2. Dr. Derakhshandeh, PhD Mutation Screening

3. 2 Quantitative PCR and Dosage

4. 3  used in a number of diagnostic applications eg.  SMA  trisomy 21  X-linked Agammaglobulinemia

5. 4 SMA

6. 5 CLASSIFICATION  SMA TYPE I (Werdnig- Hoffmann)  SMA TYPE II (Classic)  SMA TYPE III (Kugelberg-Welander)

7. 6 SMA TYPE I  Severe form of SMA  Onset : first 6 months  Death : < 2 year  Never raising the head or sitting

8. 7 SMA TYPE I

9. 8 SMA TYPE II  Less severe  Clinical appearing : < 18 months  Able to sit un aid  Death : about 9 years

10. 9 SMA TYPE III  Mildest form of SMA  Onset : > 18 months  Walking without aid

11. 10 GENETIC MAP  Three candidate genes named SMN (Survival Motor Neuron), NAIP (Neuronal Apoptosis Inhibitory Protein) and P44 were identified in this locus  Up to 95% of SMA patients (SMNI-III) are homozygously deleted for two exons (7&8) of both telomeric copy of SMN gene (SMN t )

12. 11 Deletions  Up to 5% of SMA patients have frameshift mutations, gene conversions and point mutation  Exons 5 and 6 of NAIP t gene are deleted in approximately 50% of type I SMA and 18% of types II and III SMA  P44 t is lacked or intrrupted in 73% of SMA type I patients and 7% in types II and III

13. 12 The summery of normal alleles (N), mutant alleles (M) and deletion types (D) of SMN

14. 13 MOLECULAR DIAGNOSIS & PND PPCR-SSCP or PCR-RFLP of SMN gene enables confirmation of a suspected clinical diagnosis of SMA or prenatal diagnosis TThese two techniques based on nucleotide differences of both exon 7 and exon 8 of telomeric and centromeric copy of SMN

15. 14

16. 15

17. 16

18. 17

19. 18

20. 19 Deletion Analysis of SMN gene Exones 7 and 8 of SMN gene were amplified and cut by Dra I and Dde I, respectively. (only centromeric copy is cutted) Absence of SMN t exone(s) 7 (and 8) confirm diagnosis of SMA

21. 20

22. 21 188 bp 164 bp 188 bp 123 bp 65 bp Exon 7, DraI Exon 8, DdeI

23. 22 SMN Deletion Analysis Derakhshande et al. (Farhud Genetic Lab/ NRCGEB) SMN t Exon 7 is deleted in affected child

24. 23 NAIP Gene Deletion Analysis Exones 5 & 6 of NAIP gene were amplified with exon 13 which was the internal control Absence of exon 5 and exon 6 ( which only exist within the telomeric functional copy of NAIP) was detected in ~50% of type I SMA and 18% of types II and III SMA

25. 24 NAIP Deletion analysis Derakhshande et al. (Farhud Genetic Lab/ NRCGEB)

26. 25

27. 26 The objective of this study was to genetically characterize the childhood onset spinal muscular atrophy in Iran. SMNNAIP Deletion of exon 7 & 8Deletion of exon 5 & 6 SMA type I (n=70)70(100%)61(87%) SMA type II (n=3)2(66%)1(33%) SMA type III (n=2)1(50%)0(0%)

28. 27 Various deletion haplotypes were constructed by using genotypes of SMN and NAIP genes. Haplotype A, which has the deletions of all two involved genes, were deleted in approximately 83% of type I and II SMA but not in type III and was found predominantly in the severe group with an early onset at less than 6 month of age. we report Thirty four our experiences for prenatal diagnosis Haplotypes (%) ABC SMA type I (n=70) 871000 SMA type II (n=3) 336633 SMA type III (n=2) 050

29. 28 These studies suggested that the frequency of gene deletions of SMN1 and NAIP gene is a few higher than previous reports. It is may be due to high rate of consanguine marriage by Iranian Muslims (96 % in this families). Thus, the conformation of SMA related gene deletion will also be a useful tool for the pre and postnatal diagnostic. In addition to common PCR methods for SMN exon 7 and 8 and NAIP exons 4 and 5, we also conducted multiplex PCR of exon 5, 6 and 13 of the NAIP telomere in one reaction.

30. 29 Quantitative PCR and Dosage Products can be measured by image scanning of agarose gels or Polaroid pictures Method is not sensitive to template concentration (1.3g to 2.7pg DNA used) but can be affected by template quality so preparation is important.

31. 30 More complex method SMA  This method allows quantitation of the copy number of SMNT and SMNC genes on chromosome 5q12

32. 31 The homologous gene quantitative polymerase chain reaction(HGQ- PCR)

33. 32 Detection of trisomy 21 by HGQ-PCR Lanes 1–16 Serial dilutions for a normal individual and aDown’s syndrome patient used to evaluate the optimal HGQPCR

34. 33 Sequencing

35. 34 Top and bottom strand differences for a point mutation

36. 35 Top and bottom strand differences for a heterozygous base (Click for full size image) Figure 2. Top and bottom strand differences for a heterozygous base

37. 36 Sequencing

38. 37 Forensics

39. 38 Forensics  PCR analysis PCR (polymerase chain reaction)  RFLP analysis  STR analysis Short tandem repeat (STR) technology is a forensic analysis  For example, the likelihood that any two individuals (except identical twins) will have the same 13-loci DNA profile can be as high as 1 in 1 billion or greater.

40. 39 Forensics  Mitochondrial DNA Analysis Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR  Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR  mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion

41. 40 Forensics  While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP  they can be analyzed with mtDNA  In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable.

42. 41 Forensics  Y-Chromosome Analysis The Y chromosome is passed directly from father to son  so the analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males  this technique can be very valuable if the laboratory detects complex mixtures (multiple male contributors)

43. Reverse Dot Blot for Human Mutation Detection

44. 43 Introduction Reverse dot blot (RDB) or reverse allele specific oligonucleotide (Reverse ASO) hybridization important method for genotyping common human mutations

45. 44 Commonly used in: a high mutation spectrum high frequency disorders such as: –cystic fibrosis –hemoglobin C (HbC) –hemoglobin E (HbE) –hemoglobin S (HbS) –ß-thalassemias

46. 45 Location of mutations in the  -globin gene

47. 46 RDB procedure exons (or other regions of interest) amplified by the polymerase chain reaction (PCR) using labeled oligonucleotide primers 5' biotin label on PCR primers

48. 47 Amplicons Amplification products denatured hybridized – with mutation specific DNA probes covalently bound to solid membran

49. 48 Incubation nucleic acids: incubated with an enzyme conjugated to streptavidin. enzyme-conjugated, streptavidin-biotin-nucleic acid complex is then washed incubated with –a chromogenic –or luminogenic substrate, which allows visualization of hybridized spots

50. 49 Materials and Methods Total genomic DNA extracted from peripheral blood leukocytes Amniotic fluid cells (AF) chorionic villi (CVS)

51. 50 A woman having amniocentesis

52. 51 Oligonucleotide probes A C6-amino-link phosphoramidite amino moiety on the 5' end of the product

53. 52 Oligonucleotides used for reverse dot blot (RDB)

54. 53 RDB

55. 54 Reverse dot (RDB) blot hybridization for detection of 10 common β-thalassaemia mutations

56. 55  -thalassemia Patients

57. 56 Screening for causal mutations genomic DNA from patient blood samples reverse dot blot (RDB) amplification refractory mutation system- polymerase chain reaction (ARMSPCR) DNA sequencing

58. 57 PCR from genomic DNA 720 bp

59. 58

60. 59

61. 60 Strips N M 123456789123456789 1 2 3 4 5 6 7 8 9 10

62. 61 The Blots

63. 62 Comparison of different factors determining the efficiency of ARMS and reverse hybridization in beta thalassemia diagnosis ARMSReverse hybridization Turnover timeseveral days6-8 hours EquipmentExpensive (large PCR machine, gel electrophoresis, photodocumentationsyst em Less expensive (small PCR machine, agarose gel, small shaking water bath) Number of PCR reactions per sample 8-881 DocumentationRequires documentation process after experiment Self-documented Technician time (number of patients: time in days) 1:110:1 Starting materialDepending on the number of PCR reactions 0.5 μg genomic DNA for just one PCR reaction Toxic materialsEthidium bromide (carcinogen) None

64. 63 In the late stages of muscular dystrophy, fat and connective tissue often replace muscle fibers.

65. 64

66. 65 DMD

67. 66 Orthopaedic management of patients with Duchenne's muscular dystrophy

68. 67 ~95% of deletions can be detected in males using multiplex PCR L A B C D E F G H L

69. 68 MAPH Detection of deletions/duplication mutations in Duchenne Muscular Dystrophy using: Multiplex Amplifiable Probe Hybridisation (MAPH)

70. 69 MAPH Although ~95% of deletions can be detected in males using multiplex PCR other methods must be used to determine duplications, as well as the carrier status of females The most commonly applied methods are quantitative multiplex PCR and quantitative Southern blotting The drawback of quantitative multiplex PCR is that often not all mutations are examined meaning that small and rare mutations are missed

71. 70 MAPH Using high-quality Southern blots it is possible to perform a quantitative analysis and detect duplications this technique is time consuming it is difficult to exactly determine the duplication it can be difficult to detect duplications in females and triplications will be missed Armour et al (Nucl.Acids Res. 2000)Nucl.Acids Res. 2000)

72. 71 system for analysing all 79 exons of the DMD gene for deletions and duplications MAPH is based on a quantitative PCR of short DNA probes recovered after hybridisation to immobilized genomic DNA

73. 72 1 ug of denatured genomic DNA is spotted on a small nylon filter hybridized overnight in a solution containing one of the probe mixes Following stringent washing the next day the filter is placed in a PCR tube and a short PCR reaction is performed This releases the specifically-bound probes into the solution An aliquot of this is transferred to a second, quantitative PCR reaction