Genetics paediatric postgraduate topic presentation.pptx

Genetics paediatric postgraduate topic presentation.pptx

1. Genetics GENETICS Presented by PG 1 st Year Student Dr Aye Myat Mon

2. Molecular Basics of Genetic Diseases

3. Overview of Molecular Basics of Genetic Diseases Cell biology Cell cycle Mitosis and Meiosis Nucleic Acids DNA and RNA Protein Synthesis Mutation Use of Molecular Genetics in Diagnosis Chromosomes Chromosomal Analysis Types of chromosomal abnormalities

4. An A rchetypal Human Cell

5. G1 (6-12 hours) : RNA & protein is synthesized, There is no DNA replication S (6-8 hours) : Period of DNA replication G2 (3-4 hours) : The cell has 2 complete diploid sets of chromosomes M (1 hour) Mitosis : Period of actual cell division G0 state : Non- dividing cells e.g. neurons, do not go through this cycle & remain in resting state Cell Cycle

6. Regulation of cell cycle

7. Mitosis

8. Meiosis or Sexual Cell division

9. Fertilization

10. Human Genome A segment of DNA that codes for one polypeptide chain is called Gene ~ 25,000 genes that encode the wide variety of proteins found in the human body

11. Genetic code is degenerate i.e. some amino acids are coded by > 1 triplet codon Genome : complete set of genes of an organism Human Genome Project : International research program aimed at mapping all the genes in the human genome Genotype : Genetic constitution of an individual usually at a particular locus Phenotype :observable characteristics of an individual resulting from the interaction of its genotype with the environment

12. Alleles : Alternative forms of a gene found at the same locus on a particular chromosome Germ line cells >> 1 copy (N) of this genetic complement >> haploid Somatic (non-germ line) cells >> 2 complete copies (2N) >> diploid Hybridization : Joining of complementary sequences of DNA or DNA & RNA by base pairing

13. Where Is DNA?

14. DNA (Deoxyribonucleic Acid) Polymer made up of Nucleotide units Phosphate+ Sugar + Base = Nucleotide Sugar +Base = Nucleoside Bases Pyrimidines - cytosine (C), thymine (T) Purines - guanine (G), adenine (A) Bases are linked by hydrogen bonds A always pairs with T and G with C Sugar – Deoxyribose Double – stranded Two linear but antiparallel and complementary strands Double helix

15. Each diploid cell >> 23 pairs of chromosomes >> 3.5x 10 9 base pairs ~ 90% of the cell’s DNA >> nucleoprotein called chromatin (DNA+ both histone and non histone proteins)

16. Mitochondrial DNA The cell’s energy-producing organelles Contain their own unique genome D ouble-stranded circular piece of DNA contains 16,568 base pairs of DNA present in multiple copies per cell

17. Codes for components of respiratory chain, ribosomal RNA & transfer RNA Sperm do not usually contribute mitochondria to the developing embryo All mitochondria are maternally derived and a child’s mitochondrial genetic makeup derives exclusively from the child’s biological mother

18. RNA (Ribonucleic Acid) 90% in cell cytoplasm Bases Pyrimidines - cytosine (C), uracil (U) Purines - guanine (G), adenine (A) Sugar – R ibose Single – stranded Three forms Messenger RNA (mRNA) Transfer RNA ( tRNA ) Ribosomal RNA ( rRNA )

19. RNA

20. Three forms of RNA

21. Protein Synthesis I nformation encoded in DNA , predominantly located in the cell nucleus T ranscribed into messenger RNA ( mRNA) T ransported to the cytoplasm T ranslated into protein

22. DNA Replication Original 2 strands > unwind by breaking the hydrogen bonds between base pairs Free nucleotides > new hydrogen bonds with their complementary bases on the parent strand New phosphodiester bonds by enzyme DNA polymerase S imultaneously at multiple sites Replication bubbles expand bi-directionally until the entire DNA molecule (chromosome ) is replicated

23. DNA Replication Cont. Semi-conservative E ach daughter molecule receives one strand from parent DNA Discontinuous replication One or both DNA strands may be synthesized in pieces > Okazaki fragments < which are then linked together to yield continuous DNA chain

24. DNA Replication Cont.

25. Transcription RNA copy of one strand of gene sequence (used as template) is synthesized by RNA polymerase Raw transcript is edited to produce mRNA Each nucleotide in mRNA is complementary to one in DNA template Takes place in the nucleus Synthesis in 5’ to 3’ direction

26. Transcription Cont.

27. Post-transcriptional processing The initial transcript is edited to splice out the introns (non-coding sequences) from the RNA and the exons (coding sequences) are ligated together

28. Translation Protein synthesis occurs at cytoplasmic structures called ribosomes Ribosomes = ribosomal RNA + proteins mRNA <- template for synthesis of specific sequence of amino acid, giving rise to polypeptide chain tRNA bound to specific amino acid recognizes sequence of 3 bases (codon) in the mRNA

29. Translation Cont. Four stages Amino acid activation Initiation of polypeptide chain formation begins with the amino acid, methionine (AUG) Chain elongation Chain termination Three codons, UAA, UGA & UAG are signals for chain termination

30. Translation Cont.

31. Post-translational modification Gives mature protein functional activity Includes peptide cleavage , covalent modification ( glycosylation, phosphorylation, carboxylation, hydroxylation ) Newly synthesized protein is directed to appropriate destination

32. Protein Synthesis

33. Possible faults in protein biosynthesis Gene Gene deletion (Partial or Complete) Initial mRNA Final mRNA Initial Protein Final Protein Functional Protein Transcription mRNA processing Translation Post-translational processing Transport to correct location and 3D Structure Defective regulation ( promotor mutants) Altered splice site sequence Abnormal new splice site Partial gene deletion Polyadenylation mutants Premature Stop Codon Altered Amino acid Sequence Altered Amino acid Sequence (point substitution or frameshift )

34. Mutation at Molecular Level Single base substitutions (point mutations) substitution of one base for another Missense mutations : replacement of one amino acid for another in the gene product {e.g. Sickle Cell disease – from GAG to GTG in 6 th codon - > glutamic acid to valine } Nonsense mutations : replacement of an amino acid codon with a stop codon (UAA or UAG or UGA ) Splice site mutations : single base substitution which creates or destroys an intron–exon splice site . Insertions and duplications : may arise from unequal crossing over at meiosis and can disrupt the gene

35. Deletions may vary in size from single bases to megabase lengths of DNA E.g. Dystrophin gene deletion in Duchenne Muscular Dystrophy Frameshift mutations deletions or single base substitution and as a result all the subsequent codons in the gene are misread Dynamic or unstable mutations trinucleotide or triplet repeats in a gene -> genetically unstable -> subsequent generations, e.g. fragile X mental retardation, myotonic dystrophy & Huntington disease

36. Different types of mutation affecting coding exons

37. Genetic Imprinting Only one allele is expressed while other is switched off Whether an allele is expressed or not is determined by the sex of the parent who contributed it

38. Paternal imprinting : Paternally derived allele is inactivated Maternal uniparental disomy : both copies of a chromosome are derived from mother

39. Prader-Willi Syndrome Morbid Obesity Small Extremities Small External Genitalia

40. M aternal imprinting : Maternally derived allele is inactivated P aternal uniparental disomy : both copies of a chromosome are derived from father

41. Angelman Syndrome Developmental Delay Mental Retardation Autistic features in duplications of maternal origin

42. The Use of Molecular Genetics in Diagnosis DNA extraction Polymerase chain reaction Southern blotting Gene sequencing

43. DNA Extraction Samples Mostly from blood samples Buccal Scrapes or mouth washes Dried blood spots (Neonatal screening) Chorionic villous sampling (Prenatal diagnosis) DNA Extraction Lyse the cells D igest the proteins by a proteinase P urify the DNA by serial extractions with phenol and chloroform P urity of the DNA obtained is then determined by spectroscopy Run an electrophoretic gel Extracted DNA may be stored for many years at −80°C.

44. Polymerase Chain Reaction Steps Small Sample of DNA is placed in a tube Two primers (oligonucleotides) that match region of interest are added Thermostable DNA Polymerase is added Mixture is heated just below 100˙C > DNA dissociates into two single strands Then it is allowed to cool > single strands bind to oligonucleotides which are in excess Oligonucleotide acts as primer and extended to form new double-stranded molecule

45. Repeat Cycles 20-30 times

46. Advantages of PCR Very quick Highly sensitive Uses to study DNA as well as mRNA to study very small amounts of almost any tissue , e.g. the blood spots on a Guthrie card or the cells found in a mouthwash sample molecular diagnosis of genetic disease to confirm the presence of infectious agents to identify the human leukocyte antigen (HLA) haplotype

47. Southern Blotting

48. Comparative G enomic Hybridization Microarray (CGH) Relative hybridization between Green fluorescent labeled patient’s DNA & Red fluorescent labeled reference DNA Hybridization (+) > Standard Yellow fluorescent color Deletion in patient’s DNA > Red color Duplication in patient’s DNA > Green color

49. CGH Microarray Cont.

50. Gene Sequencing

51. Chromosomes Each somatic cell has 46 chromosomes 22 pairs of autosomes, or non-sex chromosomes 1 pair of sex chromosomes (XY in a male, XX in a female) Germ cells (ova or sperm) 22 autosomes and 1 sex chromosome ( total 23 ) Fertilization >> full diploid chromosome complement of 46 in the embryo

52. Identified under microscope by Their size Pattern of banding when stained Position of centromere Centromere Located centrally : metacentric Located peripherally : acrocentric End of chromosome arm : telomere Maintain integrity of chromosome Subtelomeric regions : Chromosomal regions adjacent to telomeres are gene-rich

53. Chromosomal analysis ( Cytogenetics ) Samples that can be used : Human lymphocytes ( most used in clinical practice ) Bone marrow (e.g. in leukemias ) Fibroblasts (from skin biopsy) Amniocentesis : 12-16 weeks gestation Contains skin cells shed from fetus & amniotic membrane Takes 2-3 weeks to culture and analyze Chorionic villous samples : 9-11 weeks gestation Fetal origin but has to separate from maternal tissue under dissecting microscope

54. Preparation of chromosome karyotype from blood sample Giemsa Using Colchine Centrifugation

55. Standard Chromosome Karyotype

56. Fluorescent In Situ Hybridization (FISH) Used to look for small chromosome deletions E.g. Williams Syndrome, DiGeorge Syndrome

57. Indication for Chromosomal Studies Intellectual disability associated with physical abnormalities Multiple congenital abnormalities Intersex conditions (e.g. ambiguous genitalia) Congenital lymphedema (e.g. Turner Syndrome, Noonan Syndrome Gross failure to thrive (Prenatal origin) Childhood leukemias & malignancies (may be indicator for prognosis)

58. Types of chromosome abnormality Numerical abnormalities Structural abnormalities in chromosomes Mosaicism

59. Numerical chromosomal abnormalities Euploidy : multiples of the haploid (n) number n=(23 in man ), diploid: 2n = 46, triploid: 3n = 69 , or tetraploid : 4n = 92 Polyploidy : multiples > 2n Clinical practice -> rare T riploidy : occasionally with severe abnormalities at birth : fertilization of a single ovum by two spermatozoa ( dispermy )

60. Aneuploidy : not exact multiple of the haploid number {e.g. trisomy and monosomy } Non- dysjunction : failure of homologous chromosomes to separate & migrate to opposite poles of nucleus during cell division Major mechanism of Monosomy and Trisomy Numerical chromosomal abnormalities Cont.

61. Structural chromosomal abnormalities Deletions Ring chromosomes Inversion Isochromosomes Translocations

62. Structural chromosomal abnormalities Cont.

63. Mosaicism Mosaicism two or more genetically different cell lines in an individual derived from a single zygote C himerism fusion of two zygotes or exchange of cells between two zygotes

64. Pattern of Inheritances

65. Pattern of Inheritances Single gene disorders Multifactorial or polygenic inheritance Chromosomal disorders

66. Single gene inheritance Autosomal dominant A utosomal recessive Codominance X-linked dominant X-linked recessive Mitochondrial inheritance Gregor Mendel (1822-1884)

67. Locus : The position at which the gene lies on the chromosome Alleles : Alternative forms of a gene found at the same locus on a particular chromosome At the locus there is a pair of alleles Homozygous : an individual possesses two alleles that are the same Heterozygous : the two alleles are different Dominant disorder : Disorder which manifest in the heterozygote Recessive disorder : Disorder which only manifest in the homozygote Terms used in pattern of inheritance

68. Autosomal Dominant Both Sexes are equally affected Heterozygotes are phenotypically affected i.e. no carrier condition 50% of children are affected Risk remains the same for each successive pregnancy Variable expressivity , Reduced penetrance Exhibit Anticipation High New Mutation Rate Rare and less severe than autosomal recessive

69. Pedigree Symbols

70. Autosomal Dominant

71. Examples of Autosomal Dominant Disorders Achondroplasia Facio - scapulo -humeral dystrophy Hereditary spherocytosis & elliptocytosis Huntington’s disease Marfan’s Syndrome, Noonan’s Syndrome Myotonic dystrophy Neurofibromatosis type 1 & 2 Tuberous sclerosis von Willebrand’s disease

72. Autosomal Recessive Both Sexes are equally affected Only manifests in Homozygous state Heterozygotes are phenotypically unaffected, i.e. Carrier state exists When both parents carry the gene, 1:4 > affected, 2:4 > carriers Asymptomatic carriers produce affected children Those with disease do not usually have affected unless they marry a carrier risk of disease among offspring of consanguineous marriages Variable expressivity less than AD

73. Autosomal Recessive

74. Examples of Autosomal Recessive Disorders Alpha-1 antitrypsin deficiency Ataxia telangiectasia Beta- thalassaemia Congenital adrenal hyperplasia Cystic fibrosis Fanconi Anaemia Galactosaemia Glycogen Storage Disorders Haemochromatosis Homocystinuria Mucopolysaccharidoses (except Hunter’s Syndrome) Oculocutaneous albinism Phenylketouria Sickle Disease Wilson’s Disease

75. Codominance Codominance is the term used for two traits , which are both expressed in the heterozygote . An example of codominance is the inheritance for blood groups A and B An individual with both alleles will have the blood group AB.

76. X-linked Recessive Affected cases are usually males carrying the gene and homozygous females (rare) Half the sons of carriers are affected and half the daughters are carriers No male-to-male transmission Affected males can have only normal sons and carrier daughters Affected cases have affected brothers and affected maternal uncles

77. Affected cases have affected brothers and affected maternal uncles X-linked Recessive Half the sons of carriers are affected and half the daughters are carriers

78. Becker’s muscular dystrophy Duchenne muscular dystrophy Fabry’s disease Glucose-6-phosphate dehydrogenase deficiency Haemophilia A and B Hunter’s syndrome Lesch-Nyhan Syndrome Colour blindness Examples of X-linked Recessive Disorders

79. X-linked dominant Affects both sexes but female > male All children of affected homozygous females are affected Females pass the trait to half their sons and half their daughters All daughters of affected males are affected but none of their sons

80. XY + X D X X D Y XX XY X D X Females pass the trait to half their sons and half their daughters All daughters of affected males are affected but none of their sons X-linked Dominant

81. Vitamin-D resistant rickets Incontinentia pigmenti Rett’s Syndrome Alport’s Syndrome Examples of X-linked Dominant Disorders

82. Y-linked disorders Only males Affected males always have an affected father All sons of an affected man are affected Examples- Web toes, Hairy ears

83. Mitochondrial Disorders Rare and complex neurological conditions Mitochondrial DNA mutates 10 times > nuclear DNA No introns >> mutation will invariably affect coding sequence Maternal inheritance Normal & mutant mitochondrial DNA may coexist within one cell ( heteroplasmy )

84. Examples of Mitochondrial Inherited Disorders MELAS Mitochondrial Encephalopathy, Lactic Acidosis, Stroke-like episodes, diabetes mellitus MERRF Myoclonic Epilepsy, Ragged-Red Fibers in muscles, ataxia, sensorineural deafness NARP Neuropathy, Ataxia, Retinitis, Pigmentosa , developmental delay, lactic acidosis Chronic progressive external opthalmoplegia Ptosis, opthalmoplegia Leber’s hereditary optic neuropathy Visual loss, neurodegenerative features Hypertonic cardiomyopathy with myopathy HOCM, muscle weakness Pearson Syndrome Pancreatic insufficiency, pancytopenia, lactic acidosis

85. Multifactorial or polygenic inheritance Due to many contributing genes Environmental factors Risk increases Previous offspring affected First-degree relative affected Relatives severely affected

86. Congenital Malformations Neural Tube Defects Congenital Heart Disease ( 8 per 1000 births) Cleft lip ± Cleft palate (1 per 1000 births) Hirschsprung’s disease ( Male:Female = 3:1) Common adult disease Insulin dependent diabetes mellitus Epilepsy Essential hypertension Ischaemic heart disease Examples of Multifactorial or polygenic inheritance

87. References Forfar and Arneil's Textbook of Pediatrics 7 th Edition Nelson Textbook of Pediatrics, 20 th Edition Basic Medical Sciences (Easterbrook) 3 rd Edition [Malcolm Levene ] MRCPCH MasterCourse (Volume 1 )

88. Thank You To be continued