Hum Genet (1995) 95 : 586-588 �9 Springer-Verlag 1995

Alan Russell �9 Hisham Nazer �9 Ali Shams - Jan Sj6vall Roger Sutcliffe

No linkage to the 3 -HSD gene cluster in a kindred affected with 3 -hydroxy-AS-Ca7-steroid dehydrogenase deficiency and early onset hepatic failure

Received: 12 September 1994

Abstract We studied the segregation of the genes for 3[3- hydroxy-C19/21-steroid dehydrogenase types I and II (3[3- HSD I and It) in a consanguineous family affected with 3~-hydroxy-AS-C:7-steroid dehydrogenase (313-OH-C27- SD) deficiency. The results show that the C2v and C~,)/2L steroid dehydrogenase activities are encoded by distinct genes that are not in genetic linkage. Further kindreds would assist in screening for linkage of 313-OH-C27-SD to other members of the 3[3-hydroxysteroid dehydrogenase gene family.

Introduction

Cholesterol is converted to bile acids by a series of modi- fications to the steroid nucleus and side chain. Oxidation of the side chain is known to be disrupted in several in- born errors of metabolism affecting enzymes of the mito- chondria and peroxisomes, including Zellweger syn- drome, Refsum disease and 26-hydroxylase deficiency (Clayton 1991; Russell and Setchell 1992). Recently, Buchmann et al. (1990) described familial giant cell he- patitis associated with a deficiency of 313-hydroxy-AS-C27 - steroid dehydrogenase (3[3-OH-C:7-SD). This is a rare re- cessive cholestatic condition in which abnormal bile acid synthesis can lead to fatal liver damage. The defect in 3~3- OH-C27-SD leads to a failure to convert 3[3, 7r droxy-A -~ steroids into 7~x-hydroxy-3-oxo-A 4 intermediates and has been identified through elevated levels of abnor-

A. Russell. R. Sutcliffe ([E~) Institute of Genetics, University of Glasgow, Church Street, Glasgow G1 I 5JS, UK

H. Nazer - A. Shams Department of Paediatrics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

J. Sj6vall Department of Medical Biochemistry and Biophysics, Karolinska lnstitutet, S-171 77 Stockholm, Sweden

real metabolites of 7c~-hydroxycholesterol in bile, plasma and urine (Ichimiya et al. 1991) and the lack of 313-OH- C27-SD enzyme activity in skin fibroblasts (Buchmann et al. 1990). The gene for 313-OH-C27-SD has not been mapped or cloned. Treatment with chenodeoxycholic acid has been found to be effective, acting to replace the en- dogenous deficiency and possibly to reduce the synthesis of 7c~-hydroxycholesterol through partial inhibition of cholesterol 7~-hydroxylase (Ichimiya et al. 1990).

The genes for two other human 3[3-hydroxysteroid de- hydrogenase enzymes (3[3-HSD types I and II) convert C21 and C~9 A 5 hydroxysteroids to A 4 ketosteroids such as progesterone and androstenedione and are in close genetic linkage on chromosome lpl3.1 (Morrison et al. 1990; Russell et al. 1994). The lack of a cholestatic condition in patients with pseudohermaphroditism due to 3[3-HSD 11 deficiency shows that 313-OH-C27-SD and 3[3-HSD type II are encoded by separate genes (Laatikainen et al. 1972; Rheaume et al. 1992; Russell et al. 1994; Mendona et al. 1994), but the specificity of the type I enzyme has not been formally tested for C27 substrates. There is evidence of other members of the 313-HSD gene family. In rat and mouse four 3[3-HSD genes have been cloned to date: rat type III and mouse type IV have 3-ketosteroid reductase activity; other forms have dehydrogenase/isomerase ac- tivities, principally in adrenals and gonads, liver and kid- ney (Zhao et al. 1990; de Launoit et al. 1992; Clarke et al. 1993a, b; Mason 1993; Sutcliffe et al. 1995). In mouse Bain et al. (1993) showed that four loci map to a region of chromosome 3 that is syntenic with human chromosome lp13 (Morrison et al. 1990), suggesting that further ex- pressed members of the human 3[3-HSD gene family should be found clustered with the 3[3-HSD type I and II genes. As an initial step to defining the gene for 313-OH- C27-SD we therefore determined whether it is genetically linked to the type I and II genes of 3[3-HSD.

Fig. 1 Pedigree of a consan- guineous Saudi Arabian family affected with neonatal jaun- dice, showing genotypes and deduced haplotypes for poly- morphic alleles of the genes for 313-hydroxysteroid dehy- drogenase (3~-HSD). The alle- les of the 3~-HSD type I (BglII , + or -; codon 367, C or A) and type II (intron C mi- crosatellite alleles, 1-5) are written in two columns, di- vided by a vertical line (see key). Alleles in the same col- umn represent those that are physically located on the same chromosome homologue

IV

587

([~ 3 t 4 d 5 d 6[~[] 7 d 8 d 9~ 1"]

"lCl A C A A + -- + + + + 2 1 4 ~ 1 3 1 4 113

haplotype key

locus site 3B.HSDI{C]A. codon367Bgll,

313-HSD II 4 1 3 intron C microsat.

Materials and methods

The affected children shown in the consanguineous family (Fig. 1) were those reported in Buchmann et al. (1990). Typing of poly- morphisms in the genes for 313-HSD types I and II was carried out as described by Russell et al. (1994).

Results

Two genetic polymorphisms have been described in exon 4 of the 313-HSD type I gene (Russell et al. 1994). One is a synonymous polymorphism in codon 338 (CTG ---> TTG) leading to a BglII restriction fragment length poly- morphism (RFLP). The other is a substitution in codon 367 (AAC ---> ACC) leading to the polymorphism N367T; this is not an RFLP and amplified segments of the two al- leles were distinguished by denaturing gradient gel elec- trophoresis. In the closely linked type II gene, a polymor- phic simple sequence microsatellite repeat is located in the third intron approximately 250 bp from the 5" end of exon 4 (Russell et al. 1994).

Figure 1 shows the pedigree of the original family in which 313-OH-AS-C27-SD deficiency was described in two cousins (IV-l, IV-8) as well as two sibs (IV-4,5) who died of neonatal cholestatic jaundice. The pedigree is consis- tent with autosomal recessive inheritance. When a rare autosomal recessive disease occurs in a consanguineous family, the same mutation is expected to be inherited from the kindred by each heterozygous parent and passed to

their homozygous affected offspring. However, in this pedigree, there was minimal sharing of haplotypes in par- ents III-1,2,3,6. Affected children IV-1 and IV-8 were both heterozygous for 3~-HSD type I and II haplotypes and had the same 3[3-HSD genotype as unaffected child IV-10. This is very strong evidence of a lack of genetic linkage between the gene for 3~-OH-AS-Czv-SD and the 3[3-HSD gene cluster on chromosome lp13.

This genetic evidence also argues against the type I and II enzymes having significant 313-OH-AS-C27-SD ac- tivity. It has also been previously shown that the rat he- patic AS-Cz7 and /~5-C~9/2 ~ enzymes differed in that only the latter activity can be inhibited by using inhibitory ana- logues of androstene-3-one, arguing that these C19/21 en- z y m e s are also non-identical to the Cz7 enzyme (Bj6rk- hem et al. 1972). Nevertheless, similarity of function ar- gues that 313-OH-AS-C27-SD is likely to be a member of the 3[3-HSD gene family. As further members of the hu- man 3~-HSD gene family are cloned and mapped (McBride et al. 1994) it may be possible to identify can- didate genes for 3[3-OH-AS-CzT-SD. This process would be facilitated if other cases of 313-OH-AS-C27-SD defi- ciency could be recruited to a mapping study.

Acknowledgements This work was supported by a grant of the Scottish Hospital Endowments Research Trust to R.G.S.

588

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