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Association between the defective Pro369Ser mutation and in vivo intrahepatic α1-antitrypsin accumulation

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Editor—α1-antitrypsin (PI), the major inhibitor of neutrophil elastase in the lower respiratory tract, is a highly polymorphic glycoprotein synthesised in the liver that has several rare gene products in which serum protein levels are reduced or even undetectable.1 Early onset pulmonary emphysema, resulting from unopposed elastase activity, and neonatal cholestasis probably resulting from the retention of the defective protein in the liver,2 are the two most common clinical manifestations of PI deficiency and are mainly associated with PI*Z, the most common deficient allele. In addition, other rare alleles occasionally associated with liver injury have been shown to share with PI*Z an increased tendency for intracellular accumulation. Recently, a complete intracellular transport block has been reported for a newly identified3 4 defective Pro369Ser allele (Mwürzburg) by in vitro expression studies in human cell cultures. Adenovirus mediated transfer of the mutant gene into the mouse reproduced the consequences of this block and no traceable amounts of the variant protein could be detected in the plasma after in vivo recombinant expression.3 However, no detectable intrahepatocytic PI inclusions were found in the mice expressing the Mwürzburg mutant3 and no liver biopsy material has yet been presented from patients with this defective allele.

Case report

We report a carrier of the Mwürzburg allele with evidence for in vivo intrahepatic accumulation of PI. The patient is a white Portuguese boy who presented at the age of 1.5 months with cholestasis associated with a recent cytomegalovirus (CMV) infection. A percutaneous liver biopsy performed at the age of 2.5 months showed significant portal fibrosis with porto-portal bridging, giant cell transformation, moderate cholestasis, and an intense portal-acinar inflammatory infiltrate. Periodic acid-Schiff staining after diastase treatment (PAS-D) additionally showed the presence of positive, diastase resistant, intracellular inclusions. Immunoperoxidase staining specific for PI was positive (fig 1). Serum PI concentration, determined by automated nephelometry (Behring), was found to be 92% of normal on admission and dropped to 45% of normal at the age of 24 months, after CMV serology (IgM) and antigens became negative and following progressive decline of transaminase levels to their normal upper limit. The PI concentration in the patient's father was 49% of normal.

Figure 1

Tissue section from the patient's percutaneous liver biopsy. PAS positive diastase resistant inclusions were found in the cytoplasm of several hepatocytes (PAS-D). PI is identified by immunoperoxidase in inset (immunoperoxidase staining).

Isoelectric focusing analysis of PI types showed that the patient was heterozygous for the S allele and for a deficient variant gene product, inherited from his father, which has an isoelectric point identical to M1 and a band with decreased intensity (fig 2). These patterns were confirmed by print immunofixation (not shown). DNA sequencing of all PI coding exons (II-V), performed as previously described,6 7 has shown that the patient and his father shared a C to T transition leading to a 369Pro (CCC)→Ser (TCC) substitution in the common M1Val213 allele, as in the variant Mwürzburg.3 4 The presence of the Mwürzburg allele was also confirmed by partial PCR amplification of exon V with a mismatched primer that generates an 118 bp fragment: 5′-CCCGAGGTCAAGTTCAA CAgA-3′ (bases 10049-10069, mismatched base in lower case); 5′- GAGGAGCGAGAGG CAGTTATT-3′ (bases 10166-10146). Thirty five cycles of PCR were performed for one minute at 94°C, one minute at 58°C, and one minute at 72°C. The mismatched primer artificially introduces a NdeII restriction site in the mutated Pro369Ser allele during PCR amplification (fig 3, lanes 1 and 2). In addition, the primer also generates a furtherHinfI restriction site in the presence of the Pro369Leu mutation (fig 3, lanes 3 and 4), which characterises the severely deficient variant Mheerlen.8

Figure 2

PI patterns after polyacrylamide gel hybrid isoelectric focusing (pH range of 4.3 to 4.8) and Coomassie blue staining.5 From left (1) M2/S = patient's mother, (2) Mwürzburg/S = patient, (3) Mwürzburg/M3 = patient's father, (4) M1 = control. The major band corresponding to the Mwürzburg gene product is indicated with an arrow.

Figure 3

Detection of the Pro369Ser and Pro369Leu mutations after PCR amplification with a mismatched primer. (Lanes 1 and 2) NdeII digested fragments from the Mwürzburg/S patient (1) and from a normal M1 control (2). The Pro369Ser mutated sequence generates one 98 bp fragment and one 20 bp fragment (not visible). (Lanes 3 and 4) HinfI digested fragments from a M2/Mheerlen subject (3) and from a normal M1 control (4). The normal sequence produces one 84 bp fragment and one 34 bp fragment. The Pro369Leu mutated sequence generates an additional restriction site and 64 bp, 34 bp, and 20 bp (not visible) fragments are produced. MW= molecular weight marker. DNA fragments were visualised by silver staining after non-denaturing electrophoresis on 9% polyacrylamide gels.


To our knowledge the present case represents the first reported association between the defective α1-antitrypsin Pro369Ser mutation and in vivo intrahepatic protein accumulation. Although the S variant has been found to show increased intracellular retention and the ability to form heteropolymers with Z,9-11 this increase is only marginal and no evident inclusions of S α1-antitrypsin have been found in most pathology samples observed so far. Therefore, the observation of PI liver inclusions in the Mwürzburg/S patient is most likely to be predominantly caused by the Mwürzburg variant and provides further in vivo evidence that the severe deficiency resulting from the Pro369Ser mutation is caused by protein accumulation, as in the case of the Z allele.

Since the patient's PI type would be expected to be similar to SZ, which is not associated with increased risk of liver disease in infancy, his liver injury is probably related more to the CMV infection than to the Mwürzburg variant. However, the similar behaviour of Mwürzburg and Z both in vitro and in vivo indicates that it may lead to liver disease in Mwürzburg homozygotes or in Mwürzburg/Z heterozygous combinations.

Contrary to previous observations,3 the present detection of a faint PI band with the same isoelectric point of M1, both in the patient and his father, indicates that Mwürzburg can still be secreted in limited amounts into the plasma. However, since the variant will remain unidentified in combination with the common normal M1 allele, the PCR introduction of a NdeII restriction site is a simple alternative tool to detect the Pro369Ser mutation, especially in cases where no unusual isoelectric focusing patterns are associated with decreased PI serum levels or with intrahepatic protein accumulation.


We thank Ms Lúcia Ramires and Ms Piedade Mendonça for technical assistance. This work was supported in part by Conselho de Prevenção do Tabagismo. Susana Seixas is supported by grant BD/13885/97 from Praxis XXI.