بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Umm Al-Qura University Faculty of Medicine Department of Medical Genetics Medical Human Genetics Nasser A. Elhawary Professor of Medical Genetics

3. REVISION • Introductory • Some Definitions • Organization of the Cell • Human Chromosomes • Chromosome-Gene-DNA • DNA • The Gene • DNA History • Composition & structure of DNA • DNA & RNA Structures • Cell Cycle & DNA Replication • Transcription of DNA & Translation into Protein • The Genetic code

4. What is Genetics? • Genetics is the scientific study of Heredity. • Transmissionof Characteristics from one Generation to the next.

5. What is Medical Genetics? … Some definitions • Human Genetics: is the science of biological variations in humans.. • Medical Genetics: is the science of human biologic variations as it relates to health and disease. • Clinical Genetics is the science and art of diagnosis, prevention & treatment of genetic disease.

6. THE CELL • Cytoskeleton • Nucleus • Centriole • Mitochondria • ER • Ribosome • Nucleolus • Lysosome • Chromosomes Eukaryotic cell:has nuclear membrane separating the nucleus from the cytoplasm, e.g. in humans. Prokaryotic cell:has not nuclear membrane, e.g. in bacteria.

7. An electron micro-graph of Chr. 12 Human metaphase Human Karyotype HUMAN CHROMOSOMES • Contains DNA and Protein • 23-Pairs • Autosomes: Pairs 1-22 • Sex chromosmes: X- and Y-chromosomes. • Metaphase & Karyotyping

8. Chromosome-Gene-DNA

9. How to search in WEB? • www.google.com (search text) • www.google.com (click ‘images’, then search) • www.google.com (click ‘more’,then search) • www.google.com (click ‘videos’, then search) • www.download.com (download software of DNA, gene, genetics…) • www.pubmed.com (searching for ‘hereditary diseases’ →articles) • In www.pubmed.com Page (Click on OMIM, then search for a gene or a genetic disorder).

10. James Watson Francis Crick DNA History … • In 1869: Miescherdiscovered DNA. • In 1940: DNA was known to consist of phosphate, sugar and Bases (C, T & A, G). • In 1953: Wilkins & Rosa. Franklin conducted X-ray diffraction analysis of DNA crystal. • In 1953:Watson&Crick deduced the double helix structure of DNA. • In 1962:Wilkins, Watson and Crick received the Noble Prize for Medicine or Physiology.

11. Nucleic acids are POLYMERS (Long chains of monomers, called Nucleotides) 1- Sugar (deoxy-ribose) 2- Phosphate group (PO4) 3- Bases Cytosine (C) Thymine (T) Adenine (A) Guanine (G) DNA Composition of DNA … 3- Pyrimidines Purines

12. O O O P O Sugar Sugar-Phosphate-Sugar-Phosphate Backbone H Base deoxyribose

13. DNA RNA • Structure: Double strand • Length: Long • Bases: A, G, C, T • Sugar: Deoxyribose • Found: mainly in Nucleus and Mitochondria. • Single Strand • Short • A, G, C, U • Ribose • Cytoplasm and in high concn. in nucleolus.

14. 1. Transcription of DNA into mRNA2. Translation of mRNA into Protein What is the Point …

15. SUMMARY • DNA → mRNA → mature mRNA → Protein Splicing Capping + PolyA

16. Genetic code… • Each triplet base (codon) represents an amino acid. • There is a degeneracy in the genetic code (i.e. several codons represent the same a.a. (e.g. triplet bases UUA, UUG, CUU, CUC, CUG or CUA represent leu). • “Start codon”: ‘AUG’ represent ‘met’. • “Stop codons”: UAA, UAG or UGA that does not encode amino acid, but terminates translation.

19. Genetic Disorders • Caused by abnormal expression of one or more genes or chromosomal changes in an individual causing a clinical phenotype

20. Human Genome Project Start 1990 and finished 2003 with main goals: • identify ~ 20,000-25,000 genes in human DNA, • determine the sequences of the 3 billion base pairs, • store this information in databases, • improve tools for data analysis, • transfer related technologies to the private sector, and • address the ethical, legal, and social issues (ELSI) that may arise from the project.

21. The Human Genome • Human genome 3 billion bp • Average chromosome 150x106 bp • Average gene 50x103 bp • Average coding sequence 3 x103 bp • Unit of the genetic code 3 bp

22. Pathological variations Normal Variations Change in coding sequence No obvious phenotypic effect

23. MUTATIONS • It is an heritable alteration or change in the genetic material & results in pathological phenotypes… • Mutations arise from: • Exposure to mutagenic agents (mutagens). • Errors in DNA replication. • Errors in repair.

24. Mutations… • Somatic mutations may cause adult-onset disease (cancer), not transmitted to offspring. • Gonadal mutations can be transmitted to future generations.

25. Mutations… e.g. α-thal, β-thal, DMD, CF, PKU e.g. Fragile X, HD, FA Myotonia dystrophy e.g. β-thal, LI, DMD, PKU

26. Types of mutations… • Substitution: is the replacement of a single base (nucleotide ‘nt’) by another. - Transition substitution: C>T or A>G - Transitions occur frequently more than Transversion. • Deletions: If loss of 1 or 2 nt’s, it results to disrupt the reading frame (give severe phenotype) - If loss of 3 nt’s → maintain the reading frame.

27. Types of mutations… • Partial gene deletions (DMD, CF) or whole gene deletions (α-thalassemia).

28. Types of mutations… • Insertion: involves the addition of 1 or 2 nt’s into a gene, if insertion occurs in the coding region→ disrupt the reading frame.

29. Trinucleotide repeats • A specific DNA sequence of 3 nucleotides (e.g. CCG) that is repeated along the human genome • No. of the polymorphic repeats varies from one to another (two to several hundreds). • Trinucleotide repeat sequences are stable, and inheritfrom parents to children with the same no. of repeats.

30. Fragile X Syndrome … • Most common form of inherited mental retardation • Incidence of ~1/4000 male and ~1/8000 females. • Due to expansion of trinucleotide repeats (CGG)n at X27.3. • Mutation in the FMR1 gene due to expansion CGG repeats. • Examples: HD (CAG), FA (GAA, intronic), MD1 (CTG, 3`UTR), FRAXA (CGG, 5`UTR). CAG-repeats in Huntington disease:

31. Structural Effects of Mutations on the Protein • Synonymous or Silent mutations: This nomenclature since the old and new codon code for the same amino acid. If the mutation doesn’t alter the polypeptide product of the gene e.g. V245V silent mutation in PKU disease • Non-synonymous mutations: e.g. R261Q 1- Missense: e.g. R261Q 2- Nonsense: e.g. G272ter 3- Frameshift

32. Missense mutations …

33. Nonsense mutations …

34. Frameshift mutations …

35. SUMMARY

36. Mutations in Non-coding DNA • Mutations in non-coding DNA region are less effective in phenotypes. • Mutations in donor splice (gt) and splice acceptor (ag) sites result in loss of coding sequence (exon skipping) or retention of intronic sequence leads to frameshift mutations. ag gt

37. Cryptic splice site mutation

38. Mutations at splice sites & cryptic splice sites • Mutations that occur at the boundaries between introns and exons can lead to aberrant splicing. This could lead to an intron not being removed from the pre-mRNA, but it is more likely that a cryptic splice site would be used as an alternative. • A cryptic splice site is a genetic sequence that resembles an authentic splice site and might be selected during aberrant splicing. It is also possible for a mutation (within an intron or exon) to create a new cryptic splice site that is preferred over a genuine splice site that is not itself mutated.

39. Cryptic splice site Mutation… • Aberrant splicing might delete part of the resulting protein, add a new section of amino acids, or result in a frameshift. • Several forms of the genetic disease β-thalassemia are caused by mutations that lead to cryptic splice site selection.

40. Exon skipping/splice site Mutation… Splicing of an intron requires an essential signal: "GT........AG". If the splice ‘AG’ is mutated (e.g., A>C), the splicing machinery will look for the next acceptor site AG.  As a result, the exon between two introns is also removed.

41. Functional Effects of Mutations on the Protein • Loss-of-function mutations: 1- Hypomorph: results in reduced activity or decrease stability of gene product. 2- Null allele (Amorph): a complete loss of gene product. Haplo-insufficiency mutations: occurs in the heterozygous status giving rise to ½ normal levels of the gene product affecting the phenotypes.

42. Functional effects of mutations on protein … • Gain-of-function mutations: - Increased levels of gene expression: in HD trinucleotide repeats increase the gene product in the CNS → → → disease. • orDevelopment of a new function(s) of the gene (timing of gene expression in chromosomal rearrangement (tumor cancer). • Gain-of-function are often dominantly inherited and in some rare homozygous state gives severe phenotypes.

43. Functional effects of mutations on protein … • Dominant-negative mutations A mutant gene in the heterozygous state results in the loss of protein activity or function, because of interfering with the function of the normal gene product of the corresponding allele. e.g. Collagens in osteogenesis imperfecta