annet damhuis, vikki moye and sian ellard department of molecular genetics
Post on 28-Mar-2015
216 Views
Preview:
TRANSCRIPT
Annet Damhuis, Vikki Moye and Sian EllardDepartment of Molecular Genetics
Clinical features◦ Diabetes◦ Renal developmental abnormalities◦ Female genital tract malformations
Genetics◦ Heterozygous HNF1B mutations◦ Whole gene deletions account for 50% of
mutations
KT: Double uterus Unilateral renal agenesis
RT: Dysplastic kidneys Diabetes at 30 months
KT
RT
3 affected family members over 3 generations
quantitative multiplex PCR amplification of short fluorescent fragments
Duplication of HNF1B exon 5
Normal dosage3 4 5 6
Normal dosage
Inverted tandem duplication (tail to tail)
3 4 5 5 4 6
3 4 5 6
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
Inverted tandem duplication (tail to tail)
3 4 5 5 4 6
max. 13484 bp
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
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
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)
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
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
1. Normal control 1
2. Normal control 2
3. Normal control 3
4. Negative control
1 2 3 4
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
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
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
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
3 4 5 54 6
5 4
3500bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4 Primer
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
Intron 5 Intron 3 Exon 4 Primer
5 4
3500bp
626 bp
GCTGTGGCGGTAGATAGCCTGCTTTCAACCCCAATTCTACCACTTGGGCACACTTCTTAACTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAAGCCCCTGACTCCCAGGGCGGCATAGCCTGCTCTGTCCCTGCGTTCAGGCCCACTTCAGCCATCTTCATCAGATCACTCCATGGATTGGCCTTTTCTCTGCATAGGCCTCTCCTCCCCAGCCTTCCATTCTGCCCACGGCCCCTTCATACTCCCAACCAAGACTGCTGTGATTGTGTGTTTTTGGCCAAGCACCAACAAGTCCCCCCGCCCCCCCTCACTCACCATCTCCCCTCCATCCATTCCCAGGGCAGAATGTTTGCAGCGAGGGGTTCCCCCCCCAAAGCCCCACCCGCCT
5 4
3500bp
626 bp
Fragment size approximately 293 bp
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
CGGGCCTTCCTTACTTTCTCTAAATTTCAGTATCCTTCTCTATGAAATAGGGATAATAATACCAACCTTATAGATTATAGGTATATAAAGAACCTAGCATAGTGAAGAGACCATAAATGACAGCATTAATGTAATTCAGGCTCTCCAAATTATCATGTAAGGATCACCACTGATACCCTTGGCAGTGGGAAGAATGGCTAAATAAGGAAGCCCCATCTGGACCTTCCCCTGGACTGCCTGTCCCCGGCTTGCTCTCTTCTCCTCGAGAGCCACACCAGAGCCTAA
Intron 5 Intron 3 primers
5 4
3500bp
626 bp
293 bp
KT: Double uterus Unilateral renal agenesis
RT: Dysplastic kidneys Diabetes at 30 months
Low level mosaic duplication
RT
Heterozygous duplication
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
Michael Day Kevin Colclough
All staff at the Molecular Genetics Laboratory in Exeter
top related