Ilaria Ferrarotti Centro per la Diagnosi del Deficit di Alfa1-Antitripsina

1. BPCO e carenza ereditaria di AAT Ilaria Ferrarotti Centro per la Diagnosi del Deficit di Alfa1-Antitripsina Fondazione IRCCS Policlinico S. Matteo Dipartimento di Medicina Molecolare Università di Pavia

2. Alpha-1 antitrypsinDeficiency • Common genetic disease among Caucasians (1/5000) • Autosomalco-dominant heredity • Reduced or complete lack of alpha-1-antitrypsin secretion from hepatocytes into the bloodstream • Severe AAT deficiency is most often associated with homozygous Z-mutation (PiZZ); over 120 genetic variants of the Pi gene are currently known • Lung, liver, and skin (very rare) can be affected • Often remains unrecognized or is wrongly diagnosed as COPD

3. CASE-FINDING Severe AATD is clearly associated with disease risk accounting for 1-2% of all COPD cases.

5. Nevertheless... .... large genome-wide association studies (GWAS) on these pulmonary outcomes have so far failed to point to this chromosomal region.

6. Missingheritability Manolio TA et al, Nature 2009

7. The first GWAS on AAT serum concentration in a subset of the SAPALDIA (Swiss Cohort Study of Air Pollution and Lung Disease in Adults) general population sample • Fine mapping of the SERPINA1 locus (genotyping of additional SNPs in the whole study sample - exon sequencing in subjects with unexplained low levels of circulating AAT ). Detection ofassociatedchromosomal loci Addictionallyasssessmentofcontributionofthis locus to the trait’s variability

8. Conclusions: • GWAS on AAT blood levels, not yet present in the literature, identified a region that includes the gene SERPINA1 as the only associated locus in the genome. • Strong signals of common variants completely disappeared after conditioning on two low frequent causal variants • Rare SERPINA1 alleles much rather than common alleles contribute to the further variability of this blood marker. Manhattan-Plot of genome-wide P-values for association with blood AAT levels Manhattan-Plot of genome-wide p-values for association with blood AAT levels conditioning on S and Z alleles. p<5*10-8 14q32.1

9. Inference: • this locus is suggestive to contribute to the missing heritability of pulmonary outcomes. • The absence of this locus among the statistically significantly associated signals in corresponding GWAS may reflect • small effect sizes of the common variants in combination with insufficient sample size and stringent correction for multiple testing leading to a high risk of type II errors • poor tagging of the underlying causal variants by nearby SNPs in the chip arrays would further reduce the chance to find this locus among the significantly associated results.

10. LOSS OF FUNCTION Normallung AATD lung degradation degradation HNE HNE AAT AAT

12. Luisetti, Breathe2007

13. Alpha1-antitrypsin serumlevel, SERPINA1 genotype , and riskfor COPD Dahl & Nordestgaard, Int J COPD 2008;4:157-167

14. GAIN OF FUNCTION AATD asconformationaldisease Gooptu B, Lomas D, AnnRevBiochem, 2009

15. Polymers in the lung McElvaney, Lung 2007

17. Tanet al,

20. AnnualCongress, EuropeanRespiratory Society, Vienna, 1-5 Settembre, 2012 Effect of augmentation therapy on immune function of patients with severe Alpha1-antitrypsin deficiency Ferrarotti I1, Carroll TP2, Inghilleri S1, Ottaviani S1, Luisetti M1, McElvaney NG2 1Center for Diagnosis of Inherited Alpha1-antitrypsin Deficiency, Dept of Molecular Medicine, Section of Pneumology, IRCCS San Matteo Hospital Foundation, University of Pavia, Italy; 2Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin, Ireland. ‘We acknowledge the support of the European Respiratory Society (ERS) and the National Society (AIMAR), joint ERS/AIMAR Fellowship STRTF 87-2010’ Augmentation therapy appeared to attenuate IL-6 and IL-8 production from ZZ and Q0/Q0 monocytes. (***p<0.001, **p<0.01) Relative IL-8 and IL-6 levels measured by ELISA in monocyte supernatants collected after 24h incubation in the presence (+) or absence of LPS. A-B. Comparison among MM, ZZ and ZZ treated with aumentation therapy (ZZa.t.). C-D. ComparisonamongMM, M/Q0 and Q0/Q0treatedwithaumentationtherapy (Q0/Q0a.t.) A C D B

21. Reduced chemotactic activity of AATD individuals (PI*ZZ) receiving augmentation therapy in comparison to untreated PI*ZZ patients and controls. P< 0.005 p=0.005 p=0.009 PatientPI*ZZ: male, 42y, 20pack/y FEV1%31-FVC%60, aug.therapy 2 years PatientPI*ZZ: male, 49y, 20pack/y FEV1%29 - FVC%68 no aug. therapy MMcontrols: healthy, matchedforage and gender

22. Reduced superoxide production from AATD individuals (PI*ZZ and PI*Q0/Q0) on augmentation therapy in comparison to untreated PI*ZZ and controls.

25. AATD subjectsenrolled 5,511 (Update 20th August 2013)

26. DISTRIBUTION OF DIFFERENT CLINICAL RESPIRATORY PHENOTYPES ACCORDING TO GENOTYPES * R denotes non-Z and non-Sdeficiencyvariants

27. DISTRIBUTION OF EARLY-ONSET EMPHYSEMA ACCORDING TO GENOTYPES % ofearly-onsetemphysema * R denotes non-Z and non-Sdeficiencyvariants

28. Severe AATD ItalianRegistry 413 patients (december 2013) Genotype

29. IRCCS San Matteo Hospital Foundation, University of Pavia: Pulmonology Unit Head of Department: Maurizio Luisetti Laboratory of Biochemistry and Genetics of Lung Disease StefaniaOttaviani Marina Gorrini Michele Zorzetto Ilaria Campo SimonaInghilleri Simona Ferrari Unit of Pulmonology Francesca Mariani ZamirKadija Elena Paracchini