Diagnosis Of The Gene Deletion D/BMD Proband And Carrier By Fluorescent In Situ Hybridization

Objective:The ascertainment of the carrier status is one of the basic dilemmas in the genetic counselling of the D/BMD. In this paper, we set up the method of FISH to identify the carrier status of the female relatives of the dystrophin gene deletion probands. Besides that, the amplification of the chromosome specific repetitive probes by the method of DOP-PCR was also tried.Methods:1. We chose the cosmid as the vector of the DMD specific probes. Thanks to the help of Dr. E. Bakker of the Leiden University and Dr. Jan Van Hemel of the Erasmus University, we obtained 8 DMD cosmids, which cover the region of the exons 44-47 of the dystrophin gene and the X centromere probe XB12 respectively. We extracted the DNA from the cosmids by the method of the alkaline lysis. We also amplified a large quantity of the chromosome specific probes by DOP-PCR . Nick-translation was used to label the above probes with digoxinin and biotin and the termination method was changed according to the aim of the FISH.2. From 1997, 19 probands of the D/BMD, who asked for genetic counselling at our genetic outpatient department, were diagnosed by the method of multiplex PCR. In addition to that, 5 cases of prenatal diagnoses were also made. Within these 19 probands, we found two patients whose exon 46 of the dystrophin gene was deleted, one had a positive pedigree and the other one was a sporatic patient.3. The carrier status of the female relatives of the above two patients were identified by double-color FISH. Besides the risk probe 1919, which located at the exon 46 of the dystrophin gene, we chose the probe XB12 and the probe 1906, which located at the intron 47 of the dystrophin gene as the control. We also designed the normal male and the normal female as the control. For each case, all the metaphases and 200 interphases were observed. We controlled the error rate of the normal control as below the 5%. The hybridization rates of the probe 1919 and 1906 were calculated and compared with the normal...