NALP12, a gene responsible for periodic fever syndromes. Isabelle Jéru INSERM U.654, Paris, FRANCE

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1 NALP12, a gene responsible for periodic fever syndromes Isabelle Jéru INSERM U.654, Paris, FRANCE

2 Hereditary periodic fever syndromes (HPFs) Phenotype 6 clinical entities Fever episodes Abdominal pain Arthralgia Cutaneous signs Systemic inflammation Complication: renal amyloidosis FMF TRAPS HIDS FCAS MWS CINCA / NOMID

3 Hereditary periodic fever syndromes (HPFs) Phenotype 6 clinical entities 4 genes ID date Protein Fever episodes Abdominal pain Arthralgia Cutaneous signs Systemic inflammation Complication: renal amyloidosis FMF TRAPS HIDS FCAS MWS CINCA / NOMID MEFV TNFRSF1A MVK CIAS1 / NLRP pyrin TNFRSF1A mevalonate kinase cryopyrin / NALP3 Limits of molecular diagnosis Missense mutation functional test? Many unexplained cases genetic heterogeneity TNFRSF1A pyrin PYD B DD NALP3 cryopyrin PYD NBS LRR MVK GHMP kinase GHMP kinase

4 Choice of a candidate gene Srategy Sporadic cases + Clinical heterogeneity Candidate-gene approach

5 Choice of a candidate gene Srategy Sporadic cases + Clinical heterogeneity Candidate-gene approach Patients and candidate gene Attacks triggered by cold NALP family FCAS CIAS1 PYD *** * NBS * * * * cryopyrin / NALP3 LRR Myelo-monocytic expression Regulation of inflammatory signalling pathways Sequence homologies NALP12

6 Analysis of NALP12 I Family 1 II Ile283 Arg284/X Val285 Ile283 Arg284/X Val285 Nonsense Mutation (heterozygous) c.850c>t

7 Analysis of NALP12 Family 1 Family 2 I I II II Ile283 Arg284/X Val285 Ile283 Arg284/X Val285 exon 3 intron 3 Gln690 C A G C T g t w a r k Nonsense Mutation (heterozygous) c.850c>t Splice mutation (heterozygous) c inst

8 Analysis of NALP12 Family 1 Family 2 I I Ile283 Arg284/X Val285 exon 3 intron 3 Gln690 C A G C T g t w a r k II II Ile283 Arg284/X Val285 Ile283 Arg284/X Val285 exon 3 intron 3 Gln690 C A G C T g t w a r k Nonsense Mutation (heterozygous) c.850c>t Splice mutation (heterozygous) c inst

9 Analysis of NALP12 Family 1 Family 2 I I Ile283 Arg284/X Val285 Ile283 Arg284 Val285 exon 3 intron 3 exon 3 intron 3 A T C C G A G T T Gln690 Gln690 C A G C T g t a a g t C A G C T g t w a r k II II Ile283 Arg284/X Val285 Ile283 Arg284/X Val285 exon 3 intron 3 exon 3 intron 3 Gln690 Gln690 C A G C T g t a a g t C A G C T g t w a r k Nonsense Mutation (heterozygous) c.850c>t Splice mutation (heterozygous) c inst

10 Effect of the c inst mutation on splicing Study of NALP12 transcripts from minigenes Ex.3 Ex.4 T T C C A G G T G A T C Ex.3 G C A G C T Ex.4 A C C A G A bp L WT c inst no RNA L NALP12 WT Ex.3 Ex.4 T C A C T T C C A G A C C A G A G A G G β-actin c inst

11 Effect of the c inst mutation on splicing Study of NALP12 transcripts from minigenes Ex.3 Ex.4 T T C C A G G T G A T C Ex.3 G C A G C T Ex.4 A C C A G A bp L WT c inst no RNA L NALP12 WT Ex.3 Ex.4 T C A C T T C C A G A C C A G A G A G G β-actin c inst Consequences on transcripts Expected consequences at the protein level Ex bp 5 TTCCAG GTGATC......GCAGCT Ex.4 gttaag... aagcag ACCAGA Val Ile Val Val Ser Asn Ile Ala Ser Lys Met Glu // X TTCCAG ACCAGA Thr Arg Glu Asp Arg Ser Ala Gly Arg Leu Gln X Activation of a cryptic splice site Frameshift / Premature termination codon

12 Location of NALP12 mutations Arg284X * Val635ThrfsX12 Human NALP DPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQVQQQLLDTGRGH Chimp NALP DPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQAQQQLLDTGRGH Mouse NALP DLQTTYKDYVRRKFQLMEDRNARLGECVNLSNRYTRLLLVKEHSNPIWTQQKFVDVEWER Rat NALP DPQITYKDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPIWAQQKLLETGWEH Dog NALP12 62 DPRETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMWAQQKLLDTGWGQ Human NALP3 135 DYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIG--K 424 LRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGLWNQKILFEEQDLRK 424 LRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGIWNQKILFEEQDLRK 424 LRQTPRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFDEQDLGK 424 LRQTSRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFEEEDLGK 362 LRQTSRTTTAVYMLYLLSLMQPKPGSPILQSPPNQRGLCSLAADGLWNQKILFEEQDLRK 432 LAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRN Human NALP ARTVGHQASPIKIETLFEPDEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADGKLFQGR Chimp NALP ARTVGHQASPIKIETLFEPDEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADGKLFQGR Mouse NALP SRTRRHQTSPIQMETLFEPDEERPEPPHTVVLQGAAGMGKSMLAHKVMLDWADGRLFQGR Rat NALP SRTRGHQASPIQMETLFEPDEERPEPPRTVVLQGAAGMGKSMLTHKVMLDWADGRLFQDQ Dog NALP ARTVGHQASFIQMETLFEPDEERPEPPRTVVLQGAAGMGKSMLAHKVMLDWADGRLFQDR Human NALP3 193 TKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDR Human NALP FDYLFYINCREMNQSATECSMQDLIFSCWPEPSAPLQELIRVPERLLFIIDGFDELKPSF Chimp NALP FDYLFYINCREMNQSATECSMQDLISGCWPEPSAPLQELIRVPERLLFIIDGFDELKPSF Mouse NALP FDYVFYISCRELNRSHTQCSVQDLISSCWPERGISLEDLMQAPDRLLFIIDGFDKLHPSF Rat NALP FDYVFYISCRELNRSHTQCSVHDLLSSCWPEHGAPLEDLIRAPDRLLFIIDGFHELHPSF Dog NALP FDYLFYINCRKMNQSTAEQSAQDLISSCWPEPSVPLQELVRVPERLLFIIDGFHELKPSF Human NALP3 253 FDYLFYIHCRS-LVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAF * 484 HGLDGEDVSAFLNMNIFQKDINCERYYSFIHLSFQEFFAAMYYILDEGEGGAG HGLDGEDVSAFLNMNIFQKDMNCERYYSFIHLSFQEFFAAMYYILDEGERGAG HGLDGADVSTFLNVNIFQKGIKCEKFYSFIHLSFQEFFAAMYCALNGRE HGLDGASTFLNVNIFQKGIKCEKFYSFIHLSFQEFFTAMYCALHGRE HGLDGADVSSFLNMNIFQKDINCEKFYSFIHLSFQEFFAAMYYILDPGESRSS HGLQKADVSAFLRMNLFQKDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRL PDQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQW PDQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQW AVRRALAEYGFSERNFLALTVHFLFGLLNEEMRCYLERNLGWSISPQVKELAW AVRRALAEYGFSERNFLAHTVRFLFGLLNEEMRCYLERNLGWTISPQVKEEALAW PEHNVTRLLAEYEFSERSFLALTVRFLFGLLNEETRSYLEKSLCWKVSPHVKVELLEW 552 KLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKW Human NALP HDPQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHPRHVEIL Chimp NALP HDAQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHPRHVEIL Mouse NALP HDAQGPWCLCWEEKQPTLLGSLIRRLLLPQVSLLITTRPCALEKLHGLLEHPRHVEIL Rat NALP HDVQGPWCHCWEEKRPTELLLGSLIRRLLLPQLSLLITTRPCALEKLHGLLEHPRHVEIL Dog NALP HDPQGPWCLCWEEKRPTELLLSSLIRKKLLPELSVLITIRPTALEKLHRLLEHPRHVEIL Human NALP3 312 DEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEIL 595 IQSKAQSDGSTLQQGSLEFFSCLYEIQEEEFIQQALSHFQVIVVSNIASKMEHMVSSFCL 595 IQSKAQSDGSTLQQGSLEFFSCLYEIQK-EFTQQALSHFQVIVVSNIASKMEHMVSSFCL 588 IQNKAGSEGSTLQHGSLELLSCLYQEEDFIQQALSHFQVVVVRSISTKMEHMVCSFCA 588 IQNKARSEGSTLQHGSLELLSCLYEIQEEDFIQQALSHFQVVVVRNLSTKMEHVVCSFCA 533 IQRKAQSEGSTLQQGSLELFSCLYEIQEEDFIQQALSPFQVVVVNNIATKMEHMISSFCV 612 IKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEIN-LSTRMDHMVSSFCI Human NALP GFSEAERKEYFYKYFHNAEQAGQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQLEGGGL Chimp NALP GFSEAERKEYFYKYFHNAEQAGQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQLEGGGL Mouse NALP GFSEEARKEYFYRYFHNTGQASRVLSFLMDYEPLFTMCFVPMVSWVVCTCLKQQLESGEL Rat NALP GFSEAEREEYFYRYFHNTGQASQVFSFMRDYEPLFTMCFVPMVSWVVCTCLKQQLESGEL Dog NALP GFSEAERKEYFYKYFHNAEQAGQVFNFIRDNEPLFTLCFVPMVCWVVCTCLKQQLEDGGL Human NALP3 372 GFSEAKRKEYFFKYFSDEAQARAAFSLIQENLFTMCFIPLVCWIVCTGLKQQMESGKS 655 KRCRSAQVLHLYGATYSADGEDRARCSAGAHTLLVQL 654 KHCRSAQVLHLYGATYSADGEDRARCSAGTHTLLVQL 648 RYCRSTLHLHGSAYSTGMEDDPPEPSGVQTQSTYL 648 RYCRGTLHLYGSAYSTGAEDGPPEPPGAQTQSTHS 593 KNCRSALVLHLHGAAYSPDEDDGGRWASGPQMLPTQI 671 ENCHRVESLSLG-FLHNMPKEEEEEEKEG Mutations Deletion of highly conserved regions

13 NALP functions ligands PYD NBS LRR NALP TIR CC TLR

14 NALP functions ligands p65? PYD PYD NBS LRR CARD ASC CARD p20 p10 NALP pro-caspase1 TIR CC TLR p20 p10 caspase-1 p65 pro - IL-1β IL-1β IL-1β NF-κB signalling inhibition Activation of IL-1b secretion

15 Functional consequences NF-κB signalling pathway HEK293 transfected cells NF-κB activation (fold) NALP12-WT NALP12-Arg284X NALP12-Val635ThrfsX12 + p65 kda 98 WT Arg284X Val635ThrfsX12 64 NALP α-tubulin 50 + p65 WT: inhibition of NF-κB Mutants: loss of inhibition

16 Functional consequences NF-κB signalling pathway HEK293 transfected cells NF-κB activation (fold) NALP12-WT IL-1β secretion NALP12-Arg284X NALP12-Val635ThrfsX12 + p65 kda 98 WT Arg284X Val635ThrfsX12 64 NALP α-tubulin 50 + p65 Culture of patients monocytes (family 1) WT: inhibition of NF-κB Mutants: loss of inhibition IL-1β secretion (pg/ml) father mother x 50 x 100 child II.1 child II.2 Patients: IL-1β secretion anti-il-1 treatment

17 HRFs and autoinflammatory disorders Inflammatory / immune disorders Transmission Gene HPFs Hydatidiform mole Vitiligo-associated autoimmune disorders Crohn s disease Blau syndrome Bare lymphocyte syndrome Multiple sclerosis Biological and clinical continuum autoinflammatory / autoimmune disorders AD AR - multifactorial AD AR multifactorial NALP3 NALP12 NALP7 NALP1 NOD2 CIITA NALP3 / NALP12 PYD NBS LRR NALP1 / NALP7 PYD NBS LRR NOD2 CARD CARD NBS LRR CIITA CARD AD NBS LRR

18 Conclusions New HPF gene PYD NBS LRR NALP12 Improvement of disease management (genetic counselling, treatment) No functional redundancy NALP12 / NALP3 In vitro and in vivo demonstration of NALP12 function Mutational spectrum? Role of NALP12 in other autoinflammatory ou autoimmune disorders, Mendelian or multifactorial?

19 Acknowledgments INSERM U.654 E. Cochet P. Duquesnoy S. Amselem Clinicians G. Grateau (Hôpital Tenon, Paris) E. Grimprel (Hôpital Trousseau, Paris) V. Hentgen (Hôpital Le Chesnay, Versailles) M. Lackmy-Port-Lis (CHU Pointe-à-Pitre) J. Landman-Parker (Hôpital Trousseau, Paris) S. Marlin (Hôpital Trousseau, Paris) T. Sarkisian (Center of Medical Genetics, Armenia) Research departments L. Cuisset, C. Dodé, M. Delpech (Hôpital Cochin, Paris) T. and E. Alnemri, JW. Yu (TJU, Philadelphia, USA) J.C. Lecron (CHU Poitiers, Paris) K. McElreavey (Institut Pasteur, Paris)