Atypical 22q11 microdeletions in Iranian patients with congenital conotruncal cardiac defects.

A highly characteristic feature of 22q11DS (22q11 Deletion Syndrome) is congenital heart disease (CHD), which occurs in approximately 75% of all patients.1 Characteristics of congenital cardio vascular defects associated with DG/VCF (DiGeorge/ Velocardiofacial syndrome) are termed tetralogy of Fallot including pulmonary atresia and ventricular septal defect (VSD) in the severest type, truncus arteriosus communis and interrupted aortic arch. The great majority of patients suffering from the del22q11.2 syndrome (97-98%) have a common proximal breakpoint, while the distal breakpoint can vary, and in 90% of patients it produces a 3 Mb deletion (typical deleted region [TDR]); or in 7% of patients produces a 1.5 Mb deletion. While smaller numbers have “atypical” deletions or translocations, and could present with symptoms similar to 22q11DS. Furthermore, other chromosome defects such as 4q, 10p13, 18p21, and 17p13 with features of DiGeorge anomaly have also been described.2 The currently accepted clinical laboratory assay for 22q11DS is fluorescent in situ hybridization (FISH),3 but this technique has some drawbacks. Fluorescent in situ hybridization is mostly efficient when the breakpoints are the same in all patients. Otherwise using FISH can produce false negative results. Special expertise and equipment, which are prerequisites of FISH cannot be found easily in most laboratories.4 Based on these facts, we designed and set up a novel semi quantitative polymerase chain reaction (PCR) for detection of 22q11.2 microdeletions and microduplications. Multiplex ligation dependent probe amplification (MLPA) with P023 probe mix was used to validate our data. This study was conducted from January 2007 to January 2008 in the Molecular Genetics Laboratory, Sarem Women Hospital, Tehran, Iran. Ten patients were selected based on the presence of congenital conotruncal cardiac defects (inclusion criteria). Patients or their parents signed a consent form approved by the local ethics committee. Peripheral blood of 3 normal individuals was used as normal control. The results of FISH analysis by commercial region probe TUPLE1, carried out in the Laboratory of Cytogenetics, Markaz Tebi Hospital, Tehran, Iran, were taken into consideration while comparing molecular and FISH results. Genomic DNA was extracted from fresh peripheral blood of the patients and controls using the salting out method. Five pairs of primers were designed and verified by primer3plus program to set up multiplex PCRs (D22S944-F: CAT GTG AAA GAT GCT ACT TCC, D22S944-R: ATC CCA TGC TCC TCC CCA T; D22S936-F:CAA TCT TGG CAG CCA GTT TAG, D22S936-R: CAG CAT CTT CCT GGT GGC C; D22S636-F:AAC CTT CTG ATG GCT CCT CT, D22S636-R: CAT GGA GCT GAC ACT GAG TG). Three of the primer sets amplify established sequence tag site (STS) markers. Two of these markers reside in the 3 Mb TDR (D22S944, D22S936), and one is situated outside this region (D22S636). The last marker is used as internal control. Two remaining primer sets amplify HPRT and P15567 (HPRT-F: ACG TCT TGC TCG AGA TGT GA, HPRT-R CCA GCA GGT CAG CAA AGA AT; P15567-F: GCC AGA GGA TAG GGA GTG C, P-15567-R GTG GAA GCA GTC AAA CAG AAC) and were used as known copy number controls. The P155675 is situated on the 5’flanking region of TDR. The PCR reactions were performed in 25 μl total reaction volume containing: 0.25 unit of Taq polymerase (CinaGen, Iran), 2 mM of dNTP mix (CinaGen, Iran), 1X PCR buffer (CinaGen, Iran) (P15567-HPRT coamplification) or 2X PCR buffer (D22S936-D22S636 coamplification) and 200 nM of each primer. The D22S944-D22S636 coamplification was performed using Hot-start protocol of QIAGEN multiplex PCR kit. The PCR conditions are summarized in Figure 1a. The PCR products where verified on 7.5% acrylamide gel at 22, 25, and 28 cycles to be sure of reproducibility of results and care was taken to be in the log phase of DNA amplification. Patient/normal control ratios were calculated by TotalLab TL100 software. The MLPA technique with P023 probe set was performed according to manufacturer’s recommendations. Patient/control ratios in normalized data identified 4 patients with apparently diminished marker/control gene or marker ratio. Patients P-19, P-34, P-40, and P125 had ratios compatible with microdeletion in their corresponding region (Figures 1b & 1c). Other patients had ratios between 0.6 and 1.6. No increase in patient/ control ratio was detected. The FISH with TUPLE1 probe had identified P-40 as the only 22q11 deleted sample, whilst the other 9 showed normal cytogenetic results. Each assay was performed in 3 independent experiments. The results of cycles 22, 25, and 28 were concordant in all patients and showed reproducibility Brief Communication