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November 2000, Volume 50, Issue 11

Original Article

Phenotypes of Alpha 1 Antitrypsin in Karachi, Pakistan

Anjum Shahid  ( Pakistan Medical Research Council, Research Center, Jinnah Postgraduate Medical Centre, Karachi. )
Anwar A. Siddiqui  ( Pakistan Medical Research Council, Research Center, Jinnah Postgraduate Medical Centre, Karachi. )
Sarwar J. Zuberi  ( Pakistan Medical Research Council, Research Center, Jinnah Postgraduate Medical Centre, Karachi. )
Mohammad Waqar  ( Department of Biochemistry, The Aga Khan University of Health Sciences, Karachi. )


Objective: To determine serum level of the protease inhibitor, to identify phenotypes and determine their frequencies.
Study Design: A prospective study.
Setting: PMRC Research Centre, JPMC and the Aga Khan University Hospital Karachi.
Subjects: Healthy aduIts without history ol peptic ulcer disease and a normal endoscopy.
Methodology: Quantitive measurement of serum alpha 1 AT was carried out by radial immunodiffusion. phenotyping by iso-electric focusing and confirmation of phenotypes by immuno-fixation and DNA analysis technique.
Results: Serum alpha I AT was low in 13.4% of the subjects. Ni MM phenotype predominated followed by SZ SS, MZ and ZZ. DNA diagnosis accurately resolved the phenotypes as S and Z.
Conclusion: Frequency by phenotype associated with total and intermediate deficiency is less in the population (JPMA 50: , 2000).


Serine protease inhibitor, alpha I AT is secreted y liver cells1. The deficiency, a common autosomal recessive disorder is characterized by reduced serum levels2 and aminoacid substitutions of alpha I AT due to gene variation3. It is associated with premature development of emphysema4, chronic liver disease and hepatocellular carcinoma5. The deficiency state is caused by mutations in the alpha I AT gene6. Apha I AT locus is polymorphic and 75 genetic variants have been identified7 in different populations with a variable prevalance8,9. The commonest variants is M consisting of at least six types. MI[Va 213]. Ml[Ala213]. M2, M3, M4 and M510. The most frequent variants causing alpha l AT deficiency are Z and S, formed by two different point mutations11,12. Studies conducted in various population indicate that gene frequencies of variants vary for different racial groups13. Almost complete absence of data regarding the genetic varints of alpha l AT in our population prompted us to determine serum level of the protease inhibitor, to identify phenotypes and determine their frequencies. The present findings were compared with those reported earlier13,19,26,27.

Subjects and Methods

Blood sample from 269 healthy adults were collected and their sera atored at -70C until analyzed. There were 173 males and 96 fermales. The age range was 18-82 years (mean:36.94+0.81). All subjects gave informed consent to participate in this study wich was approved by the ethical committee of Jinnah Jostgraduate Medical Centre, Karachi. Quantitative measurement of serum alpha l AT was carried out by single radial immunodiffusion technique14 using M paritgen immundiffusion plates (Behring Diagnostic, Marburg, Germany). Phenotyping was performed by ultrathin layer polyacrylamide gel isoelectric focusing15. Further confirmation of alpha l AT phenotypes was done by immunofixation15. IEF is a simple technique but interpretation of the banding pattern obtained by IEF is difficult at times, hence cinfirmation of phenotyping was done by DNA analysis technique16. A combination of polymerase chain reaction (PCR) and restriction enzyme digestion was then applied to confirm the deficient variants.
DNA was extracted from freshly drawn blood samples by the standard method17 and was then subjected to a non radioactive PCR assay of genomic DNA for the detection of S and Z mutations in the alpha l AT gene, followed by restriction enzymes digestion18.


Serum alpha l AT concentration by RID showed a mean value of 2.63+0.05g/1 (range: 0.52-5. 51g/1). It was observed that 13.4% of the subjects mainfested low levels. Any value less then 2.0g/1 was considered as lower then normal. These low levels appear to have no diagnostic significance in most of the cases as only a few sera exhibited abnormal patterns when subjected to IEF, wich is a procedure of choice in evaluating the various phenotypes.It was found that MM predominates followed by SZ,SS, MZ, and ZZ (Figure 1).

PiM Piz and PiS were identified in both the homozygous and heterozygous states. IEF findings were also detected by DNA based diagnostic technique, DNA diagnosis has more accurately resolved the phenotypes as S and Z. Thus genetic deficiency was confirmed by the DNA based methods which appears to be most direct and accurate way of confirming the deficient phenotypes. The results ol the typing for the S and Z mutations were in all subjects ill concordance with those of the IEF USSii\\\\. Normal subjects and those h omozygous or heterozygous for the 1 or S mutations were d istingu ished unambigously (Figure:2).

The frequency of phenotypes is presented in Table 1.

PIM was normal alpha I AT phenotype existing either alone or m combination with S or z variants. Liomozygous 7 variants was also identified in one subject.

Table.2 illustrates the present Findings in Pakistani population with those for other populations.


The present study indicates that MM oyerwhelm inglv predominates all other less common phenotypes described in the literature 19-22 and thus could be regarded as the normal type in Pakistan. Since our sample selection of population contained a certain number of representative from various ethnic groups living in Pakistan. it can be safely assumed that it truly represents Pakistani population in general. In another study conducted in school children where 300 samples were analyzed. MM was also found to be dominant phenotype The present findings also supports the notion that the frequency of phenotypes associated with total and those linked with intermediate deficiency of alpha I AT is substantially less in this population in comparison to European and American caucacians23,24.
Several studies conducted in a number ol populations have shown that gene frequencies of Pi variants vary for ditlerent racial groups. The variants demonstrated in different population have a variable prevalence8,9 The most common from is PIMM which exists in most populations at frequencies ranging from 0.8798 to 0.995825 This holds true in the study where frequency is 0.9766. The highest frequency of piS allele (0. 116) has been reported in Spanish population26 followed by 0.0800 in the British27. Present findings show a frequency of 0.0074 which is lower than that cited for Spanish26. ltal ians20 and French19. The piZ variant was encountered at a gene frequency of 0.0037 which was higher than Greek2 K and lower than that reported from ltalians20, French19 and Saudis13. The present findings show that alpha I AT polymorphism exist in Pakistani population as well and that geographical variations plays a role in the existence of various alpha 1 AT phenotypes.


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