1Human disease genes summary1. Goals: discover the basis for disease, understand keyprocesses, and develop diagnostics and cures.2. Finding human disease genes -- OMIM3. Sickle Cell Anemia4. Inheritance and linkage5. RFLPs and chromosome “walking”6. Huntington’s disease -- Scientific suicide7. FutureSome examples of single-gene diseasesCommon?2Find disease genesAt OMIM (Online Mendelian Inheritance in Man)http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMThis database catalogs human genes and genetic disorders.The database contains textual information andreferences. It also contains links to MEDLINE andsequence records in the Entrez system, and links toadditional related resources.Sequencing HbS proteins revealed a single change: Glu6Val in the β chain. Fiber formation (R) at low [O2] causes sickling of RBCs (center).Gene finding 1. Sequence candidate genes or proteins3Autosome: not a sex chromosomeX, Y: sex chromosomes= Male (XY) = Female (XX)InheritanceMarkers separated by 1 centimorgan have a 1% chance ofbeing separated in meiosis.1 centimorgan corresponds to ~750,000 bp in humans!Linkage--Recombination during meiosisseparates genes1. Genes on differentchromosomes assortindependently2. Genes on the samechromosome arelinked3. This linkage is notabsolute4Gene finding 2. RFLP analysisLook for restriction fragment length polymorphism (RFLP) thatcorrelates with the inheritance pattern of the disease.Fig. 9-46. Three alleles of a RFLP onchromosome 5 in 14 individuals in 3generations. Each lane corresponds tothe individual above it.Can a gene be located by RFLP linkage? A “crazy” approach:1. Collect DNA from 100s of related individuals with and without thedisease.2. Establish their pedigrees without errors.3. Digest their DNA with various restriction enzymes.4. Probe Southern blots with RANDOM probes.5. Look for an RFLP that is inherited with the same pattern as thedisease.5Markers separated by 1 centimorgan have a 1% chance ofbeing separated in meiosis.1 centimorgan corresponds to ~750,000 bp in humans!For a “fully penetrant”, single-gene disease:Linkage of a RFLP to a disease in 99/100 patients impliesthe RFLP may be within 750 kbp of the disease mutation.In practice, many more patients are needed to get reliablelinkage statistics.Linkage mapping requires large patientpopulationsJim Gusella commits “scientific suicide” 1980: Gusella starts his first faculty job at Massachusetts General Hospitalwith the aim of finding an RFLP marker for Huntington’s disease.No one had ever found an RFLP marker for an unmapped disease gene.The approach was to screen for RFLPs using random human DNA probes. Asmany as 300 probes might be needed to cover the genome.At the time, there were two RFLP markers mapped in the entire humangenome. The largest accessible HD family had 27 members--too few toestablish tight linkage.David Botstein, an originator of the RFLP concept, estimated it would take10 years to find a marker linked to the HD gene!6More patients: HD families in Venezuela1952: Biochemist and physician, Dr. Americo Negrette diagnosesHuntington’s disease at Lake Maracaibo in Venezuela.1963: Negrette published Corea de Huntington: Estudio de unasola familia a través de várias generaciones (Huntington’sChorea: Study of a Single Family Through Several Generations)1972: Dr. Ramon Avila-Giron, a studentof Negrette’s, attended the CentennialSymposium on HD in Columbus, OH. Heshowed the 146 participants from 14countries a startling 20-minute, black-and-white film of several communitiesaround Lake Maracaibo ravaged by HD.Patient advocacy: funding to collect DNA in Venezuela1981: Nancy Wexler leads a US/Venezuelan project to definepedigrees and collect blood samples from HD families in thetowns on Lake Maracaibo in western Venezuela.--Genetically isolated--Large families--High HD incidence--All cases are believe to arise from asingle “founder” individual who settledin the area in the 1870s.PanamaColombia Venezuela7Linking genotype and phenotypeMarch 10, 1983: “…The meeting room in the modulo takes on a slightlycarnivalesque atmosphere as people from the barrio drift in, children dartingunderfoot, staring over shoulders, while the adults shoo them outside, where theypeer through the doorway or huddle at the windows. . . Taped around the walls ofthe room is the pedigree chart, a computer-generated system of lines, circles andsquares, like a Mondrian mural, that traces the relationships of all the local familieswith Huntington’s. . . Alice Wexler, Mapping FateLake Maracaibo, VenezuelaHalf fishing village, half urban slum, San Luis hugs the shore of Lake Maracaibo onthe sandy southern outskirts of the city, unremarkable except for Huntington’sdisease, which haunts almost every home. . .Today is a “draw day”, when those who have comeearlier for a neuorological exam will return to giveblood and a small skin sample. . . . The “draw” takesplace on the other side of the modulo, in a closet-sized, air-conditioned examining room. Fidela Gomez,an emergency room nurse who lives in Miami but wasborn in the Canary Islands and grew up in Argentina, iskey to this operation. She is so quick-witted andskillful with the needle that she draws blood beforeanyone knows what is happening. Fidela clips a tinysquare of skin from someone’s arm and draws the sixtubes of blood while bantering in rapid-fire Spanish,slowly shaking each dark red tube before handing theit over to an assistant, who applies identifying labelsand packs them gently into a Styrofoam box.8The 12th probe, G8, is linked to HDApril 1983: Ginger Weeks, a technician in the Gusella lab at MGH, developeda new human DNA probe. The probe comprised a unique 17.6-kb fragmentfrom an unknown location in the human genome.G8 showed an RFLP in HindIII-digested DNA. The RFLP gave a 65:1 chanceof being linked to the HD gene in an Iowa family of 27 members.July 1983: G8 revealed a 106:1 chance of being linked to the HD gene in ananalysis of RFLPs in a pedigree of 75 individuals from Lake MaracaiboNovember 1983: Results reported in Nature, Gusella appears on the TodayShow.HD gene: Ten years after1984-1992: 6.2 Mb of DNA from the short arm of chromosome 4 is cloned andmapped.February 1993: HD gene sequenced. Gusella names the protein Huntingtin.3144 amino
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