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GENOMIC REARRANGEMENTS and MENDELIAN DISEASES

GENOMIC REARRANGEMENTS and MENDELIAN DISEASES. Eric LEGUERN Département de génétique, cytogénétique et embryologie ; INSERM U289 . GH Pitié-Salpêtrière. GENOMIC REARRANGEMENTS AND HUMAN PATHOLOGIES. Common size. Comments. SYNDROMES. Rearrangements. Gene(s). of rearrangements.

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GENOMIC REARRANGEMENTS and MENDELIAN DISEASES

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  1. GENOMIC REARRANGEMENTS and MENDELIAN DISEASES • Eric LEGUERN • Département de génétique, cytogénétique et embryologie; • INSERM U289. • GH Pitié-Salpêtrière

  2. GENOMIC REARRANGEMENTS AND HUMAN PATHOLOGIES Common size Comments SYNDROMES Rearrangements Gene(s) of rearrangements Proximal region / CMT1A Smith-Magenis del(17) (p11.2) ~ 4 Mb RAI1 On paternal chromosome Prader-Willi del(15q)(q11q13) ~ 4 Mb [SNRPN] On maternal chromosome Angelman del(15q)(q11q13) ~ 4 Mb UB3A CATCH22 (Velo-cardio facial / del(22)(q11) ~1.5 Mb ? DiGeorge) Williams-Beuren syndrome del (7) (q11.23) ~ 2 Mb ELN + LIM kinase 1 distal region / deletion of Charcot-Marie-Tooth type 1A dup(17)(p11.2) ~ 1.5 Mb PMP22 Smith-Magenis distal region / deletion of Hereditary neuropathy with del(17)(p11.2) ~ 1.5 Mb PMP22 Smith-Magenis liability to pressure palsies

  3. (Stankiewicz et Lupski, 2002)

  4. MENDELIAN PATHOLOGIES and GENOMIC REARRANGEMENTS Common size MENDELIAN PATHOLOGIES Rearrangements Gene(s) Transmission of rearrangement AR Familial juvenile nephronophtisis Del 2q13 ~ 290 kb NPHP1 AD NF1 Neurofibromatosis type 1 Del 17q11.2 ~1,5 Mb XL Ichtyosis Del Xp22.32 ~ 1,9 Mb STS XL Del Xq28 ~300-500 kb F8 Hémophilia A AR Spinal muscular atrophy Inv/Dup 5q13.2 ~ 500 kb SMN AD Charcot-Marie-Tooth type 1A Dup 17p11.2 ~ 1.5 Mb PMP22 Hereditary neuropathy with AD Del 17p11.2 ~ 1.5 Mb PMP22 Liability to pressure palsies

  5. GENOMIC REARRANGEMENTS AND HUMAN PATHOLOGIES In these syndromes, a majority of patients have rearrangements of the same size. 1- What are the chromosomal mechanisms leading to recurrent genomic rearrangements ? 2- Is this recurrence due to a particularstructure of the genome in these regions ?

  6. CHARCOT-MARIE-TOOTH DISEASE Hereditary motor and sensory neuropathy characterized by: • Slowly progressive weakness and atrophy of distal limbs; • Distal sensory loss; • Decreased or absent tendon reflexes; • Pes cavus; • Scoliosis.

  7. Morphological features in demyelinating CMT (CMT1) • Demyelination : hypomyelination, hyperproliferation of Schwann cells • (« onion bulbs») • - ± axonal loss (Bouche et Oueslati, 1992)

  8. Charcot-Marie-Tooth DOMINANT FORMS VCM > 40m/s 30 < VCM ≤ 40m/s VCM ≤ 30m/s CMT1A CMT1B CMT1C CMT1D ? CMTX CMT2A CMT2B CMT2C CMT2D CMT2E CMT2F ? 7p14 7q11 12q23 17p11.2 1q23 16p13 10q21 Xq13-21 1p35 3q13 8p21 PMP22 P0 LITAF EGR2 Cx32 RAB7 GARS NEFL ? MFN2 ?

  9. CMT1A: duplication of the PMP22 gene Genetics - Autosomal dominant, incomplete penetrance; - 70% of CMT1 patients: duplication of a 1,5 Mb region within 17p11.2 (CMT1A) 3 copies of PMP22 gene 1.5 Mb 17p(Normal) 17p(Duplicated) PMP22 PMP22 3 Mb

  10. HEREDITARY NEUROPATHY WITH LIABILITY TO PRESSURE PALSIES Clinical features • Transient and painless episodes of nerve paresis and/or paresthesias • Frequently after mild trauma or compression • Isolated or numerous • Nerves most frequently implicated : peroneal, ulnar, radial, median • Recovery over days to months, partial or complete • Residual motor deficit in 10% of cases Electrophysiological features • Prolonged distal motor latencies • Slowing of MNCVs at common entrapment sites • Diffusely reduced SNAP amplitudes • Detects asymptomatic mutation carriers

  11. Neuropathological features in HNPP Patients (Poujet et al., 1992)

  12. HEREDITARY NEUROPATHY WITH LIABILITY TO PRESSURE PALSIES Genetics - Autosomal dominant, incomplete penetrance - In 87% of patients : deletion of the 1,5 Mb region in 17p11.2 1 copy of the PMP22 gene - Point mutations in PMP22

  13. Unequal recombinations between chromosome 17 homologues CMT1A-REP distal chimeric CMT1A-REP 1.5 Mb region PMP22 CMT1A-REP proximal Centromere HNPP CMT1A

  14. de novo duplication of paternal origin due to a crossing-over between 2 chomosome 17 homologues De novo Duplication 1 3 4 2 1/2 3/4 3 2 Duplication 2/3/4 4 1 2 3 4

  15. Images of recombinations during meiosis

  16. Questions • Reciprocal duplications and deletions : the same mechanism ? • recombination between two chromosome 17 homologues And • Parental origin : paternal meiosis • The CMT1A-REPs : mediators of recombination in 17p11.2 ? To study the chromosomal and molecular mechanisms leading to 17p11.2 rearrangements

  17. Two mechanisms of de novo duplication Paternal Maternal (A) (B) 1 /2 2/3 D17S122 3/5 D17S122 2/1 7/7 8/8 4 /3 D17S839 D17S839 4/3 D17S955 1/1 1/2 2 /3 D17S955 1/1 D17S921 1/5 1/5 2/ 1 1 3 5 /2 4/ 4 4 7 7 /8 1/ 2 2 1 1 /1 1 5 /1 interchromosomal rearrangement intrachromosomal rearrangement

  18. 7 6 3 6 5 1 3 2 1 1 4 2 1 3 2 2 2 6 4 5 5 5 6 6 2 2 3 5 1 2 2 2 2 1 2 2 1 2 6 4 SAL-902 De novo deletion 5 2 6 7 2 2 3 5 1 2 3 4 3 3 1 2 2 8 4 4 1 6 4 6 5 1 5 2 4 2 2 4 3 5 1 6 4 6 5 1 5 2 2 1 4 2 2 3 2 2 4 6 3 5 5 6 6 6 2 1 5 2 2 1 2 4 1 1 2 2 2 2 4 4 5 6 6 6 2 1 5 2 2 1 2 2 1 3 2 2 2 6 4 5 2 6 7 6 2 1 5 2 4 3 2 8 4 5 De novo deletion of maternal origin caused by a recombination in one chromosome 17

  19. intrachromosomal mechanisms : 2 models Dist. Dist. Prox. CMT1A HNPP + Prox. HNPP Centromere CMT1A HNPP

  20. Sister chromatid exchanges

  21. Parental origin and chromosomal mechanisms in 63 de novo rearrangements Parental origin Paternal Maternal Total Mechanism 49 5 54 Interchromosomal (CMT1A) (CMT1A) 1 6 7 Intrachromosomal (CMT1A) (4 CMT1A; 2 HNPP) 1 (HNPP) 1 (HNPP) 2 ND

  22. Common size of rearrangements Chromosomal mechanisms in other syndromes with deletions Syndromes Rearrangements Genes Smith-Magenis del 17p11.2 5 Mb ? CATCH22 del 22q11 3 Mb ? Williams-Beuren del 7q11.23 2 Mb ELN, LIMK1 SNRPN, PAR1, Prader-Willi del 15q11-q13 4 Mb PAR5 et IPW Angelman del 15q11-q13 4 Mb UBE3A

  23. Chromosoml mechanisms in the Williams-Beuren syndrome Dutly (1996) - Baumer (1998) Paternal Maternal Total 23 Interchr. 11 12 7 7 14 Intrachr. Chromosomal mechanisms in CATCH22 syndrome Baumer (1998) Paternal Maternal Total 8 Interchr. 5 3 1 1 2 Intrachr.

  24. NF1 gene (Lopez-Correa et al., 2000) Origin of deletions in 17q11 responsible for Neurofibromatosis type 1 • NF1:: • 5-10% of patients: deletion • of 1.5 Mb; • NF1-REP~100 kb, 98% homol. The deletions in the NF1 region are predominantly of maternal origin and inter-homologue

  25. Chromosomal mechanisms in other syndromes with deletions The 4 types of mechanisms are found in the other syndromes The influence of the gender of the transmitting parent is not observed Only the chromosomal mechanisms occurring in 17p11.2 depend on the gender of the transmitting parent

  26. Second part : Study of genomic rearrangements within the CMT1A-REPs

  27. Distribution of breakpoints within CMT1A-REPs Proximal Cen. Distal NHHP CMT1A Cen. Distal Proximal 8.8 4.6 3.2 (kb) 9.7 zone 4 zone 2 zone 3 zone 1 23.7% 0% 75% 1.3% CMT1A (n=76) 21% 3% 76% 0% HNPP (n=38)

  28. A new tool for molecular diagnosis Southern blot EcoRI/SacI, pJ7.8P SacI Prox. 7.8 kb NHHP CMT1A Dis. EcoRI 3.2 kb 3.2

  29. Study of rearrangements in CMT1A-REPs The 17p11.2 rearrangements are due to unequal recombinations between CMT1A-REPs 75% are localized in a 3.2 kb region

  30. Study of DNA exchanges in the 17p11.2 region ~75% of rearrangements are localized in a 1.7 kb region Zone 1 Zone 2 Zone 3 Zone 4 Tél. 23% 1% Cen. 1% 75% 75% SacI 3.2 EcoRI 74% NsiI EcoRI 1.7 Localisation of 28 breakpoints by sequencing in the 1.7 kb region in 25 CMT1A et 3 HNPP

  31. Localisation of 28 de novo rearrangements in the 1.7 kb region Prox. A C G A Transmitting parent C T T G Dist. Chimeric A T G C Child Breakpoint

  32. The 1.7 kb region of the proximal and distal CMT1A-REPs in the transmitting parent NsiI T T - G A C C G T C G C - A G C C A - C Prox. Dist. C C T T G T T A C T A G A T T T T A AAA GATAG EcoRI The 1.7 kb region of the chimeric CMT1A-REP in the child with a de novo CMT1A NsiI EcoRI GATAG T T - G G T T A C T A G A T T T T A AAA Chim. Crossing over

  33. PmeI PmeI NsiI Proximal a K c T R T C G C Y T A G C C A C Telomere Centromere G C C T K A T T T T A R C T A g t t Distal GATAG 309 M AAA EcoRI 524 M 283 P 3-9 P 26 P 75 P 284 P 1264 M 412 P Paternal P 1169 P 138 P Maternal M 387 P HNPP 406 M 150 P Interchromosomal 460 M 631 P Intrachromosomal 1-3 P 112 P 457 P 1139 P 271 P 160 P 21-1 P 270 P 397 M 902 M 395 P 373 P 100 bp 741 bp

  34. 1.9 kb A « hot spot » of recombination in the NF1-REPs (Lopez-Correa et al., 2001)

  35. NsiI Proximal y y r k r T T C G C T A G C C A C Cen. Distal G C C T k A T T T T A r C T A GATAG G T T EcoRI AAA c 271 P c *309 M c *21.3 P c 397 M s 1262 M s 283 P s 26 P s 412 P s 1169 P 138 P s 387 P s s 406 P 460 M s 284 P s s 150 P c 1-3 P c 395 P 631 P c 112 P s 457 P s 75 P c 160 P c 1139 P s 270 P s 373 P s c *902 M s *524 M s 259 P

  36. The 1.7 kb region of the proximal and distal CMT1A-REPs in the transmitting parent (LF 397 M): (conversion + crossing-over) NsiI T T - G A C C G T C G C A G C C A - C - Prox. 1 2 Dist. C C T G G T T A C T A G A T T T T A AAA GATAG EcoRI The 1.7 kb region of the chimeric CMT1A-REP in the child with a de novo CMT1A (LF 398) NsiI EcoRI T T T G G T T G C T A G A T T T T A AAA GATAG Chim.

  37. Distal Proximal Proximal Distal n=10 (9 CMT1A + 1 HNPP) n=18 (16 CMT1A + 2 HNPP) Crossing-over Crossing-over + conversion Which model explains crossing-over + conversion ?

  38. Double-strand break repair model G T T C CMT1A-REP proximal 5 ’ 3 ’ 3 ’ 5 ’ A C A G 1 A G T G 3 ’ 5 ’ CMT1A-REP distal 3 ’ 5 ’ T C A C HO G T C 3 ’ 5 ’ 5 ’ 3 ’ OH A G A 2 G A G T 5 ’ 3 ’ 3 ’ 5 ’ C A C T HO G T C 3 ’ 5 ’ 5 ’ 3 ’ 3 G A G A T G A 5 ’ 3 ’ 3 ’ 5 ’ C A C T . . C G T C 3 ’ 5 ’ 5 ’ 3 ’ 4 G T G G . A A G 5 ’ T 3 ’ 3 ’ 5 ’ A C C T

  39. Double-strand break repair model (2) . . C G T C 5 ’ 3 ’ 5 ’ 3 ’ G T G G 5 . G A A T 5 ’ 3 ’ 5 ’ 3 ’ A C T C Correction of mismatched bp using the 3 ’ 5 ’ strand as template b C G T C 5 ’ 3 ’ 3 ’ 5 ’ a G A C G c a c 6 A G G T 3 ’ 5 ’ 5 ’ 3 ’ A T C C b Breakpoints in a et b Breakpoints in a et c C G C C G T 3 ’ T 5 ’ T 5 ’ 3 ’ III I 5 ’ 3 ’ 3 ’ 5 ’ A A C A G C G G 7 5 ’ A G G G G T T G 3 ’ 3 ’ 5 ’ IV II 3 ’ 5 ’ 5 ’ 3 ’ A C C C A C T C Products with crossing-overs Products without crossing-over

  40. Study of mechanisms of DNA exchange in the 17p11.2 région • Conversions are associated with crossing-overs (36%) Double-strand break repair model • This model can be applied to inter- or intrachromosomal rearrangements, to paternal as well as maternal meiosis The double-strand break repair model is a universal mechanism for DNA exchange from Yeast to Humans

  41. Part 3 - Perspectives : The identification of factors promoting recombinations in 17p11.2

  42. Factors which promote recombinations Homology between sequences:proximal et distal CMT1A-REPs (98,7%) the NsiI-EcoRI region do not have a greaer homology The chromatin structure: the sites for initiating recombination are often located in the 5’ promoter region of genes (S. cerevisiae) the NsiI-EcoRI region is located in intron V of the COX10 gene

  43. Candidate sequences that promote recombination in the 1.7 kb region Position from NsiI site (bp) Name Consensus sequence Homology Species hypervariable 270 GGG TG GGAGG Human G)G GGGCAGG A(A/ minisatellites 380 GGGC G G A AAG 440 GG A CAGG G GG 480 GGGCAG TC AG hypervariable 540 Human C AG T GGTGG T T G CAG T GGGCAGG GG AGAGGT T minisatellites Mice GGG C GGAAAGGGT 380 Seq. MT (289 pb) Seq. of 1649 pb in Lmp2 hot spot Mice 750 CCTCTTCTTTCCTGG Chi seq. E. coli 450 G G TGGTGG GCTGGTGG

  44. Localisation of candidate sequences that promote recombination in the 1.7 kb region Proximal NsiI y y r r T C k C G A G C C A C T T Tél. Cen. G C T C k A T T T T A r C T A GATAG G T T Distal AAA EcoRI Sequence homologous to minisatellites Sequence homologous to Chi seq. 13 or 15 bp sequences Homologos to MHC seq

  45. And what about the rearrangements in other genomic regions…?

  46. ~4 Mb BP2 BP3 Cen. Tel. BP3A BP3B ~5 Mb ~3 Mb SMS-REPP SMS-REPM SMS-REPD Cen. Tel. Cen. Tel. VCFS-REPP VCFS-REPD ~2 Mb GTF2IP1 GTF2I Cen. Tel. WBS in 7q11.23 Cen. GTF2IP1 GTF2I VCFS in 22q11 PWS/AS In 15q11-q13 SMS in 17p11.2

  47. ~ 4 Mb ~ 0.2 Mb Tel Cen Median SMS-REP SRPD-TRED-KERD-CLPD SRPP-KERP-TREP-CLPP Distal SMS-REP Proximal SMS-REP REPEATS and CHROMOSOMAL REARRANGEMENTS The Smith-Magenis syndrome (Chen et al., 1997): The same junction fragment is detected by PFGE in 90% of patients; In these patients, the deletion is limited by CLPD and CLPP.

  48. The deletions responsible for FamilialJuvenileNephronophtisis (FJN) • FJN: • kidney; • AR; • 70% of patients: 200 kb deletion in 2q13 (Saunier et al., 2000)

  49. THE MECHANISMS RESPONSIBLE FOR GENOMIC REARRANGEMENTS (3) The incriminated mechanisms have an axe of symmetry Where are the duplications ?

  50. SYNDROMES ASSOCIATED WITH DUPLICATIONS • 7 patients with a dup(17)(p11.2p11.2)(Potocki et al., 2001) : • Heterogeneous phenotype : • Very mild facial dysmorphy : tooth defects (6/7); • Mental retardation from weak to moderate; • Behaviour abnormalities: autism, hyperactivity, attention deficit.... • Detection by PFGE of the same junction fragment detectedin the vast majority of patients with SMS. • 13 patients with a dup(22)(q11.2q11.2) (Ensenauer et al., 2003): • Variable facial dysmorphy of skull, eyes, ears… • Velo-pharyngeal deficit (7/10), urogenital malformations (5/11), deafness (5/12), …..; • Cognitive impairment (11/11); • 3 Mb duplicated region overlapping the deleted region in the catch22 syndrome

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