Prenatal diagnosis of X-linked ichthyosis

Clinica Chimica Acta, 120 (1982) 143-152 Elsevier Biomedical Press

143

CCA 2066

Prenatal diagnosis .of X-linked ichthyosis R. HSihnel*, Department

E. Htinel,

S.J. Wysocki,

S.P. Wilkinson

and A. Hockey

of Obstetrics and Gynaecologv, University of Western Austrcrlia, und King Edward Memorial Hospitalfor Women, Subiaco, 6008 Western Austruliu (Australia) (Received

July 20th; revision November

27th, 1981)

Summary Prenatal diagnosis of X-linked ichthyosis in a case of steroid sulfatase deficiency was made at 16 weeks by the demonstration of (1) high levels of dehydroepiandrosterone sulfate in amniotic fluid; (2) gross deficiency of steroid sulfatase activity in cultured amniotic fluid cells; (3) very low estriol concentrations in maternal blood and urine; (4) increased maternal plasma dehydroepiandrosterone sulfate; and (5) a characteristic maternal urinary steroid profile with greatly increased levels of 16a-hydroxydehydroepiandrosterone. The latter method is particularly useful since it requires no invasive procedures for the patient and is very specific.

Introduction Evidence has been presented that X-linked ichthyosis is due to steroid sulfatase deficiency [ 1,2]. Cultured fibroblasts of individuals with X-linked ichthyosis had no sulfatase activity; fibroblasts from an obligate heterozygote had sulfatase activity intermediate between that for normal controls and for affected individuals. By contrast, a patient with ichthyosis vulgaris had high levels of sulfatase activity indistinguishable from normal controls. This made the prenatal diagnosis of X-linked ichthyosis potentially possible. In this report we wish to present studies on a patient with a family history of X-linked ichthyosis in whom the diagnosis of steroid sulfatase deficiency was made at 16 weeks gestation by the quantitation of dehydroepiandrosterone sulfate (DHEAS) in amniotic fluid, assays of DHEAS-sulfatase activity in cultured amniotic fluid cells, maternal plasma estriol assays and maternal urinary steroid profiles by gas chromatography-mass spectrometry.

Case history The patient

S.C. is a 22-year-old primigravida. Her father and one of his brothers, as well as three males on the father’s side in the previous generation were all * To whom correspondence

0009-8981/82/0000-0000/$02.75

should be addressed.

0 1982 Elsevier Biomedical

Press

144

PEDIGREE

@ &

q

OBLIGATE X - LINKED

0 8

Fig.

S. C.

CARRlEA

OF X-

LlNKED

ICHTHYOSIS.

MOTHER

OF AFFECTEO

NEWBORN

ICHTHYOSIS.

DOWN’S SYNDROME ADULT

ONSET DIABETES.

I Pedigree of patient SC.

affected, see pedigree (Fig. 1). In addition, the patient has two first paternal cousins with Down’s syndrome, who were both born when the mother was in her twenties. Amniocentesis was performed on SC. at 16 weeks gestation. Analysis of cells grown from this sample showed the fetus was a male with normal karotype. The baby was delivered at term. Labour was augmented with oxytocics and delivery was by forceps. The newborn weighed 2660 g and had an apgar of 9; he had dry defoliating skin especially on the trunk. Congenital ichthyosis was diagnosed in consultation with a dermatologist. Materials 7-3H(N)-dehydroepiandrosterone sulfate, ammonium salt (New England Nuclear, Boston, MA, USA); 2,4-3H(N)-estriol (Radiochemical Centre, Amersham, UK). Dehydroepiandrosterone sulfate sodium salt (Ikapharm, Ramat-Gan, Israel). Antiserum to estriol-C6-conjugate (Prof. E. Kuss, Munich, FRG). Antiserum to dehydroepiandrosterone sulfate (Endocrine Sciences, Tarzana, CA, USA). Methoxylamine hydrochloride, pyridine (Pierce, Rockford, IL, USA); Sylon BTZ, a 3 : 2 : 3 mixture of bis- trimethylsilylacetamide, trimethylchlorosilane, trimethylsilylimidazole (Supelco, Bellefonte, PA, USA); hexamethyldisilazane, n-C,,, n-C,, and n-C3z alkanes, Chromosorb W-HP (Applied Science Laboratories, Waltham, MA, USA); Lipidex-5000, Instagel, Permablend (Packard, Downers Grove, IL, USA); Amberlite XAD-2 (Rohm and Haas, Philadelphia, PA, USA); Sue d’Helix pomatia, an enzyme preparation containing P-glucuronidase and sulfatase activity (Industrie Biologique Francaise, Clichy, France); SE-30 (Alltech Associates, Deerfield, IL, USA);, dextran T70 (Pharmacia, North Ryde, N.S.W. Australia); NoriteA charcoal (Pfanstiehl Laboratories, Waukegan, IL, USA); bovine serum albumin (Commonwealth Serum Laboratories, Perth, Australia). All other chemicals used were of A.R. quality.

145

Methods Dehydroepiandrosterone sulfate (DHEAS) in plasma and amniotic fluid DHEAS was quantitated by radioimmunoassay similar to Furayama Samples were assayed in quadruplicate.

et al [3].

DHEAS-suljatase in cultured amniotic jluid cells Amniotic fluid cells from the pregnancy with steroid sulfatase deficiency were very slow to grow in culture and unusual in appearance. The cells were large and their cytoplasm appeared to be filled with inclusions. The cells from two monolayered Falcon flasks were frozen, thawed and homogenised in 0.3 ml of 0.1 mol/l Tris-HCl buffer, pH 7.2. Duplicate 100~~1 aliquots were incubated with radioactive substrate as described [2] but all of the reaction mixture (0.5 ml) was extracted with benzene (1.0 ml) by gently rocking to minimise emulsion formation. 0.5 ml of the benzene layer was added to 6 ml of Instagel and counted by scintillation spectrophotometry. The limit of sensitivity of DHEAS-sulfatase in cultured amniotic fluid cells was about 20 pmol . hh’ . mg - ’ protein. Estriol in plasma and urine Estriol was measured by radioimmunoassay. For the plasma assays the method of Goebel and Kuss [4] was used. This method has also been adopted for urine [5]. Protein Protein

was estimated

by the method

of Lowry et al. [6].

Extraction of steroids from urine Steroids were isolated from urine by adsorption to Amberlite XAD-2 resin [7], eluted and hydrolyzed with Sue d’Helix pomatia at 50°C [8]. After cooling, sodium chloride was added to the hydrolysate and the steroids were extracted with diethyl ether (3 X equal volume). Estrogens were separated from the other steroids by partitioning between aqueous sodium hydroxide and ether [9]. Derivatisation and gas chromatography of steroids Methoxime-trimethylsilyl (MO-TMS) derivatives of the steroids including estrogens were prepared as described by Thenot [lo]. Before gas chromatography the derivatives were purified by Lipidex [ 111. The MO-TMS derivatives were finally dissolved in 50 ~1 of cyclohexane/pyridine/hexamethyldisilazane (98 : 1: 1, v/v/v) containing 2 mg of n-C,, and 8 mg of n-C,, alkane in 10 ml. Gas chromatography was carried out on fused silica SCOT capillaries (50 m long, 0.2 mm i.d.) coated with methyl silicone fluid (Hewlett Packard No. 19091-60050) using a Hewlett-Packard 5840 instrument. The column oven was programmed from 150-250°C at 30”C/min and maintained at 250°C for 120 min. Gas chromatography-mass spectrometry was carried out using a Varian 2740 chromatograph coupled to a Varian MAT 311 mass spectrometer via a WatsonBiemann separator. Spectra were acquired repetitively with a Varian spectrosystem 100 MS computer.

146

Results DHEAS in amniotic fluid The concentration of DHEAS in amniotic fluid collected from the pregnancy where the fetus was at risk for X-linked ichthyosis is shown in Table I. There was an elevation of some 4 to 5 times in DHEAS concentration when compared to the mean for liquor specimens collected from normal pregnancies at 16 weeks gestation. The value for the pregnancy at risk was clearly outside the normal range.

DHEAS in maternal plasma Persistently high values were found for DHEAS in maternal plasma sampled at 34-37 weeks gestation from the pregnancy at risk (Table II). The plasma DHEAS was nearly twice as high as the top of the normal range for pregnancies of similar duration. DHEAS-sulfatase in cultured amniotic fluid cells DHEAS-sulfatase was barely detectable in cultured amniotic fluid cells from the pregnancy at risk for X-linked ichthyosis (Table III). The level of the enzyme was at the limit of sensitivity of the assay and was less than 5% of the mean activity of normal cells grown from liquors collected at 16 weeks gestation. Unfortunately, the placenta of the patient was not available for steroid sulfatase determination.

Urinary steroid profiles

Metabolic profiles of the urinary steroids in normal pregnancies were compared with those of the patient with a fetus at risk for X-linked ichthyosis (Fig. 2). The urinary steroid profiles of the patient contained two large peaks (18,19) that were barely visible in the normal profile. These two peaks were identified as stereoisomers of 16a-hydroxy-dehydroepiandrosterone by their position on the chromatogram and by their mass spectra. In addition, excretion of dehydroepiandrosterone (peak 6), 5-androstene-3/3,16cu,17Ptrio1 (peak 27), etiocholanolone (peak 2) and 16ahydroxyetiocholanolone (peak 5) were markedly increased in the patient. In contrast, the following steroids were ,found in decreased amounts in the patient compared with normal controls: pregnanolone (peak 17), pregnanediol (peak 22) 5fl-pregnane-

TABLE

I

DEHYDROEPIANDROSTERONE I6 weeks gestation

DHEAS

Patient Normal

SULFATE

IN AMNIOTIC

@g/l)

593 controls

(n = 10)

145232

(SD), range 80-185

FLUID

147 TABLE

II

DEHYDROEPIANDROSTERONE

SULFATE

IN THIRD

DHEAS Patient Patient Patient

at 34 weeks gestation at 36 weeks gestation at 37 weeks gestation

Normal

controls

TABLE

III

MATERNAL

PLASMA

(1.18/l)

3 830 4060 4050 I68Ok620

(n = 7)

DEHYDROEPIANDROSTERONE-SULFATE CELLS

(SD), range 680-2360

SULFATASE

DHEAS Patient

TRIMESTER

sulfatase

(pmol h-

IN CULTURED

‘. rnk

AMNIOTIC

FLUID

’ protein)

(20

Normal

controls

TABLE

IV

(n = 5)

455 i- 399 (SD), range

164-l 008

QUANTITATIVE COMPARISON OF URINARY STEROID PROFILES OF PATIENT WITH FETUS AT RISK FOR X-LINKED ICHTHYOSIS AND OF 19 NORMAL CONTROL PREGNANCIES The data shown are peak areas in relation Peak No.

1

Patient

to the internal

standard

Normal controls mean * standard

deviation

2 3 5 6

2.65 4.36 1.14 0.94 3.29

2.18k2.05 1.46* 1.73 0.69 *0.60 0.26t0.24 0.83 k-o.75

7 9 II 12 15 16 17 18 19 21 22

0.69 0.96 0.42 0.29 1.31 0.38 1.71 4.64 3.80 0.86 1.38

0.54f0.53 1.29”0.97 0.86’1.81 0.66 -to.67 2.28% 1.84 1.31*1.29 4.30-t 2.26 0.59kO.30 0.62*0.44 1.45* 0.87 9.03*5.18

24 25 27 28

0.65 1.17 2.77 1.87

1.43-tl.10 0.76kO.78 1.15*0.44 4.46 f 2.43

29 31 35 37

3.15 1.71 0.50 0.40

3.36) 1.46 5.35kO.84 0.99*0.50 0.54io.30

(peak 26) Comparison of patient with controls

increased increased increased

decreased increased increased decreased

increased decreased decreased

(a)

(b)

Fig. 2. Urinary steroid profiles of the patient with a fetus at risk for X-linked normal pregnant patient (b). Gestation was 36 weeks in both cases. Conditions:

ichthyosis capillary

(a) and of a column 50 m

149 TABLE V ESTRIOL

LEVELS

IN PLASMA

Gestation

Normal

Plasma 18 weeks 31 weeks 33 weeks 36 weeks

6 26 31 40

Urine RIA

GC

18 32 36 18 32

weeks weeks weeks weeks weeks

36 weeks RIA, GC, l

AND

URINE

controls

nmol/l nmol/l nmol/l nmol/l

radioimmunoassay. gas chromatography. peak area relative to 0.4 ug of added

area

a-C,,

% of normal

I .7 nmol/l 7.0 nmol/l

28% 28%

3.6 nmol/l

9%

0.49 3.00 4.95 0.25 0.13

32.9 nmo1/24 h 43.5 nmo1/24 h 0.88 * relative area _ 3.43 relative

Patients

nmol/24 h nmo1/24 h nmo1/24 h relative area relative area

0.07 relative

area

9% 11% 28% _ 2%

alkane.

3/3,21-diol-20-one (peak 28) and 5&pregnane-3/3,16cY,20/34riol (peak 31) *. These differences were observed at 18, 32 and 36 weeks gestation. The results are summarised on Table IV. In normal pregnancy urines peaks 18/19 (16~~ hydroxydehydroepiandrosterone) and 27 (5androstene-3/3,16a,17&triol) were measurable in only about half the cases. The profile of urinary estrogens of the patient showed a greatly diminished estriol peak in comparison with a normal control of the same gestation (Fig. 3). This deficiency was confirmed by radioimmunoassays of estriol in plasma and urine (Table V). * Detailed evidence for the identity of the steroids in urinary steroid profiles and their quantitative estimation is given in a separate paper (E. H&hnel, S.P. Wilkinson, R. H&hnel, Urinary steroid profiles in pregnancy by gas chromatography, in preparation). long, 0.2 mm inner diameter, coated with silicone fluid; temperature 150 to 250°C at 30’C/min, then 25O’C for 120 min; carrier gas nitrogen, linear flow velocity I7 cm/s. Identification of major peaks: (1) androsterone, (2) etiocholanolone, (3) 16a-hydroxyandrosterone, (5) 16a-hydroxyetiocholanolone, (6) dehydroepikndrosterane, (7) epiandrosterone, (8) androstenediol, (9) 1 1-oxoandrosterone and 1 Ioxoetiocholanolone, (IO), (1 I), (12), (13), (14) unknown, (15) pregnanedidlone and I l/3hydroxyandrosterone, ( 16) 3/I-pregnanolone, ( 17) pregnanolone, ( 18), ( 19) 16a-hydroxydehydroepiandrosterone, (21) allopregnanediol, (22) pregnanediol, (24) pregnanetriol, (25) 5-androstene-3/3,17@-diol-l6-one. (26) internal standard (7-metbylandrosta-4,6-dione-lJ?-ol-3-one), (27) 5-androstenetriol-3/3,16a,l7& (28) pregnanediolone, (29) 5/3-pregnanetriol-3~,16a,20a, (30) 5-pregnene-3&16a,2Oatriol, (31) S/3pregnanetriol-3/3,16a,208, (35) 16a-hydroxypregnenolone, (36) 5a-pregnane-3/3,16a-dial-20-one, (37) 5pregnene-3/J,17a,20ar-trio1 and THE (tetrahydrocortisone), (39) THA, (40) THB (tetrahydrocorticosterone), (41) Sa-pregnane-3B,16a,20a-triol, (42) THF (tetrahydrocortisol).

150 (a)

(b)

VI _

I !+

0

*F w

Fig. 3. Urinary estrogen profiles of the patient with a fetus at risk for X-linked ichthyosis (a) and of a normal pregnant patient(b). Gestation was 36 weeks in both cases. Conditions as given in Fig. I. Note the massive peak of estriol in the normal urine and the insignificant quantity of estriol in the patient.

Discussion The data in this communication X-linked ichthyosis at an early

confirm the potential for prenatal stage of pregnancy. The increase

diagnosis of in DHEAS

151

concentration reported here (4 to 5 times) is not as striking as that reported by braunstein et al ([ 121 7 to 18 times) and Osathanondh et al ([ 131 22 times) but these authors were examining specimens collected at term. Shapiro et al [15] have demonstrated a deficiency in steroid sulfatase activity in amniotic fluid cells at term from a pregnancy in which the fetus had X-linked ichthyosis. We found that the steroid sulfatase deficiency is manifest already at 16 weeks gestation in amniotic fluid cells from an affected pregnancy. Our observation of an elevated level of DHEAS in maternal plasma in the last few weeks of an affected pregnancy is in accord with the data of Osathanondh et al [ 131 who reported a plasma DHEAS of 1300 pg/l (normal range 240-l 100 pg/l) in an affected pregnancy of 41 weeks duration. Very low values for urinary estriol excretion in a case of placental sulfatase deficiency were first described by France and Liggins [16] and have since been confirmed by other authors. More recently, urinary steroid profiles for patients with placental sulfatase deficiency have been described [ 17,181. Both groups found raised levels of 5-androstene-3/3,16a,l7fl-triol, 3/i,17P-dihydroxy-5-androsten-16-one and of 16a-hydroxydehydroepiandrosterone. In addition, an increase in the urinary concentration of 16a-hydroxypregnenolone and dehydroepiandrosterone was observed [ 181. Significant amounts of 5-pregnen-3P,17a,20a-triol, 5-pregnene-3fi,16a,20a-triol, 5/3-pregnane-3a,16a,20a-triol and 5-androstene-3/3,15a,16a,17/3-tetrol were also found in the nine patients, but these steroids were below the level of detection in normal pregnancy urine [ 181. In our patient the most outstanding feature of the steroid profile were the two peaks of 16a-hydroxydehydroepiandrosterone, which were already very marked at 18 weeks gestation. In agreement with the two published papers [ 17,181 androstenetriol, dehydroepiandrosterone and androstenediolone were also raised in our case. In addition, we found significantly increased levels of etiocholanolone and 16a-hydroxyetiocholanolone. Furthermore, a few urinary steroids in the patient were consistently lower than in normal controls. These included progesterone metabolites like pregnanediol, allopregnanediol and pregnanolone, in contrast to published papers (e.g. [16]) which report normal pregnanediol excretion in cases of placental sulfatase deficiency. 16a-Hydroxypregnenolone (peak 35), which was reported to be increased 33 times in patients with placental sulfatase deficiency [ 181, was present in the urinary steroid profile of our patient but its concentration was similar to that found in normal pregnancy. We did not detect 5-androstene_3/3,15a, 16a,l7&tetrol in either the urines of the patient or of normal controls. Taylor and Shackleton [18] measured some other steroids in placental sulfatase deficiency, which were below the detection limit in normal controls. In contrast, we have been able to identify these steroids i.e. 5-pregnene-3b, 16a,20a-trio1 (peak 30), 5-pregnene-3/3,17a,20a-trio1 (peak 37) and 5~-pregnane-3~,16a,2Oa (peak 29) in both normal urines and that of the patient with X-linked ichthyosis. It is not known whether the observed differences in the urinary steroid profile between our patient and the other patients reported [18] are due to inherent differences between X-linked ichthyosis-placental sulfatase deficiency and other forms of placental sulfatase deficiency. Taylor and Shackleton [ 181 did not observe ichthyosis in any of the nine infants whose mothers were found

152

to have placental sulfatase deficiency. Likewise the infants of other cases were also not reported to have ichthyosis [ 12,13,15,17.19,20]. But it has been observed [l] that in some individuals in whom ichthyosis developed later, the skin had appeared normal at birth and first appeared abnormal at about 3 months of age. On the other hand, Kubilus et al [2] who selected nine patients representing eight families on the basis of having the skin disease rather than as a member of a family with placental steroid sulfatase deficiency, found the enzyme deficiency in cultured fibroblasts of all patients. References I Shapiro LJ, Weiss R. X-linked ichthyosis due to steroid-sulphatase deficiency. Lancet 197X: i: 70-72. 2 Kubilus J, Tarascio AJ, Baden HP. Steroid sulfatase deficiency in sex-linked ichthyosis. Am J Hum Genet 1979; 31: 50-53. 3 Furayama S, Mayes D. Nugent CA. A radioimmunoassay for plasma testosterone. Steroids IY70: 16: 415-428. 4 Goebel R. Kuss E. Oestrogene in der Schwangerschaft. I. Eine einfache und zuverlksige radioimmunologische Methode zur Bestimmung des estriols im Schwangeren-Serum. Geburtsh Fraucnheilk 1974; 34: 329-338. 5 Masters AM, Giles PFH, H%hnel R. Assay of urinary oestriol in infertility patients by radioimmunoassay. Austr NZ J Obstet Gynaecol 198 I ; 2 I : I8 I - 183. 6 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 7 Bradlow HL. Extraction of steroid comugates with a neutral resin, Steroids 1968; I I : 265-272. 8 Scholler R, Metay S, Herbin S, Jayle MF. Hydrolyse enzymatique rapide des oestrogenes conjuguea urinaires. I. Oestrogenes totaux. Eur J Steroids 1966; I : 373-388. 9 Brown JB. A chemical method for the determination of oestriol, oestrone and oestradiol in human urine. Biochem J 1955; 60: 185-193. IO Thenot JP, Homing EC. MO-TMS derivatives of human urinary steroids for GC and GC-MS studies. Anal Lett 1972; 5: 21-33. I I Axelson M, Sjiivall J. Separation and computerized gas chromatography-mass spectrometry of unconjugated neutral steroids in plasma. J Steroid Biochem 1974; 5: 733-738. 12 Braunstein GD, Ziel FH, Allen A, Van de Velde R. Wade ME. Prenatal diagnosis of placental steroid sulfatase deficiency. Am J Obstet Gynecol 1976: 126: 716-719. 13 Osathanondh R, Canick J. Ryan KJ, Tulchinsky D. Placental sulfatase deficiency: a case study. J Clin Endocrinol Metab 1976; 43: 208-214. 14 Matalon R, Dorfman A. The antenatal diagnosis of mucopolysaccharidoses. In: Dorfman A, ed. Antenatal diagnosis. Chicago: University of Chicago Press, 1972: 213. I5 Shapiro LJ, Cousins L, Fluharty AL, Stevens RL, Kihara H. Steroid sulfatase deficiency. Pediat Res 1977; I I : 894-897. I6 France JT, Liggins GC. Placental sulfatase deficiency. J Clin Endocrinol Metab 1969; 29: l38- 141. 17 Phillipou G, Seamark RF, Sweet R. Facile prenatal diagnosis of placental sulphatase deficiency. Aust NZ J Obstet Gynaecol 1979; 19: 233-236. 18 Taylor NF, Shackleton CHL. Gas chromatographic steroid analysis for diagnosis of placental sulfatase deficiency: a study of nine patients. J Clin Endocrinol Metab 1980: 49: 78-86. I9 Fliegner JRH, Schindler I, Brown JB. Low urinary oestriol excretion during pregnancy associated with placental sulphatase deficiency or congenital adrenal hypoplasia. J Obstet Gynaecol Br Cmw 1972; 79: 810-815. 20 Oakey RE, Cawood ML, MacDonald RR. Biochemical and clinical observations in a pregnancy with placental sulphatase and other enzyme deficiencies, Clin Endocrinol 1974: 3: I3 I - 148.