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Introduction to Gene Mapping Techniques Lecture 2

Introduction to Gene Mapping Techniques Lecture 2. Background Readings : Chapter 5 & 6 (190-193) of An introduction to Genetics, Griffiths et al. 2000, Seventh Edition.

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Introduction to Gene Mapping Techniques Lecture 2

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  1. Introduction to Gene Mapping TechniquesLecture 2 Background Readings: Chapter 5 & 6 (190-193) of An introduction to Genetics, Griffiths et al. 2000, Seventh Edition. This class has been edited from several sources. Primarily from Terry Speed’s homepage at Stanford and the Technion course “Introduction to Genetics”. Changes made by Dan Geiger. .

  2. Observed 1,339 151 154 1,195 The pair AB stick together more than expected from Mendel’s law. “Exceptions” to Mendel’s Second Law Morgan’s fruit fly data (1909): 2,839 flies Eye color A: red a: purple Wing length B: normal b: vestigial (not fully developed) AABB x aabb AaBb x aabb AaBb Aabb aaBb aabb Expected 710 710 710 710

  3. A A a a  B B b b a a A a  b B b b a a A a a a A a b b b b B B b b Crossover has taken place Morgan’s explanation F1: F2: 1,339 151 154 1,195

  4. The proportion of recombinants between the two genes (or characters) is called the recombination fraction between these two genes. It is usually denoted by RF or r or. For Morgan’s traits: r = (151 + 154)/2839 = 0.107 If r < 1/2: two genes are said to be linked. If r = 1/2: independent segregation (Mendel’s second law).

  5. Recombination Phenomenon(Happens during Meiosis) Recombination Haplotype Male or female תאי מין: ביצית, או זרע

  6. Homolog chromosomes showing Chaismata כרומוזומים הומולוגיים המראים כיאסמתה Sister chromatids הכיאסמה היא הביטוי הציטולוגי לשחלוף. Chaisma(ta) is the cellular expression of recombination.

  7. O A2/A2 A A1/A1 1 2 A A2/A2 A A1/A2 3 4 A | O A2 | A2 A O A1 A2 O O A2 A2 O O A1 A2 Recombinant O A1/A2 5 Example: ABO, AK1 on Chromosome 9 Phase inferred Recombination fraction is 12/100 in males and 20/100 in females. One centi-morgan means one recombination every 100 meiosis. One centi-morgan corresponds to approx 1M nucleotides (with large variance) depending on location and sex.

  8. צבע פלפל: אינטראקציה בין 4 גנים

  9. צבע פלפל: אינטראקציה בין 4 גנים Y : removal of green chlorophyll from fruit y : green chlorophyll in fruit R : Red carotenoid pigment r : yellow carotenoid pigment C1; C2 : Two genes with the same function, determine amount of carotenoids. c1; c2 : Recessive mutations, lower the amount of carotenoids. GenotypePhenotype r/r C1/C1 C2/C2 y/y green R/R C1/C1 C2/C2 Y/Y red R/R C1/C1 C2/C2 y/y brown r/r C1/C1 C2/C2 Y/Y yellow R/R C1/C1 c2/c2 Y/Y orange r/r c1/c1 c2/c2 Y/Y white

  10. אי התאמת רקמות:אינטראקציה בין 2 גנים קיימים 2 גנים HLA-A ו- HLA-B הקובעים את הטיפוס האימינולוגי של התא והרקמה. בגןHLA-A קיימים 8 אללים שונים: A1, A2, A3, A9, A10, A11, A28, A29 בגן HLA-B קיימים 8 אללים שונים: B5, B7, B8, B12, B13, B14, B18, B27 כאשר תורמים רקמה, קבלת השתל תלויה בכך שלתורם לא יהיו אללים שאינם נמצאים בנתרם. אללים אלו יצרו אנטיגנים שבנתרם יגרמו לתגובה אימונית, יצירת נוגדנים, ודחית השתל. דוגמאות: תוצאהגנוטיפ תורםגנוטיפ נתרם דחיה A1 A1 B5 B7A1 A2 B5 B5 דחיה A1 A2 B7 B7A2 A3 B7 B12 קבלה A2 A2 B7 B7A1 A2 B5 B7 קבלה A2 A3 B5 B5A2 A3 B5 B7

  11. Purpose of human linkage analysisTo obtain a crude chromosomal location of the gene or genes associated with a phenotype of interest, e.g. a genetic disease or an important quantitative trait.Examples: Cystic fibrosis (found), Diabetes, Alzheimer, and Blood pressure.

  12. LinkageStrategies I Traditional (from the 1980s or earlier) • Linkage analysis on pedigrees • Association studies: candidate genes • Allele-sharing methods: Affected siblings • Animal models: identifying candidate genes • Cell – hybrids Newer (from the 1990s) • Focus on special populations (Finland, Hutterites) • Haplotype-sharing (many variants)

  13. Linkage analysis

  14. D A2/A2 H A1/A1 1 2 H A2/A2 H A1/A2 3 4 H | D A2 | A2 H D A1 A2 D D A2 A2 D D A1 A2 Recombinant D A1/A2 5 Fictitious Example for Finding Disease Genes Phase inferred We use a marker with codominant alleles A1/A2. We speculate a locus with alleles H (Healthy) / D (affected) If the expected number of recombinants is low (close to zero), then the speculated locus and the marker are tentatively physically closed.

  15. Association Studies

  16. Healthy/Affected versus a bi-allelic Marker (X,x) So healthy status seems independent of marker X.

  17. The Chi-Square test Expected means under assumption of independence of H/A versus X/x. Using 2tables, with one degree of freedom, the assertion of independence is not rejected in this example; the probability of 2is much higher than 0.05.

  18. Morgan’s fruit fly data: Can we just be unlucky ? AABB x aabb AaBb x aabb AaBb AabbaaBbaabb Expected 710 710 710 710 Observed 1,339 151 154 1,195 Perhaps genes A and B segregate according to Mendel’s law but we happen to see data that does not support this rule ? What if the answer were: Fictitious 794 625 625 795

  19. The Chi-Square test for Morgan’s data Expected means under assumption of independence of the loci A and B. Use with care; the conversion to probability encodes technical assumptions. This translates to a tiny probability not appearing in the tables; so independence is strongly rejected.

  20. Allele-sharing methods Affected siblings that share an allele more than expected indicate that this allele is near a disease locus. Expected sharing is 1 (out of 2 alleles).

  21. Animal/Plant Breeding Methods Inappropriate for humans. Not practical for large mammals. Not covered in this course, which focuses on computation related to human genetics.

  22. מיפוי גנים לכרומוזוםבשיטות מעבדתיות דוגמא: איחוי בין תאי אדם לעכבר וירוס מסוים, המטופל בקרני- uv למניעת פעילותו, נקשר בו זמנית ל-2 תאים שונים, וגורם לממברנות התאים, אחד מהאדם ואחד מהעכבר, להתאחות. נוצר תא המכיל 2 גרעינים, ולאחר מכן הגרעינים מתאחים ונוצר תא היברידי בו גרעין המכיל את שני הסטים של הכרומוזומים (אדם ועכבר).

  23. המשך: איחוי בין תאי אדם לעכבר מסיבות שאינן ברורות, רוב כרומוזומי האדם נעלמים באופן רנדומלי, וכל תא היברידי מכיל סט שלם של כרומוזומי עכבר, ובין 1-4 כרומוזומי אדם. ניתן לעצור העלמות של כרומוזומי אדם ספציפיים במידה והם מכילים גן המייצר חלבון העמיד לתנאי התמיסה והאלל של לוקוס גן זה בעכבר אינו מייצר את החלבון הנדרש. בצורה זו מזוהה הגן עם אחד הכרומוזומים שנותרו בתא ההיברידי.

  24. על מצע מסוים (HAT medium) תאים נדרשים ליצר שני אנזימים כדי לגדול (TK,HGPRT). TK מיוצר כאשר לוקוס הגן המתאים הינוtk+. ו- HGPRT מיוצר כאשר לוקוס הגן המתאים הינו hgprt+. נבחר תאי עכבר הומוזיגוטים: tk- / tk- ; hgprt+ / hgprt+ ונבחר תאי אדם הומוזיגוטים: tk+ / tk+ ; hgprt- / hgprt- כדי שהתאים ההיברידיים יגדלו מתחייב שיישאר לפחות כרומוזום אנושי אחד ובו הגן המקודד את TK. צביעת הכרומוזומים נותנת דגם פספוס המאפשר זיהוי כל כרומוזומי האדם הספציפיים שנשארו בכל תא היברידי. המשך בשקופית הבאה

  25. Hybrid Cell line 1 2 3 4 5 6 7 8 A B C D Human chromosome present gene product - - + + 23 - - + + 24 - + + + 25 ניתן להשתמש בהיברידים אלו למפות גנים לכרומוסומים עי שימוש במגוון של מצעים. יכולת המיפוי תלויה ביכולתנו לזהות תוצר גן המאפשר גידול על מצע ספציפי (חלבון, אנזים) . למשל כאשר ידוע גן המקודד לחלבון המאפשר עמידות לתרופה, נבחר עכברים שאינם עמידים ותאים אנושיים עמידים. מסקנות: גן A: ממוקם לכרומוזום 5. גן B: ממוקם לכרומוזום 3. גן C: אינו ממוקם לכרומוזומים 1-7. גן D: ממוקם לכרומוזום 1.

  26. Linkage Strategies II On the horizon (here) • Single-nucleotide polymorphism (SNPs) • Functional analyses: finding candidate genes Needed (starting to happen) • New multilocus analysis techniques, especially • Ways of dealing with large pedigrees • Better phenotypes: ones closer to gene products • Large collaborations

  27. Horses for courses • Each of these strategies has its domain of applicability • Each of them has a different theoretical basis and method of analysis • Which is appropriate for mapping genes for a disease of interest depends on a number of matters, most importantly the disease, and the population from which the sample comes.

  28. The disease matters Definition (phenotype), prevalence, features such as age at onset Genetics: nature of genes (Penetrance), number of genes, nature of their contributions (additive, interacting), size of effect Other relevant variables: Sex, obesity, etc. Genotype-by-environment interactions: Exposure to sun.

  29. Example: Age at onset

  30. Example: Y-linked disease

  31. Example: X-linked disease נורמלי המופיליה עיוורון צבעים המופיליה + עיוורון צבעים שני הגנים בתאחיזה לכרומוזום X, וקיימת תאחיזה חלקית ביניהם.

  32. The population matters History: pattern of growth, immigration Composition: homogeneous or melting pot, or in between Mating patterns: family sizes, mate choice Frequencies of disease-related alleles, and of marker alleles Ages of disease-related alleles

  33. 106 years 105 years Bottleneck Effects

  34. Complex traits Definition vague, but usually thought of as having multiple, possibly interacting loci, with unknown penetrances; and phenocopies. Affected only methods are widely used. The jury is still out on which, if any will succeed. Few success stories so far. Important: heart disease, cancer susceptibility, diabetes, …are all “complex” traits. We focus more on simple traits where success has been demonstrated very often. About 6-8 percent of human diseases are thought o be simple Mendelian diseases.

  35. Design of gene mapping studies How good are your data implying a genetic component to your trait? Can you estimate the size of the genetic component? Have you got, or will you eventually have enough of the right sort of data to have a good chance of getting a definitive result? Power studies. Simulations.

  36. Genotyping A person is said to be typed if its markers have been genotyped. Choice of markers: highly polymorphic preferred. Heterozygosity and polymorphism information content (PIC)value are measures commonly used. Reliability of markers important too Good quality data critical: errors can play a surprisingly large role.

  37. Preparing genotype data for analysis Data cleaning is the big issue here. Need much ancillary data…how good is it?

  38. Analysis A very large range of methods/programs are available. Effort to understand their theory will pay off in leading to the right choice of analysis tools. Trying everything is not recommended, but not uncommon. Many opportunities for innovation.

  39. Interpretation of results of analysis An important issue here is whether you have established linkage. The standards seem to be getting increasingly stringent. What p-value or LOD should you use? Dealing with multiple testing, especially in the context of genome scans and the use of multiple models and multiple phenotypes, is one of the big issues.

  40. Replication of results This has recently become a big issue with complex diseases, especially in psychiatry. Nature Genetics suggested in May 1998 that they will require replication before publishing results mapping complex traits. Simulations by Suarez et al (1994) show that sample sizes necessary for replication may be substantially greater than that needed for first detection.

  41. Topics not mentioned Exclusion mapping, homozygosity mapping, interference, variance component methods, twin studies, and much more. Some of these topics plus others are covered in two books: Handbook of Human Genetic Linkageby J.D. Terwilliger & J. Ott (1994) Johns Hopkins University Press. Ordered, not available at the library. Analysis of Human Genetic Linkageby J. Ott, 3rd Edition (1999), Johns Hopkins University Press.

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