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THALASSEMIA is caused by genetic defects leading to reduced amount of globin chains.

Four Novel Deletions in Globin Genes Revealed by Multiplex Ligation Dependent Probe Amplification (MLPA) Technology Dvora Filon, Marion Phylipsen, Piero C Giordano, Ariella Oppenheim, Deborah Rund and Cornelis L Harteveld Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

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THALASSEMIA is caused by genetic defects leading to reduced amount of globin chains.

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  1. Four Novel Deletions in Globin Genes Revealed by Multiplex Ligation Dependent Probe Amplification (MLPA) Technology Dvora Filon, Marion Phylipsen, Piero C Giordano, Ariella Oppenheim, Deborah Rund and Cornelis L Harteveld Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Hemoglobinopathies Laboratory, Human and Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands

  2. THALASSEMIA iscaused by genetic defects leading to reduced amount of globin chains. b-thalassemia is caused by any of several hundred point mutations in the critical regions of the b-globin gene: promoter, splice sites, exons (nonsense or frameshift), polyadenylation. a-thalassemia is caused mainly by large deletions: phenotype determined by how many genes are missing

  3. LCR e Gg Ag yb d b HS40 z yz ya2 ya1 a2 a1 q b-globin cluster on chromosome 11 a-globin cluster on chromosome 16

  4. a-globin gene deletions

  5. From: Schouten, JP. Nucleic Acids Res. 2002; 30:e57

  6. MSLN NARFL C160rf23-25 C16orf20-22 RHBDL C16orf39 C16orf15-19 WFIKKN1 RAB40C PIGQ C16orf11 SOLH KIAA0665 DECR2 C16orf43 AXIN PDIP RGS11 C16orf9 LUC71 Alpha cluster C16orf35 MPG C16orf8 C16orf33 0 100 200 300 400 500 600 700 (kb) probe nr1 2 345 6 7-18 19 20 2122 2324 252627 28 29 3031 32 33 34 35 180 kb 130 140 150 160 170 probe nr 7 8 9 10 11 1213 14 15 1617 18 L1 L0 2 inter-HV 1212 1  3’HVR Chromosome16p13.3; 750 kb region from the telomere From: Harteveld CL. Journal of Medical Genetics, 2005; 42: 922

  7. Chromosome 11p15.4; 500 kb region 5.45 5.35 5.30 5.25 5.20 5.15 5.10 5.05 5.00Mb 5.40 OR51B4 OR51B2 OR51B5 OR51B6 OR51M1 OR52D1 21-25 1 23 45 6 7891011121314151617181920 2627 2829 303132 3334 OR52A4 OR52A5 OR52A1 OR51A1P OR52Z1P OR51V1    A G  HS5-1 From: Harteveld CL. Journal of Medical Genetics, 2005; 42: 922

  8. Age 42y 33y 14 mo 30y RBC 5.51 5.85 5.68 4.49 Hb 9.7 11.4 10.8 13.1 MCV 56.7 62.6 60.4 85 MCH17.5 19.4 19 29.1 Hb H6% 0% 0% 0% -a3.7/ ? Family MF- Hematological results

  9. 180 kb 130 140 150 160 170 probe nr7 8 9 10 11 1213 14 15 1617 18 L1 L0 2 inter-HVR 1212 1  3’HVR Family MF MLPA results 6-11 kb 2 1,5 Ratio 1 0,5 0 0 5 10 15 20 25 30 35 40 Probe Number

  10. RBC 4 Hb 11.4 MCV 87 MCH 28.5 RBC 6.08 Hb 13.5 MCV 71 MCH 22.2 Age 10y 8y 5y 2y RBC 5.51 5.91 4.11 4.67 Hb 11.1 11.9 11.5 13 MCV 63 65 84.6 83.8 MCH20.120.1 27.727.9 Family C- Hematological results

  11. 2 MSLN NARFL C160rf23-25 C16orf20-22 RHBDL C16orf39 C16orf15-19 WFIKKN1 RAB40C PIGQ C16orf11 SOLH KIAA0665 DECR2 C16orf43 AXIN PDIP RGS11 C16orf9 LUC71 Alpha cluster C16orf35 MPG C16orf8 C16orf33 104-120 kb 1,5 Ratio 1 0,5 0 0 5 10 15 20 25 30 35 40 Probe Number probe nr1 2 345 6 7-18 19 20 2122 2324 25 2627 28 29 3031 32 33 34 35 0 100 200 300 400 500 600 700 (kb) Family C- MLPA results

  12. Family K- Hematological results Age 61y 33y 61y RBC 5.6 6.63 5.22 Hb 16.1 14.1 15.7 MCV 84.5 69.4 91 MCH 28.8 21.3 30

  13. 2 170-240 kb MSLN NARFL C160rf23-25 C16orf20-22 RHBDL C16orf39 C16orf15-19 WFIKKN1 RAB40C PIGQ C16orf11 SOLH KIAA0665 DECR2 C16orf43 AXIN PDIP RGS11 C16orf9 LUC71 Alpha cluster C16orf35 MPG C16orf8 C16orf33 1,5 Ratio 1 0,5 0 0 5 10 15 20 25 30 35 40 0 100 200 300 400 500 600 700 (kb) probe nr1 2 34 5 6 7-18 19 20 2122 2324 25 2627 28 29 3031 32 33 3435 Family K – MLPA Results Probe number

  14. Family M- Hematological results Age 38y RBC 5.46 Hb 10.7 MCV 63.9 MCH 19.6 Age 51y RBC 5.0 Hb 13.3 MCV 80.3 MCH 26.2 Age 17y 13y 11y 4y RBC 4.85 5.43 4.43 6.32 Hb 14.2 10.6 12.2 11.2 MCV 86.7 62.3 83.4 56.7 MCH 29.3 19.5 27.4 17.7

  15. 2 > 289 kb Ratio 1,5 1 0,5 Probe number 0 0 5 10 15 20 25 30 35 40 5.45 5.40 5.35 5.30 5.25 5.20 5.15 5.10 5.05 5.00Mb OR51B4 OR51B2 OR51B5 OR51B6 OR51M1 OR52D1 21-25 1 23 45 6 78 91011121314151617181920 2627 2829 303132 3334 OR52A4 OR52A5 OR52A1 OR51A1P OR52Z1P OR51V1    A G  HS5-1 Family M –MLPA results

  16. In conclusion, Large deletions of the alpha or beta globin cluster can present a serious problem in diagnosis, potentially leading to erroneous genetic counseling. Molecular identification of such deletions can allow accurate genetic counseling and prenatal diagnosis in appropriate families. Molecular analysis of the deletionslead to a better understanding of globin regulation and expression. MPLA technology is invaluable in characterizing these types of deletions which escape detection using more conventional techniques.

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