027n-91~9/~2/0204-n4fi3$02.00/0 HEPATOLOGY Copyright 0 1982 by the American Association for the Study of Liver Diseases

Vol 2, No. 4, pp 463-466, 1982 Printed in l i .SA .

HLA in Chileans with Intrahepatic Cholestasis of Pregnancy

HUMBERTO REYES, M A R ~ A E. WEGMANN, NELLY SEGOVIA, MIGUEL CUCHACOVICH, ENRIQUE JADRESIC, MONICA CONTADOR, CECILIA FUENTES, AND MIREYA MELENDEZ

Departments of Medicine and Experimental Medicine, University of Chile School of Medicine (Eastern Campus), and the Department of Immunology, Instituto de Salud Publica, Santiago

de Chile

A possible association between intrahepatic cholestasis of pregnancy (ICP) and human-leukocyte histocompatibility (HLA) antigens-used as genetic markers-was studied in 100 women with ICP compared to 100 multiparous women without a past history of the disease. Because we previously found a higher frequency of ICP in women with an overt Araucanian Indian descent than in Chilean Caucasoids, women from both ethnic groups were studied. Among the 37 specificities of the HLA system studied (17 of HLA-A, 16 of HLA-B, and 4 of HLA-C series), only HLA-BW16 showed a tendency to be more frequent in women with ICP rather than in control women. This finding appears to be related with ethnic origin and not ICP, HLA-BW16 was significantly more frequent in women with Araucanian Indian descent (43.4%) than in Chilean Caucasoids (16.3%) (p < 0.01). The high frequency of HLA-BW16 in the predominantly Caucasoid population in Chile, in compar- ison with Caucasians in Europe and in North America, may be another indicator of their ethnic admixture with aborigine groups. The high frequency of HLA-BW16 reported in North American Indian-admixed groups (16%) suggests that HLA-BW16 may be a genetic characteristic common to some aboriginal populations in North and South America.

Although the cause of intrahepatic cholestasis of preg- nancy (ICP) is unknown, familial aggregation of cases (1-5) and geographic distribution of the disease, with a high prevalence in certain countries and ethnic groups (6-10), suggest that genetic factors are involved in its pathogenesis. In Chile, the prevalence of the disease is higher in women with Araucanian Indian descent (27%) than in Caucasoids (14%) (8), and higher in Chilean Caucasoids than in the population of neighboring coun- tries or in Caucasians in North America, Europe, and Scandinavian countries (6, 10). We postulated that the high prevalence of ICP in Chile is influenced by genetic susceptibility related to ethnic admixture with Araucan- ian Indian groups (6, 8-11).

The present study investigates the possible association between ICP and the distribution of human-leukocyte histocompatibility (HLA) antigens in Chilean Caucasoids and in women of Araucanian Indian descent.

MATERIALS AND METHODS POPULATION STUDIED

Two hundred patients were selected from all pregnant women who underwent parturition at the Department of

Received danuary 26, 1982; accepted March 26, 1982. This study was supported by the Servicio de Desarrollo Cientifico,

Universidad de Chile (Grant M039). Address reprint requests to: Dr. Humberto Keyes, Universidad de

Chile, Departmento de Medicina Experimental Oriente, Casilla 16038, Santiago 9, Chile.

Obstetrics, Hospital del Salvador, Santiago, Chile. The patients were divided into four groups with the following characteristics. Group 1: Multiparous Caucasoid Chilean women with- out a past history of ICP.

This group included 50 women with Caucasian facial traits who were born in Chile and whose parents and grandparents had European surnames. None of their pregnancies was affected by ICP, and physical examina- tion did not disclose signs of chronic liver disease. The mean age was 31.9 years (range 20 to 41); mean parity was 5.8 (range 3 to 12). Group 2: Caucasoid Chilean women with a past history

Fifty women with the same ethnic characteristics as Group 1 who had clinical and laboratory findings of ICP (6) were identified during pregnancy. Direct-reacting hy- perbilirubinemia established the diagnosis of cholestatic jaundice of pregnancy (the icteric form of ICP) in 17 patients; the other 33 were classified as pruritus gravi- darum (i.e., anicteric ICP). Among 36 multiparous women, 32 had a past history of ICP in previous preg- nancies (recurrent ICP). The mean age was 28.2 years (range 17 to 41); mean parity was 2.6 (range 1 to 9). Group 3: Multiparous women with Araucanian Indian descent without a past history of ICP.

This group included 50 women with Indian facial traits who were born in the central and southern zones of Chile

of ICP.

463

464 REYES ET AL. HEPATOLOGY

which were originally inhabited by Araucanian Indians. In their immediate ancestry, at least the mother and two grandparents had surnames derived from Araucanian Indians. Similarly to women in Group 1, none of their pregnancies were affected by ICP, and no signs of liver disease were detected. The mean age was 30.3 years (range 18 to 49); mean parity was 3.9 (range 2 to 10). Group 4: Women with Araucanian Indian descent and a past history of ICP.

This group consisted of 50 women with the same ethnic characteristics as in Group 3, but in whom clinical and laboratory alterations typical of ICP were detected dur- ing their present pregnancy. Direct-reacting hyperbili- rubinemia was detected in eight cases (cholestatic jaun- dice of pregnancy); the other 42 were classified as pruritus gravidarum. Among 36 multiparous women, 20 had a history of ICP in previous pregnancies. The mean age was 27.2 years (range 15 to 40); mean parity was 2.4 (range 1 to 5). GENETIC MARKERS AND HLA TISSUE TYPING

ABO blood groups and Rh factor were determined by standard laboratory techniques. Shovel-shaped teeth was identified by experienced observers. Typing for HLA antigens was performed with the standard microlympho- cytotoxicity method of the National Institutes of Health 17 antigens of the HLA-A series, 16 of the HLA-B series, and 4 of the HLA-C series were analyzed. STATISTICAL ANALYSIS

Comparison between groups was done by the x2 test with Yates’ correction (12). In the HLA evaluation, p values were corrected for the number of specificities studied (13).

RESULTS Using facial traits, surnames, and birthplace as ethnic

markers, similar to previous studies (8-lo), women clas-

sified as Caucasoids or admixed Araucanian Indian had differences in the distribution of ABO blood groups and in the frequency of shovel-shaped teeth (Table 1). Women with Araucanian Indian descent had a higher frequency of 0 blood group than did Caucasoids (74.2% in pooled Groups 3 and 4 vs. 56.7% in pooled Groups 1 and 2; p c 0.01) and a higher frequency of shovel-shaped teeth (70.5 vs. 25.8%; p c 0.01). These results support the assumption that these women are of partially different ethnic origin.

The distribution of the 37 HLA specificities studied was similar in the four groups of women (Table 2) except that HLA-BW16 appeared more frequently in women with ICP than in control women, and in women of Araucanian Indian descent than in Caucasoids.

The higher frequency of HLA-BW16 in women with ICP was not significant when Groups 2 and 4 were independently compared with their respective ethnic controls without the disease (Groups 1 and 3) nor when women from Groups 2 and 4 were pooled (HLA-BWl6 = 35.7%) and compared with pooled Groups 1 and 3

The frequency of HLA-BW16 in women of Araucanian Indian descent (43.4% in pooled Groups 3 and 4) was significantly higher than in Caucasoids (16.3% in pooled Groups 3 and 4) (p < 0.01).

A more elaborate ethnic comparison was attempted by stratifying the 100 women of Araucanian Indian descent into two subgroups, according to their probability of “ethnic purity.” Twenty women from Groups 3 and 4 were considered closer to “ethnically pure” Araucanian Indians because of the 0 blood group, Rh positive factor, shovel-shaped teeth, and both parents and the two grand- mothers having Araucanian surnames. They were com- pared with the remaining 80 women from Groups 3 and 4 who lacked one of these genetic and socio-cultural characteristics. The frequency of HLA-BW 16 was similar in the two subgroups (47.3 vs. 43%; p > 0.1).

(HLA-BW16 = 24.2%; p < 0.05).

TABLE 1. FREQUENCY OF ETHNIC MARKERS IN CHILEAN WOMEN WITH ARAUCANIAN INDIAN DESCENT AND IN CAUCASOIDS WITH OR WITHOUT

ICP“

Caucasoids Araucanian Indians

Group 1 Group 2 Group 3 Group 4

n = 50 n = 50 n = 50 n = 50 (with ICP) (without ICP) (with ICP) (without ICP)

Blood groups AB 2 4.2 0 2 A 24 33.3 20.7 18.6 B 14 10.4 6.3 4 0 60 94 96 98

Rh factor (D) 96 94 96 98 Shovel-shaped teeth 14.8 (27) 35.5 (32) 57.7 (26) 80 (35)

x 2 Test for 0 blood group Shovel-shaped teeth

I‘ Between Caucasoid Groups 1 and 2 p > 0.05 p > 0.05

p > 0.05 p < 0.05 Between Araucanian Groups 3 and 4

Between Caucasoids (Groups 1 and 2) and Araucanians (Groups 3 p < 0.01 p < 0.01 and 4)

” Figures in parentheses indicate the number of individuals in whom upper frontal teeth remained undamaged and the presence or absence of shovel-shaped teeth could be established in them.

Vol. 2, No. 4, 1982 HLA AND CHOLESTASIS OF PREGNANCY 465

TABLE 2. FREQUENCY OF HLA ANTIGENS IN CHILEAN WOMEN WITH

ARAIJCANIAN INDIAN DESCENT AND IN CAUCASOIDS WITH OR WITHOUT I c P

Caucasoids Araucanian Indians

Antigens Group 1 Group 2 Group 3 Group 4 (without ICP) (with ICP) (without ICP) (with ICP)

n = 50 n = 50 n = 50 n = 50 <-%,

HLA-A1 A2 A3 A9 A23 A24 A10 A25 A26 A l l AFwl9 A29 A30 A:i 1 A92 A83 A28

Blank"

HLA-B5 B7 B8 B12 B13 B14 B15 Bw16 B17 B 18 Bw2i Bw22 B25 Bw35 B40 B42

Blank

HLA-Cwl CW2 CW3 c w 4

Blank

10 46 8

28 2

16 10 0 0 4

20 4 4 2 2 0

14

12

22 12 10 18 4

10 6

12 2 8 6 4 6 6 4 2

14

9.1 4.5 4.5 0

84.1

16.6 31.2 10.4 37.5 6.2

27.1 2.1 0 0

12.5 16.7 0 0

10.4 2.1 0

18.8

6.3

16.7 18.8 8.3

10.4 0

10.4 10.4 20.8 4.2

10.4 4.2 4.2 8.3 6.3 6.3 2.1

10.4

8.5 4.3 6.4 6.4

76.6

12.2 42.9 10.2 20.4

2.0 14.3 14.3 6.1 0 2.0

26.5 4.1 6.1 4.1 6.1 0

14.3

4.1

2.0 6.1 8.2 8.2 2.0 8.2 8.2

36.7 8.2 4.0 0 4.1 8.2 8.2

12.2 0

16.3

12.5 2.1 0 6.3

79.2

18 48 10 20 0

14 16 4 4 4

26 0 0 6 4 2

18

4

12 4 8 8 4 4 6

50 4 4 2 2 6

16 12 2

6

4.2 6.3 4.2 2.1

85.4

" Blank = per cent of unidentified specificities in the HLA pheno- type.

DISCUSSION The distribution of 37 specificities of the HLA system

was not significantly different in Chilean women with ICP than in controls without a past history of the disease. Women with ICP tended to have a higher frequency of HLA-BW16; this finding may be related to ethnic origin and not to ICP.

Although Caucasoid women without a Dast historv of

population of pregnant women, their physical and socio- cultural characteristics coincide with the predominantly Caucasoid population in Chile. The distribution of ABO blood groups was similar in 633 Caucasoid women pre- viously studied at random in the same obstetric wards (8) and the distribution of HLA antigens was similar to the results obtained in 186 healthy blood donors in San- tiago (14). The frequency of HLA-BW16 in Caucasoid Chilean women without a past history of ICP (12%) or with ICP (20.8%) and in healthy blood donors (16.1%) is higher than the frequency in Caucasians in Europe (2.0 to 3.5%) (15) and the United States (5.0%) (16).

Women of overt Araucanian Indian descent (Groups 3 and 4) had a significantly higher frequency of HLA- BW16 than did Chilean Caucasoids; other HLA specific- ities showed no differences in these ethnic groups. A high frequency of HLA-BW16 (13.7 to 14.5%) was reported in admixed North American Indians (15) and may be an- other ethnic characteristic common to aborigines from North and South America; it is associated with shovel- shaped teeth and 0 blood group. HLA-BW16 may also indicate Indian admixture in the predominantly Cauca- soid population in Chile. Our attempt to stratify women with Araucanian Indian descent according to their "ethnic purity" produced two subgroups with grossly dissimilar size, which complicates interpretation of the frequency of HLA-BW16 in both subgroups.

ABO blood groups, Rh factor, and shovel-shaped teeth have been used in several studies as genetic markers of ethnic characteristics and differences in population sam- ples (8-10). Although the prevalence of ICP appears to be different in some ethnic groups, no positive association was demonstrated between any single genetic marker and the disease. Similarly, none of the HLA antigens which were studied was related to the genetic predispo- sition to develop ICP. These observations do not exclude the possibility of finding an association between ICP and other specificities of the HLA antigen system which were not included in our study (17).

Acknowledgments: We are grateful to our colleagues in the Department of Obstetrics, Hospital del Salvador for permission to investigate their patients. Miss Alicia Vicente C. typed the manuscript.

REFERENCES

1. Svanborg A. A study of recurrent jaundice in pregnancy. Acta Obstet Gynecol Scand 1954; 33:434-444.

2. Thorling L. Jaundice in pregnancy. A clinical study. Acta Med Scand 1955; 15l(supp1302):1-123.

3. Holzbach RT, Saunders HH. Recurrent intrahepatic cholestasis of pregnancy. Observations on its pathogenesis. JAMA 1965; 193:542-544.

4. Dalen E, Westerholm B. Occurrence of hepatic impairment in women jaundiced by oral contraceptives and in their mothers and sisters. Acta Med Scand 1974; 195:459-463.

5. Reyes H, Ribalta J, Gonzalez-Ceron M. Idiopathic cholestasis of pregnancy in a large kindred. Gut 1976; 17:709-713.

6. Reyes H. The enigma of intrahepatic cholestasis of pregnancy: lessons from Chile. Hepatology 1982; 297-96.

7. Haemrnerli UP, Wyss HI. Recurrent intrahepatic cholestasis of pregnancy. Report of six cases and review of the literature. Medi- cine (Baltimore) 1967; 46299-321.

8. Reves H. Gonzdez MC. Ribalta J. et al. Prevalence of intraheoatic ICP (GroGp 1) were not chosen at random-from the tbtal cholestasis of pregnancy in Chile. Ann Intern Med 1978; 88:487:493.

466 KEYES ET AL. HEPATOLOGY

9. Ruiz-Esquide M, Ribalta J , Reyes H. Prevalencia de la colestasia intrahepatica de la embarazada en Los Angeles, Chile. Rev Med Chile 1978; 106507-510.

10. Reyes H, Taboada G , Ribalta J . Prevalence of intrahepatic choles- tasis of pregnancy in La Paz, Bolivia. J Chron Dis 1979; 32:499-504.

11. Reyes H, Ribalta J, Gonzalez MC, et al. Sulfobromophthalein clearance tests before and after ethinyl estradiol administration, in women and men with familial history of intrahepatic cholestasis of pregnancy. Gastroenterology 1981; 81:226-231.

12. Armitage P. Statistical methods in medical research. Oxford: Black- well Scientific Publications, 1971.

13. Svejgaard A, Ryder LP. Associations between HLA and disease.

In: Dausset J, Svejgaard A, eds. HLA and disease. Copenhagen: Munksgaard, 1977: 46-71.

14. Melendez M, Vargas-Tank L, Fuentes C, et al. Distribution of HLA histocompatibility antigens, ABO blood groups and Rh antigens in alcoholic liver disease. Gut 1979; 20288-290.

15. Joysey VC, Wolf E. HLA-A, -B and C-antigens their serology and cross-reaction. Br Med Bull 1978; 34:217-222.

16. Richman AV, Mahoney JJ, Fuller TJ. Higher prevalence of HLA- B12 in patients with IgA nephropathy. Ann Intern Med 1979; 90201.

17. McDevitt HO. Regulation of the immune response by the major histocompatibility system. N Engl J Med 1980; 303:1514-1517.