Alpha 1 antitrypsin (AAT) is an acute phase glycoprotein predominantly derived from the liver, and its major biological function is to inhibit neutrophil elastase 1. AAT, a highly polymorphic protein with more than 120 variants known to date 2 is coded by a gene, called SERPINA1, located on chromosome 14q31-32.3 within the SERPIN cluster 3. The SERPINA1 PI*M alleles code for the commonest normal AAT variants, whereas PI*S and PI*Z are the most common deficiency alleles associated with reduced concentrations of plasma AAT. Nevertheless, there are at least 30 SERPINA1 alleles rarely detected, other than the PI*Z and the PI*S alleles, which are associated with significantly reduced AAT levels 456.

Inherited deficiency of AAT (AATD) is one of the most common genetic disorders in the world, and is associated with an increased risk of developing lung and, to a lesser extent, liver disease 7. Individuals homozygous for the PI*Z allele usually have an AAT level in the region of 0.35 g/L 3 and are at high risk for the development of emphysema 8, asthma 9, chronic bronchitis and bronchiectasis 10. However, heterozygous individuals with PI*S and PI*Z mutation (or rare mutations) showing a more protective AAT level (> 0,8 g/L) 3 can also be at increased risk for lung disease, depending on multiple environmental factors, such as smoking, occupational exposure, and environmental exposure.

Epidemiology studies showed that highest prevalence of PI*ZZ related AATD is recorded among Northern Europeans and populations with North European background 11. Notwithstanding, during the last few years, based on estimates from allele frequencies obtained in available cohort studies, it has been suggested that the Z variant is not only common in Caucasians, but also among other ethnic groups worldwide 1213.

A total of 30 cohorts have been investigated for AATD in the African continent. Twenty four cohorts, having a total of 4,718 individuals, were in Sub-Saharian Africa 14, 6 cohorts with a total sample size of 1,735 have been investigated in the north African populations 151617181920, Three of them in Tunisia: in the Tunisian population, the PI*Z allele, previously considered as virtually absent 1516, has then been detected once on the heterozygous state in a total cohort of 1,168 individuals (allele frequency 0.04%) 17. However, none of the previous surveys in Tunisia has looked at rare, non-S and non-Z SERPINA1 variants, which have been hypothesised to be particularly frequent in the Mediterrenean area, where PI*Z allele frequency is reduced 4. We therefore aimed this paper at investigating AATD variants in a Tunisia area, targeting a cohort of obstructed individuals, in which the diagnosis of AATD has been recommended accoding to the ATS/ERS document 21.

One hundred twenty subjects with OLD were enroled in this study. There were 115 males and 5 females, mean age (SD) 49.2 (9.5) years (range 34–81). The large majority of patients were smokers (84.1%). As far as the OLD phenotype was concerned, there were 49 COPD (40.8%), 48 emphysema (40%), and 23 asthma patients (19.2%). Mean (SD) plasma AAT level was 1.74 g/L (0.63) (range 0.81 to 3.3). Plasma AAT level distribution is shown in figure 1.

SERPINA1 S and Z allele genotyping, performed in all OLD subjects, allowed the detection of one PI*MZ individual (plasma AAT level: 0.81 g/l, consistent with the detected genotype).

Nevertheless, some subjects negative for PI*S and PI*Z allele detection, displayed plasma AAT levels consistent with intermediate AATD; in particular, 4 subjects displayed plasma AAT level < 1 g/L [mean (SD) 0.91 g/L (0.02)]. According to our protocol for detection of rare AATD variants, such samples with inconsistent AAT level/genotype were suitable for sequencing 424. In the absence of reference values of plasma AAT in the general population from Tunisia, we decided to submit to sequencing, DNA samples from subjects with plasma AAT levels < 1.5 g/L, rather than the usual cut-off of 1.13 g/L 25. We therefore sequenced 24 DNA samples (plasma AAT level range: 0.89–1.48). We found that 5 subjects were heterozygous for rare deficiency variants: 3 subjects carried the PI* MMmalton, 1 the PI*MPlowell, and 1 the PI*MMwurzburg genotypes. Figure 2 summarises the results of genotyping/sequencing.

Meta-analysis of available data showed that SERPINA 1 PI*S and PI*Z deficiency alleles are extremely rare in North African populations 1213. Recent investigations in the Tunisian population confirmed the low frequency of AATD common deficiency variants in the general population and healthy subjects (1078 subjects investigated: 15 PI*MS detected (PI*S: 0.7%; PI*Z: 0%)) 151617, as well as in COPD patients (90 subjects investigated: 1 PI*SS and 1 PI*MZ detected (PI*S: 1%; PI*Z: 0.5%)) 17. The present study, performed in 120 patients with OLD, confirms these data: PI*S allele was not seen, whereas PI*Z was detected only once on the heterozygous state (frequency 0.4%).

In this study, we extended the SERPINA1 gene investigation to variants not detectable by rapid PI*S and PI*Z genotyping: by this strategy, we found 5 more deficiency variants (frequency as a whole 2%) detected on the heterozygous state. Such a condition of a relatively higher frequency of rare than common AATD variants, is shared by Central – Southern regions of Italy, in which PI*Mmalton and PI*Mprocida variants seem to prevail over PI*Z 4. Interestingly, the PI*Mmalton variant, detected in three unrelated individuals in this paper, is the commonest AATD variant in Sardinia, where the PI*Z variant is detected very rarely 426. A novel SERPINA1 Null mutation, first described in an Egyptian family, and for that reason called Q0*cairo 27 has been repeatedly detected in unrelated individuals from regions of Southern Italy. Population admixture due to migration occurred in ancestral periods, as well as contacts for commercial purposes in more recent centuries are likely to be responsible for dissemination of rare AATD variants in the Southern Mediterrenean basin. Table 1 summarizes the rare variants reported in the Mediterranean basin, as well as the mutations types, the cellular defect and the related clinical data 4282930.

This study strengthens the concept that for a correct laboratory diagnosis of AATD, there is a need of a combination of biochemical and biomolecular methods 24, otherwise rare AATD variants will be missed 5. These preliminary data also confirm the usefulness of enrolling patients with OLD in a screening programme for AATD. According to the ATS/ERS statement's evidence based recommendations, all subjects with COPD and asthma should be submitted to diagnostic testing for AATD 21. In this paper, although no subjects with severe AATD deficiency were detected, we found six out of 120 subjects (5%) carrying the so called "intermediate deficiency", that means heterozygosity with one normal PI*M allele and one severe AATD allele [mean (SD) plasma AAT level: 0.93 g/L (0.12)]. These findings are consistent with the hypothesis that intermediate AATD, such as PI*MZ or equivalent genotypes, represent a risk factor for developing COPD 20. Recent meta-analysis 31 reported the increase in risk of COPD in PI*MZ heterozygous individuals (OR for PI*MZ versus PI*MM (normal genotype) was 2.31 (95% CI 1.60 to 3.35)).

Severe AATD diagnosis in the North African countries might be beneficial to the treatment of patients with the introduction of AAT replacement therapy in these populations. Furthermore, AATD carrier diagnosis might provide genetic counseling to persons who are planning a pregnancy or are in the prenatal period 21. In addition, awareness of carrying a gene that may increase the susceptibility to COPD may be an additional factor for a successful enrolment of patients in smoking cessation programmes 32.

This investigation, performed for the first time with current diagnostic standards in a North African population, highlights the implication of AATD in development of OLD in this area, whereas previous data excluded a role for AATD in the African continent. This would suggest that, similarly to what happens in the Caucasians, also in Northern Africans AATD is an underdiagnosed condition, and therefore investigations in larger sample sizes would be advisable. It seems also likely that targeted detection strategy to identify affected individuals produces a rate of detecting disease higher than the population-based screening programs. Such approach may also contribute to change the widespread concept concerning the AAT deficiency epidemiology in North African and others populations.

The authors declare that they have no competing interests.

SD, MZ, IF and IC designed the study and drafted the Manuscript. FA and JK participated in data collection. SO, RS and MG participated in laboratory investigations. JBC, AHK and ML supervised the study process, revised and edited the final manuscript.