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1 Quantitative analysis of SMN gene copies in spinal muscular atrophy 孫建峰

2 脊髓肌肉萎縮症第一型 (Werdnig-Hoffman disease), 一般在六個月前發病第二型又稱中間型 (Dubowitz disease ), 一般在一歲半前發病第三型又稱為庫格勃 - 韋蘭德症 (Kugelberg- Welander disease), 約在一歲半後發病

3 疾病的確定診齗血中肌肉酵素的高低肌電圖肌肉切片基因檢查 The type 2 SMA-like mouse tail(a,b) Normal control(c,d)

4 Figure. Genetic mechanisms in three motor neuron diseases. (B) Spinal muscular atrophy (SMA). In SMA, there are two copies of the SMN (survival motor neuron), NAIP (neuronal apoptosis inhibitory polypeptide), and p44c genes. In this condition, the telomeric copy of SMN, SMN T, is deleted or converted to SMN C. The NAIP and p44c genes may also be mutant. From: Orrell: Neurology, Volume 57(1).July 10,

5 Table 2 Chromosomes in the general population Chromosomes with No. in general population Frequency No SMN % 1 SMN % 2 SMN % Total %

FIGURE 1. Schematic diagram of SMN construct containing C6U mutation within exon 7 and the preferred splicing pathways. Tra2-ESE is located in the middle of exon 7 (Hofmann et al.

6 FIGURE 1. Schematic diagram of SMN construct containing C6U mutation within exon 7 and the preferred splicing pathways. Tra2-ESE is located in the middle of exon 7 (Hofmann et al. 2000), whereas SF2/ASF-ESE is located toward the 3' ss of exon 7 (Cartegni and Krainer 2002) and overlaps with hnrnp A1-ESS (Kashima and Manley 2003). Earlier reports suggest that C6U (indicated by a star) abrogates SF2/ASF-ESE and/or creates hnrnp A1-ESS, producing exon 7-skipped product in SMN2. We have recently shown that C6U creates an extended inhibitory context (Exinct) that causes exon 7 skipping (Singh et al. 2004). (ss) Splice site.

7 SMN1/SMN2

8 SMN1/SMN2 Exon 6 Exon 7-6 Intron Intron Exon 8 SMN1 G C A A G SMN2 A T G G A

9 The topographic distribution of the SMN protein domains encoded by the SMN gene and various microrearrangements and missense mutations identified in the SMN gene. NBD=nucleic acid binding domain, NLS=nuclear localisation signal, (P)5=stretch of five prolines, (P)10=stretch of 10 prolines, "Y-G"="Y-G box".

10 Mutations involved in SMA J Med Genet 1999;36:1 8

11 The PCR products of exons 7 of the SMN gene digested with restriction enzyme DraI

12 The PCR products of exons 7 of the SMN gene digested with restriction enzyme DraI

13 Quantitative Real-Time PCR of SMN1 and SMN2 Copy Numbers

15 M Feldkotter et al. Am J Hum Genet 2002;70: copy SMN2 2 copy SMN2 3 copy SMN2 4 copy SMN2 Total Type I 13(6.9%) 138(73.4%) 37(19.7%) Type II 0 12(10.9%) 90(81.8%) 8(7.3%) 110 Type III 0 3(3.9%) 39(50.6%) 35(45.5%) 77 V Chan et al. J Neurol 2004;51: copy SMN2 2 copy SMN2 3 copy SMN2 4 copy SMN2 Type I Total 2 Type II Type III * 13

17 M Feldkotter et al. Am J Hum Genet 2002;70: copy SMN1 2 copy SMN1 3 copy SMN1 4 copy SMN1 Total Carrier 119(96.0%) 5(4.0%) Noncarrier 0 61(93.8%) 4(6.2%) 0 65 Controls 4(2.9%) 132(94.3%) 3(2.1%) 1(0.7%) 140

19 Protocol DNA extraction PCR DHPLC DATA analysis

20 Heteroduplex Analysis wild type mutant heteroduplexes homoduplexes A T G C A C G T A T G C

21 DHPLC

22 Examples of Partial Denaturing Operation for Mutation Detection

24 Chromatography of DHPLC SMN2 SMN1 SMA carrier SMA affected Normal pattern Normal pattern SMN1:SMN2=1:2 SMN1:SMN2=0:2 SMN1:SMN2=2:2 SMN1:SMN2=2:1

29 Conclusions It is important to identify carriers of the SMN1 absence for diagnostic purposes and genetic counseling. DHPLC is a fast and reliable tool for detection of carriers of SMA and SMA-affected patient with SMN1 exon 7 deletion.

30 Misdiagnosis of SMA carrier SMN 2 SMN 2 SMN 1 SMN 1 Wild Type SMN 1 SMN 2 Carrier SMN 1 SMN 2 SMN 1 SMN 2

31 Prenatal Diagnosis of Thalassemia 孫建峰

32 α-thalassemia genotype Two gene deletion(--/αα) SEA type (85%~96%) Philippine type(1~3%) others(<1%) One gene deletion (-α/αα) -3.7 deletion(1~3%) -4.2 deletion(1~2%) Point mutation(αα/αα T ) Hb Constant Spring(1~3%) Hb Quong Sze(<1%) Hb H disease ( / α) hydrops carrier normal 1/4 1/2 1/4

33 Screening for thalassemia CBC, Hb EP, Hb A 2 quantitation, Serum ferritin α-thalassemia prevalence in Taiwan 3~7% MCV < 80 fl, Hb A 2 <3.5% β-thalassemia prevalence in Taiwan 1~3% MCV< 80 fl, Hb A 2 >3.5% or Hb F >2%

34 Thalassemia 檢驗流程 MCV<80 Hb A2<3.5 % and Hb F<2% Hb A2>3.5 % or Hb F>2% Hb A2>5% α -thalassemia test β -thalassemia sequencing α -thalassemia test β -thalassemia sequencing G γ + ( A γδβ) 0 -thal

35 Prevalence of αthalassemia and β thalassemia in people in different areas of the Asia. α1 α2 β Taiwan Guangzhou 8.3 NA 3.4 South-East Asia NA > Thailand Japan NA NA 0.1 J Formos Med Assoc 1998; 97: 5-15 (Ko et al. 1998)

36 Prenatal diagnosis of α-thalassemia by PCR 0 Kb 10 Kb 20 Kb 30 Kb ζ2 ψζ2 ψα2 ψα1 α2 α1 θ1 A B F G C SEA deletion Philippine deletion Primers A+C A+B F+G 偵測基因 SEA N ormal N ormal N ormal Carrier H ydrops + - -

37 α-thalassemia-1 of Southeast Asia (SEA) type 0 Kb 10 Kb 20 Kb 30 Kb ζ2 ψζ2 ψα2 ψα1 α2 α1 θ1 A B F G C SEA deletion

38 α-thalassemia-1 of Philippine deletion type 0 Kb 10 Kb 20 Kb 30 Kb ζ2 ψζ2 ψα2 ψα1 α2 α1 θ1 A C B Philippine deletion

39 α-thalassemia-2 of -α 3.7 deletion type

40 α-thalassemia-2 of -α 4.2 deletion type

41 Hb Constant Spring (Hb CS)

42 Hb Quong Sze(Hb QS)

43 β-thalassemia 基因型分析 Y3 Y5 Exon 1 Exon 2 Exon 3 Y1 Y2 Y4 Y7

44 β-thalassemia of IVS-II-654(C T)

45 β-thalassemia of CD41/42(-TCTT)

46 β-thalassemia of -28(A G)

47 β-thalassemia of C17(A T)

48 β-thalassemia of Hb E C26(GAG AAG)

49 β-thalassemia of Initial codon(t G)

50 β-thalassemia of CD27/28(+C)

51 β-thalassemia of IVS-I nt 1(G T)

52 α-thalassemia 六型基因型分析

53 β-thalassemia 基因型分析

54 6 common types of β thalassemia in Taiwan Zhang et al Lin et al Ko et al Our lab. (87~93) 6 common types in Taiwan (in order) C41/42, nt654, C17, P28, IVS-I nt5, C71/72 nt654, C41/42, P28, C17, C27/28, P29 nt654, C41/42, P28, C17, C27/28, C26 (HbE) C41/42, nt654, P28, C17, C26 (HbE), C27/28 and Initial codon)

55 近三年內重大更動時期甲型 (HbA2< 3.5) 乙型 (HbA2> 3.5) ~ ~ ~ 先做 SEA+PHI 若 SEA (-), PHI(-) 加作其他四型做常見六型 RFLP 常見五型 Betasequencing

56 特殊案例 -1: 林子文 Date Hb MCV A2 F Ferritin Result * α-thal. (-) C43 (+), nt654 (+) # P28 (+) * After transfusion # After stem cell transplantation

57 特殊案例 -2: 溫玉如 Date Hb MCV A2 F Ferritin Result α-thal. (-) 5 common β (-) Sequencing: Initial codon (ATG AGG)

58 特殊案例 -3: 陳韋杉 Date Hb MCV A2 F Ferritin Result α-thal. (-) 5 common β: nt654 (+) Sequencing: codon 67 (-TG): (+)

59 特殊案例 -4: 許嘉玲 Date Hb MCV A2 Ferritin Result Sequencing: Hb Siriraj (+) [β-gene codon 7 : GAG AAG (Glu Lys)]

60 以及時定量 PCR 的方法可避免誤診同合子東南亞型甲型海洋性貧血

Primer S1+S2(normal) M 1 2 3 4 5 Primer S1+S3(SEA) M 1 2

61 α-thalassemia-1 1 of SEA type Detection 0 Kb 10 Kb 20 Kb 30 Kb ζ2 ψζ2 ψα2 ψα1 α2 α1 θ1 S1 S2 S3 SEA deletion Primer S1+S2(normal) M Primer S1+S3(SEA) M bp bp Hum Genet,1992, 88: TM Ko et al

62 Real-Time PCR : α-thalassemia-1 1 of SEA type Detection ς 2 ϕς 1 ϕα 2 ϕα 1 α 2 α 1 θ 5 HVR 1 3 HVR W W Taqman probe S1 S2 S3 SEA deletion

63 Probe Design 2 fluorescent dyes: R = Reporter Q= Quencher 3' end blocked (so it is not extended like a primer) High T m (primers = 60 C; probe = 70 C)

64 Principle of Real-Time PCR Polymerization Strand displacement Forward Probe Primer 5' R Q 3' 3' 5' Q = Quencher R = Reporter Cleavage 5' 3' 5' R Reverse Primer Q 3' 5' 5' 3' 3' 5' 5' 5' 3' 5' 5' 3' 5' R Q 3' 5' 3' 5' Polymerization completed R Q 5' 3' 5'

65 Theoretical PCR Amplification Curve Sample Threshold ΔR n R n C T No Template Control cycle number

66 the C T (threshold cycle) a measure of when

Sensitivity of traditional PCR and Real time PCR M 30 3 0.

67 Sensitivity of traditional PCR and Real time PCR M ng C T y= x correlation coefficient= DNA (ng)

68 Real-time Quantitative PCR Analysis for α- thalassemia-1 of Southeast Asian Type Deletion 1: S1+S3 for SEA 2 : S1+S2 for normal 3 : No template control

69 Real-time Quantitative PCR Analysis for α- thalassemia-1 of SEA Type Deletion SEA homozygote SEA Carrier SEA Negative Normal SEA

70 Real-time Quantitative PCR Analysis for fetus samples SEA carrier SEA carrier SEA homozygote SEA homozygote SEA homozygote SEA negative SEA negative S1+S2 for normal S1+S3 for SEA

71 α-thalassemia-1 1 of SEA type Detection by traditional PCR M : 100 bp marker 1 : cultured cells M : placenta 3 : placenta 4 & 6 : SEA carrier 5 : SEA negative Normal SEA 7 : H 2 O

72 α-thalassemia-1 1 of SEA type Detection by Real time PCR SEA Normal M

73 如果 SEA homozygote 的胎兒檢體被 SEA carrier 母體細胞污染 SEA homozygous DNA mixed with SEA carrier DNA

74 SEA homozygous sample mixed with SEA carrier sample Homo : carrier 1 : 1/10 1 : 1/20 1 : 1/40 1 : 1/100 1 : 1/200 1 : 1/ : 1/ : 0 S1+S2 for normal S1+S3 for SEA ΔC T

626 139 genotype phenotype

thalassemia Watson Crick 1953 neucleic acid 3 10 9 46 mitochondria Mckusick 2208 2136 4344 1988 2208 1433 626 139 genotype phenotype peptide chain 1 2 2 HbA 2 HbA2 thalassemia (1) (2) (3) (4) MCV mean