Genetic Research Of 4q35 In Facioscapulohumeral Muscular Dystrophy(FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is a kind of progressive muscular disorder. It often occurs before 30 years old The face is initially involved, then the shoulder girdle, and finally the pelvic muscles Asymmetry of weakness is not unusual. Lifespan is not significantly affected. It shows a penetrance of 95% for muscular symptoms by the age of 20 years The incidence is 1:20, 000.FSHD is an autosomal dominant hereditary disease. In more than 80% of the families, the FSIID phenotype is linked to 4q35 (FSHD1~) with locus order, centromere-D4S163-D4S1~-DY1o4S1-FSHD~telomere It was discovered by C Wijimenga et al in 1990 for the first time. The other gene locus of familial patients is not clear The FSHD gene locus at 4q35 is called D4Z4. In FSHD there is partial deletion of 3 3kb Kpn I tandem repeat units in D4Z4 DNA. In normal person the number of the tandem repeat units is above12-100.But in FSHD it is less than 11.80% of FSHD is located in 4q35 This may brings the genetic diagnosis of FSHD convenient. In normal person, probe p13E-11(D4F1~S1) detects an EcoR I DNA polymorphism of 50-320kb consisting of multiple copies of a 3. 3kb Kpn I tandem repeat In FSHD , when there is partial deletion of3 3kb Kpn I randem repeat units, the molecular weight of the EcoR I fragment is decreased below 38-41kb This fragment can be detected with probe pl3E-11 But there is also an isogeny sequence of 4q35 EcoR I fragment in loqter That EcoR I fragment in l0qter also contains a pl3E-11 site And the molecular weight of that of 4q35 and loqter are similar To purely observe the size of 4q35 EcoR I in FSHD, Bln I is used to cut4the pl3E-ll lOqter EcoR I fragment into pieces Then it will be disappear from the electrophoresis result. Even though there is also a decrease of 3kb of the 4q35 pl3E-ll EcoR I fragment under the use of Bin I, But there is no influence to the judgment of the decrease of the size of 4q35 EcoRI.So in most references the genetic diagnosis of FSHD is under the EcoR I/Bin I double enzyme digestion of the genome DNA, electrophoresis, Southern blot hybridization with probe pl3E-11 to observe the decrease of the size of 4q35 EcoRI The standard of normal in the genetic test is various. Most references believe that 38kb is a better standard In FSHD the pl3E-l1 EcoR I/Bin I fragment is less than 38kb The normal is greater than 38kbIn this article, genomic DNA is extracted from lOml of peripheral blood Phen-chloroform---isoamyl alcohol method and low melting point agarose gel block method are used to extract DNA. After EcoR I IBm I double digestion, pulsed field gel electrophoresis is used to separate the large molecular weight genome DNA fragment of double digestion. The DNA fragment is transmitted to nylon membrane. After UY fixing, Southern blot hybridization is done with probe pl3E-1l The P32 on the pl3E-l1 will show the band of related EcoR I fragment on Fuji X ray film. In this research there are modifications in PFGE, multiplication of pl3E-ll and Southern blot procedures. If the molecular weight of the band is less than 38kb, the diagnosis will be FSHD If that is greater than 38kb, the diagnosis is normal person.In this article, the FSHD group has 26 cases. In 20 cases (76 92%), the pl3E-ll EcoR I IBm I fragment is smaller than 38kb The FSHD relatives group has 12 cases. In 2 cases (16 67%) that fragment is smaller than 38kb The control group has 22 cases. In 21 cases(95.45%) that fragment is greater than 38kb.The result coincide with that of the5references. In genetic test in 7692% of FSHD patients, the 4q35 EcoR I fragment is smaller than 38kb In this research, probe pl3E-ll is cloned successfully It makes the base of genetic diagnosis of FSHD in our lab later The suitable parameters are found in various processes to make the result clearly seen. This method can be a genetic diagnosis method for FSHD members, carriers, and even prenatal FSHD fetus...