherited Colon Cancer e. Germline Mutation and Truncated Protein

106. In germline mutations there are two hot spots at codons 1061 and 1309, with the most common mutation being an AAAAG deletion at codon 1309. These mutations leave the truncated APC protein unable to regu­late  -catenin. 3. Inherited Colon Cancer e. Germline Mutation and Truncated Protein

107. Genotype-phenotype correlations exist for some APC mutations. Patients with truncating mutations at the extreme 5`-end of the gene (codons 1 to 163) or mutations at the carboxyl-terminal end of the gene (codons 1860 to 1987) have the attenuated form of the disease, developing a smaller number of polyps (<100). 3. Inherited Colon Cancer f. Genotype-phenotype correlations

108. A severe phenotype is observed in patients with mutations between codons 1250 and 1464, and patients with truncating mutations between codons 1403 and 1578 are at an increased risk for extra­colonic disease. 3. Inherited Colon Cancer f. Genotype-phenotype correlations

109. The PTT is most often used to test for mutations in a family because the majority of FAP mutations result in a truncated protein. Once a possible mutation has been detected, the region is sequenced to determine the precise identity of the mutation. 3. Inherited Colon Cancer g. DNA Testing

110. Other screening or direct detection methodologies can be used, and collectively a sen­sitivity of about 87% can be achieved. If the mutation within the family can be identified, it is recommended that the family be referred for genetic counseling. DNA testing should be performed in at-risk family members as young as 10 to 12 years of age. Genetic testing can significantly alter the 50% pretest risk for disease to a risk of 0% or 100%. 3. Inherited Colon Cancer g. DNA Testing

111. 3. Inherited Colon Cancer

112. The most common inherited CRC susceptibility syn­drome is HNPCC, which represents about 2% to 3% of all CRC cases. In contrast to FAP, HNPCC is characterized by a few polyps that possess an accelerated transformation poten­tial to carcinoma in as little as 1 to 2 years. HNPCC is inher­ited as an autosomal dominant disorder with a penetrance of 80% to 85%. HNPCC patients have a lifetime risk of 70% to 80% for developing CRC, thereby suggesting a role for other factors in this disease process. 3. Inherited Colon Cancer h. HNPCC

113. Mutations in six mismatch repair genes have now been linked to HNPCC. The first was mapped to 2p 15-16. Simultaneously, MSI was noted in a subset of sporadic CRC. One such gene, hMSH2, mapped to 2p15-16 and in fact was found to be altered in HNPCC kindreds . 3. Inherited Colon Cancer i . Genes Linked to HNPCC

114. Subsequently, mutations in genes hMSH6 (2pI5-16), hMLHI (3p21), hPMSI (2q3l), hPMS2 (7p22), and hMLH3 (14q24.3) have been identified in HNPCC families, although more than 90% of HNPCC mutations are observed in hMSH2 and hMLHI . 3. Inherited Colon Cancer i . Genes Linked to HNPCC

115. HNPCC mutations are diverse and are located through­out these genes. Almost all errors made during DNA repli­cation are repaired through the proofreading 3'-to-5' exonuclease activity of DNA polymerase. Uncorrected errors of mismatched bases between the two strands are repaired before cell division through the MMR proteins 3. Inherited Colon Cancer j. HNPCC and Errors in Mismatch Repair

116. Testing of tumor tissue for MSI is the initial laboratory step in investigation of HNPCC patients because MSI is a measure of MMR deficiency and indicates probable defects in MMR genes through germline and somatic changes, International guidelines for analysis of MSI in CRC recommend a panel of five markers: BAT25, BAT26, D5S346, D2S123, and D 17S250. MSI is characterized by the expansion or contraction of DNA sequences through the insertion or deletion of repeated sequences. 3. Inherited Colon Cancer l. MSI Testing, Sequence Analysis and PTT

117. THANK YOU