Validation of A Diagnostic Score For Molecular Analysis of Hereditary Auto inflammatory Syndromes With Periodic Fever.

Transcription

1 Validation of A Diagnostic Score For Molecular Analysis of Hereditary Auto inflammatory Syndromes With Periodic Fever. Silvia Federici 2 Division of Pediatrics, G. Gaslini Institute, Genoa, ITALY Rome, 4-8 April 2008

2 Autoinflammatory syndromes Disease Gene/chromosome Protein Transmission Familial Mediterranean Fever Hyper IgD syndrome TRAPS FCAS, MWS, CINCA Blau s syndrome PAPA syndrome Majeed s syndrome CRMO (murine) MEVF 16p13.3 MVK 12q24 TNFRSF1A 12p13 CIAS1 1q44 CARD15/NOD2 16q12 PSTPIP1 15q24-q25.1 LPIN2 18p PSTPIP2 18p Pyrin Mevalonatokinase TNF p55 receptor Cryopyrin CARD15/ NOD2 PSTPIP1 LPIN2 PSTPIP2 Autosomal recessive Autosomal recessive Autosomal dominant Autosomal dominant Autosomal dominant Autosomal dominant Autosomal recessive Autosomal recessive

3 Recruitment: January 2002-January Patient with suspected autoinflammatory syndromes Patients with at least one mutation (20,48%) Roma. 68 Patients with relevant mutations (9,67%) : at least a couple of families sent MEFV TNFRSF1A MVK CIAS-1

4 PFAPA syndrome (Periodic Fever, Aphthous stomatitis, Pharyngitis, Adenitis) Recurrent fever Early onset (< 5 years) At least one of the following symptoms without upper respiratory infections - Aphthosis - Cervical adenitis - Pharyngitis Exclusion of cyclic neutropenia Free intervals within febrile attacks Normal development Marshall et al, 1987 Low incidence of familial cases. No genetic etiology is known. Self-remitting. Very similar to Hyper IgD!!

5 Rationale of the study: additional arguments supporting the study Only 10-25% of patients with clinical manifestations consistent with periodic fever turn out to be positive for mutations in already known causative genes in few patients genetic tests are useful for the diagnosis. Hight costs related to the genetic testing possibility for reducing the number of tests required.

6 Aim of the study Is it possible to identify some clinical variables able to detect a subset of patients with periodic fevers with higher probability to result positive at genetic analysis for already known genes?

7 Inclusion criteria Periodic or recurrent fever attacks of unknown origin Exclusion of infections, immunodeficiency, autoimmune disorders Free intervals within febrile attacks At least two of the following symptoms during attacks: pharyngitis, lymphadenopathy, gastrointestinal symptoms (vomiting, diarrhea, abdominal pain), mucocutaneous manifestations (erythematous macules and papules, stomatitis), muscle-skeletal manifestations (arthritis/arthralgia, myalgias), splenomegaly.

8 Data collection ITALIAN PERIODIC FEVER STUDY G ROUP Institution.. Patient code:.. Sex M F Date of birth /../. Father : affected yes Mother affected yes no no Ethnic group (caucasic, jewish, armenian):.. Age at onset of fever attacks.... Date of the present evaluation. Clinical characteristic of febrile episodes: Mean duration of episodes (days ) < > 10.. Temperature >38 C yes no N. of episodes/year < >12 Fever -free intervals Regular (periodic) Irregular (non periodic) Mean duration (days ) Chills at feve r onset yes n o Fever episodes during summer period yes no

9 Data collection Fever-associated manifestations Muco-cutaneous Localized erythematous patches * Generalized erythematous serpiginous pathces Maculo -papular rash * Urticarial r ash * Aphtous stomatitis Aphthous ulcers at genitalia Exudative pharyngitis Erythematous pharyngitis Others Ever Often Sometime Never At onset Present Onset Present Onset Present Onset Present *Specify the prevalent localization... Muscular -skeletal system Ever Often Sometime Never Onset Present Onset Present Onset Present Onset Present Arthralgia Myalgia s Monoarthritis * Oligoarthritis * (! 4 joints ) Polyarthritis * ("4 joints ) Other * Specify the prevalent localization..

10 Data collection Recruitment: Jan Sept Patients enrolled: 353 Not included: 119 (not fulfilling inclusion criteria, incomplete clinical data, screened for CIAS-1 only.) Patients analyzed: 234 (complete clinical information) Ethnicity: 3 Jews, 1 Brazil, 2 Arab, 1 Mauritius, 1 India

11 Molecular analysis Screening of genomic DNA extracted from peripheral blood lymphocytes by means of denaturing high-performance liquid chromatography (DHPLC) and direct sequence analysis MVK gene (exons 1-10) MEFV gene (exons 2, 3, 5, 10) TNFRSF1A gene (exons 1-6)

12 18 22 MVK TFNRSF1A Results genetics (234 pts) Homozygous/comp. heterozygous 5 cystein mutations (C52Y, C43R, C55Y, C88Y, C29Y) 1 T50M 1 deletion of exon 6 15 R92Q 52 MEFV 12 homozygus/comp. heterozygous, 40 heterozygous 142 Negative

13 Question 1 Are there any already existing clinical criteria of potential usefulness to distinguish between positive and negative patients?

14 Testing the existing criteria Patients positive for FMF criteria (121/234) 14/18 HIDS 0/7 TRAPS 8/15 R92Q 12/12 homoz. for MEVF 5/5 heteroz. for M694V (MEFV) 17/35 heteroz. for MEFV 66/142 negative FMF Criteria Major criteria 1-4 Typical attacks Peritonitis (generalized) Pleuritis or pericarditis Monoartritis Fever alone Incomplete abdominal attacks Minor criteria 1-2 Incomplete attacks involving: - Chest - Joint 3. Exertional leg pain 4. Favorable response to colchicine A&R 40: ; 1997 Patients positive for PFAPA Criteria (110/234) 15/18 HIDS 4/7 TRAPS 5/15 R92Q 1/12 homoz. for MEFV 25/40 heteroz. for MEFV 61/142 negative PFAPA criteria 1. Recurrent fever. Early onset (< 5 years) 2. At least one of the following symptoms without upper respiratory infections - Aphthosis - Cervical adenitis - Pharyngitis 3. Exclusion of cyclic neutropenia 4. Free intervals within febrile attacks 5. Normal development Marshall et al, 1989

15 Question 2 Is it possible to identify a set of variables that may predict the probability for a single patient to display significant mutations of the known genes?

16 Statystical analysis Clinical manifestations (234 pts) Periodic fever MVK TNFRSF1A MEFV Negative Severe* R92Q Homoz. Heteroz. N Age at onset (months; mean ± SD) 10.4 ± ± ± ± ± ± 102 Days of fever 4.3 ± ± ± ± ± ± 10.3 Family history (%) Periodicity Aphthosis Pharyngitis Rash Cervical lymph nodes Abdominal pain Diarrhea Arthralgias Myalgias Thoracic pain

17 Step 1: univariate logistic regression VARIABILE OR (95% CI) P Age at onset 0,96 (0,94-0,99) 0,005 Days of fever 1,01 (0,97-1,05) 0,73 Positive family history 4,6 (1,8-11,50) 0,001 Aphthosis 0,5 (0,2-1,1) 0,09 Thoracic pain 3,9 (1,32-11,6) 0,01 Rash 1,50 (0,70-3,20) 0,29 Erythematous Pharyngitis 1,1 (0,5-2,4) 0,74 Exudative Pharyngitis 0,6 (0,3-1,3) 0,19 Conjunctivitis 0,9 (0,3-2,6) 0,85 Periorbital edema 2,7 (0,7-10,30) 0,13 Cervical lymph node enlargement 1,4 (0,6-2,92) 0,44 Cervical lymph node pain 2,7 (1,3-5,7) 0,009 Splenomegaly 2,6 (1,1-6,1) 0,037 Pleurisy 5,3 (1,5-18,5) 0,09 Abdominal pain 21,33 (4,9-92,10) 0,001 Diarrhea 5,0 (2,3-10,8) 0,001 Vomiting 2,3 (1,10-4,90) 0,026 Arthralgia 1,9 (0,9-3,8) 0,1 Myalgia 1,5 (0,7-3,1) 0,29 Dependent variable positive vs negative status Variables with a high discriminant power were included in a multivariate model (p<0.20) Headache 0,9 (0,4-1,94) 0,81

18 Step 2: multivariate logistic analysis Set of factors that indipendently affect the probability to be positive, each weighted according to the parameters estimated by the model Var i Coding β i Age at onset months Abdominal pain Apthosis Thoracic pain Diarrhea Family history Never = 0 Sometimes or Often = 2 Always = 3 Never = 0 Sometimes or Often =1 Always = 2 Absent = 0 Present = 1 Never = 0 Sometimes = 1 Often = 2 Always = 3 Negative = 0 Positive = Diagnostic score = (0.067 x Age) + (1.494 x Abd. pain) - (1.504 x Aphtosis) + (1.958 x Thoracic pain) + (0.901 x Diarrhea) + (1.503 x Family history)

19 Sensitivity ROC curve Specificity 82.7% Sensitivity 94.6% Cut off 15% 1-Specificity Gattorno et al, A&R (in press)

20 Total number of patients: /2 24/35 70% Percentage of subjects 60% 50% 40% 30% 20% 10% 0% 0% 20% 40% 60% 80% 100% Probability to be positive Cut off 15% NEGATIVE POSITIVE

21 Validation study (independent data set) Italian patients received after September 2005 and not included in the previous data-set Patients from other European Centers All the clinical variables in already diagnosed (genetics) pediatric patients has been collected retrospectically by filling the same form used for the training set.

22 ROC curve Specificity 72% Sensitivity 87 % Sensitivity Cut off 15% 1-Specificity

23 Conclusions (i) Some clinical variables associated with periodic fever are strongly associated to the presence of relevant mutations of one gene related to autoinflammatory disorders: - early age at onset - positive family history - abdominal pain - diarrhoea - thoracic pain - aphthosis

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25 Are there some variables able to distinguish the three different forms (HIDS, TRAPS, FMF) among positive patients?

26 Regression tree analysis (37 positive patients of training set) 2 days High risk patients (score > 15%) Fever duration (p < 0.001) 2-7 days 7 days MEFV Vomiting (p = 0.004) TNFRS1A No Yes Splenomegaly (p = 0.05) MVK No Yes MEFV MVK

27 Whole patients Group (234 patients) Validation group (7 7 patients) Diagnosis HIDS Severe TRAPS FMF Mild TRAPS Heterozygous MEFV Negative HIDS Severe TRAPS FMF Mild TRAPS Heterozygous MEFV Negative # of patients Positive family history Oral Aphthosis Erythematosus pharyngitis Exudative pharyngitis Rash Genital aphthosis Conjunctivitis Periorbital edema Cervical lymph nodes Pain at cervical lymph nodes Splenomegaly Thoracic pain Pleurisy Pericarditis Abdominal pain Diarrhea Vomiting Arthralgia Myalgia Arthritis Headache Age at onset (months) mean (SD) Fever duration (days) mean (SD) 10.4 (8.3) 4.3 (1.4) 17.9 (17. 1) 15.3 (7.8) 16.6 (11.2) 3.0 (1.9) 58.1 (64.4) 4.7 (3.7) 29.6 (44.6) 5.9 (9.1) 49.5 (58.2) 5.4 (7.5 ) 12.0 (6.3) 4.2 (1.0) 45.0 (38.0) 14.4 (9.3) 35.1 (28.9) 2.2 (0.9) 48.0 (24.0) 9.0 (5.2) 24.3 (20.2) 42.9 (56.8) 4.0 (2.6) 5.7 (3.8)

28 Validation of the regression tree Fever duration 27 positive patients at the diagnostic score (Validation set) - 12 HIDS - 4 TRAPS - 11 FMF 2 days 2-7 days MEFV (5/11) No Splenomegaly Vomiting Yes MVK (6/12) + 2 FMF TNFRS1A (3/4) No Yes MEFV (4/11) + 1 TRAPS MVK (6/12)

29 Conclusions (ii) Some clinical variables could differentiate among different diseases - duration of episodes (TRAPS, FMF) - splenomegaly (Hyper IgD) - vomiting (Hyper IgD) This evidence-based score has been demonstrated to provide good sensibility and specificity. This tool could help general pediatricians to select those patients with higher probability to be positive to the genetic analysis.

30 Patient with periodic fever other causes) Diagnostic score* (exclude Sensitivity to cold exposure? Urticarial rash? Hearing loss? Exclude NALPs-related diseases High probability (> 15%) Low probability (< 15%) Go for genetic testing Duration of episodes Follow-up (6-12 months) 2 days 3-6 days 7 days Persistence (new symptoms?) Resolution or improvement MEFV No Splenomegaly Yes No Yes Vomiting TNFRS1A MVK If negative test Go for genetic testing Follow-up (6-12 months)

31 Acknowledgement G. Gaslini Institute (Genoa) 2nd Division of Pediatrics A. Martini M. Gattorno N. Solari M.A. Pelagatti Lab. Of Molecular Genetics I. Ceccherini A.d Osualdo F. Caroli Ospedale Galliera (Genoa) M. Baldi M. Cecconi Italian centers A. Meini, L. Obici (Pavia), F. Zulian (Padova) A. Tommasini, G. Bossi (Pavia) L. Breda (Chieti), S. Martino (Torino) Other centers P Woo (London), I. Kone-Paut (Paris), J Frenkel, A Govers (Utrecht), I Touitou (Paris) Dept. of Statistics, Univ. of Genoa M.P. Sormani

32 Patient with periodic fever (exclude other causes) Diagnostic score* Sensitivity to cold exposure? Urticarial rash? Hearing loss? Exclude NALP-related diseases High risk (>15%) Low risk (< 15%) Ethnicity & positive FMF Criteria No Go for genetic testing Duration of episodes Follow-up (6-12 months) Yes 2 days 3-6 days 7 days Persistence (new symptoms?) Resolution or improvement MEFV No Splenomegaly No Yes Vomiting Yes MVK TNFRSF1A Go for genetic testing Follow-up (6-12 months) If negative test

33 How to calculate the diagnostic score

34 As an example of the score calculation, a patient (patient A) with an age at onset of 18 months, abdominal pain (sometimes), aphtosis always present, no thoracic pain, no diarrhea and negative family history (suggestive for a PFAPA) will have a score: score =! 3.587! 0.038" " 2! 1.275" " " " 0 =! 3.65 Diagnostic score = (0.038 x Age) + (1.581x Abd. pain) - (1.275 x Aphtosis) + (1.597 x Thoracic pain) + (0.665 x Diarrhea) + (2.18 x Family history) The score calculated for each patient can be converted in a probability to be positive to the genetic test according to the formula: e P( + ) = 1+ e score score For example the patient A, with a score=0.36, will have a probability to be positive to the genetic test: P! e ) = 1+ e 3.65 ( +! = = 2.5%

35 Variables Valore weight code SCORE Costant -3,587 Age at onset (months) 7-0,04 7-0,266 Abdominal pain never 1, Aphtosis never -1, Thoracic pain never 1, Diarrhea never 0, Family history never 2,18 1 2,18 SCORE= -1,673 PROBABILITY'= 15,8%

36

37 Patient with periodic fever (exclude other causes) Diagnostic score* High probability (> 15%) Low probability (< 15%) Ethnicity & positive FMF Criteria No Go for genetic testing Duration of episodes Follow-up (6-12 months) Yes 2 days 3-6 days 7 days Persistence (new symptoms?) Resolution or improvement MEFV No Splenomegaly No Yes Vomiting Yes MVK TNFRS1A Go for genetic testing Follow-up (6-12 months) If negative test

38 Patient with periodic fever (exclude other causes) 234 Diagnostic score 59 High probability (> 15%) Low probability (< 15%) Go for genetic testing Follow-up (6-12 months) Positive family history Duration 7 days Age at onset < 1.5 anni Duration: 3-7 days Duration 3 days Abdominal pain Chest pain Persistence Resolution or improvement TNFRS1A MVK MEFV Positive Test Negative test Go for genetic testing Follow-up (6-12 months)

39 Patient with periodic fever (exclude other causes) 234 Diagnostic score 59 High probability (> 15%) Low probability (< 15%) Go for genetic testing Follow-up (6-12 months) Duration 7 days Duration: 2-7 days Vomiting Splenomegaly Duration 2 days Persistence Resolution or improvement TNFRS1A MVK MEFV 6/7 16/17 12/12 Positive Test Negative test Go for genetic testing Follow-up (6-12 months)

40 Node 0 Category % n HIDS T RAPS vere F M F vere HIDS 48,6 18 T RAPS vere 18,9 7 F M F vere 32,4 12 T otal 100,0 37 Duration Durata2 P-value=0,000, Chi-square=32,982, df=4 <= 2,00 (2,00, 7,00] > 7,00 Node 1 Node 2 Node 3 Category % n Category % n Category % n HIDS T RAPS vere 14,3 1 0,0 0 HIDS T RAPS vere 73,9 17 4,3 1 HIDS T RAPS vere 0,0 0 85,7 6 F M F vere 85,7 6 F M F vere 21,7 5 F M F vere 14,3 1 T otal 18,9 7 T otal 62,2 23 T otal 18,9 7 Regression tree analysis Vomiting VOM IT O P-value=0,004, Chi-square=10,895, df=2 <= 0 > 0 Node 4 Category % n HIDS 50,0 5 T RAPS vere 0,0 0 F M F vere 50,0 5 T otal 27,0 10 Splenomegaly SPLENOM EGbin P-value=0,050, Chi-square=3,855, df=1 Node 5 Category % n HIDS 92,3 12 T RAPS vere 7,7 1 F M F vere 0,0 0 T otal 35,1 13 <= 0,00 > 0,00 Node 6 Category % n HIDS 20,0 1 T RAPS vere 0,0 0 F M F vere 80,0 4 T otal 13,5 5 Node 7 Category % n HIDS 80,0 4 T RAPS vere 0,0 0 F M F vere 20,0 1 T otal 13,5 5

41 Patient with periodic fever (exclude other causes) Diagnostic score High probability (> 15%) Low probability (< 15%) Go for genetic testing Follow-up (6-12 months) Positive family history Duration 7 days Age at onset < 1.5 anni Duration: 3-7 days Splenomegaly, vomiting Duration 3 days Abdominal pain Chest pain Persistence Resolution or improvement TNFRS1A MVK MEFV Go for genetic testing Follow-up (6-12 months) Positive Test Negative test

42 Table VI: Global model performance (score + regression tree) on the validation set Predicted diagnosis MKD MKD TRAPS Mild TRAPS 12* 0 0 Genetic results FMF 2 (NCC) Heterozygous MEFV Negative Total 1(NCC) 6 (FP) 21 TRAPS 0 2* (FP) 5 FMF 0 1 (NCC) 0 9* 0 3 (FP) 13 Negative 1 (FN) 1 (FN) 3 2 (FN) 2 28* 37 Total *correctly classified patients FP= false positive FN= false negative NCC= true positive not correctly classified

43 Case 1 Male, 7 year-old, caucasian No family history Age at onset: 7 months Duration: 5 days (n. episodes: 9) Aphtousis: often Pharyngitis: often Arthralgia: sometime Abdominal pain: often; diarrhea & vomiting: sometime Thoracic pain: sometime Splenomegaly: sometime MVK: V377I/V377I

44 Case 2 10 years old girl, caucasian No family history Age at onset: 24 months Duration: 3 days (n. episodes: ) Abdominal pain: always Vomiting: often Thoracic pain: sometime MEFV: M680I/M680I

45 Case 3 12 years old girl, caucasian Family history: yes Age at onset: 10 years (120 months) Duration: 15 days (n. episodes: 6 ) Periodicity: yes Aphtousis: sometime Pharyngitis: sometime Lymphoadenopathy: sometime Arthralgia: often Myalgia: sometime Headace: often MVK, MEFV, TNFRSF1A: negative

46 Case 4 1 year old boy, indian Family history: yes Age at onset: 1 month Duration: 9 (n. episodes: 10) Periodicity: yes Exudative pharyngitis: sometime Erythematous pharyngitis: often Lymphoadenopathy: sometime Abdominal pain: sometime TNFRSF1A: C29Y

47 Giovanni, 7 year-old Mom, dad. I really don t care if vomiting is so important for Dutch children may I have some more pasta, PLEASE!?

48 Analisi multivariata TIPO HIDS T a. b. a The Thi I [SPLEN [SPLENO [VOMIT [VOMITO DURAT I [SPLENOMEGbin= [SPL [VOMITObin=.0] [VOMI DURATA B Lower b b b b

49 Classi predette s Cou TIPO Tot HIDS T FM Cou clas TIPO Tota T F Total ?

50 Correlazione dei sintomi Case Number CONGIUNTbin FARINGITESSUDbin DOLLINFCERVbin u X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Faringite eritem Splenomegalia LAML Vomito Rash

51 Molecular Diagnosis Patients with mutations out of % screened MEFV* MVK TFNRSF1A 13 Homozygous, 39 heterozygous 9 CIAS-1 6 CINCA, 1 MWS, 2 FCAS

52 Age at onset/disease n. patients < 1a >

53 Internal cross-validation leave-one-out 75% positive negative SCORE = Abd pain Apht Thor pain Diar Age Family H.

54 Cross-validation leave-one-out 5% positive negative SCORE = Abd pain Apht Thor pain Diar Age Family H.

55 Children with periodic or recurrent fever?

56 Case 1 Male, 7 year-old, caucasian No family history Age at onset: 7 months Duration: 5 days (n. episodes: 9) Aphtousis: often Pharyngitis: often Arthralgia: sometime Abdominal pain: often; diarrhea & vomiting: sometime Thoracic pain: sometime Splenomegaly: sometime

57 Case 2 10 years old girl, caucasian No family history Age at onset: 24 months Duration: 3 days (n. episodes: ) Abdominal pain: always Vomiting: often Thoracic pain: sometime

58 Case 3 12 years old girl, caucasian Family history: yes Age at onset: 10 years (120 months) Duration: 15 days (n. episodes: 6 ) Periodicity: yes Aphtousis: sometime Pharyngitis: sometime Lymphoadenopathy: sometime Arthralgia: often Myalgia: sometime Headace: often

59 Case 4 1 year old boy, indian Family history: yes Age at onset: 1 month Duration: 9 (n. episodes: 10) Periodicity: yes Exudative pharyngitis: sometime Erythematous pharyngitis: often Lymphoadenopathy: sometime Abdominal pain: sometime

60 Monogenic fevers in Italy

61 Patient recruitments: Jan Feb Pazienti con mutazioni su 572 screenati 22.7 % MEFV MVK TFNRSF1A CIAS-1 16 omoz./etero. comp, 48 eteroz. omozigoti/eterozigoti composti 8 severe, 22 R92Q, 2 D12E, 1 P46L 8 CINCA, 1 MWS, 2 FCAS

62 CUT OFF: probability to be positive 15% TRUE Positive Negative Positive Total POS_NEG_PRED2.00 Count % within VERIPOS 82.7% 5.4% 66.5% 1.00 Count % within VERIPOS 17.3% 94.6% 33.5% Total Count % within VERIPOS 100.0% 100.0% 100.0% Specificity Sensitivity

63 Criteri FFM (Tel-Hashomer) Criteri maggiori 1. Peritonite 2. Pleurite o pericardite 3. Monoartrite 4. Febbre isolata tipica Criteri minori 1-3 Attacchi incompleti con coinvolgimento di - Addome - Torace - Articolazioni 4. Dolori da sforzo 5. Risposta alla colchicina Criteri di supporto 1. Storia familiare FMF 2. Origine etnica 3. Esordio < 20 anni 4-7. Caratteristiche degli attacchi 4. Severi, riposo a letto 5. Remissione spontanea 6. Intervallo libero 7. Elevazione transitoria indici fase acuta 8. Proteinuria/ematuria episodica 9. Laparotomia o appendicectomia non patologica 10. Consanguneità 1 o più criteri maggiori 2 o più criteri minori 1 criterio minore e 5 criteri di supporto

64 Validation set A total of 77 patients have been enrolled 31 were positive at genetic test, 6 were with an undefined diagnosis (3 patients with low-penetrance TNFRSF1A mutations, 3 heterozygous for MEFV) 40 were negative

65 Validation set: Results: genetics Validation set (total number 77 patients)** Gene (n. of patients) MVK (13) Severe TNFRSF1A (5) Composite MEFV (13) Mild TNFRSF1A (3) Heterozygous MEFV (3) Mutations (n. of patients) I268T/V377I (6) V377I/V377I (2) L264F/V377I L315V /V377I V337I/H20P A334T/A334T I268T/P167L T50M C43S C73W N65I C55Y M694V/M694V (4) M694V/V726A (3) M694V/M694I (2) M694V/E148Q (2) M694V/M680I M680I/V726A R92Q (2) P46L M694V (2) A744S **Three patients (2 FMF and 1 HIDS patients coming from Morocco) had an Arab origin, 4 FMF patients were Jewish, 1 negative patient came from Brazil. The remaining patients declared a Caucasian origin. Mutations never reported before

66 Results: clinical manifestations Validation group (7 7 patients) Diagnosis HIDS Severe TRAPS FMF Mild TRAPS Heterozygous MEFV Negative # of patients Positive family history Oral Aphthosis Eryth ematosus pharyngitis Exudative pharyngitis Rash Genital aphthosis Conjunctivitis Periorbital edema Cervical lymph nodes Pain at cervical lymph nodes Splenomegaly Thoracic pain Pleurisy Pericarditis Abdominal pain Diarrhea Vomiting Arthralgia Myalgia Arthritis Headache Age at onset (months) mean (SD) Fever duration (days) mean (SD) 12.0 (6.3) 4.2 (1.0) 45.0 (38.0) 14.4 (9.3 ) 35.1 (28.9) 2.2 (0.9) 48.0 (24.0) 9.0 (5.2) 24.3 (20.2) 42.9 (56.8) 4.0 (2.6) 5.7 (3.8)

67 Performance of the diagnostic score in the validation set Validation set Predicted positivity Diagnosis HIDS 12 (92%) TRAPS 4 (80%) FMF 11 (85%) Negative Not defined* + - Total 12 (30%) 1 (17%) 1 (8%) 1 (20%) 2 (15%) 28 (70%) 5 (83%) Cut off 15%

68 Step 2: multivariate logistic analysis Variables Beta (SE) OR (95% CI) P value Positive family history 1,5 (0,8) 4,1 (1,1-16,0) 0,039 Age at onset -0,067 (0,02) 0,94 (0,91-0,98) 0,003 Abdominal pain 1,494 (0,34) 33,1 (6,1-178,5) 0,001 Oral aphthosis - 1,504 (0,55) 0,2 (0,1-0,7) 0,007 Diarrhea 0,901 (0,34) 3,3 (1,1-9,8) 0,028 Thoracic pain 1,958 (0,79) 4,6 (1,0-22,5) 0,02 A diagnostic score, able to give the probability to be positive at the genetic tests, was computed combining the variables selected by the multivariate model, each weighted according to the parameters estimated by the model