In India, AAT deficiency is being diagnosed on the basis of low serum AAT levels, Periodic Acid Schiff (PAS) positive diastase resistant granules on liver histology and absent or faint alpha 1 globulin band on serum electrophoresis. Based on these criteria, up to 8% of children were suspected to have AAT deficiency associated CLD(5-8). However, these techniques have issues of low specificity with high false positivity(9) and no confirmed case based on either Isoelectric focusing (IEF) or PCR based assay has ever been reported in India. In view of this background, we attempted to determine prevalence of AAT deficiency among pediatric chronic liver disease patients in North India using diagnostic tests (IEF and genotyping) that are considered gold standard.

The study (retrospective: 1991-1999, prospective: 2000-2004) was conducted in pediatric liver disease patients (up to 15 years) mostly residents of North India, at the Pediatric Gastroenterology Clinic, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India The caretakers of the patients who were worked up for AAT genotype, gave consent to participate in the study.

CLD was diagnosed on the basis of clinical, biochemical, ultrasound picture, endoscopic evidence of varices and when feasible liver histology. Subsequent diagnostic workup was for specific etiologies. Neonatal cholestasis was defined as conjugated bilirubin 2 mg/dL or more than 20% of total bilirubin (whichever is less)(10). For neonatal cholestasis, workup was done as previously described(11). The investigation protocol is shown in Fig.1

Fig.1. Investigation protocol for chronic liver disease and neonatal cholestasis.

Screening: Reduction of serum levels(12) and/or a faint or absent alpha-1-globulin band on routine gel electrophoresis and/or histopathological evidence of PAS positive diastase resistant granules on liver biopsy were indicators of suspected AAT deficiency state.

Genotypic characterization: The Pi Z (14) and S(15) genotypic characterization was done by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP)(16) on DNA extracted from whole blood. Sequencing was done commercially for confirmation.

Serum and genomic DNA from five control blood samples were obtained from adults volunteers aged 26-49 years with no history or evidence of CLD. Their bilirubin levels, liver enzymes, serum AAT levels and total proteins were within normal limits. All had a PiMM phenotype.

Family screening: Father (n-43), mother (n-48) and siblings (n-32) of the 50 suspected AAT deficiency patients (undergoing genotypic characterization) were also investigated for their AAT status (Pi phenotyping and mutation screening).

The clinical and laboratory records of all patients were entered in a database on a regular basis and appropriate consistency checks with reference ranges put in for quality assurance purposes.

Analysis was done using "STATA" version 8.0 (STATA Corporation, Texas, USA) statistical packages.

The serum samples from 1700 patients were available for investigating AAT deficiency. Out of these, 682 patients were recruited retrospectively (1991-1999) and 1018 prospectively (2000-2004). A total of 840 subjects had evidence of underlying CLD and 410 had NC. Remaining 450 subjects, who had either no CLD (n-404) or NC (n-24) or were Hepatitis B surface antigen (HBsAg) carrier (n-22) with no histological or biochemical evidence of ongoing hepatic inflammation, were also part of this study and labeled as "without liver disease", and served as controls.

Etiology of CLD and NC: Among 840 patients with definite CLD (3 months – 15 years), chronic viral hepatitis (HBV/ HCV/ mixed infection) (n-238; 28.3%) and metabolic liver diseases (MLD) (n-159; 18.9%) were the most common etiologies. In 237 patients (28.2%), no etiologic label could be assigned (Table I).

Table I



Etiologic Factors Associated with CLD in North Indian Children [1991-2004]

A total of 410 patients fulfilled the diagnostic criteria of NC (Table II). Obstructive causes contributed to over one-third (141; 34.4%) cases followed by infective etiology (66; 16%). Almost one-third (30.7%) children presenting with NC could not be assigned any specific etiology.

Table II



Etiological Factors Associated with Neonatal Cholestasis [1991-2004] [N=410]

Suspected Alpha 1 antitrypsin deficiency: Ninety eight (CLD-78; NC-20) of 1250 (7.8%) liver disease patients were suspected to be AAT deficient on the basis of screening tests. Forty-nine liver biopsies were possible in these 98 cases (CLD: 34; NC: 15). There were 9 biopsies with histopathological suggestion of AAT deficiency with presence of PAS positive diastase resistant granules in the liver biopsy. Four of these biopsies had evidence of lipofuscin material and in one PAS positive granules were present in the cytoplasm. On follow up liver biopsies, PAS positive granules were not observed in three of five [CLD: 1; NC: 2] study patients. A faint/ absent alpha-1-globulin band was documented in 88 children (CLD: 72; NC: 16) and 20 children had low serum AAT levels. Five of these 20 children with low AAT serum levels had severe malnutrition at the time of presentation.

Underlying conditions in the group of children with suspected AAT deficiency: CLD cases suspected to be AAT deficient (n-78) were also worked up in parallel for other etiologies. They had chronic viral hepatitis B/C (n-18); autoimmune hepatitis (n-5); Wilson’s disease (n-3); other MLD’s [Gaucher’s disease (n-1), hemochromatosis (n-1), presumed bile acid metabolic defect (n-1). Byler’s disease (presumptive; n-1) and hereditary fructose intolerance (n-1)]; biliary cirrhosis (histological diagnosis, n-2); hepatic venous outflow tract obstruction (HVOTO; n-1); miscellaneous hepatic disorders (n-5) and unknown etiology (n-39). Similarly, NC patients (n-20) were diagnosed as: extra hepatic biliary atresia (EHBA) (n-3); cytomegalovirus (CMV) with neonatal hepatitis (n-3); hepatitis B associated (n-1); suspected MLD’s (n-1); sepsis (n-1), Down syndrome (n-1); and unknown etiology (n-10). This etiological spectrum was very similar to the overall etiological profile of CLD and NC patients. Clinical and biochemical profile of the patients suspected to be AAT deficient in the initial screen (78 CLD and 20 NC) and the rest of the liver disease patients (762 CLD and 390 NC) were similar and did not help to differentiate the two groups.

Genotypic characterization for AAT Deficiency: Genetic workup was done for 50/98 children suspected to be AAT deficient on screening. Two of the 98 patients were siblings and hence both were included for complete genetic workup. Of the remaining 96 suspected AAT deficient patients, 48 unrelated children (CLD-32, NC-16) were selected through a computer generated random process. S or Z mutation was not found in any of the 50 patients by PCR. Two patients (siblings) showed a shift in band pattern in exon V on SSCP and sequencing confirmed a single base substitution (G to A) at position 333. Index ‘1’ (younger; female) had homozygous mutation and her sibling ‘2’ (elder; male) had hetero-zygous base substitution. The mutation converts valine to methionine at position 333 in exon V.

Clinical details of two patients with novel mutation at position at 333 in exon V: The index cases (1 and 2) had low serum AAT levels (Index 1:126mg/dL and Index 2: 108mg/dL) and absence of alpha-1 globulin band on serum agarose electrophoresis. Both of them had portal hypertension along with histological evidence of fibrosis and inflammation without PAS positive diastase resistant granules. However, immunohistochemistry revealed numerous rounded deposits of AAT in index 1 with homozygous mutation (Fig. 2). In index 2 (heterozygous mutation), there were bands of fibrosis extending from central and portal regions and immunohistochemistry was weakly positive for AAT. Neither of them had other known etiological factor associated with liver disease.

Fig. 2 Index 1(female, 120 months, homozygous mutation at position 333 in exon V); Immunohistochemistry (X20) showing numerous rounded alpha 1 antitrypsin (AAT) bodies.

The siblings were born out of non-consanguineous marriage. The mother of these patients had normal band pattern on SSCP for both exon III and V. The father had expired at the age of 40 years in 1999 at our hospital. He was admitted with portal hypertension, grade II hepatic encephalopathy and hepatorenal syndrome. The liver biopsy could not be carried out but in the background of history of regular intake of alcohol for 16 years, he was labeled as having alcoholic liver disease. Genetic studies were not done for the father.

Quality assurance: At the inception of this study, no expertise was available to interpret IEF gels. Thus, 10 IEF gels including the gels containing M1E, MC and MP phenotypes and gel strip of patient with homozygous mutation at position 333 were sent to Alpha-1-Foundation Research Professor, University of Florida, School of Medicine, Florida. Also, five randomly chosen DNA samples (of the patients suspected to be alpha-1-antitrypsin deficient and chosen for detailed genetic analysis) were sent to Genetics and IVF Institute, Virginia, USA.

In comparison to studies done among South Asian children, the pediatric liver disease data from Caucasian population shows several AAT alleles (homozygous and heterozygous states) which are associated with liver diseases. In California, ZZ phenotype was found in 4.5% children with neonatal cholestasis(2) In Serbia(3), Pi*Z and Pi*S phenotypes were found to be 15 and 3 times higher respectively in newborns with liver disease as compared to their healthy counterparts in the population. Absence of AAT deficient alleles in highly selected North Indian child population with liver disease in the present study further confirmed the findings of epidemiologic surveys done in this region(4) . The results of the present study are also consistent to a previous study done in India(23).

For our patient population, we cannot be certain about occasional presence of mutations in exons and introns other than exon III and V that were not screened as part of current investigation. Such mutations associated with liver disease are however not reported so far.

In conclusion, the study indicated that the AAT deficiency alleles are uncommon in our population. In regions with very low prevalence of abnormal AAT alleles, diagnosis of AAT deficiency based on screening tests is not helpful to identify occasional patient with AAT deficiency alleles. In strongly suspected patients, IEF and molecular techniques should be used to diagnose the condition. We detected a single base substitution mutation at position Val333 Met in exon V in two siblings. The role of this novel mutation in etiology of liver disease could not be completely ruled out. In view of our findings, we do not recommend routine screening for AAT associated liver disease in our region. Further study in adult emphysema patients may clarify the role of AAT deficiency in Indians, although the possibility of it being a significant etiologic factor appears unlikely.

We are indebted to Prof Mark Brantly, Head, Alpha 1 Research Foundation for helping us interpret IEF gels, Dr Anne Maddalena, Genetics and IVF Institute, Virginia, USA for cross validating our DNA samples for Z and S mutation and Dr Magne K Fagerhol, Oslo, Norway for providing us with reference sera.

Contributors: NKA: Study design, protocol preparation, results interpretation and manuscript editing; SA: Protocol preparation, manuscript writing, IEF standardization, analysis of data and other laboratory work; AA: Protocol preparation, PCR-SSCP, analysis of data and other laboratory work; PM: Study design and patients recruitment; MM: Laboratory support; MKD and VB: Patient screening and recruitment; MK: Laboratory resource, genetic analysis and technical guidance; RK and MA: Serum agarose electrophoresis standardization; AK: Laboratory Support; SDG: Pathology; and SV: Special investigations for CLD

Funding: DBT, Govt. of India.

Competing Interests: None stated.

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