The study included 30 pregnant females each with single fetus. They were divided into two groups. The first group consisted of 10 pregnants with previous history or family history of one or more affected child with MPS VI seeking prenatal diagnosis. The second group consisted of 20 pregnants seeking prenatal diagnosis by amniocentesis for other indications.
The age of the MPS VI group (ranged from 20 to 42 years with a mean of 32.3 years) is comparable to that of the control group (ranged from 22 to 40 years with a mean of 31.6 years).
Seven patients in the MPS VI group have affected sibling/s, and three patients have an affected family member (one patient has an affected sister and two patients have an affected brother in law). Consanguinity was positive in 100% of MPS VI couples.
Amniocentesis was done in previous pregnancies in 3 out of the 10 MPS VI group by 2-DEP of the extracted GAGs.
Our prospective cohort case control study was performed in the Prenatal Diagnosis and Fetal Medicine Department and the Biochemical Genetics Department, NRC, National Research Centre.
History taking and prenatal counseling were done to each patient at her first visit. A detailed medical history, determination of gestational age and history of viral exposure/illness, bleeding, or use of medication during the pregnancy were obtained. Parental past medical history, consanguinity and 3-generation pedigree analysis were documented. All patients were medically free.
All patients were instructed about the research objectives and methodology. The steps of the work and the details of the procedures were explained to them as well. Then, an informed consent was obtained from each patient.
A detailed ultrasound scan was done to check the viability of the fetus, its development compared to the gestational age, any abnormal features, malformations or anomalies, the site of the placenta and the amount of the amniotic fluid and its index. The ultrasound scans were carried out using General Electric Voluson P8 real-time scan system with a capacity of simultaneous B-mode and M-mode scanning. The carrier frequency was obtained using a broad-band probe which covers a range of frequencies from 5 to 7.5 megahertz (MHz). The real-time obstetric ultrasound examination was performed to the pregnant ladies in the supine position.
Amniocentesis was done at 16 weeks of gestational age under ultrasonographic guidance to withdraw 10 cc of amniotic fluid (using a spinal needle size 22G) for biochemical diagnosis by both 2-DEP of extracted GAGs and mass spectrometry.
The amniotic fluid samples were centrifuged, and the supernatants were taken and used for the extraction of GAGs by formation of complexes with alcian blue 8GX. In this procedure, 3 ml of centrifuged amniotic fluid was mixed with 160 alcian blue reagent containing 50 mmol \({\mathrm{MgCl}}_{2}\). Blue complex was formed and after centrifugation it was separated and precipitated. The complex was dissociated by shaking with 4 mol/l NaCl and methanol. Free alcian blue was precipitated by the addition of 0.1 mol/l \({\mathrm{Na}}_{2}{\mathrm{Co}}_{3}\) and water. GAGs were precipitated from clear supernatant by the addition of ethanol, and the precipitate after centrifugation was taken up in 20 µl water [10].
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Qualitative analysis of glycosaminoglycans (GAGs) using two-dimensional electrophoresis (2-DEP) was done. 0.1–2 µl of the extracted GAGs were applied on cellulose acetate sheets. The separation proceeded into two phases. The first phase (pyridine, glacial acetic acid, water) for 1 h. The second phase (barium acetate buffer) for 3 h. Staining for 1 h with 0.05% alcian blue solution containing 50 mmol/l \({\mathrm{MgCl}}_{2}\) in 50 mmol/l sodium acetate buffer (pH 5.8) and washed with 5% acetic acid solution. Clear blue spots on a white background were obtained [10, 11].
According to the pattern of the detected spots, the diagnosis was made. MPS VI was diagnosed by a big dermatan sulfate spot.
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Quantitative analysis of the GAGs by using Mass spectrometer, (XEVO-TQD, waters, USA).
Dermatan sulfate (DS) stock solution, containing 3 mg/ml, was separately prepared in water and stored at − 20 °C until analysis. Calibration curve was prepared, covering a concentration range from 1.56 to 100 µg/ml for DS using seven different levels.
The Ion spray voltage was set to 5500 V, and the temperature was 300 °C.
The parent ion of DS is 510.4. The daughter ion of DS is 278.2. The collision energy for DS is 13 V in positive mode.
Chromatographic separation was carried out using a Kinetex Biphenyl column 2.6 µm 100 × 2.1 mm, (Phenomenex, Torrance, CA) maintained at 40 °C with a flow rate of 0.2 ml/min. Acquisition time was 11 min; the elute was introduced into the ESI interface without splitting. The mobile phase A consisted of 0.1% formic acid in water, while solvent B was 0.1% formic acid in acetonitrile. Total run time was 21 min with gradient elution: 0.0–2.0 min (15% B); 2.1–14.1 min (35% B); 14.2–17.1 min (100%); 17.2–21.0 min (15% B). The injection volume was 1 µl [12].
In MPS VI, affected fetuses dermatan sulfate is elevated.
Pregnants with nonaffected fetuses results in the MPS VI group were seen 1 month after delivery, and the newborns were tested and proved to be nonaffected with MPS VI.
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) Statistical Software. Qualitative data were presented as frequencies and percentages. The Chi-square was used to determine the presence of significant differences between studied groups. P values less than 0.05 will be considered as statistically significant. Quantitative data were presented as mean ± standard deviation (SD), and student t-test was used for statistical analysis.
