Genotyping of intron 22-related rearrangements of F8 by inverse-shifting PCR in Egyptian hemophilia A patients.

Hemophilia A (HA) is the most common severe bleeding disorder in humans, affecting one in 5,000 male births. In severe HA, intron 22 inversion of F8 is the most prevalent mutation, accounting for 40-50% of all mutations; however, little is known about the disease-causing mutations among Egyptian hemophiliacs. We aimed at genotyping all possible known DNA rearrangements of intron 22 of F8 in Egyptian HA patients. Peripheral blood samples were collected from 30 Egyptian HA patients (13 severe, ten moderate, and seven mild cases). Genotyping of F8 intron 22 rearrangements was performed by inverse-shifting PCR (IS-PCR). Our study revealed that seven patients (23.3%) had inversion 22, three patients (10%) had deletion 22, and 20 patients (66.7%) carried the wild-type allele. No intron 22 duplication was detected. The relative proportion of inversion 22-type 1 to inversion 22-type 2 was 85.7% and 14.3%, respectively, whereas the relative proportion of deletion 22-type 1 to deletion 22-type 2 was 33.3% and 66.7%, respectively. A statistically highly significant relation was found between disease severity and F8 intron 22 rearrangements (p = 0.008). Among severe cases, 46.1% had inversion 22, 23.1% had deletion 22, and 30.8% carried the wild-type allele. We conclude that F8 intron 22 inversion/deletion is responsible for about one third of disease-causing mutations among Egyptian hemophiliacs and for nearly 70% in severe cases. In addition, F8 intron 22 inversion/deletion by IS-PCR has proven to be a rapid and robust technique and might be the recommended tool for genetic analysis of HA patients specially with severe cases in developing countries.

Comparative study of three amniotic fluid markers in premature rupture of membranes: prolactin, beta subunit of human chorionic gonadotropin, and alpha-fetoprotein.

OBJECTIVE: To evaluate whether prolactin, alpha-fetoprotein (AFP) or B-human chorionic gonadotropin (BHCG) is the most effective marker in vaginal fluid for diagnosing prelabor rupture of membranes (PROM). These proteins are present in amniotic and vaginal fluid and have been reported to be potent markers of PROM, but have not been used clinically nor compared to each other. STUDY DESIGN: A total of 100 pregnant women between 28 and 37 weeks of gestation were recruited for the study. Patients were divided into 2 groups. The first group consisted of 50 pregnant women diagnosed with ruptured membranes. The second group consisted of 50 normal pregnant women seen during routine antenatal clinic visit (control) group. All women underwent speculum examination aiming to sample prolactin, BHCG and AFP in the vaginal fluid. Ultrasonographic examination for gestational age and amniotic fluid index measurement was performed. The electrochemoluminescence (ECLIA) method was used for quantitative measurement of the three proteins (the total duration of the assay was 18 min). RESULTS: Vaginal fluid concentrations of the three markers were significantly higher in the PROM group than in the control group (p < 0.001). Receiver operator curve analysis indicated that AFP had 94% specificity, sensitivity, positive and negative predictive values, and efficiency. The other two markers have lower specificity, sensitivity, positive and negative predictive values, and efficiency: 70, 76, 71.7, 74.5 and 73% for prolactin and 72, 84, 75, 81.8 and 78% for BHCG, respectively. CONCLUSION: This work demonstrates that of the three markers investigated AFP has the highest diagnostic performance. Using the ECLIA method it can be an ideal marker for diagnosing PROM particularly in equivocal cases. The technique could be introduced into laboratory tests to meet clinical needs. Basel.