Ferrarotti I

Rare AATD mutations
Challenging target
Italia Experience
Ilaria Ferrarotti
Centro per la Diagnosi del Deficit di Alfa1Alfa1-Antitripsina
Fondazione IRCCS Policlinico S. Matteo
Dipartimento di Medicina Molecolare
Università di Pavia
SERPINA1 is a Highly Polymorphic Gene : The
Variants
• Normal AAT plasma levels within general
population normal levels
• Deficient AAT plasma levels reduced, but
detectable (increased risk for COPD and liver
disease)
• Null
No detectable plasma AAT (increased risk
for COPD)
• Dysfunctional AAT Pittsburgh (NE → thrombin)
MILANO
BRESCIA
GUSSAGO
TRENTO
ORBASSANO
BUSSOLENGO
TRIESTE
Pavia
MODENA
SIENA
CISANELLO
CHIETI
SCREENING FOR AATD IN ITALY
NUORO
CASSANO DELLE
MURGE
ROMA
MILAZZO
CATANIA
Diagnosis of AATD - the
approach at Pavia Lab
•Quantitative test: nephelometry (AAT and CRP)
•Qualitative test: genotyping S and Z alleles ( TaqMan probes)
phenotyping (IEF )
sequence analysis (exons II,III, IV, V)
Sequence analysis (introns IA-IB-IC)
Q0isola di procida allele detection
Sample collection (DBS)
•AAT protein level: nephelometer
•CRP protein level: ELISA
•S and Z variant genotype: TaqMan probes
AAT > 113 mg/dl
S Z genotype: negative
AAT < 113 mg/dl
S Z genotype: negative
S Z genotype: positive
•Phenotype (IEF)
• Sequencing
Phenotype (IEF)
S Z genotype: positive
Phenotype (IEF)
PI*ZZ or PI*SS ?
Homozygous for deficient allele?
1
1 = normal
yes
2
no
no
yes
AAT < 70mg/dl?
2 = intermediate AATD
Q0 isola di procida
detection
3 = severe AATD (compound heterozygous)
no
yes
4 = severe AATD (homozygous)
Sequencing
1- 2 - 3
Ferrarotti et al, Transl Res, 2007 (modified)
2-3-4
2
2-3
Target
screening AATD
at Pavia Lab
700
600
500
AlphaKit
400
No deficit
AATD intermedio
300
AATD severo
200
100
0
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
Italian Registry of AATD
422 severe AATD
Genotype
SZ
17%
100%
90%
SS
1%
80%
Null/Null
11%
70%
60%
RR
ZZ
16%
R
20%
50%
ZZ
40%
62%
SZ
ZR
61%
30%
20%
10%
0%
1996-2000
2001-2005
2006-2010
2011-2014
R
SR
12%
Rates of R mutations in Italy
(2006--2015) 3132 alphakits
(2006
Severe AATD
SR
5%
RR
6%
ZR
21%
ZZ
50%
SZ
18%
Severe
Deficiency
10%
Intermediate AATD
Normal
57%
Intermediate
deficiency
33%
MR
27%
MS
17%
Italian Registry of AATD
PI*ZZ
PI*SZ
PI*R
MZ
56%
Italian Registry of AATD –
rare variants
Deficient and Null)
Z
S
Mmalton
Mprocida
Mwurzburg
I
Plowell
Pbrescia
Yorzinuovi
Smunich
Mbologna
Mheerlen
Rare variants
Q0cairo
Q0mattawa
Q0granite falls
Q0pordenone
Q0isoladiprocid
Q0brescia
Q0lampedusa
Q0clayton
Q0ourem
Mvarallo
Alleles will be designated by capital letters corresponding to their relative positions in
IEF……..
And place of origin names (i.e., birthplace-earliest generation)
should be used for rare alleles.
The names of AAT variants come
from their electrophoretic mobility
Fast
Medium
Slow
Very slow (Z)
From Pittschieler, Verducci Ed. 1993
How many
many??
??
100
• papers, abstracts, communications
• databanks :http://www.ncbi.nlm.nih.gov/SNP
http://www.pdb.org/
http://www.hgmd.cf.ac.uk/
http://www.hgvs.org
How many
many??
• 114 variants from papers, abstracts…
• 30 new /unpublished variants (just in our experience)
•184 refSNP (rs)
328
120
150
SERPINA1 gene
Transcription
promoter
(macrophage)
Transcription
promoter
TAA
ATG
(hepatocyte)
3’
5’
Ia
II
Ib Ic
Signal peptide
III
IV
V
Active site
(Met358)
Null
Deficient
Normal
Dysfunctional
16,000 bp
Lung diseases:
diseases:
Age according to genotype (422 severe AATD)
Age at diagnosis (years),
mean (IC)
Age at onset (years),
mean (IC)
PI*SZ
55.4 (49.2-61.6)
47.9 (37.5-58.2)
PI*ZZ
52.5 (50.1-54.4)
45.0 (42.9-47.1)
PI*R/Q0SZR
52.6 (49.2-56.0)
44.2 (39.3-49.1)
PI* Q0/Q0SZR 45.8 (36.3-55.3) §
34.6 (23.2-45.9) §§
PI*Q0Q0
32.0 (20.2-43.8) **
40.9 (33.1-48.6)*
§
p=0.092 vs PI*ZZ
vs PI*ZZ
§§p=0.014
*p=0.013 vs PI*ZZ
**p=0.004 vs PI*ZZ
Augmentation therapy
Genotype (N)
Mean age at first therapy± SD (years)
PI*SZ (6)
53.67 ± 17.50
PI*ZZ (73)
54.48 ± 9.67
PI*R (21)
48.76 ± 11.38
Foreign patients in the Italian Registry (N=23)
Genotypes
ZR
30%
ZZ
57%
SZ
13%
Pathological variants of AAT
Mprocida
Z
S
Mmalton
V
I
Siiyama
Mwur.
F
Plowell
Take home message
The Italian Targed screening
program has supported by:
NO
AATD is not
always Z and S…
look for rare
variants !
YES