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LongRange PCR and breakpoint sequence analysis for an HNF1B duplication

LongRange PCR and breakpoint sequence analysis for an HNF1B duplication. Annet Damhuis, Vikki Moye and Sian Ellard Department of Molecular Genetics. Renal Cysts And Diabetes syndrome (RCAD). Clinical features Diabetes Renal developmental abnormalities Female genital tract malformations.

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LongRange PCR and breakpoint sequence analysis for an HNF1B duplication

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  1. LongRangePCR and breakpoint sequence analysis for an HNF1B duplication Annet Damhuis, Vikki Moye and Sian Ellard Department of Molecular Genetics

  2. RenalCysts And Diabetes syndrome (RCAD) • Clinical features • Diabetes • Renal developmental abnormalities • Female genital tract malformations

  3. RenalCysts And Diabetes syndrome (RCAD) • Genetics • Heterozygous HNF1B mutations • Whole gene deletions account for 50% of mutations

  4. Family MY2491RT KT: • Double uterus • Unilateral renal agenesis RT: • Dysplastic kidneys • Diabetes at 30 months KT RT

  5. HNF1B MLPA results RT

  6. HNF1B duplications • 3 affected family members over 3 generations • quantitative multiplex PCR amplification of short fluorescent fragments • Duplication of HNF1B exon 5

  7. HNF1B MLPA results KT

  8. HNF1B MLPA results KT

  9. 3 4 5 6 Possible scenarios for duplication • Normal dosage

  10. 3 4 5 6 3 4 5 5 4 6 Possible scenarios for duplication • Normal dosage • Inverted tandem duplication (tail to tail)

  11. 3 4 5 6 3 4 5 5 4 6 Possible scenarios for duplication • Normal dosage • Inverted tandem duplication (tail to tail) • Direct tandem duplication (tail to head) 3 4 5 4 5 6

  12. 3 4 5 5 4 6 Primer design • Inverted tandem duplication (tail to tail) max. 13484 bp

  13. 3 4 5 5 4 6 max. 6620 bp 3 4 5 4 5 6 Primer design • Inverted tandem duplication (tail to tail) • Direct tandem duplication (tail to head) max. 13484 bp

  14. 3 4 5 5 4 6 max. 6620 bp 3 4 5 4 5 6 Primer design • Inverted tandem duplication (tail to tail) • Direct tandem duplication (tail to head) max. 13484 bp Control primerset: HNF1B fragment 1000 bp

  15. QiagenSeqTargetLongRange PCR Kit • LongRange PCR buffer 2.5 ul • dNTP mix 1.25ul • Primer (10pmol/ul) 2.0 ul • H2O 9.05 ul • Enzyme mix 0.2 ul • 15ul PCR mix + 10 ul DNA (40ng/ul)

  16. QiagenSeqTargetLongRange PCR Kit • Primers designed with annealing temp 62°C • Elongation time: 1 min/kb • PCR program • 3 min 93°C • 15 sec 93°C • 30 sec 62°C • 10 min 68°C • ∞ 4°C

  17. 1 2 3 4 5 6 7 3 4 5 5 4 6 ResultsTail to tail orientation • Proband RT 1 • Proband RT 2 • Mother KT • Normalcontrol 1 • Normalcontrol 2 • Normalcontrol 3 • Negativecontrol

  18. 1 2 3 4 • Normal control 1 • Normal control 2 • Normal control 3 • Negative control ResultsControl primer set

  19. 1 2 3 4 5 6 7 3 4 5 5 4 6 ResultsTail to tail orientation • Proband RT (1) • Proband RT (2) • Mother KT • Normalcontrol 1 • Normalcontrol 2 • Normalcontrol 3 • Negativecontrol

  20. 3 4 5 4 5 6 ResultsTail to head orientation • Proband RT • Mother KT 1 • Mother KT 2 • Normal control 1 • Normal control 2 • Normal control 3 • Negative control 1 2 3 4 5 6 7

  21. 3 4 5 4 5 6 ResultsTail to head orientation • Proband RT • Mother KT 1 • Mother KT 2 • Normal control 1 • Normal control 2 • Normal control 3 • Negative control • Fragment size approximately 3500 bp 1 2 3 4 5 6 7

  22. 3 4 5 4 5 6 ResultsTail to head orientation • Proband RT 1 • Proband RT 2 • Mother KT 1 • Normal control 1 • Normal control 2 • Normal control 3 • Negative control • empty 1 2 3 4 5 6 7 8

  23. 5 4 3 4 5 4 5 6 ResultsSequencing LongRange fragment 3500bp 3500bp

  24. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT

  25. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT

  26. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5

  27. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5

  28. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5 Intron 3

  29. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5 Intron 3

  30. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5 Intron 3 Exon 4

  31. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5 Intron 3 Exon 4 Primer

  32. ResultsSequencing LongRange fragment 3500bp 5 4 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT Intron 5 Intron 3 Exon 4 Primer

  33. 5 4 ResultsPrimer design for junction fragment 3500bp 626 bp GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT • Fragment size approximately 293 bp

  34. ResultsShort fragment PCR 1 2 3 4 5 6 7 8 Proband RT Proband RT Mother KT Mother KT Normal control 1 Normal control 2 Normal control 3 Negative control

  35. ResultsSequenceanalysis short fragment 5 4 3500bp 626 bp 293 bp CGGGCCTTCCTTACTTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAA Intron 5Intron 3 primers

  36. Family MY2491RT Low level mosaic duplication RT Heterozygous duplication KT: • Double uterus • Unilateral renal agenesis RT: • Dysplastic kidneys • Diabetes at 30 months

  37. Conclusions • Direct tandem duplication (tail to head) • Junction fragment assay (293bp) identified duplication in mother KT • LongRange PCR is a useful tool to determine junctions in duplications found by MLPA • Easy to use • Not always necessary to redesign primers

  38. Acknowledgements • Michael Day • Kevin Colclough • All staff at the Molecular Genetics Laboratory in Exeter

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