Značaj određivanja statusa prenosioca kod Dišenove i Bekerove mišićne distrofije u populaciji Srbije

Abstract

Distrofinopatije su bolesti koje nastaju kao posledica mutacija u genu zadistrofin. Dišenova mišićna distrofija (DMD) predstavlja najteži oblik iz ove grupebolesti. Karakteriše je rani početak bolesti, progresivna mišićna slabost koja dovodi dogubitka pokretljivosti bolesnika i kardio-pulmonalne slabosti zbog zahvatanja srčanog irespiratornih mišića. Bekerova mišićna distrofija (BMD) se javlja kasnije, ima blaži tokbolesti, ali sa velikom varijabilnošću u kliničkoj slici. Nasleđujuju se X-vezanorecesivno, oboljevaju muškarci dok su žene uglavnom zdravi prenosioci bolesti.Procenjeno je da su 2/3 majki nosioci, 5-10% ima gonadni mozaicizam, dok 25-30%nema mutaciju. Gen za distrofin (DMD gen) je najveći opisani gen u humanom genomui često je podložan promenama. Najčešće su prisutne intragenske delecije (65%-70%) iduplikacije (5-15%) jednog ili više egzona, dok su tačkaste mutacije prisutne u 20%slučajeva. 1/3 bolesnika ima de novo mutaciju. Za sada nema uspešne terapijedistrofinopatija, pa je utvrđivanje statusa nosioca kod ženskih članova u familiji odznačaja za davanje genetskog saveta i prenatalnu dijagnozu.Cilj rada je bio da se utvrde i analiziraju delecije i duplikacije u genu za distrofin kodprobanada; da se u slučajevima potvrđenih delecija i duplikacija kod probanada utvdistatus prenosioca kod njihovih ženskih srodnka; u slučajevima bez dokazanih delecija iduplikacija kod probanada, da se ispita mogućnost indirektne genetičke analize zaodređivanje statusa ženskih prenosioca; da se u indikovanim slučajevima izvršiprenatalna molekularno genetička analiza DMD gena, primenom adekvatne metode zadatu porodicu.Material i metode: Uzorak su činila 72 DMD/BMD probanda, 69 ženskih članova iz44 porodice probanada i 11 trudnica (15 trudnoća). Genomska DNK za analizu jeizolovana iz limfocita periferne krvi ispitanika metodom isoljavanja prema standardnojproceduri, a za prenatalnu dijagnozu, DNK je izolovana iz uzorka horionskih resica,plodove vode ili krvi pupčanika ploda primenom komercijalnog kita. Za detetekcijudelecija i duplikacija u DMD genu kod probanda primenjene su metoda lančane reakcijepolimeraze (PCR) i metoda istovremenog umnožavanja vezanih proba (MLPA); zadetekciju ženskih nosioca primenjene su MLPA metoda i analiza vezanosti; zaprenatalnu dijagnozu primenjene su PCR metoda, analiza vezanosti i MLPA metoda...

Dystrophinopathies are diseases that result from mutations in thedystrophin gene. Duchenne muscular dystrophy (DMD) is the most severe form of thisgroup of diseases. It is characterized by an early onset of the disease, a progressivemuscle weakness that leads to loss of mobility of the patient, spreading to the heart andrespiratory muscles, causing cardio-pulmonary weakness. Becker muscular dystrophy(BMD) occurs in late childhood or adolescence, has a milder course of the disease, butwith widely variable clinical presentations. It has an X-linked recessive inheritedpattern, whereby males are affected, while females are mostly healthy carriers of thedisease. It is estimated that 2/3 of the mothers are carriers, 5-10% have gonadalmosaicism and 25-30% have no mutation. The gene for dystrophin (DMD gene) is thelargest known gene in the human genome and is often subject to change. The mostcommon changes are intragenal deletions (65% -70%) and the duplication (5-15%) ofone or more exons, as well as point mutations in 20% of cases. 1/3 of patients have denovo mutation. There is no successful therapy for dystrophinopathy, therefore thedetection of female carriers in a family is important for genetic counseling and prenataldaignosis.The aim of work was to determine and analyze the deletions and duplications of thedystrophin gene in probands; in the cases of confirmed deletions and duplications in thetrial, to determine the status of the carrier in their female relatives; in the cases with noproven deletions and duplications in the trial, the possibility of indirect genetic analysisfor determining the status of female carriers; in the indicated cases to perform prenatalmolecular genetic analysis of the DMD gene, using an appropriate method for aparticular family.Material and methods: The sample consisted of 72 DMD/BMD probands, 69 femalemembers from 44 proband families and 11 pregnant women (15 pregnancies). Thegenomic DNA for analysis was isolated from the peripheral blood lymphocytes of thesubjects, according to the standard procedure, and for prenatal diagnosis, the DNA wasisolated from the sample of chorionic villi, amniotic fluid or blood of the umbilical cordusing a commercial kit. For the detection of deletions and duplications in the DMDgene, polymerase chain reaction (PCR) method and multiplex ligation-dependent probeamplification (MLPA) method were applied; for the detection of female carriers, theMLPA method and linkage analysis were used; for prenatal diagnosis, the PCR method,linkage analysis and MLPA methods were applied...