Mutation Spectrum And Phenotypic Manifestation In FSHD Chinese Patients

Background and objectives Facioscapulohumeral muscular dystrophy is one of the most prominent autosomal dominant muscular dystrophies with an estimated prevalence of 1:20000,second only to the Duchenne muscular dystrophy(DMD)and myotonic dystrophy(DM).It usually affects muscles in a typical pattern of initially facial,shoulder griddle and upper arm muscles,progressively local lower limb muscles.The clinical specific signs include asymmetry weakness,scapular wings and Beevor's sign.20 % of patients over the age of 50 require full-time wheelchair use.Some infantile FSHD have extramuscular manifestations,like retinal vascular changes,hearing loss and epilepsy.95% of FSHD patients carry a heterozygous contraction of the macrosatellite of D4Z4 repeats(DRs)from 1 to 10 repeats in the subtelomeric region of chromosome 4q35,which yield the change of methylation and chromosome structure,causing the disorder of gene function.The upstream of DRs,SSLP,and the downstream,4q A,are both linked to the stable abnormal expression of DUX4 transcript.We have confirmed that the genotype of Chinese FSHD is mainly4 q A161PAS.Accurate detection of the genotype is the base of the FSHD genetic diagnosis.Hence the genetic diagnosis process of FSHD is different from the other monogenic genetic diseases detected by next generation sequencing(NGS).It requires specially complex methods to isolate the klenow fragment and do hybridization,and the domestic research center of FSHD is few.Therefore,the clinical and genetic analysis of Chinese FSHD is rare.Meanwhile,phenotype heterogeneity exists in extra-families and inter-families of FSHD.Atypical patients also occupied the certain part,mainly facial-sparing FSHD(SHD),which is easily to be misdiagnosed.Our team has done research in the FSHD diagnosis for decades and established formal diagnostic process.Today we analyze the 242 FSHD families to demonstrate the clinical and genetic features of Chinese FSHD,and summarize the relationship between phenotype and genotype,and further study the special type of FSHD in order to enhance the understanding of FSHD.Patients and Method1.Patients: We recruited 242 consecutive patients and 90 relatives genetically diagnosed with FSHD.Patients come from 23 provinces with 170 southeastern ones and 72 northeastern ones.121 patients have positive history,containing 71 males and 50 females.111 sporadic cases consist of 68 males and 43 females.Thirty patients are infantile FSHD.The clinical diagnosis of FSHD was based on the criteria proposed by Padberg GW etc.All patients are evaluated by Clinical Severity Scores(CSS).This study was approved by the hospital ethics committee for medical research.Informed consent was obtained from the patients.2.Fragment detection process: DNA was gently extracted from the peripheral lymphocytes according to standard protocols.For Southern blot analysis,5ug DNA plugs were digested with Eco RI and Eco RI/Bln I and Hind III as well as Not I if necessary.DNA was then separated by pulsed field gel electrophoresis.DNA fragments were then transferred to the membranes.Membranes for D4Z4 array sizing were hybridized with prepared probes p13E-11,4q A and 4q B.Finally,the DRs was calculated according PFGE marker and the haplotype was identified between 4q A and 4q B.The composite genotypic-severity scale was further calculated for the FSHD mosaic.3.The clinical and genetic features of Chinese FSHD: The onset age,Eco RI length and biomedical markers et al.are all in accordance with normal distribution,which are described by means and standard deviation.The correlation between the clinical severity score(CSS)and the Eco RI length is analyzed by spearman rank correlation.The comparisons of the onset age distribution,muscle weakness proportion,Eco RI fragment level are used independent-samples t test.P value to less than 0.05 was statistically significant.All data are analyzed by SPSS16.0.4.Clinical and genetic analysis of Facial-sparing Facioscapulohumeral muscular dystrophy(SHD): 20 patients from above database were recruited.There were 14 males and 6 females with age from 22 to 53 years.The offsprings of 4 patients behave typical phenotype.All patients were clinically evaluated.The methylation level of SHD families was detected by Pyrosequencing for global average methylation of DR1.Result1.The initial symptoms of onset variably differ,generally in the second and third decades with the onset age 17.8±7.6 years.The penetrance is 71% at age 20,and91% at age 30.42 patients were asymptomatic carriers.69.3% were firstly involved in the scapulo-humeral muscle,and 22.4% were involved in facial muscles,and 7.9% showed weakness from the lower limbs.About half of patients were asymmetric.The proximal muscles like biceps,triceps and deltoid were mostly suffered,but wrist flexor and gastrocnemius were almost spared.Among242 FSHD probands,199(82.2%)patients have positive scapular wing.Positive Beevor's sign was seen in 56% typical patients but 35% atypical ones.Only 3.7%patients depend on the wheelchair with the age 25.7±13.5 years.The CSS of 242 probands were 229.48±121.1.There are significant difference between the CSS in the northeast and the southeast.15 patients present the muscle weakness became more severe after pregnancy or childbirth.2.242 patients and 90 relatives all have contracted EcoRI fragments with 4qA allele by PFGE,which genetically diagnosed as FSHD.There are 121 positive family history and 102 sporadic cases.26 patients were confirmed as de novo because their parents' samples are available.The general Eco RI fragment was 20.0±5.2kb.And the Eco RI fragment of infantile FSHD was 18.2±5.3kb.The Eco RI fragments of families in infantile FSHD were longer than that of sporadic cases.FSHD mosaic was found about 7%,whose average Eco RI fragment was 18.4±4.6kb.Also,the CSS of female mosaic was significantly different from that of male mosaic.3.It was generally reverse correlation between DUX4 fragments and clinical severity(r=-0.278,P<0.05),but the moderate reverse correlation was found in the sporatic cases that was higher than familiar ones.It was moderate positive correlation between CSS of mosaic patients and genotypic-severity scale(r=0.529,P<0.05).The male mosaic patients were more severe than the female ones,but the next generation of the female mosaic showed more serious muscle weakness.4.Biochemical markers including creatine kinase(CK)was increased to three times of the normal value and myoglobin(MYO)were slightly increased.However,creatinine was decreased.Most limbs EMG(171/187)showed myopathy while facial EMG(11/48)has less myopathy results.Almost all biopsy results(54/66)of FSHD patients were myopathic changes.The MRI in double lower limbs muscle showed asymmetric muscle weakness and atrophy,which was same to the manual muscle testing(MMT).The most common abnormality of ECG is incomplete right bundle branch block(IRBBB).Pulmonary function tests of 7 patients(6.3%)showed severe restrictive lung ventilation dysfunction,whose CSS means was3.9±0.2.The average Eco RI fragment was 15.7±5.2kb.5.20 patients were all confirmed to have D4Z4 deletion.The onset age was25.97±8.2 years,ranged from 22 to 53 years.All patients were followed for2.3±2.8 years.Almost all patients had mild muscular weakness initially presented with the upper limb weakness and slow development.The positive proportion of Beevor's sign was relatively lower than that in the typical FSHD.The next generation of SHD had more severe symptom with identical D4Z4 RUs.The auxiliary examination generally showed mild myopathic changes,especially facial EMG that was almost normal.Genetic analysis revealed that 20 patients had4 q A-type FSHD-related Eco RI fragments,ranging from 18 kb to 33 kb(mean,26.7±4.3kb;4-8 D4Z4 repeat units).Six patients carry 4q-10 q complicate translocations.The methylation of SHD families was not consistently changed.Conclusion1.The initial symptoms of Chinese FSHD are almost scapular weakness and atrophy,generally in the adolescent.2.The muscle involvement of Chinese FSHD has its own pattern that biceps,triceps and deltoid are easy to be involved.The positive rates of myopathic face,scapular wing,and Beevor's sign are relatively high.3.The Eco RI fragment of Chinese FSHD ranges from 9kb to 34.5kb(means,20.0±5.2kb),which suggests the difference of race.4.The Eco RI fragment of infantile FSHD is smaller than that of typical FSHD(18.2±5.3kb).The smaller Eco RI fragment is,the more severe phenotype is.The sporadic infantile ones are more susceptible to Eco RI fragment length.It hints that identification of the pathogenic fragment early is benefit for the disease assessment.5.The mosaic FSHD exits,which reveals that somatic mutation is also involved in the pathogenic mechanism.it is concerned that the offsprings of female mosaic patients are possible to show more severe FSHD symptoms.The higher genotypic-severity scale is,the more severe phenotype is.6.For those who have shorter EcoRI fragments or severe phenotype,the cardiac and pulmonary functions are obviously affected,which should be regularly monitored.7.SHD,as a number of special FSHD,has milder phenotype.The D4Z4 deletion is less than that of typical FSHD.Clinical assessment including scapular wing,Beevor's sign and asymmetry may help clinicians do a correct clinical diagnosis.The methylation is not the main reason of heterogeneity between SHD patients and the children.