Diagnosis of a deletion of steroid sulfatase by polymerase chain reaction and high-performance liquid chromatography

Clinica Chimica Acta 263 (1997) 25–32

Diagnosis of a deletion of steroid sulfatase by polymerase chain reaction and high-performance liquid chromatography Teruo Sugawara*, Masanori Iwaki, Seiichiro Fujimoto Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Kita-Ku Kita 15 Nishi 7, Sapporo 060, Japan Received 22 November 1996; revised 25 January 1997; accepted 8 February 1997

Abstract X-linked ichthyosis is an inherited skin disorder caused by deficiency of steroid sulfatase activity. We studied the possibility of diagnosing the defect in patients and carriers by using polymerase chain reaction (PCR) and high-performance liquid chromatography (HPLC). We chose the usual PCR procedure of 25 temperature cycles. PCR products were resolved by HPLC and quantified by measurement of absorbance at 260 nm. The optimal amount of DNA template was 50 ng using either steroid sulfatase (STS) or b -globin (internal control) primer. The results show that the amount of STS in ichthyosis patients was null. The amount of STS DNA in mothers of patients was half of that in normal females. By this HPLC-PCR method we will be able to diagnose not only ichthyosis patients but also carriers before birth.  1997 Elsevier Science B.V. Keywords: X-linked ichthyosis; Steroid sulfatase; PCR-HPLC

1. Introduction X-linked ichthyosis (XLI) is an inherited skin disorder due to deficiency of steroid sulfatase (STS) activity. A deficiency of placental STS results in low estriol level in the urine and plasma of mothers of affected fetuses. Most XLI *Corresponding author. Tel.: 1 81 11 7161161, ext. 5941; fax: 81 11 7276006. 0009-8981 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0009-8981( 97 )06552-2

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fetuses are delivered by cesarean section because of pseudo-placental dysfunction. XLI has been diagnosed by assaying STS activity in placenta or lymphocytes of the patient after birth [1]. The STS gene has been cloned [2] and found to contain ten exons [3]. The functional STS gene was mapped to Xp22.3-Xpter and there was a pseudogene on Yq [3]. It was reported that most patients have a large deletion of the STS gene [2], probably generated by inaccurate recombination at the STS locus [4]. We reported that XLI in most Japanese patients was also caused by an extensive deletion, as demonstrated by polymerase chain reaction (PCR) [5]. The detection of the carriers of Duchenne muscular dystrophy by combined PCR and high-performance liquid chromatography (PCR-HPLC) has been reported [6]. In the present study, we demonstrate the feasibility of detecting XLI patients and carriers using PCR and HPLC. The use of this procedure in cases of apparent placental deficiency will identify XLI carriers, and thereby prevent unnecessary cesarean section in these individuals.

2. Method

2.1. Patients Six Japanese patients, already diagnosed as suffering from XLI by Southern blotting [5], took part in this study. Four mothers of these patients were also examined.

2.2. Isolation of DNA DNA was isolated from human leukocytes according to an established method minimizing the content of RNA [7]. The concentration of DNA was estimated by absorbance at 260 nm.

2.3. PCR Primers synthesized by a DNA synthesizer (ABI, type 380B-02) were: D5 (59-GGGAATTCTGGTGAGCTTTGCCCACAT-39, sense) and D6 (59-ACATCCATCTGGAAGGCCTT-39, antisense) for the amplification of exon 10 of STS; KM29 (59-GGTTGGCCAATCTACTCCCAGG-39, sense) and PC04 (59-CAACTTCATCCACGTTCACC-39, antisense) for the b -globin (BGB). DNA was dissolved in 10 mmol / l Tris–HCl (pH 7.4), 1 mmol / l EDTA buffer. Coamplified PCR was performed in triplicate. The final reaction volume was 100 m l and it contained 10 mmol / l Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mmol / l MgCl 2 , 0.001% gelatin, 160 m mol / l dNTPs, 0.4 m mol / l each primer and 2 units

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of Ampli Taq DNA polymerase (Takara, Tokyo, Japan). The sizes of the target gene sequences were 414 bp for the STS primers and 205 bp for the b -globin primers. PCR was performed, as previously described by Asakawa et al. [5] with a slight modification. The first cycle of the PCR reaction was denaturing at 948C for 550 s, followed by annealing at 568C for 900 s, and then extension at 728C for 60 s. Subsequent cycles were at 948C for 85 s, 568C for 150 s and 728C for 45 s.

2.4. HPLC The HPLC system consisted of a Shimadzu LC-9A pump and an SPD-6A UV detector. Elution was monitored by UV absorbance at 260 nm, and peak areas of PCR products were determined with a Chromate Pack CR-4A integrator. Column dimensions were 35 3 4.6 mm I.D. (Toso, Japan). The column packing consisted of non-porous spherical hydrophilic resin particles (TSK Gel DEAENPR) of 2.5 m m diameter. Analyses were performed as previously described by Asakawa et al. [6]. Briefly, 20 m l of PCR reaction mixture was injected into the column. Products were eluted with a liner salt gradient. The buffer was NaCl from 0.2 to 1.0 mol / l in a 20-mmol / l Tris–HCl buffer (pH 9.0) at a flow rate of 1.0 ml / min. All elution buffers were filtered through a 0.22-m m (Millipore) membrane filter. Peak area indications of the quantities of specific products are expressed in units of microvolt-seconds ( mV? s). Each sample was amplified and analyzed in triplicate. To examine the reproducibility of HPLC we analyzed 20 m l of a standard sample ten times. The mean (6S.D.) of the peak was 17 3726485 mV? s (n 5 10); the co-efficiency of variation (C.V.) was 2.8%. The mean (6S.D.) of the retention time was 6.6760.013 (n 5 10); the C.V. was 0.2%. Thus, the reproducibility of PCR-HPLC was excellent.

3. Result

3.1. Optimal conditions for PCR with STS and BGB 3.1.1. Correlation of PCR product quantity with template DNA quantity We examined the correlation between PCR product quantity and template quantity using STS primers, with b -globin amplification as an internal control. PCR was performed for 26 cycles. HPLC provided good separation of PCR products from both normal female and normal male subjects (Fig. 1). The PCR product ratio of female vs. male was 1:1 for BGB (an autosomal gene), but 2:1 for STS (an X-linked gene). The correlation of the amount of template DNA was good with 25 and 50 ng of template, but not so good for STS with 100 ng of template DNA. It was better to amplify using 50 ng or less of template.

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Fig. 1. Separation of PCR products in a normal female and a normal male. The left chromatography is that of a normal female. The right is the chromatography of normal male. Separation was carried out with a linear salt gradient. The buffer was NaCl from 0.2 to 1.0 mol / l in a 20-mmol / l Tris–HCl buffer (pH 9.0) at a flow rate 1.0 ml / min. BGB, b -globin; STS, steroid sulfatase.

3.1.2. Correlation with the number of PCR cycles We studied the correlation of PCR product quantity with the number of PCR cycles. We used 50 ng of DNA template, and obtained a good correlation from 25 to 26 cycles. The STS ratio of female vs. males was 2:1 (Table 1). The correlation was not as good using fewer or more cycles. Thus, optimal conditions for PCR were 25 temperature cycles and 50 ng of DNA template.

3.2. Ichthyosis patients and carriers We examined the quantities of STS in ichthyosis patients and in their mothers, who are obligate carriers. The quantity of template DNA was 50 ng and the number of PCR cycles was 25. At the same time we amplified BGB for internal control. The separation of PCR products in ichthyosis patients and ichthyosis carriers was good (Fig. 2). There was no STS in ichthyosis patients. The amounts of BGB were the same in patients and controls. In XLI carriers, the amounts of DNA were half that in normals (Table 2).

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Table 1 Relationship between the PCR products and the number of PCR cycles DNA

Female

Male

No. of cycles

24 25 26 27 24 25 26 27

Quantities of the products ( mV? s, mean6S.D.) BGB (%)

STS (%)

348.5615.8 (52) 668.3624.6 (100) 1372.2663.5 (205) 3236.36138.8 (484) 280.062.7 (41) 692.8629.7 (103) 1384.8637.0 (207) 2760.56257.4 (413)

719.0651.8 (71) 1001.9623.0 (100) 2036.26148.0 (203) 5703.3686.3 (569) 221.366.3 (22) 531.1623.1 (53) 1185.56113.9 (118) 2223.06716.5 (222)

We amplified 50 ng of DNA template, using STS and BGB primers. The results are presented as mean6S.D. Values in parentheses are percentages of the PCR products relative to normal female BGB products and normal female STS.

Fig. 2. Separation of PCR products in a ichthyosis patient and a ichthyosis carrier. The left chromatography is that of patient. The right is the chromatography of carrier. Separation of the buffer mixture was carried by the linear gradient elution. The buffer was NaCl from 0.2 to 1.0 mol / l in a 20-mmol / l Tris–HCl buffer (pH 9.0) at a flow rate 1.0 ml / min. BGB, b -globin; STS, steroid sulfatase.

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Table 2 The results of ichthyosis patients and carriers Quantities of the products ( mV? s)

Case

Ichthyosis

Normal female Carrier

1 2 3 4 5 6 1 2 3 4

BGB

STS

588 1067 430 747 752 967 744.6612.3 624 663 700 792

0 0 0 0 0 0 764.5614.4 288 458 344 361

STS / BGB

0 0 0 0 0 0 1.0260.02 0.46 0.69 0.49 0.45

We amplified the STS exon 10 for 25 cycles, using 50 ng DNA. The results are represented means. The results are represented of normal female are presented as mean6S.D. (n 5 3). STS / BGB are presented as the ratio of STS to b -globin.

4. Discussion Many inherited diseases can be diagnosed by Southern blotting. Recently, diagnosis using PCR has become popular [8,9]. These PCR methods rely on amplification of gene sequences that are deleted in the case of inherited disease. Southern blotting and PCR are useful to diagnose patients, but there are some problems diagnosing heterozygous carriers of inherited disease. With Southern blotting, heterozygous carriers display the same band as normals. The density of the band is half that in a normal sample. The PCR method also results in the same band as in normals. Since these techniques are qualitative or only semi-quantitative, it is difficult to discriminate heterozygous disease carriers from normals [10]. Generally, using PCR alone it is difficult to measure the amount of product [11]. When PCR and HPLC are combined, it becomes possible to obtain precise quantification of PCR product rapidly [12–14]. The degree of DNA amplification by PCR was approximately 100 million times. To be able to compensate for small variations in the amount of starting DNA template, we co-amplified with b -globin as an internal control gene. In PCR with two primers, the correlation of product formation with the amount of template DNA was not good. The reason for this was a ‘plateau’ phenomenon caused by factors that included substrate saturation of the enzyme, product strand re-annealing, and incomplete product strand separation. It is important to optimize the cycle number and the amount of template DNA to ensure that the amplifications are within the exponential range. In the present study, for

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diagnosis using this PCR-HPLC method, the optimal conditions were determined to be 50 ng of template DNA and 25 PCR cycles. Our previous study shows that PCR is an effective method to diagnosis X-linked ichthyosis [5]. The HPLC-PCR method presented here can diagnose not only ichthyosis patients but also carriers. We often encounter low estriol levels in the urine and plasma of pregnant women and their fetuses. With the PCR-HPLC method, we will be able to discriminate ichthyosis from other diseases (placental dysfunction, adrenal agenesis and congenital lipoid adrenal hyperplasia) rapidly and accurately before birth. The PCR-HPLC approach does not require radioisotopes, making it useful in clinical laboratories. It should be possible to detect other sex-linked and autosomal-linked deletions using similar methodology.

Acknowledgments We thank Dr. William Johnson for his critical review of the manuscript.

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