annet damhuis, vikki moye and sian ellard department of molecular genetics
TRANSCRIPT
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Annet Damhuis, Vikki Moye and Sian EllardDepartment of Molecular Genetics
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Clinical features◦ Diabetes◦ Renal developmental abnormalities◦ Female genital tract malformations
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Genetics◦ Heterozygous HNF1B mutations◦ Whole gene deletions account for 50% of
mutations
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KT: Double uterus Unilateral renal agenesis
RT: Dysplastic kidneys Diabetes at 30 months
KT
RT
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3 affected family members over 3 generations
quantitative multiplex PCR amplification of short fluorescent fragments
Duplication of HNF1B exon 5
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Normal dosage3 4 5 6
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Normal dosage
Inverted tandem duplication (tail to tail)
3 4 5 5 4 6
3 4 5 6
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3 4 5 54 6
Normal dosage
Inverted tandem duplication (tail to tail)
Direct tandem duplication (tail to head)
3 4 5 5 4 6
3 4 5 6
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Inverted tandem duplication (tail to tail)
3 4 5 5 4 6
max. 13484 bp
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Inverted tandem duplication (tail to tail)
Direct tandem duplication (tail to head)
3 4 5 5 4 6
max. 13484 bp
3 4 5 54 6
max. 6620 bp
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Inverted tandem duplication (tail to tail)
Direct tandem duplication (tail to head)
3 4 5 5 4 6
max. 13484 bp
3 4 5 54 6
max. 6620 bp
Control primerset: HNF1B fragment 1000 bp
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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)
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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
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1.Proband RT 12.Proband RT 23.Mother KT4.Normal control 15.Normal control 26.Normal control 37.Negative control
3 4 5 5 4 6
1 2 3 4 5 6 7
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1. Normal control 1
2. Normal control 2
3. Normal control 3
4. Negative control
1 2 3 4
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1.Proband RT (1)2.Proband RT (2)3.Mother KT4.Normal control 15.Normal control 26.Normal control 37.Negative control
3 4 5 5 4 6
1 2 3 4 5 6 7
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1. Proband RT2. Mother KT 13. Mother KT 24. Normal control 15. Normal control 26. Normal control 37. Negative control
3 4 5 54 6
1 2 3 4 5 6 7
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1. Proband RT2. Mother KT 13. Mother KT 24. Normal control 15. Normal control 26. Normal control 37. Negative control
Fragment size approximately 3500 bp
3 4 5 54 6
1 2 3 4 5 6 7
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1. Proband RT 12. Proband RT 2 3. Mother KT 14. Normal control 15. Normal control 26. Normal control 37. Negative control8. empty
3 4 5 54 6
1 2 3 4 5 6 7 8
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3 4 5 54 6
5 4
3500bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4 Primer
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4 Primer
5 4
3500bp
626 bp
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GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
Fragment size approximately 293 bp
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1. Proband RT2. Proband RT3. Mother KT4. Mother KT5. Normal control 16. Normal control 27. Normal control 38. Negative control
1 2 3 4 5 6 7 8
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CGGGCCTTCCTTACTTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAA
Intron 5 Intron 3 primers
5 4
3500bp
626 bp
293 bp
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KT: Double uterus Unilateral renal agenesis
RT: Dysplastic kidneys Diabetes at 30 months
Low level mosaic duplication
RT
Heterozygous duplication
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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
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Michael Day Kevin Colclough
All staff at the Molecular Genetics Laboratory in Exeter