NALP12, a gene responsible for periodic fever syndromes Isabelle Jéru INSERM U.654, Paris, FRANCE
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
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 / NLRP3 1997 1999 1999 2001 pyrin TNFRSF1A mevalonate kinase cryopyrin / NALP3 Limits of molecular diagnosis Missense mutation functional test? Many unexplained cases genetic heterogeneity TNFRSF1A pyrin PYD B30.2.. DD NALP3 cryopyrin PYD NBS LRR MVK GHMP kinase GHMP kinase
Choice of a candidate gene Srategy Sporadic cases + Clinical heterogeneity Candidate-gene approach
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
Analysis of NALP12 I Family 1 II Ile283 Arg284/X Val285 Ile283 Arg284/X Val285 Nonsense Mutation (heterozygous) c.850c>t
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.2072+3inst
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.2072+3inst
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.2072+3inst
Effect of the c.2072+3inst 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 650 500 L WT c.2072+3inst 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 650 500 β-actin c.2072+3inst
Effect of the c.2072+3inst 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 650 500 L WT c.2072+3inst 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 650 500 β-actin c.2072+3inst Consequences on transcripts Expected consequences at the protein level Ex.3 170 bp 5 TTCCAG GTGATC......GCAGCT Ex.4 gttaag... aagcag ACCAGA 3 635 646 1061 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
Location of NALP12 mutations Arg284X * Val635ThrfsX12 Human NALP12 124 DPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQVQQQLLDTGRGH Chimp NALP12 124 DPQETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMQAQQQLLDTGRGH Mouse NALP12 124 DLQTTYKDYVRRKFQLMEDRNARLGECVNLSNRYTRLLLVKEHSNPIWTQQKFVDVEWER Rat NALP12 124 DPQITYKDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPIWAQQKLLETGWEH Dog NALP12 62 DPRETYRDYVRRKFRLMEDRNARLGECVNLSHRYTRLLLVKEHSNPMWAQQKLLDTGWGQ Human NALP3 135 DYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQQEREQELLAIG--K 424 LRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGLWNQKILFEEQDLRK 424 LRQTSRTTTAVYMLYLLSLMQPKPGAPRLQPPPNQRGLCSLAADGIWNQKILFEEQDLRK 424 LRQTPRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFDEQDLGK 424 LRQTSRTTTAVYMFYLLSLMQPKPGTPTFKVPANQRGLVSLAAEGLWNQKILFEEEDLGK 362 LRQTSRTTTAVYMLYLLSLMQPKPGSPILQSPPNQRGLCSLAADGLWNQKILFEEQDLRK 432 LAQTSKTTTAVYVFFLSSLLQPRGGSQEHGLCAHLWGLCSLAADGIWNQKILFEESDLRN Human NALP12 184 ARTVGHQASPIKIETLFEPDEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADGKLFQGR Chimp NALP12 184 ARTVGHQASPIKIETLFEPDEERPEPPRTVVMQGAAGIGKSMLAHKVMLDWADGKLFQGR Mouse NALP12 184 SRTRRHQTSPIQMETLFEPDEERPEPPHTVVLQGAAGMGKSMLAHKVMLDWADGRLFQGR Rat NALP12 184 SRTRGHQASPIQMETLFEPDEERPEPPRTVVLQGAAGMGKSMLTHKVMLDWADGRLFQDQ Dog NALP12 122 ARTVGHQASFIQMETLFEPDEERPEPPRTVVLQGAAGMGKSMLAHKVMLDWADGRLFQDR Human NALP3 193 TKTCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARKMMLDWASGTLYQDR Human NALP12 244 FDYLFYINCREMNQSATECSMQDLIFSCWPEPSAPLQELIRVPERLLFIIDGFDELKPSF Chimp NALP12 244 FDYLFYINCREMNQSATECSMQDLISGCWPEPSAPLQELIRVPERLLFIIDGFDELKPSF Mouse NALP12 244 FDYVFYISCRELNRSHTQCSVQDLISSCWPERGISLEDLMQAPDRLLFIIDGFDKLHPSF Rat NALP12 244 FDYVFYISCRELNRSHTQCSVHDLLSSCWPEHGAPLEDLIRAPDRLLFIIDGFHELHPSF Dog NALP12 182 FDYLFYINCRKMNQSTAEQSAQDLISSCWPEPSVPLQELVRVPERLLFIIDGFHELKPSF Human NALP3 253 FDYLFYIHCRS-LVTQRSLGDLIMSCCPDPNPPIHKIVRKPSRILFLMDGFDELQGAF * 484 HGLDGEDVSAFLNMNIFQKDINCERYYSFIHLSFQEFFAAMYYILDEGEGGAG------- 484 HGLDGEDVSAFLNMNIFQKDMNCERYYSFIHLSFQEFFAAMYYILDEGERGAG------- 484 HGLDGADVSTFLNVNIFQKGIKCEKFYSFIHLSFQEFFAAMYCALNGRE----------- 484 HGLDGASTFLNVNIFQKGIKCEKFYSFIHLSFQEFFTAMYCALHGRE----------- 422 HGLDGADVSSFLNMNIFQKDINCEKFYSFIHLSFQEFFAAMYYILDPGESRSS------- 492 HGLQKADVSAFLRMNLFQKDCEKFYSFIHMTFQEFFAAMYYLLEEEKEGRTNVPGSRL 537 --PDQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQW 537 --PDQDVTRLLTEYAFSERSFLALTSRFLFGLLNEETRSHLEKSLCWKVSPHIKMDLLQW 533 -----AVRRALAEYGFSERNFLALTVHFLFGLLNEEMRCYLERNLGWSISPQVKELAW 533 -----AVRRALAEYGFSERNFLAHTVRFLFGLLNEEMRCYLERNLGWTISPQVKEEALAW 475 --PEHNVTRLLAEYEFSERSFLALTVRFLFGLLNEETRSYLEKSLCWKVSPHVKVELLEW 552 KLPSRDVTVLLENYGKFEKGYLIFVVRFLFGLVNQERTSYLEKKLSCKISQQIRLELLKW Human NALP12 304 HDPQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHPRHVEIL Chimp NALP12 304 HDAQGPWCLCWEEKRPTELLLNSLIRKKLLPELSLLITTRPTALEKLHRLLEHPRHVEIL Mouse NALP12 304 HDAQGPWCLCWEEKQPTLLGSLIRRLLLPQVSLLITTRPCALEKLHGLLEHPRHVEIL Rat NALP12 304 HDVQGPWCHCWEEKRPTELLLGSLIRRLLLPQLSLLITTRPCALEKLHGLLEHPRHVEIL Dog NALP12 242 HDPQGPWCLCWEEKRPTELLLSSLIRKKLLPELSVLITIRPTALEKLHRLLEHPRHVEIL Human NALP3 312 DEHIGPLCTDWQKAERGDILLSSLIRKKLLPEASLLITTRPVALEKLQHLLDHPRHVEIL 595 IQSKAQSDGSTLQQGSLEFFSCLYEIQEEEFIQQALSHFQVIVVSNIASKMEHMVSSFCL 595 IQSKAQSDGSTLQQGSLEFFSCLYEIQK-EFTQQALSHFQVIVVSNIASKMEHMVSSFCL 588 IQNKAGSEGSTLQHGSLELLSCLYQEEDFIQQALSHFQVVVVRSISTKMEHMVCSFCA 588 IQNKARSEGSTLQHGSLELLSCLYEIQEEDFIQQALSHFQVVVVRNLSTKMEHVVCSFCA 533 IQRKAQSEGSTLQQGSLELFSCLYEIQEEDFIQQALSPFQVVVVNNIATKMEHMISSFCV 612 IKAKAKKLQIQPSQLELFYCLYEMQEEDFVQRAMDYFPKIEIN-LSTRMDHMVSSFCI Human NALP12 364 GFSEAERKEYFYKYFHNAEQAGQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQLEGGGL Chimp NALP12 364 GFSEAERKEYFYKYFHNAEQAGQVFNYVRDNEPLFTMCFVPLVCWVVCTCLQQQLEGGGL Mouse NALP12 364 GFSEEARKEYFYRYFHNTGQASRVLSFLMDYEPLFTMCFVPMVSWVVCTCLKQQLESGEL Rat NALP12 364 GFSEAEREEYFYRYFHNTGQASQVFSFMRDYEPLFTMCFVPMVSWVVCTCLKQQLESGEL Dog NALP12 302 GFSEAERKEYFYKYFHNAEQAGQVFNFIRDNEPLFTLCFVPMVCWVVCTCLKQQLEDGGL Human NALP3 372 GFSEAKRKEYFFKYFSDEAQARAAFSLIQENLFTMCFIPLVCWIVCTGLKQQMESGKS 655 KRCRSAQVLHLYGATYSADGEDRARCSAGAHTLLVQL 654 KHCRSAQVLHLYGATYSADGEDRARCSAGTHTLLVQL 648 RYCRSTLHLHGSAYSTGMEDDPPEPSGVQTQSTYL 648 RYCRGTLHLYGSAYSTGAEDGPPEPPGAQTQSTHS 593 KNCRSALVLHLHGAAYSPDEDDGGRWASGPQMLPTQI 671 ENCHRVESLSLG-FLHNMPKEEEEEEKEG-------- Mutations Deletion of highly conserved regions
NALP functions ligands PYD NBS LRR NALP TIR CC TLR
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
Functional consequences NF-κB signalling pathway HEK293 transfected cells NF-κB activation (fold) 600 400 200 0 NALP12-WT NALP12-Arg284X NALP12-Val635ThrfsX12 + p65 kda 98 WT Arg284X Val635ThrfsX12 64 NALP12 50 64 α-tubulin 50 + p65 WT: inhibition of NF-κB Mutants: loss of inhibition
Functional consequences NF-κB signalling pathway HEK293 transfected cells NF-κB activation (fold) 600 400 200 0 NALP12-WT IL-1β secretion NALP12-Arg284X NALP12-Val635ThrfsX12 + p65 kda 98 WT Arg284X Val635ThrfsX12 64 NALP12 50 64 α-tubulin 50 + p65 Culture of patients monocytes (family 1) WT: inhibition of NF-κB Mutants: loss of inhibition IL-1β secretion (pg/ml) 10000 1000 100 10 1 father mother x 50 x 100 child II.1 child II.2 Patients: IL-1β secretion anti-il-1 treatment
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
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?
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)