Génétique des DCP. Deciphering the molecular bases of ciliopathies. Estelle Escudier, INSERM U 681 Serge Amselem, INSERM U 654

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1 Génétique des DCP Estelle Escudier, INSERM U 681 Serge Amselem, INSERM U 654 Deciphering the molecular bases of ciliopathies Linkage analyses Candidate gene approaches Chromosome abnormalities Comparative genomics

2 Molecular bases of PCD? Linkage analyses Candidate gene approaches Chromosome abnormalities Comparative genomics Candidate gene approach based on Chlamydomonas mutants Normal strains (adapted from Witman, 1992) Immotile strains 9 and 6 mutants (IC78, IC69)

3 Isolation of human sequences orthologous to IC78 and IC69 Primers Chlamydomonas IC78 See urchin IC2 conserved regions Template = total RNA from human adult tissues: testis and trachea RT-PCR / Cloning / Sequencing Tissue expression of and DNAI2 estomac thyroïde moelle épinière ganglion lymphatique trachée glande surrénale moelle osseuse rate thymus prostate testicule uterus intestin grèle colon leucocytes estomac thyroïde moelle épinière ganglion lymphatique trachée glande surrénale moelle osseuse rate thymus prostate testicule uterus intestin grèle colon leucocytes 2.5 kb 2.4 kb DNAI2 expression restricted to trachea eand testis Pennarun et al. Am J Hum Genet 1999

4 Chromosomal localization of and DNAI p p p p q q 25 q Chromosome 9 Chromosome exons 53 kb DNAI2 14 exons 39 kb Pennarun et al. Am J Hum Genet 1999 mutation spectrum in PCD/KS W568S 1 G515S 699 ATG nt 1 WD1 WD2 WD3 WD4 WD TGA nt insT insAATA (fsh118x) W436X del W568X G>A Pennarun et al. Am J Hum Genet 1999 Guichard et al. Am J Hum Genet 2001 Zariwala et al. Am J Respir Cell Mol Biol 2001

5 Molecular bases of PCD? Linkage analyses Candidate gene approaches Chromosome abnormalities Comparative genomics Involvement of DNH5 in PCD/KS (absence of ) Linkage analysis (Homozygosity mapping) 5p14-14 (null mutations) Patient Control Omran et al. Am J Respir Cell Mol Biol 2000 Chung et al. Nature Genet 2002

6 Molecular bases of PCD? Linkage analyses Candidate gene approaches Chromosome abnormalities Comparative genomics RPGR IFT Moore et al. J Med Genet 2006 Molecular bases of PCD? Linkage analyses Candidate gene approaches Chromosome abnormalities Comparative genomics R2852X F508 R2852X R2852X F DNAH11 Normal EM SI F508 Bartoloni et al. PNAS 2002 RPGR IFT

7 Thioredoxins and Nucleoside diphosphokinases NDK family Group I NDK Enzymatic activity + Group II NDK enzymatic activity? mainly expressed in the testis (except NME6) NDK domain of IC1 TRX TXNDC3 () TXNDC6 NDK TRX domain NDPK domain1 NDPK domain2 TXNDC TXNDC3 gene defects Duriez et al PNAS 2007

8 TXNDC3 gene defects and related products Duriez et al PNAS 2007 Expression of the TXNDC3fl and TXNDC3d7 isoforms Duriez et al PNAS 2007

9 Impact of the c c>t variant on splicing of TXNDC3 transcripts Duriez et al PNAS 2007 Conservation of TXNDC3 and TXNDC3 exon7 throughout evolution Human Chimpanzee Dog Cow Mouse Rat Chicken Frog Pufferfish Tetraodon Sea urchin Ciona Overall identity 100% 97% 67% 67% 63% 61% 46% 38% 40% 39% 38% 34% A Conservation of the human regions encoded by TXNDC3 exon 7 and TXNDC6 exon 5 TXNDC3 E7 NGKIIEKIQGANAPLVNKKVINLIDEERKIAAGEMARPQ TXNDC6 E5 GGELVAVVRGANAPLLQKTILDQLEAEKKVLAEGRERKV B Phylogenetic conservation of the region encoded by TXNDC3 exon 7 Human NGKIIEKIQGANAPLVNKKVINLIDEERKIAAGEMARPQ Chimpanzee NGKIIEKIQGANAPLVNKKVINLIDEERKIAAGEMARPQ Dog NGKIIARINGANAPLVNKKITNLINEEKKIAAGEMVRPQ Cow NGTIVAKIQGANAPLVNQKIIALVNEERKIAAGEMVRPQ Mouse NGKIIAKIQGANAPLINRKVITLIDEERKIVAGEMDRPQ Rat NGKIIAKIQGANAPLINRKVIALIDEEKKIAAGEMARPQ Chicken NGKIIAIVRGANAPLLSKKITELVQEEREILAGQKERPE Frog GGELVAVVRGANGPLLQKTIIEQLAAEKKVLSQGSERHV Pufferfish GGELVGVLRGANAPLLQRMIVQKLGEEKMVLEKGVERKV Tetraodon GGELVGVLRGANAPLLQKMIVQKLSEEKMVLEKGGERKV Zebrafish GGELVSVLRGPNAPLLQKTIQEELSNEKNVLEHGGARRA Ciona GGELVAAVRGCNAPLVQETIQETLKNEHKILSGEMERKV Duriez et al PNAS 2007

10 Comparison of the binding of of TXNDC3fl and TXNDC3d7 to microtubules Duriez et al PNAS 2007 Molecular bases of PCD? Linkage analyses Candidate gene approaches Comparative genomics Chromosome abnormalities R2852X F508 R2852X R2852X F DNAH11 Normal EM F508 RPGR IFT TXNDC3 + SA

11 Ciliary defects in PCD n=273 10% 17% 29% 15% 8% 21% alone both DA IDA? IDA alone CC Kartagener with normal cilia Gene identification in PCD n=273 10% 10% 53% 17% 29% 15% 8% 21% alone both DA IDA? IDA alone CC Kartagener with normal cilia

12 Gene identification in PCD n=273 genes? alone both DA IDA? IDA alone CC Kartagener with normal cilia