2. HUMAN MOLECULAR GENETICS N7-2006 L. Duroux Slides assembled from diverse sources

3. Recommended reading list - textbooks  Human Molecular Genetics 3 Strachan & Read Strachan & Read Garland Publishing, ISBN 0-8153-4182-2Garland Publishing, ISBN 0-8153-4182-2  Principles of Medical Genetics Gelehrter, Collins & Ginsburg Gelehrter, Collins & Ginsburg Lippincott, Williams & Wilkins, ISBN 0683034456Lippincott, Williams & Wilkins, ISBN 0683034456  Genetics in Medicine Nussbaum, McInnes & Willard Nussbaum, McInnes & Willard Elsevier, ISBN 0721602444Elsevier, ISBN 0721602444

4. Journals  Nature Genetics http://www.nature.com/ng/index.html http://www.nature.com/ng/index.html http://www.nature.com/ng/index.html  Nature Reviews Genetics http://www.nature.com/nrg/index.html http://www.nature.com/nrg/index.html http://www.nature.com/nrg/index.html  Trends in Genetics http://www.trends.com/tig/default.htm http://www.trends.com/tig/default.htm http://www.trends.com/tig/default.htm

5. Lecture Plan 1. Examples of genetic diseases in Humans 2. Meiosis & Recombination 3. Mendelian Genetics 4. Modes of Heredity 5. Glossary and Standards

6. 1. Genetic Diseases in Humans

7. Role of Genes in Human Disease  Most diseases / phenotypes result from the interaction between genes and the environment  Some phenotypes are primarily genetically determined Achondroplasia Achondroplasia  Other phenotypes require genetic and environmental factors Mental retardation in persons with PKU Mental retardation in persons with PKU  Some phenotypes result primarily from the environment or chance Lead poisoning Lead poisoning

8. 100% Environmental Struck by lightning Infection Weight Cancer Diabetes Height Sex, Down syndrome, achondroplasia 100% Genetic Hair Colour

9. Clinical Genetics Consultant Cytogenetics Lab Molecular Genetics Lab A Medical Genetics Unit Clinical diagnosis Genetic counselling Genetic counselling Risk assessment Risk assessment Prenatal & presymptomatic diagnosis Prenatal & presymptomatic diagnosis Medical genetics in the health service

10. Types of Genetic Disorders 1. Chromosomes and chromosome abnormalities 2. Single gene disorders 3. Polygenic Disorders 4. Mutation and human disease

11. Chromosomal disorders  Addition or deletion of entire chromosomes or parts of chromosomes  Typically more than 1 gene involved  1% of paediatric admissions and 2.5% of childhood deaths  Classic example is trisomy 21 - Down syndrome

12. Down Syndrome KARYOTYPE

13. Single gene disorders  Single mutant gene has a large effect on the patient  Transmitted in a Mendelian fashion  Autosomal dominant, autosomal recessive, X-linked, Y-linked  Osteogenesis imperfecta - autosomal dominant  Sickle cell anaemia - autosomal recessive  Haemophilia - X-linked

14. Neonatal fractures typical of osteogenesis imperfecta, an autosomal dominant disease caused by rare mutations in the type I collagen genes COL1A1 and COL1A2 A famous carrier of haemophilia A, an X-linked disease caused by mutation in the factor VIII gene Sickle cell anaemia, an autosomal recessive disease caused by mutation in the  -globin gene

15. Autosomal dominant pedigree

16. Polygenic diseases  The most common yet still the least understood of human genetic diseases  Result from an interaction of multiple genes, each with a minor effect  The susceptibility alleles are common  Type I and type II diabetes, autism, osteoarthritis

17. Polygenic disease pedigree

18. 2. Meiosis & Genetic Recombination

19. DNA abc genes unreplicated pair of homologs Are long stable DNA strands with many genes. Occur in pairs in diploid organisms. The two chromosomes in a pair are called “homologs” Homologs usually contain the same genes, arranged in the same order Homologs often have different alleles of specific genes that differ in part of their DNA sequence. Chromosomes & Genes

20. From Griffiths et al. Introduction to Genetic Analysis W. H. Freeman 2000 The number of chromosomes per cell varies in different species

21. Chromosome Structure a a unreplicated chromosome telomeres centromere replicated chromosome sister chromatids Each chromatid consists of a very long strand of DNA. The DNA is roughly colinear with the chromosome but is highly structured around histones and other proteins which serve to condense its length and control the activity of genes.

22. Telomeres Centromere Specialized structures at chromosome ends that are important for chromosome stability. A region within chromosomes that is required for proper segregation during meiosis and mitosis. Key chromosomal regions

23. Mitosis Goal is to produce two cells that are genetically identical to the parental cell. Meiosis Goal is to produce haploid gametes from a diploid parental cell. Gametes are genetically different from parent and each other. Two types of cell divisions

24. Homologs and Sisters a Sister chromatids unreplicated homologs replicated homologs

25. In mitosis the homologs do not pair up. Rather they behave independently. Each resultant cell receives one copy of each homolog. Mitosis a 2n 4n 2n

26. In meiosis the products are haploid gametes so two divisions are necessary. Prior to the first division, the homologs pair up (synapse) and segregate from each other. In the second meiotic division sister chromatids segregate. Each cell receives a single chromatid from only one of the two homologs. Meiosis a 2n4n 2n1n III

27. Meiosis/perfect linkage a a PL p l PL p l PL p l PL PL p l p l PL p l p l PL only parental-type gametes

28. Meiosis w/recombination a a PL p l PL p l PL Pl p L p l Pl p L p l PL In some meiotic divisions these recombination events between the genes will occur resulting in recombinant gametes.

29. chiasma Chiasma Meiotic recombination in a grasshopper: Chiasma

30. Mitosis vs Meiosis One Division Homologues do not pair Centromeres divide Each cell inherits both homologues Mitosis is conservative producing daughter cells that are like parental cell. Two Divisions Homologues Pair up In meiosis I, centromeres do not divide Homologues segregate from each other. Meiosis is not conservative, rather it promotes variation through segregation of chromosomes and recombination

31. 3. Mendelian Genetics The laws of heridity

32. Gregor Mendel: “Father of Genetics” Augustinian Monk at Brno Monastery in Austria (now Czech Republic) Not a great teacher but well trained in math, statistics, probability, physics, and interested in plants and heredity. While assigned to teach, he was also assigned to tend the gardens and grow vegetables for the monks to eat. Mountains with short, cool growing season meant pea (Pisum sativum) was an ideal crop plant.

33. Contributions in 1860s (US Civil War Era) Discovered Genes as Particles of Inheritance Discovered Patterns of Inheritance Discovered Genes Come from Both Parents  Egg + Sperm = Zygote  Nature vs Nurture  Sperm means Seed (Homunculus) Discovered One Form of Gene (Allele) Dominant to Another Discovered Recessive Allele Expressed in Absence of Dominant Allele http://academic.evergreen.edu/v/vivianoc/homunculus.gif

34. Mendel worked with peas (Pisum sativum) Good choice for environment of monastery Network provided unusual varieties for testing Obligate self-pollination reproductive system  Permits side-by-side genetic barriers  Cross-pollinations require intentional process Crosses meticulously documented Crosses numerically/statistically analyzed Work lost in journals for 50 years! Rediscovered in 1900s independently by 3 scientists Recognized as landmark work!

35. Tall P Dwarf x F1 All Tall Phenotyp e One Example of Mendel’s Work Clearly Tall is Inherited… What happened to Dwarf? F1 x F1 = F2 F2 3 / 4 Tall 1 / 4 Dwarf Dwarf is not missing…just masked as “recessive” in a diploid state… there IS a female contribution. 1.Tall is dominant to Dwarf 2.Use D/d rather than T/t for symbolic logic DD dd Dd Genotype Homozygous Dominant Homozygous Recessive Heterozygous DwarfddTallDdd TallDdTallDDD dD Punnett Square: possible gametes

36. 1. The Law of Segregation: Genes exist in pairs and alleles segregate from each other during gamete formation, into equal numbers of gametes. Progeny obtain one determinant from each parent. 2. The Law of Independent Assortment Members of one pair of genes (alleles) segregate independently of members of other pairs. Two fundamental laws derived from Mendel’s work

37. After rediscovery of Mendel’s principles, an early task was to show that they were true for animals And especially in humans

38. Problems with doing human genetics: Can’t make controlled crosses! Long generation time Small number of offspring per cross So, human genetics uses different methods

39. Chief method used in human genetics is pedigree analysis I.e., the patterns of distribution of traits in kindreds

40. Pedigrees give information on: Dominance or recessiveness of alleles Risks (probabilities) of having affected offspring

41. Standard symbols used in pedigrees

42. 4. Modes of Heredity

43. Autosomal Dominant First pedigree of this type: Farabee 1903 Brachydactyly

44. Autosomal Dominant Most dominant traits of clinical significance are very rare So, most matings that produce affected individuals are of the form: Aa x aa

45. Autosomal Dominant Requirements for ideal auto. dom. pedigree: Every affected person must have at least 1 affected parent

46. Autosomal Dominant Requirements for ideal auto. dom. pedigree: Both males and females are affected and capable of transmitting the trait

47. Autosomal Dominant Requirements for ideal auto. dom. pedigree: No skipping of generations

48. Autosomal Dominant Requirements for ideal auto. dom. pedigree: No alternation of sexes: we see father to son, father to daughter, mother to son, and mother to daughter

49. Autosomal Dominant Requirements for ideal auto. dom. pedigree: In the usual mating, expect 1/2 affected, 1/2 unaffected

50. Example: Achondroplasia  Short limbs, a normal-sized head and body, normal intelligence

51. Caused by mutation in the FGFR3 gene  Fibroblast growth factor receptor 3 Inhibits endochondral bone growth by inhibiting chondrocyte proliferation and differentiation Inhibits endochondral bone growth by inhibiting chondrocyte proliferation and differentiation  Mutation causes the receptor to signal even in absence of ligand

52. extracellular intracellular Normal FGFR3 signaling FGFR3 FGF ligand

53. extracellular intracellular Normal FGFR3 signaling Inhibition of bone growth

54. extracellular intracellular Achondroplasia Receptor signals in absence of ligand Receptor signals in absence of ligand Bone growth attenuated Bone growth attenuated Gly380Arg mutation in transmembrane domain *

55. Autosomal recessive Affected persons must be homozygous for the disease allele These are likely to be more deleterious than dominant disorders, and so are usually very rare Thus, the usual mating is: Aa x Aa

56. Autosomal recessive Features of recessive pedigrees: Both parents are normal, but may see multiple affected individuals in the sibship, even though the disease is very rare in the population

57. Autosomal recessive Features of recessive pedigrees: Usually see “skipped” generations. Because most matings are with homozygous normal individuals and no offspring are affected

58. Autosomal recessive Features of recessive pedigrees: Expect increased consanguinity between the parents. That is, the parents are more likely to be relatives

59. Examples of autosomal recessive diseases Sickle-cell anemia Cystic fibrosis

60. X-linked Recessive Features of X-linked recessive inheritance: Act as recessive traits in females, but dominant traits in males

61. X-linked Recessive Features of X-linked recessive inheritance: An affected male cannot pass the trait on to his sons, but passes the allele on to all his daughters, who are unaffected carriers

62. X-linked Recessive Features of X-linked recessive inheritance: A carrier female passes the trait on to 1/2 her sons

63. X-linked Recessive About 70 pathological traits known in humans Examples: Hemophilia A, fragile X syndrome, Duchenne muscular dystrophy, color blindness

64. Summary of mutations which can cause a disease  Three principal types of mutation Single-base changes Single-base changes Deletions/Insertions (indels) Deletions/Insertions (indels) Unstable repeat units Unstable repeat units  Two main effects Loss of function Loss of function Gain of function Gain of function

65. 5. Genetic Linkage Mapping a disease Locus

66. Although Mendel's Law of Independent Assortment applies well to genes that are on different chromosomes. It does not apply well to two genes that are close to each other on the same chromosome. Such genes are said to be “linked” and tend to segregate together in crosses. Linkage

67. Why map and characterize disease genes?  Can lead to an understanding of the molecular basis of the disease  May suggest new therapies  Allows development of DNA-based diagnosis - including pre-symptomatic and pre-natal diagnosis

68. First question to ask in a mapping exercise  Are there functional or cytogenetic clues? Functional Clues Osteogenesis imperfecta(OI) Collagen I Haemophilia A Factor VIII Haemophilia B Factor IX Cytogenetic Clues Duchenne muscular dystrophy Translocation at Xp21 Polyposis coli Deletions in 5q

69. If there are clues, then one can target a particular gene or a particular chromosomal region If there are no clues, then one needs to conduct a genome-wide linkage scan

70. Linkage analysis  The mapping of a trait on the basis of its tendency to be co-inherited with polymorphic markers Recombination  The exchange (crossing over) of DNA between members of a chromosomal pair, usually in meiosis

71. Basic rules of linkage  Loci on different chromosomes will not be co- inherited i.e. locus A on chromosome 1 will not be co-inherited with locus B on chromosome 2 i.e. locus A on chromosome 1 will not be co-inherited with locus B on chromosome 2  Loci on the same chromosome will be co- inherited****  The closer two loci are on the same chromosome the greater the probability that they will be co-inherited i.e the likelihood of recombination is small i.e the likelihood of recombination is small

72. Consider the following pair of genes from the sweet pea that are located on the same chromosome: Trait affectedAlleles Phenotype PurpleFlower color p Ppurple red LongPollen lengthLLong l short Gene Purple Sweat Pea Purple & Long

73. Test cross - more clearly reveals what gametes (and how many) were contributed by the F1 dihybrid. P/P L/L X p/p l/l F1 P/p L/lXp/p l/l "tester" F2Score progeny (total = 2840) Test cross

74. Recombination is very precise -- During meiosis chromosomes pair and align with homologous genes in exact opposition. This allows crossovers between genes at the exact same nucleotides. abcdef abcdef ------AGCCCGTTAAGC------ Note: this diagram does not represent the actual molecular mechanism of recombination-- only the result. ------AGCCCGTTAAGC------ Recombination precision

75. Recombination mapping abc ABC Consider a chromosome segment with three genes that can be followed in a cross: Will there be more recombination between A and B or between B and C ?Mapping

76. Recombination mapping Recombination frequency is a direct measure of the distance between genes. The higher the frequency of recombination (assortment) between two genes the more distant the genes are from each other. A map distance can be calculated using the formula: # recombinant progeny /total progeny X 100 = map distance (% recombination) 1 map unit = 1% recombination = 1 centimorgan Calculation of Recombination Frequency

77. CentiMorgan (cM)  Thomas Hunt Morgan  cM is the unit of genetic distance Loci 1cM apart have a 1% probability of recombination during meiosis Loci 1cM apart have a 1% probability of recombination during meiosis Loci 50cM apart are unlinked Loci 50cM apart are unlinked

78. Logarithm of odds (LOD) score  The logarithm (in base 10) of the odds of linkage the ratio of the likelihood that loci are linked to the likelihood that they are not linked the ratio of the likelihood that loci are linked to the likelihood that they are not linked  A LOD of 3.0 = odds of 1000/1 in favour of linkage Equivalent to a 5% chance of error Equivalent to a 5% chance of error

79. gameteszygote phenotype observed P LP/p L/lPurple long1340parental type P lP/p l/lpurple short 154 recombinant p Lp/p L/lred long 151recombinant p lp/p l/lred short 1195parental type 2840TOTAL Calculation of map distance between the P and L genes # recombinant progeny /total progeny X 100 = map distance 305 were recombinants (154 P l + 151 p L) 305/2840 X 100 = 10.7 map units or 10.7% recombination frequency

80. Recombination frequencies for a third gene (X) were determined using the same type of cross as that used for P and L.. P to L10.7 map units P to X13.1 map units X to L2.8 map units Map 13.1 units P-------------------------------L--------------X 10.7 units2.8 units We can deduce from this that L is between P and X and is closer to L than it is to P. Thus it is possible to generate a recombination map for an entire chromosomes. Build a map

81. Chromosomes and Linkage The maximum frequency of observed recombinants between two genes is 50%. At this frequency the genes are assorting independently (as if they were on two different chromosomes) dpy bw ho 413104 The dpy and bw are 91 map units apart. How frequently will these alleles become separated (total % nonparentals types in a test cross)? DpyHoBw

82. Chromosomes and Linkage The maximum frequency of observed recombinants between two genes is 50%. At this frequency the genes are assorting independently (as if they were on two different chromosomes). A a B b 50% parental gametes (AB, ab) 50% non-parental gametes (aB, Ab) A a B b If on the same chromosome, but greater than 50 map units apart, crossovers will actually occur > 50% of the time but multiples will cancel each other out. A a B b parental gametes (AB, ab) non-parental gametes (aB, Ab)

83. A B a b Infinite Genetic Distance Genes that are greater than 50 map units apart undergo frequent recombination events and thus segregate randomly in relation to each other. Their position on the same chromosome is determined by constructing a map with multiple genes that are more close linked. Bottom line: Two genes can be on the SAME chromosome but will behave as if they are unlinked in a test cross.

84. Molecular markers are most often variations in DNA sequence that do not manifest a phenotype in the organism. However they can be used to map genes in the same way that markers affecting visible phenotypes are. An example of this would be a restriction fragment length polymorphism restriction sites Gene of interest Recombination mapping using molecular markers

85. Human linkage map From Griffiths et al. Introduction to Genetic Analysis W. H. Freeman 2000

86. Determine the genotype of each family member for polymorphic markers across the genome Practicalities of Linkage Analysis Chrom. 1 Chrom. 2 Chrom. 3 etc

87. A polymorphic marker A marker that is frequently heterozygous in the population One can therefore distinguish the two copies of a gene that an individual inherits They are not themselves pathological - they simply mark specific points in the genome

88. Polymorphic markers used in mapping studies  Variable number tandem repeats (VNTRs)  Microsatellites  Single nucleotide polymorphisms (SNPs)

89. VNTRs Changes in the numbers of repeated DNA sequences arranged in tandem arrays ACGTGTACTC 3-repeat allele 4-repeat allele

90. Microsatellites Particular class of VNTR with repeat units of 1-6bp in length Also known as short tandem repeats (STRs) and sometimes as simple sequence repeats (SSRs) The most widely used are the CA n microsatellites CACACACACACA CACACACACACACACA 6 (CA) allele 8 (CA) allele

91. SNPs A polymorphism due to a base substitution or the insertion or deletion of a single base TCGAGAGGCTAGGCTAGGA TCGAGAGGCCAGGCTAGGA Substitution T-allele C-allele TCGAGAGGCTAGGCTAGGA TCGAGAGGCAGGCTAGGA Insertion/deletion (+) allele (-) allele

92. 6 (CA) allele 8 (CA) allele The individuals genotype is (6 8) The genotype for a microsatellite marker on chromosome 1 Paternal copy Maternal copy **

93. 6 66 66 66 6 6 8 6 6 6 86 86 86 8 6 86 86 86 8 6 8 6 86 86 86 8 6 86 86 86 8 9 10 8 9 6 10 Uninformative Completelyinformative Uninformative and informative meioses 6 66 66 66 6 6 6 6 66 66 66 6

94. 1 Disease gene An autosomal dominant disease for which the gene resides on chromosome 1 But you don’t know that!

95. Disease gene

96. 5 6 4 7 2 3 Marker studied

97. Disease gene 2 3 1 5 4 4 Marker studied

98. Disease gene 1 5 3 5 6 7 Marker studied

99. Disease gene 2 4 2 5 2 7 Marker studied

100. Disease gene 1 3 1 2 4 5 Marker studied

101. Disease gene 2 4 2 5 2 7 Marker studied

102. (4) (24) (5) (25) (7) (27) (3) (23)(16) (14) (6) (26) (46) (34)(13) (33)(14) (58) (1) (12) (18) (13)(78) (18) (6) (26)(47) (4) (24)(46)(67) Genotype data for the whole family

103. Disease gene The next step - define the maximal region of linkage Gene resides here

104. And then? Make a list of the genes within the interval www.ensembl.org

105. Gene content of chromosome 1

106. Genes within a linkage region

107. And finally? Find the mutation! Target candidate genes within the interval or Target all genes by DNA sequencing

108. Two important considerations for single-gene disorders  Allelic heterogeneity The existence of many different disease- causing alleles at a locus The existence of many different disease- causing alleles at a locus  Locus heterogeneity Determination of the same disease or phenotype by mutations at different loci Determination of the same disease or phenotype by mutations at different loci

109. What about mapping polygenic disorders? Gene1 Gene 2 Gene 3 Gene 4 PHENOTYPE Environment

110. Disorder Frequency (%) SchizophreniaAsthma Hypertension (essential) Osteoarthritis Type II diabetes (NIDDM) 1 4 5 5 6 Polygenic diseases are common Unrelated affected individuals share ancestral risk alleles

111. Affected individual joining the family, emphasizing the common nature of the disease An affected individual with unaffected parents A polygenic phenotype No clear inheritance pattern

112. Summary  Mapping single gene disorders Use clues Use clues If none, genome-wide linkage analysis If none, genome-wide linkage analysis A large pedigreeA large pedigree Several smaller pedigree - but beware locus heterogeneity!Several smaller pedigree - but beware locus heterogeneity! DNA sequence analysis of linked region DNA sequence analysis of linked region  Mapping polygenic disorders Model-free genome-wide linkage analysis Model-free genome-wide linkage analysis Now being superseded by genome-wide association analysisNow being superseded by genome-wide association analysis Functional analysis of associated polymorphisms within the refined genomic interval Functional analysis of associated polymorphisms within the refined genomic interval

113. Conclusions  For a single gene disease identifying the causal mutation is now relatively straightforward  Technological and analytical advances are also making polygenic diseases tractable  Genetics is going to play an ever increasing role in medical diagnosis and in the development of improved treatment regimes

114. Additional Material

115. The chromosomal basis of Mendel’s laws Yellow-round seeds (YYRR) Green-wrinkled seeds (yyrr) Meiosis Fertilization Gametes All F 1 plants produce yellow-round seeds (YyRr) P Generation F 1 Generation Meiosis Two equally probable arrangements of chromosomes at metaphase I LAW OF SEGREGATION LAW OF INDEPENDENT ASSORTMENT Anaphase I Metaphase II Fertilization among the F 1 plants 9: 3 : 1 1414 1414 1414 1414 YRyr yR Gametes Y R R Y y r r y R Y yr R y Y r R y Y r R Y r y rR Y y R Y r y R Y Y R R Y r y r y R y r Y r Y r Y r Y R y R y R y r Y F 2 Generation Starting with two true-breeding pea plants, we follow two genes through the F 1 and F 2 generations. The two genes specify seed color (allele Y for yellow and allele y for green) and seed shape (allele R for round and allele r for wrinkled). These two genes are on different chromosomes. (Peas have seven chromosome pairs, but only two pairs are illustrated here.) The R and r alleles segregate at anaphase I, yielding two types of daughter cells for this locus. 1 Each gamete gets one long chromosome with either the R or r allele. 2 Fertilization recombines the R and r alleles at random. 3 Alleles at both loci segregate in anaphase I, yielding four types of daughter cells depending on the chromosome arrangement at metaphase I. Compare the arrangement of the R and r alleles in the cells on the left and right 1 Each gamete gets a long and a short chromosome in one of four allele combinations. 2 Fertilization results in the 9:3:3:1 phenotypic ratio in the F 2 generation. 3 Physical basis of Mendel’s laws chromosome theory segregation independent assortment

116. Glossary & Definitions I  Character - a structure, function, or attribute determined by a gene or group of genes i.e. the appearance of the seed coat in Mendel’s garden pea studies i.e. the appearance of the seed coat in Mendel’s garden pea studies  Trait - the alternate forms of the character i.e “smooth” or “wrinkled” peas i.e “smooth” or “wrinkled” peas

117. Glossary & Definitions II  Phenotype - the physical description of the character in an individual organism i.e a green pea i.e a green pea  Genotype - the genetic constitution of the organism

118. Glossary & Definitions III  Locus - a chromosomal location  Alleles - alternative forms of the same locus  Mutation - a change in the genetic material, usually rare and pathological  Polymorphism - a change in the genetic material, usually common and not pathological

119. Glossary and Definitions IV  Homozygote - an organism with two identical alleles  Heterozygote - an organism with two different alleles  Hemizygote -having only one copy of a gene  Hemizygote - having only one copy of a gene Males are hemizygous for most genes on the sex chromosomes Males are hemizygous for most genes on the sex chromosomes

120.  Dominant trait - a trait that shows in a heterozygote  Recessive trait - a trait that is hidden in a heterozygote Glossary and Definitions V

121. A common misconception is that genes are dominant or recessive However, it is the trait that is dominant or recessive, not the gene

122. Standard pedigree symbols Male,affected Female,unaffected Male,deceased Mating Consanguineousmating Pregnancy Male, heterozygous for autosomal recessive trait Female, heterozygous for Autosomal or X-linked recessive trait recessive trait Dizygotic(non-identical)twins Monozygotic(identical)twins Spontaneous abortion or still birth