Samples
Peripheral blood leukocytes from the symptomatic pregnant woman (II1), the affected nephew (III4) and his mother (II3), and chorionic villus sampling (CVS) from the fetus (III1) were referred to our laboratory (Figure 1A).
Whole blood was drawn by venipuncture with 5% ethylene-diamine tetraacetic acid (EDTA) as anticoagulant. Genomic DNA (gDNA) from blood samples was isolated using the cetyl-trimethyl-ammonium bromide (CTAB) method (Murray & Thompson, 1980). For the CVS, gDNA was isolated by QIAGEN DNeasy Blood and tissue kit [Redwood City, California (www.qiagen.com)]. DNA concentration and quality were measured by absorbance at 260nm and by the ratios A260nm/A280nm and A260nm/A230nm, respectively.
The protocol was approved by the institutional ethics committee. Informed consent was obtained for all study subjects prior to the molecular studies.