With this information, the twins were conclusively diagnosed with FA. gene sequencing, chromosome breakage analysis and comet assays were performed. The results revealed double heterozygous mutations in the Fanconi Anemia Complementation Group D2 gene of the twins, therefore providing a conclusive diagnosis of FA. The case highlights how difficulties in clinical diagnosis may be overcome by including genetic screening assessments into the range of diagnostic assessments, which may also reveal unexpected results. and have been identified to cause FA. Among these, mutations in the and genes are the causative factor in 5% of all cases of this Tlr2 disease (6C12). The present case report describes a pair of twins whom suffered recurrent upper respiratory tract infections for 2 years prior to the study. They were identified to have FA, caused by a double heterozygous mutation of gene in the two patients (Tables ICIII; Fig. 1). Additional diagnostic evidence obtained by chromosome breakage analysis. Chromosome culture was performed using the standardized method of the International Atomic Energy Agency 405 technical report (14). Mitomycin C (0, 50 and 100 g/l; Kyowa Hakko Bio Co., Ltd, Tokyo, Japan) was added after 24 h and cells were cultured for 48 h at 37C, harvested, Furosemide added a potassium chloride hypotonic (0.188%) solution for 20 min at 37C, pre-fixed with glacial acetic acid-methanol fixed fluid (consisting of glacial acetic acid and methanol at a ratio of 1 1:3) for 15 min at room temperature. A total of 1C2 drops was decreased onto slides from 40C50 cm and dried naturally prior to observation using a light microscope (magnification, 100). The number of chromosome aberration cells and the chromosome breakage number Furosemide were counted under the microscope, and the chromosome aberration rate and breakage rate was analyzed. The breakage rate in 100 cells was defined as (the number of broken chromosomes/100) 100%, aberration rate was defined as (the number of cells which appeared to exhibit broken chromosomes/100) 100%. The chromosome aberration rate and breakage rate of the experimental group were considered to be positive if they were higher compared with that of the control group. A total of 100 mitotic images were analyzed per specimen. The results are presented in Table IV. A comet assay was also performed. Cytochalasin B (6 g/ml, at 37C for 28 h), used to induce cell DNA damage, and ethidium bromide were purchased from Furosemide Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). A Nikon 90i fluorescence microscope was purchased from Nikon Corporation (Tokyo, Japan). The chromosome image analysis system GK-1303 was purchased from Leica Microsystems GmbH (Wetzlar, Germany) and the Sanyo MCO-20AIC CO2 incubator was purchased from SANYO Semiconductor Manufacturing Co., Ltd. (Sakata, Japan). The normal-melting-point agarose was obtained from Biowest USA (Riverside, MO, USA) and the low-melting-point agarose was from Promega Corporation (Madison, WI, USA). Tris-HCl, dimethyl sulfoxide (DMSO) and Triton X-100 were purchased from Sigma-Aldrich, Merck KGaA. Lymphocyte-separated medium (lymphoprep) was purchased from Axis Shield Furosemide Diagnostics, Ltd. (Dundee, UK). The horizontal-strip electrophoresis apparatus was from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The comet slides were from Bio-comet (Institute for Surgical Research and Hospital Management, Basel, Switzerland). The digital imaging system DMS300 was purchased from Leica Microsystems GmbH. Open in a separate window Physique 1. Fanconi Anemia Complementation Group D2 gene sequence diagram of the twin patients. (A) Genome sequence of Allele 1 for the patients and their parents. (B) Genome sequence of Allele 2 for the patients and their parents. Table I. Mutation of in the patients. allele (allele Furosemide 2) of the twins. (15), but with a slight modification; specifically, special comet slides were used rather than general slides. There are gaps in comet slides to contain the agarose. Furthermore, less agarose was used in the procedure than originally described by Banath yielding a thinner gel agarose to enable clearer viewing under a fluorescence microscope. First, the comet slides was coated with 100 l normal-melting-point agarose (0.075%); then, once the first agarose layer was coagulated, a mixture of 75 l low-melting-point agarose (0.075%) and 25 l lymphocyte suspension was applied as the second layer. The comet slides were immersed in cold fresh lysis solution (2.5 M NaCl, 1% N-sodium lauryl sarcosinate, 30 mM Na2EDTA, 10 mM Tris, 1%.