| Dystrophinopathy is a group of inherited diseases caused by the defect ofdystrophin protein. All of these diseases are caused by mutations in the DMDgene at Xp21.2which encodes the protein dystrophin. Both male and femalecan have symptom, but male patients accounted for the vast majority of. Thedystrophinopathy spectrum include: Duchenne muscular dystrophy (DMD),Becker muscular dystrophy (BMD)(inclusive of asymptomatic hyperCKemia,cramps and myalgia, quadriceps myopathy), X-linked dilated cardiomyopathy(XLDCM), and manifesting/nonmanifesting DMD/BMD carriers. DMD is themost common and severe type of the dystrophinopathies. The disease onsets at5-6year old presented with walking slowly, easy to fall and abnormal walking,followed by progressive loss of lower leg muscle strength and ambulation,loss of ambulation at10-12year old, ultimately succumb to restrictive lungdisease or cardiac death at20-30year old. Patients with BMD present laterthan those with DMD and they ambulate independently until at least16yearsof age, with a mean age of30years. Progression is slower and there is alonger life expectancy. Carriers and females with contiguous gene deletionsyndromes may exhibit the entire spectrum of muscle weakness, from a fullDuchenne phenotype to mild proximal muscle weakness, calf hypertrophywith mild pathological findings in muscle, to essentially normal musclefunction.The DMD gene remains the largest gene yet discovered, contains79exons, and2.2million base pairs of genomic DNA encodes dystrophin protein.The dystrophin protein has four distinct domains. The amino-terminusassociates with actin or an actinlike protein. The rod domain has long flexiblerows of24spectrin-like a-helical repeats. There is a cysteinerich region, andfinally a unique carboxy-terminus. The dystrophin protein links thecytoskeleton to the basal lamina. It links with dystroglycan and sarcoglycans to form dystrophin glycoprotein complex (DGC) and to maintain themembrane constancy. Mutations lead to breakdown of the entire criticalcomplex, resulting in fragility of the sarcolemma. Intense muscle contractionintensifies this damage and leads to calcium influx and accelerated damage tomuscle fibers.In this research, we selected patients as followed from skeletalmuscle genetic resources specimens library:1) molecular pathological andgenetic analysis of some patients with dystrophinopathy, then choose twofemale MCs families to research their clinical and genetic features;2) lowerlimb skeletal muscle MRI study of42patients with confirmeddystrophinopathy diagnosis;3) glucocorticoid treatment analysis of DMDpatients. We did clinical, pathological and molecular analysis among thesepatients in order to obtain the clinical, pathological, molecular character andobservation of glucocorticoid treatment in dystrophinopathy.Part1Molecular biological study of DystrophinopathyObjective: Dystrophinopathy is a group of inherited muscular disorderscaused by disfunction of dystrophin. The dystrophinopathy spectrum include:Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD),X-linked dilated cardiomyopathy (XLDCM), and manifesting/nonmanifestingDMD/BMD carriers. Most heterozygous female carriers of DMD mutationsare asymptomatic; however, between8and22%of these carriers aremanifesting carriers (MCs). The pathogenesy of female MCs is unclear bynow, skewed X chromosome inactivation(XCI) pattern is the hot spot ofpresent studies. Beause the number of female carrier is little, the relationshipof gene mutation, XCI and clinical manifestation is indistinct. In this research,we studied the the clinical, muscle pathological and genetic data ofdystrophinopathy and two female carrier families, to investigate the charactersand possible pathogenesy of female carrier.Methods:1Inclusion criteria of selected patients:1) Disease onset at juvenile; weaknessand atrophy at proximal muscles, calf pseudohypertrophy, Gower’s sign(+);2) serum creatine kinase increased; electrophysiology study showed myogenicchanges;3) Histochemical stains of biopsied muscle specimen showedmuscular atrophic change; Anti--dystrophin-N，-C，-R monoclon antibodyimmunohistochemical stains showed dystrophin protein absolutely absent ordecreased severely.2Histochemical and immunohistochemical stains of muscle biopsy: All theselected patients underwent open biopsy of biceps brachii, histochemical andanti-dystrophin-N、-C、 R immunohistochemical stains of sample andpathologic analysis.3DMD gene sequencing: Multiplex PCR and denaturing high performanceliquid chromatography (DHPLC) were used to detect exon deletion andduplication mutation. Direct sequencing was used to detect point mutation.4Clinical and genetic analysis of two female MCs and families4.1Family data: selected two families including female MCs with mucleweakness and wasting of proximal limb, and male patients of DMD/BMD.4.2The proband and relatives of family underwent ECG, myocardial perfusionimaging and muscle MRI of lower limb to assess cardial conduction,myocardial perfusion, left ventricular function and muscle involvement；4.3Histochemical and immunohistochemical stains of muscle biopsy: asabove.4.4Karyotype analysis: Female MCs and female asymptomatic carriersunderwent conventional GRQ karyotype analysis.4.5DMD gene sequencing: as above.4.6XCI analysis: Methylation of the highly polymorphic HpaII restrictionendonuclease site in the androgenreceptor(AR) locus correlates with XCI. Weused HpaII digestion followed by PCR to determine the methylation status ofboth the maternal and paternal X chromosomes. Alleles that are active will bedigested while the inactive alleles are not. The ratio of undigested parentalalleles gives the pattern of inactivation.Results:1Among80dystrophinopathy patients, there were67DMDs,10BMDs,2 female MCs,1asymptomatic hyperCKemia.2Biopsied muscle pathology: Histochemical stains of biopsied musclespecimen showed muscular atrophic change; Anti--dystrophin-N，-C，-Rmonoclon antibody immunohistochemical stains showed dystrophin proteinabsolutely absent(67cases) or decreased(13cases).3DMD gene sequencing: We did gene sequence in14patients,1patient withBMD found no pathogenic variations,1DMD found point mutationc.4178T>A,12patents found exon deletion.4Female MCs study4.1Clinical manifestation: Both two families were X-linked recessiveinherited, two female MCs had different degree of muscle weakness andwasting of proximal limb, without calf pseudohypertrophy. Proband of family1had lightly weakness and late onset age, while proband of family2hadmarkedly weakness and early onset age.4.2Cardiac involvement of patients: The ECG and myocardial perfusionimaging of proband of family1were normal. Myocardial perfusion imagingof proband of family2showed decreased perfusion in cardiac apex, ejectionfraction was70%, left ventricular structure and function were normal.4.3Muscle MRI: Proband of family1: asymmetric muscle involvement,selected involvement was silmilar as DMD; proband of family2: almost allmuscles were severely damaged and replaced by connective or fat tissue, onlygracilis, extensor hallucis longus and flexor digitorum longuswere relativelypreserved; Ⅳ-4of two family2: selected involvement was silmilar as BMD.4.4Histochemical and immunohistochemical analysis of muscle biopsy: twoproband and Ⅳ-4of two family2had muscular dystrophic changed inhistochemical stains, immunohistochemical stains showed dystrophinabsent/decreased in some sarcolemma, a mosaic pattern of dystrophinexpression was seen in proband of family1, while dystrophin-N,-R wasabsent and-C was decreased in two patients of family2.4.5Karyotype analysis: Proband of two families and Ⅱ-5、Ⅲ-7of family2were normal female karyotype. 4.6DMD gene sequencing: DMD gene exon61heterozygous deletion wasfound in proband and Ⅱ-7of family1. DMD gene exon12-43homozygousdeletion was found in proband, Ⅱ-5and Ⅲ-7of family2, heterozygousdeletion was found in Ⅳ-4of family2. Point mutation was not found.4.7XCI analysis: Proband of two families and Ⅲ-7of family2wereuninformative.Conclusions:1Histochemical and immunohistochemical stain with anti-dystrophinmonoclonal antibody of skeletal muscle is an important method to clinicalscreens for dystrophinopathy, guide gene sequencing and determine clinicalphenotype and prognosis.2Most of dystrophinopathy have DMD gene deletion/duplication,deletion/duplication muation should be conducted first, then screen pointmutation.3The clinical manifestation degree of female MCs is related with age ofonset, the clinical symptom is corresponding with histochemical expression ofskeletal muscle biopsy, but is independent with dystrophin protein expressionin immunohistochemical stain.4Both two families have exon deletion of DMD gene, XCI analysissometimes is necessary. The relationship between clinical phenotype and genemutation in male patients consists with the reading frame theory, but femaledon’t.Part2The lower limber muscle MRI study of dystrophinopathyObjective: The dystrophinopathy spectrum include: Duchenne musculardystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilatedcardiomyopathy (XLDCM), and manifesting/nonmanifesting DMD/BMDcarriers. The pathogenesy is the absence of dystrophin, as a cytoskeletalprotein, results in structural fragility, membrane permeability, metabolic crisis,and progressive myocyte degeneration, necrosis and regeneration, connectivetissue markedly increased. Over time, the supply of regeneration cells isexhausted and damage to muscle fibers progresses, the number of muscle cells per unit volum reduced, replacement by connective tissue and fat contributesin part. Research has indicated that increased signal intensity on T1imagesmay reflect increased fibrous tissue or adipose tissue, and fat suppressionimaging showed decreased signal. Few pelvis and thigh MR imaging studiesin DMD patients have shown a characteristic pattern of fatty infiltration thatspares the gracilis, sartorius, and semimembranosus muscles, the quadricepsfemoris, gastrocnemius and soleus muscles are predominantly affected,peroneal muscles is a characteristic feature of boys with DMD.We analysed the clinical, laboratory, and MRI data of42patients withdystrophinopathy who visited our department in2004-2012, to summarymuscle injury, the relationship between muscle choice and the muscle strength,and to investigate diagnosis and different diagnosis point at molecular imaginglevel.Methods:1Inclusion criteria of selected patients:1) onset at teenage，with a historyof progressive muscle weakness, calf pseudohypertrophy, Gower sign(+);2)significantly elevated serum creatine kinase levels, myogenic change inelectromyogram;3) Muscle biopsy showed muscular dystrophy pathologicchanges, In anti-dystrophin-N,-C, and-R monoclonal antibodyimmunohistochemical staining, dystrophin complete absence in DMD andpartial absence in BMD.2Summarized analysis of42cases of patients enrolled in clinicaldata, including: sex, onset age, blood CK, semi-quantitative assess andstatistical analysis proximal and distal muscle strength and muscle injury inthigh and lower leg MRI.3Analysis and discuss the relationship of the clinical data and lowerextremity muscle injury in MRI of dystrohpinopathy.Results:1Clinical findings: There were33cases of DMD,7case of BMD,1caseof asymptomatic hyperCKemia and one female carrier. Thirty-three boys withDMD between2and12years of age participated in the study. Negative correlations existed between proximal and distal muscle strength and patientage (proximal: r=-0.550, P=0.00<0.05; distal r=-0.400, P=0.021<0.05). Thatmeans muscle strength is getting worse with age. There was a significantpositive correlation between proximal muscle and distal muscle strength(r=0.642, P <0.01).2Muscle MRI findings: The MRI changes in the thigh were more severethan the lower leg (P=0.000<0.001). In addition, the anterior group was moresevere than posterior group in thigh, whereas in the lower leg, the posteriorwas more severe than the anterior. Of the thigh muscles, the vastus lateraliswas the most involved, and the gracilis was the least involved. In the lower legmuscles, the long fibular muscle was the most involved, and the anterior tibialmuscle was least involved. BMD patients had similar characteristics in muscleMRI as DMD patients. The patient with asymptomatic hyperCKemia and theDMD/BMD carrier were slightly affected in the quadriceps femoris andgastrocnemius, but the rest of their muscles were well preserved.3Relationship between muscle MRI and clinical data: There was nostatistically significant correlation between serum CK levels and MRI muscleinjury scores (P=0.266). There was a significant positive correlation betweenthe MRI muscle injury scores in the thigh and lower leg and patient age (thigh:r=0.720, P=0.009; lower leg: r=0.680, P <0.01). There was a strong negativecorrelation between proximal and distal muscle strength compared with thedegree of muscle injury in the thigh and lower leg in MRI analyses (thigh:r=-0.611, P <0.01; lower leg: r=-0.462, P=0.012).Conclusions:1The lower limb MRI showed selective muscle involvement indystrophinopathy, the selective involvement character was corresponding withclinical manifest. The mucle injury even can appear in subclinical period.2Muscle involvement of dystrophinopathy in MRI is regular, whichcontribute to diagnose dystrophinopathy at molecular imaging level and isessential for the diagnosis and differential diagnosis of progress musculardystrophy, also can guide mutate gene sequencing. 3The severity of muscle involvement in MRI can reflect clinical severity,that is important for pathogenetic condition evaluation, rehabilitativetreatment instruction and curative effect evaluation.Part3The study of glucocorticoid treatment of DMD patientsObjective: DMD is the most common and severe type of thedystrophinopathies, loss of ambulation at10-12year old, ultimately succumbto restrictive lung disease or cardiac death at20-30year old, with significantlyelevated serum creatine kinase levels, myogenic change in electromyogram.Currently, there is no effective cure for DMD, mainly to savethe patient’s motor function, prevention of complications for treatmentpurposes, including glucocorticoid treatment, appropriate rehabilitationexercises and orthopedic management and so on. Number of large-scaleclinical trials had shown that glucocorticoid treatment can increase musclestrength in short term,and slow down the rate of loss of muscle strength,preservation movement function and improve the quality of life. Retrospectiveanalysis the data of96cases of DMD to investigate retrospectively the clinicaland laboratory appearance of DMD and evaluate therapeutic efficacy ofglucocorticoid.Methods:1Inclusion criteria of selected patients: Progressive muscle wasting andweakness of lower limb, calf pseudohypertrophy; significantly elevated serumcreatine kinase levels, myogenic change in electromyogram; biceps brachiibiopsy showed muscular dystrophy pathologic changes, In anti-dystrophin-N,-C, and-R monoclonal antibody immunohistochemical staining showeddystrophin complete absence in sarcolemma.2Grouping and glucocorticoid therapy: The patients was grouped base onage of first diagnosis. All the patients were given dexamethasone5-10mgintravenous infusion for10-15days base on weight, age and serum CK level,then rechecked serum CK and given oral prednisone0.5-0.75mg/kg.dmaintenance treatment continuely(prednisone was given as0.75mg/kg.d topatients≤8year old, gradually with the weight gain and up to the dosage of 30mg,0.5mg/kg.d was given to patients>8year old), rechecked serum CKone month later. All the patients accepted glucocorticoid therapy signed aninformed consent.3Statistical analysis the serum CK and lower limb motor funcion beforeand after glucocorticoid therapy.4Assess the myocardiac invlovement and intelligent level of DMDpatients.Results:1Clinical findings: The age of DMD patients visited hospital first were10month to16year old(mean6.61士2.68year). The reasons that patient visithospital were: hyperCKemia and family medical history in≤3year old group;abnormal walking posture in4-5year group; easy to fall and squat difficultiesin6-8year group. In EMG myogenic changes were found in upper and lowerlimbs, espically in proximal muscles.2Serum CK: The level of serum CK had three peaks at the≤3years,5years and8years respectively,and significantly decreased after10-15days’dexamethasone(5-10mg) intervenous drop infusion, and increased againafter1month’s prednisone acetate(0.5-0.75mg/kg.d) oral administration. Theserum CK level of24cases who accepted glucocorticoid therapy repeatedlydecreaed after dexamethasone intervenous drop infusion, increased one monthlater.3Lower limb motor ability: The motor ability improved in50cases ofDMD patients with long-term oral prednisone(P=0.032, P=0.014), including24cases receiving intravenous dexamethasone intermittently.4Radionuclide imaging of cardiac muscle: The myocardial perfusionimaging of37cases of DMD showed significantly uneven ventricularradionuclide distribution, was “spotted like” change. There were21casesslightly injuried,9cases moderately injuried and7cases markedly injuried.The infer-septa(14cases) was most involved and anterior septa(3cases) wasinvolved least. There was positive relation between cardiac injury and age（P<0.01，rs=0.685）. 5Intelligence assessment: The speech and operate ability, intelligencequotients in24DMD patients were lower than normal population（P<0.01）.There was not significant difference between speech and operate ability（P=0.259>0.05）.Conclusions:1DMD was the most common type of progressive muscular dystrophy,patients usually visit hospital at6-7year old, onset with abnormal walkingposture, followed with progressive lower limb weakness.2There are high CK hyperlipidemia and myocardial damage in thesubclinical stage of DMD, myocardium impairments are positively correlatedwith age. The patient have intelligence involvement, intelligence quotientswere lower than normal population.3Glucocorticoid treatment can increase muscle strength in short term andglucocorticoid intervenous drop infusion reduced the serum CK level, but oralglucocorticoid can not reduced the serum CK level.4Glucocorticosteroid therapy has an important effect on the protection ofmotor and cardiac funcions. |
PDF Full Text Request