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Title of Thesis

Anjum Rehman
Institute/University/Department Details
Department of Biochemistry/ University of Karachi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
alpha 1 antitrypsin deficiency, emphysema, hepatitis, cirrhosis, duodenal ulcer, gastrointestinal tract, liver, lungs, serum pepsinogen, transaminases, alkaline phosphatase, total protein, albumin, bilirubin

Due to scarcity of data which could accurately indicate whether there exist an association between alpha, antitrypsin (α1 AT) deficiency and diseases of gastrointestinal tract (duodenal ulcer), liver (hepatitis and cirrhosis) and lungs (emphysema)'. It appears appropriate to establish a possible relationship between them, more so because duodenal ulcer is common among local population and exact- etiology is yet to be determined.

For this study, patients were first identified as α1, AT deficient by screening their serum concentration using radial immunodiffusion (RID) and phenotypes were determined by isoelectric focusing (IEF), At the end of IEF run, pH at each I cm segment across the gel length was measured using a surface pH electrode. Further confirmation of α1 AT was done by immunofixation. Those found with reduced levels and associated with deficiency state were further analyzed by polymerase chain reaction (PCR). The amplified product was later digested with respective restriction enzymes i.e Asp-700 and Taql for the Sand Z mutations respectively. The digested product was analyzed on agarose gel for the accurate identification of the phenotype. We have included PCR based methods because respective mutations can be distinguished unambiguously by observing the band pattern on agarose gel and a normal, homozygous or heterozygous individual could be clearly differentiated.

A comparison of mean values of α1 AT levels by RID in normal individuals and duodenal ulcer patients showed statistically significant difference (p<0.05). It was observed that 13% of the control subjects and 30% patients manifested low levels, But these low levels appear to have no diagnostic significance as not all of these sera exhibited abnormal patterns when subjected to IEF Isoelectric focusing reveals that most or the subjects had phenotype resembling MM found in 70% of the healthy controls, followed by M1M2,(28%) and FM3(2%), regarding the phenotyping in duodenal ulcer patients, it was observed that MM predominates, followed by M1M2, MZ, ZZ and SS. MZ, SZ and SS represents partial and ZZ complete deficiency of this protease inhibitor. Patients identified with deficiency variant had relapse and duodenitis persisted inspite or long term follow up and treatment with H2, receptor antagonist. Immunofixation showed that in the sera of these patients, very fain bands were, present in the same region.

Traditionally the diagnosis of α1 AT deficiency has been based on RID and IEF determinations. IEF is rather simple but interpretation or the bands obtained by IEF can be difficult at times. Alternative method of diagnosis involves determination of the disease causing mutations In DNA sample by PCR-based assays. The result of the typing for the. S and Z mutations were identical as observed previously by IEF. Normal subjects and individuals homozygous or heterozygous for the respective Sand Z mutations were distinguished unambiguously by the assays based on the principle of using PCR-mediated mutagenesis.

In patients with liver disease, it was observed that almost 98% of the patients with acute viral hepatitis (A VH) had values within the normal expected range while 12% cirrhotics manifested low levels. MM was the most common phenotype among the patients with A VB (72%) and cirrhosis (52%), while MM, was found in 28% and 48% of the patients respectively.

Among the school going children, the mean values were iden1ical to those observed in adults with 10% manifesting low levels. Two children showed intermediate deficiency variant, corresponding to MZ pattern on IEF. Deficiency was also found to exist in 13% of the children with A VB, two of them manifesting intermediate deficient phenotype FZ.

In patients with pulmonary emphysema, 10% had serum concentration below the normal range, but they too had the normal phenotype MM. It is the phenotype and not the serum concentration alone that is of importance in predisposition to lung disease.

The present finding supports the notion that the frequency of phenotype associated with total and those linked with intermediate deficiency of α1 AT is substantially less in this population in comparison to other population. IEF should be the method of choice for phenotyping and further confirmation of doubtful cases be carried out by molecular techniques like PCR.

Download Full Thesis
1965.96 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
127.26 KB
2 1 Introduction 4
344.91 KB
  1.1 Classification of the α 1 antitrypsin variants 7
  1.2 Structure of the protein 9
  1.3 α 1 antitrypsin gene 12
  1.4 Biosynthesis 12
  1.5 Physiological function 14
  1.6 Genetic basis 17
  1.7 Serum α 1 AT in emphysema 21
  1.8 Serum α 1 AT in liver disease 23
  1.9 Serum α 1 AT in duodenal ulcer 26
3 2 Patients and methods 29
321.19 KB
  2.1 Chemicals 30
  2.2 Collection of blood 31
  2.3 Healthy Control subjects 31
  2.4 Duodenal ulcer 31
  2.5 Liver disease 32
  2.6 Emphysema 32
  2.7 Experimental 33
  2.8 Isolation of high molecular weight DNA 37
  2.9 Amplification by polymerase chain reaction 39
  2.10 Serum pepsinogen 42
  2.11 Transaminases 44
  2.12 Alkaline Phosphatase 48
  2.13 Total protein 49
  2.14 Albumin 50
  2.15 Bilirubin 51
  2.16 ABO antigen system 56
  21.7 Statistical methods 57
4 3 Results 58
510.61 KB
  3.1 Serum α 1 AT in controls 59
  3.2 Serum α 1 AT and duodenal ulcer 64
  3.3 Serum α 1 AT in liver disease 89
  3.4 Serum α 1 AT in healthy children and patients with hepatitis 101
  3.5 Serum α 1 AT and emphysema 106
5 4 Discussion 109
174.49 KB
  4.0 Genetic variants of serum α 1 AT 110
  4.1 Serum α 1 AT and healthy subjects 110
  4.2 Serum α 1 and duodenal ulcer 112
  4.3 Serum α 1 AT and liver disease 118
  4.4 Serum α 1 AT and pulmonary emphysema 119
6 5 Conclusions 121
257.17 KB
  5.1 Bibliography 123
7 6 Appendices 139
437.08 KB
  6.1 Papers Published 152