Journal of Hepatology, 1986; 2:65-72 65 Elsevier

HEP 0073

A Swedish Family with al-Antitrypsin Deficiency, Haemochromatosis, Haemoglobinopathy D and

Early Death in Liver Cirrhosis

Sten Eriksson 1, Bertil L indmark I and Lad Hanik 2

1Department of Medicine, University of Lund, Malta6 General Hospital, S-214 01 Malrn6, and 2Department of Medicine, Angelholm Hospital, S-262 O0,4ngelholrn (Sweden)

(Received 26 February, 1985) (Accepted 7 May, 1985)

Summary

We report a unique family with chronic liver disease associated with three differ- ent inborn errors, a~-antitrypsin deficiency, hereditary haemochromatosis and hae- moglobinopathy Hb-D Punjab. The probability of acquiring these three rare genes is less than 1/109. In one generation 4 of 5 individuals have died of liver failure be- tween 51 and 63 years of age.

Introduction

al-Antitrypsin (AAT) deficiency Pi ZZ is a hereditary condition characterized by decreased ability of the liver to secrete functionally active AAT, which is the ma- jor serine protease inhibitor in human plasma. The gene for AAT is located on chromosome 14 [1] and the different alleles can be identified by isoelectric focusing. The Z allele in double dose predisposes to early emphysema, neonatal hepatitis and adult liver disease with a high incidence of malignant hepatoma [2,3]. Recent stud- ies suggest that the Pi MZ state predisposes to chronic cryptogenic liver disease [4].

This study was supported by a grant from the Swedish Life Insurance Companies Council for Medical Research.

Address reprint requests to Sten Eriksson, Department of Medicine, Malta6 General Hospital, S-214 01 Malm6, Sweden, Tel. 040-332309.

Abbreviation: AAT = ctt-antitrypsin.

0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

66 S. ERIKSSON et al.

Pi Z gene frequency is estimated to be 0.024 and that of the heterozygous form to 0.047 in North European countries [5]. In the Pi ZZ AAT there is a GluLys substi- tution in the 342 position associated with decreased plasma levels of the protein. The abnormal protein accumulates in the rough endoplasmic reticulum and can be seen as PAS-positive globules by light microscopy [6].

Iron overload is seen in a variety of diseases. In idiopathic or hereditary hae- mochromatosis there is a progressive increase in the total body iron content. The disease was classically described as the combination of cirrhosis, diabetes mellitus and skin pigmentation caused by parenchymal iron overload of the liver and pan- creas. Common features are cardiomyopathy, arthropathy and pituitary dysfunc- tion. The pathophysiology is not yet fully understood. The disease is thought to be inherited in an autosomal recessive manner by two alleles close to the HLA-locus on chromosome 6 [7]. There is partial expression of the gene in heterozygotes but no clinical disease. The prevalence of the homozygous form has been estimated to between 0.1% and 0.3% [8,9].

Haemoglobin D is characterized by a GluGly substitution in position 121 of the fl- chain [10]. The gene for the fl-chain is located on chromosome 11 [11]. The result- ing abnormal haemoglobin can be detected on paper electrophoresis where it has mobility equal to that of Hb S but can be distinguished from Hb S by the absence of sickling. It is infrequent in most parts of the world, but occurs in 2% of the Sikh pop- ulation in the Indian district Punjab [12].

This communication describes a Swedish family with AAT deficiency, hereditary haemochromatosis and haemoglobinopathy Hb-D. All 5 siblings in the second gen- eration have developed liver cirrhosis at an early age and four of the five have died between 51 and 63 years of age.

Tr

rrr

1 2 I - - / - \ I I I t

- C_1 "

w __ __ I

.I. 9 / %

i I \ J

D Hemoglobin D [ ] Normal, male

Fe Hemochromatosis O Normal, lemale ] I - - I

AAT-def ic iency (PiZZ) ,_ 2 Male, not invest igated

[ ] AAT-deficiency (PiMZ) ( - ) Female, not investigated

Fig. 1. Family pedigree.

AAT DEFICIENCY, HAEMOCHROMATOSIS AND Hb D 67

Material and Methods

The family pedigree is presented in Fig. 1 and in separate case reports. Roman symbols refer to the pedigree in Fig. 1.

Members of the second generation have been seen at the Maim6 General Hospi- tal during the period 1955-1983. The available laboratory methods have changed during these years. Plasma protein analysis was earlier performed by paper electro- phoresis. During the seventies agarose gel electrophoresis with determination of in- dividual plasma proteins by electroimmunoassay was introduced.

AAT phenotyping was performed according to Pierce et al. [13]. The haemoglo- bin mobility was determined by paper electrophoresis according to Marengo-Rowe [14].

Serum iron and TIBC were measured in accordance with standard laboratory techniques at the Department of Clinical Chemistry at the Maim6 General Hospi- tal. Serum ferritin was analyzed using a commercial radioimmunometric assay (Becton-Dickinson). The autopsy specimens were stained for iron according to standard procedures. AAT globules were studied in diastase-treated PAS-stained liver biopsies.

Family and Case Histories

The mother's parents were cousins and reportedly healthy. The mother (12) died at the age of 59 of liver cancer. Whether primary or secondary is unknown. No de- tailed data concerning her medical history are available. The father (I1) is still living and healthy at the age of 92.

The first son B ( I I l ) , born in 1917, was healthy unti ! 1969. There was no history of excessive alcohol ingestion. After an upper respiratory tract infection a routine check-up showed increased sed. fate, and abnormal liver function tests including elevated bilirubin. Liver cirrhosis was suspected. Plasma protein analysis disclosed a moderate polyclonal hypergammaglobulinemia, a slight decrease in albumin and no visible al-band. AAT was 17% of normal. Cholecystography showed cholecystopathia but no gallstones were seen. In 1970 pedal edema, ascites, spleno- megaly and skin pigmentation were noted. Needle biopsy of the liver showed side- rosis with fibrosis. Transferrin saturation was above 83% in several samples. Hae- moglobin electrophoresis showed 50% haemoglobin D. A peroral glucose toler- ance test was normal. Uro- and coproporphyrines in urine were within normal lim- its. In the autumn of 1971 he became increasingly ill with progressive ascites. He died of liver failure. Autopsy showed diffuse panacinar emphysema, atrophic mi- cronodular hepatic cirrhosis with PAS-positive inclusions, severe iron overload in liver and pancreas.

The second son L (II2), born in 1918, had a greyish skin pigmentation and liver cirrhosis diagnosed in 1967. He had no history of alcoholism. In 1968 splenomegaly ensued and thrombocytopenia was noted. There was massive iron loading of the he- patocytes demonstrated in a percutaneous needle biopsy of the liver performed in 1972. He was heterozygote for Hb D. There were no signs of AAT deficiency and

68 S. ERIKSSON et al.

phenotyping showed that he was Pi MM. In 1973 he had his first episode of hepatic encephalopathy and he died from decompensated cirrhosis in 1976. The autopsy confirmed classical haemochromatosis.

M-B (IIa), the only girl in this generation, was born in 1920 and had no history of excessive alcohol ingestion. She had recurrent leg ulcers and presented in 1981 with abnormal liver function tests, thrombocytopenia and a slightly elevated bilirubin. There were no clinical signs of liver disease except for two spider naevi. Antinu- clear factors and mitochondrial autoantibodies were lacking. Plasma protein analy- sis demonstrated AAT deficiency with a plasma level of 0.32 g/l (0.9-1.79/1), hypo- albuminemia and hypergammaglobinemia with IgG reaching 42 g/I. She was a Pi Z homozygote on electrofocusing and a heterozygote for haemoglobin of the D type. The transferrin saturation was 85% and the ferritin value 4224pg/1 (normal < 400). In 1982 she developed fluid retention with ascites leading to death in January 1983. Autopsy showed a complete derangement of the hepatic structure, proliferating bile ducts, in the majority of the hepatocytes an abundance of PAS-positive gran- ules and iron pigment. The pancreas and liver showed massive iron loading (Fig. 2). The lungs were emphysematous.

The third son O (II4), born in 1922, is non-alcoholic and has been investigated at another hospital. He is still living but has a decompensated cirrhosis. The transfer- fin saturation is 18% and the ferritin value 28 pg/1. Biopsy of the liver has not shown iron overload. He has normal haemoglobin. He is characterized as Pi MZ.

The fourth son K (IIs) was born in 1923 and had a history of excessive alcohol in- take. In 1954 he was admitted because of hematemesis and melena. There were

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AAT DEFICIENCY, HAEMOCHROMATOSIS AND Hb D 69

Fig. 2. Liver histology in II 5. A: Haematoxylin-eosin-stained preparation showing a typical pattern of hepatic cirrhosis with fibrous septa and regeneration nodules x 125. B: PAS-staining after diastase treat- ment: A A T globules in abundance x500. C: Perls stain: iron loading of hepatocytes x250.

7 0 S. E R I K S S O N e t a l .

signs of liver disease with spider naevi, hepatomegaly and X-ray showed esopha- geal varices. He was anemic with a haemoglobin value of 68%. The skin had a grey- ish pigmentation. Further evaluation showed splenomegaly. An increased portal pressure was measured by percutaneous intrasplenic puncture, but was normalized by a porto-caval anastomosis. A peroperative liver biopsy showed no signs of AAT globules or increased iron content but fibrosis and altered architecture as in cirrho- sis. The serum level of at-globulins was 0.35 g/100 ml which is within the normal range. In 1968 the serum transferrin saturation was 85%, the Hb being 137 g/l. Hae- moglobin electrophoresis and the red cell appearance were normal. After the por- to-caval shunt there was an improvement in the general condition and the spider naevi disappeared. In 1970 insulin-dependent diabetes mellitus was diagnosed. In 1973 he had fluid retention and he died from hepatic failure in 1974. The transferrin saturation just before death was 100%. No autopsy was performed.

The third generation has been studied to a limited extent. Several members, III 6- 11I 9 refused to participate. The findings are summarized in Table 1. No individual in this generation was homozygote for AAT deficiency. We did not find biochemical evidence for abnormal iron metabolism in any of the individuals that were investi- gated, nor did we find Hb D trait.

Discussion

The combination of AAT deficiency, hereditary haemochromatosis and Hb D disease has hitherto never been described. The probability of acquiring the individ- ually rare genes is extremely low. Assuming a maximal homozygote frequency of

T A B L E 1

S C H E M A T I C P R E S E N T A T I O N O F T H E F I N D I N G S I N T H E F A M I L Y

C a s e A A T H H H b D C i r r h o s i s E m p h y s e m a D i a b e t e s A g e a t L i v i n g a t

d e a t h ( y r ) a t a g e ( y r )

II~ Z Z + + + + -

I I 2 M M + + + - -

I I 3 Z Z + + + + -

I I 4 M Z - - + - -

I I 5 M M + - + - ,-I-

I I I l M Z . . . . .

I l l 2 M M m . . . .

II13 M Z . . . . .

I I I 4 M Z . . . . .

I I I 5 m . . . . .

1116 m m m - - -

I I I 7 m m m - - -

I I I s m m m - - -

I I I 9 m m m - - -

54

58

63

51

61

44

24

30

27

30

27

38 36

32

A A T = a t - a n t i t r y p s i n p h e n o t y p e ; H H = h e r e d i t a r y h a e m o c h r o m a t o s i s ; H b D = h e t e r o z y g o u s H b D .

+ = p r e s e n c e , - = a b s e n c e , m -- d a t a m i s s i n g .

AAT DEFICIENCY, HAEMOCHROMATOSIS AND Hb D 71

1/1000 for each disease the chance would be 1/109 . The genetic background to the different diseases affords no ground for a common explanatory mechanism. Both AAT deficiency and hereditary haemochromatosis are known causes of liver cir- rhosis. Although the genes are located on different chromosomes, several reports on the concomitant existence of these two conditions have appeared [15-17]. Hae- mochromatosis and AAT deficiency were described in one patient by Anand et al. [15]. Rigas and Finch published in 1959 an electrophoretic study of 17 patients with idiopathic haemochromatosis [16]. In these patients the mean value of the al-globu- lin fraction was 2.7 g/l as compared to the normal of 4.0 g/l (P < 0.01) indicating at least partial AAT deficiency. Janus and Carrell reported in 1975 10 patients with Pi MZ phenotype [17]. Hepatocyte iron overload was demonstrated in 6 of them.

The concomitant occurrence of two conditions known to cause liver cirrhosis is likely to accelerate liver injury. The iron overload in hepatocytes is known to cause lysosome fragility [18] which is normalized by venesection [19]. The pathogenesis of liver disease in AAT deficiency is poorly understood. In the homozygous Pi ZZ state the abnormal protein is accumulated in the rough endoplasmic reticulum as PAS-positive inclusion bodies [6]. These inclusions are so abundant that normal metabolism may beimpaired and cell death ensued. In addition, the low plasma lev- el of AAT is possibly inadequate for protection against leaking lysosomal proteoly- tic enzymes. Both AAT deficiency and haemochromatosis predispose to hepatocel- lular carcinoma which was not present in any of our patients. We do not know whether the liver cancer in 12 was primary or secondary.

The Hb D disease is extremely rare in most parts of the world. In a Swedish pop- ulation screening of 4 171 individuals not a single case was found [20]. In homozy- gous haemoglobin D disease there are decreased levels of haemoglobin A1, target cells are present and a mild degree of haemolytic anemia may occur [21]. Haemo- globin D is a stable haemoglobin with a slightly increased oxygen affinity [22]. Hae- mochromatosis has to our knowledge never been described in homozygous Hb D disease. The heterozygous Hb D disease is not known to cause any symptoms [21]. Since our cases were heterozygotes the haemoglobinopathy per se is unlikely to have contributed to the liver disease. The haemochromatosis is therefore thought to be primary. One of our cases (II5) had biochemical evidence of haemochromato- sis and normal haemoglobin electrophoresis. However, this individual had been op- erated with a porta-caval anastomosis and is at risk of developing postshunt haemo- siderosis [23] which is clinically indistinguishable from primary haemochromatosis.

Acknowledgement

We thank Dr. Lennart Bondesson for helping us with the microphotographs.

References

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72 S. ERIKSSON et al.

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