11MCB 140 12-4-06Nosce te ipsum:the human genomePart II: genetics, diagnostics, and gene therapy of inherited disease2MCB 140 12-4-06Important distinction1. “Monogenic disorders” –human diseases whose etiology can in some more or less linear fashion be traced to a single-locus genetic lesion.2. Diseases with a “genetic component” or a “genetic predisposition” – disorders that mankind is known to be genetically polymorphic for (in terms of susceptibility) at multiple loci.3. All other disease (that may or may not be transcription based).1. Phenomena affecting ploidy (e.g., aneuploidies such as Down, Edwards, Turner, Klinefelter).2. Phenomena affecting chromosome structure (e.g., translocations in leukemia).3. Phenomena affecting single loci (genes or relatively small chromosomal segments).3MCB 140 12-4-06Archibald Garrod (1902)Higher frequency of children with alkaptonuria (urine turns dark on standing and alkalinization) from consanguineous marriages.Why?“There is no reason to suppose that mere consanguinity of parents can originate such a condition as alkaptonuria in their offspring, and we must rather seek an explanation in some peculiarity of the parents, which may remain latent for generations… It has recently been pointed out by Bateson that the law of heredity discovered by Mendel offers a reasonable account of such phenomena. …””http://www.esp.org/foundations/genetics/classical/ag-02.pdfGarrod (1902) Lancet 2: 116.4MCB 140 12-4-06Garrod (1902) Lancet 2: 116.5MCB 140 12-4-06Sickle-cell anemia – a brief history“In the western literature, the first description of sickle cell disease was by a Chicago physician, James B. Herrick, who noted in 1910 that a patient of his from the West Indies had an anemia characterized by unusual red cells that were sickle-shaped.”By 1923, it was realized the condition is hereditary.In 1949, Neel realized that patients with SCA are homozygous, and heterozygous carriers have a much milder condition (sickle cell trait).6MCB 140 12-4-06Sickle cell anemiaNIH:“Sickle cell anemia is the most common inherited blood disorder in the United States, affecting about 72,000 Americans or 1 in 500 African Americans. SCA is characterized by episodes of pain, chronic hemolytic anemia and severe infections, usually beginning in early childhood.”27MCB 140 12-4-06Linus Pauling, 1949: HbS has different charge!!8MCB 140 12-4-06V. Ingram, Nature 1956“On [the existing] evidence alone, it is not possible to decide whether the difference between the proteins, which is in any event small, lies in the amino-acid sequences of the polypeptide chains, or whether it lies in the folding of these chains leading to the masking of some amino-acid side chains.”Experiment:1. Digest Hb A and Hb S with trypsin (protease – cuts hemoglobin into ~30 peptides).2. Separate resulting fragments by electrophoresis, and then by chromatography.3. Trace the peptide map.V. Ingram (1956) Nature 178: 792.9MCB 140 12-4-06V. Ingram (1956)Nature 178: 792.10MCB 140 12-4-06“One can now answer at least partly the question put earlier, and say there there is a difference in the amino-acid sequence in one small part of one of the polypeptide chains. This is particularly interesting in view of the genetic evidence that the formation of hemoglobin S is due to a mutation in a single gene.”V. Ingram (1956) Nature 178: 792.11MCB 140 12-4-0611.712MCB 140 12-4-06SCA1. Fairly homogeneous genetic basis – an A-to-T transversion in the sixth codon of the HBB gene that leads to a glu → val substitution (→ RFLP!)2. In North America, heterozygosity for mutant allele is largely asymptomatic (sickle cell trait), because concentration of hemoglobin S is not high enough for the erythrocytes to sickle.3. In areas with high incidence of malaria, the fitness of heterozygotes is greater than of noncarriers or affected individuals (overdominance) because carriers are relatively malaria-resistant, explaining the high frequency of this allele.4. Therapy – hydroxyurea (wakes up embryonic and fetal globin genes), morphine for the pain, and prophylactic penicillin. “Sickle cell anaemia. A simple disease with no cure” (Nature 1989).313MCB 140 12-4-06“Functional cloning”In the case of alkaptonuria, sickle cell anemia, and blood clotting disorders such as hemophilia , the disease genes is identified based on some biochemical or other defect exhibited by the patient.What if the defect cannot be traced in a simple way to a biochemical phenomenon?14MCB 140 12-4-06Cystic fibrosis• Most common monogenic autosomal human genetic disorder – 1 in every 2000 live births.•q2=0.05%; q=2.2%; p=97.8%; 2pq=4% carriers.• Complex dysfunction of the lungs and the pancreas.15MCB 140 12-4-06Life expectancy of CF patients16MCB 140 12-4-06Mapping by linkage(“positional cloning”)If a given marker is linked (=is on the same chromosome as) to the gene mutations in which cause a certain disease, then one should be able to observe coinheritance of some allelic form of that marker to the occurrence of the disease.“Coinheritance” = occurrence in genotype of two loci with a frequency higher than Mendel’s second law allows.17MCB 140 12-4-0611.1718MCB 140 12-4-06Mendel’s second law419MCB 140 12-4-064.1720MCB 140 12-4-0621MCB 140 12-4-06Very simple and astonishingly influential consequence of all this stuffTwo markers located on different chromosomes will segregate away from each other in one out of two meioses.Two markers that are on the same chromosome will tend to stay together.22MCB 140 12-4-06Fig. 1 NolanNolan et al. Nat. Genet. (2000). 25: 440.a, Nanomouse (Nano), GENA50. b, Dominant spotting (KitW-39H), GENA133. c, A microphthalmia mutant, GENA163. d,e, Batface, a craniofacial mutant, GENA123.23MCB 140 12-4-06William Ernest Castle – founder of mouse genetics1. Inbreeding as a tool for making genetically uniform strains of mice that are homozygous for every allele in the genome.2. Brother-sister matings – makes 12.5% of all loci in the genome homozygous (Clarence Little).After 40 generations of brother- sister mating, >99.98% of genome is homozygous. By F60, mice are considered genetically identical to one another.24MCB 140 12-4-0611.12525MCB 140 12-4-06A B C DA B C DE F G HE F G HI J K LI J K L1 2 3♂♀26MCB 140 12-4-06A B C DA B C DE F G HE F G HI J K LI J K L1 2
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