| Backgroud and objectivesFacioscapulohumeral muscular dystrophy is the third common inherited neuromu scular disorder after Duchenne muscular dystrophy and myotonic dystrophy, wit h an estimated prevalence of about 1 in 20,000 individuals. 95% patients beco me symptomatic by the age of 20.The disease is characterized by progressive weakness and atrophy of muscles in the face, shoulder girdle and upper arms, subsequently, spreading to others body regions. Cognitive impairment, hearing l oss and retinal vasculopathy also have been reported to associate with some se vere affected FSHD cases. The disease is associated with contractions of tande m 3.3-kb D4Z4 repeats located on chromosome 4q35. More than 95% of FSH D patients have been detected to carry a small(<38 kb) Eco RI fragment on c hromosome 4q35.Recently, it was reported that there is a relatively stable simpl e sequence-length polymorphism(SSLP) located 3.5 kb proximal to D4Z4 was related to the disease. On the basis of the proximal SSLP and distal 4q A/4q B, the subtelomeric 4q35 region can be subdivided into different haplotypes and some of haplotypes were associated with FSHD. It was shown that the pathoge nic haplotypes carried the(ATT/CAAA) PLAM sequence, which was identified as the potential poly(A) signals necessary for the distal DUX4 transcript and DUX4 may play a important role in pathogenesis of FSHD. The present study aims to systematically summarize the clinical feature Chinese han FSHD patie nts and analyze the its association with the size of the Eco RI fragment in pati ents. We further investigated the genetic structure of 4q35 region(SSLP-D4Z4-4q A/4q B-PLAM) in FSHD Patients in order to assess the correlation between t he special signature of patients and the impact of DUX4 expression on the pat hogenesis of FSHD.Methods:1. The study concerned 110 unrelated healthy individuals and 177 patients(94 males and 83 females) from 138 unrelated families and 63 unaffected members were recruited in our study. All patients included in the present study fulfilled the clinical criteria proposed by the international FSHD consortium. Clinical se verity of FSHD patients was assessed referred to the report of Ricciet al.(Clin ical Severity Scoring) Other clinical evaluation include:(1)creatine kinase(CK) l evels, electromyography(EMG),muscle biopsy(2) Cardiopulmonary involvements: electrocardiogram, Pulmonary ventilation function.2. High molecular weight DNA was obtained from peripheral blood lymphocyte s using the standard protocols. The DNA plugs were digested with Eco RI(E), Eco RI/Bln I(B), and HINDIII(H), after digestion, DNA was then separated by pulsed field gel electrophoresis on a 1.0% agarose gel. DNA fragments in the gel were then transferred to the membranes by southern blotting. Membranes for D4Z4 array sizing were hybridized with radioactive probes p13E11. For dis tal A/B typing, HINDIII digestion membranes were hybridized with probes 4q A or 4q B.ol. We calculate the Eco RI fragment length according to the hybridization of P13 E probe. The SSLP alleles were cut from the the agarose gel in a small slice containing Eco RI-digested chromosomal DNA fragments of appropriate size aft er PFGE analysis. The position of the D4Z4 repeat in the gel was determined using a molecular size standard after which the fragments were sliced out of t he gel and the DNA was isolated. SSLP fragments were amplified using the pr imers with HEX, and PCR products are then analyzed using an ABI Prism Gene tic Analyzer to identify SSLP size differences. At last, we have to combine wit h the SSLP data with PFGE data for all D4Z4 repeats and 4q A/4q B data to get the genotype of SSLP. To define the SSLP and the p LAM SNP(AT(T/C)AAA) sequences flanking the D4Z4 repeat units, pulsed field gel electrophoresis(PFG E) of Eco RI-digested DNA was used to isolate each D4Z4-reduced allele.Results:1. The mean size of Eco RI fragment in all patients is 19.9±5.06kb(ranging fr om 9kb to 35kb), and only two cases carried larger than 30 kb Eco RI fragment. 70% patients became symptomatic by 20 years, 91% became symptomatic by 30 years. Typical Facial or scapulo-humeral muscle weakness was the predomin ant clinical manifestation at onset. Unusual presentation included: proximal lo wer limb weakness in 14 patients and 3 of them with foot drop,pelvic gird le weakness(abnormal walking posture) was also noticed in 5 cases. Facial we akness involvement was often presented early-onset in the patients who usually be complaint to have pouting appearance(smile unnaturally).2. 14 cases(11 females and 3 males) were asymptomatic or minimally affecte d with age range 21–56 years(mean age 40.54±10 years). Their mean Eco RI f ragment was 19.3±3.99 kb, ranging from 14 to 30 kb. Of the 177 subjects with an affected allele, the prevalence of asymptomatic carriers in all affected indivi duals approximately is 8.0% and the proportion of them in all affected relative s is 35.6%.3. Besides 14 asymptomatic carriers, it was observe the reverse correlation bet ween the Eco RI fragment size and age-corrected clinical severity score in our study. When separately analysis of men and women, only significant inversely correlation was observed in male. Obviously significant correlation between Eco RI fragment size and Age-corrected CSS score was observed in the sporadic cases(r=-0.769,P<0.02). Compared to the correlation in total sample, the coeff icient R rises to 0.769 in sporadic cases. Analysis of the correlation in the fa mily cases, it showed closely matched the coefficient in total sample(r=-0.274 P<0.05). There was no statistical correlation between the D4Z4 fragment size and the age of onset in both genders.4. Patients had received muscle biopsy previously which revealed several com mon dystrophic and inflammatory changes in FSHD specimen, such as variatio n of fiber size, necrotic regenerating fibers, and variable degree of inflammator y cellular infiltration. The abnormality of routine ECGs was reported in 14 cas es and the incomplete right-bundle branch block is common associated with FS HD. 9 of 22 participants had pulmonary function testing that was suggestive of restrictive ventilatory defect. 4 of them had moderate-severe restrictive respirat ory involvement.5. Genotyping of 4q and 10 q haplotypes in 80 Chinese han healthy control, 4A161 and 4B163 are the most prevalent haplotypes on chromosome 4, respecti vely 93% in 4q A and 94% in 4q B.The haplotype of chromosome 10 predomin ately was consisted of 10A166 and 10A164,respectively 76% and 23%.6. Analysis of 177 FSHD patients, all patients carried shorten Eco RI fragment size linked with 4q A,two cases(one male and one female) have been genetical ly confirmed were compound heterozygosity. 28/177 patients display non standa rd repeat array configuration(translocation between chromosome 10 and the 4q35 region), the prevalence of translocation approximately is 15.8%.7. Of the 177 FSHD patients, 7 cases(four females and 3 males) were confir med as somatic mosaicism and the proportion is approximately 4.0%. Three of them display translocation between the 4q35 region and 10q26. 3 of four fem ales somatic mosaicism were asymptomatic carriers or minimally affected, and their offspring inherited shorten Eco RI fragment from them with more severe p henotype. In contrast, 3 males somatic mosaicsim present more severe phenotyp e than females.8. Genotyping of D4Z4-reduced allele in 138 patients, we found that all of th em carried shorten Eco RI fragment size associated with 4A161PASConclusion:1. All of patients carried Eco RI fragment size smaller than 38 kb exclusively a ssociated with 4q A in Chinese han population. A relatively high proportion of asymptomatic carrier in affect relatives approximately is 35.6%.The high freque ncy translocation between chromosome 10 and the 4q35 region was associated with mitotic rearrangement mechanism in somatic individuals. Mosaic patients clinical severity is closely related to the fraction of peripheral blood lymphocyte s(PBLs) carrying the disease allele, instead of the residualrepeat length. Mosai c female subjects were almost exclusively detected in asymptomatic carriers, ea sily be neglected in clinical practice.2. Roughly inversed correlation between the clinical severity and Eco RI fragme nt size was observed in Chinese han FSHD patients. It is revealed that poor c orrelation between clinical phenotype of female and their fragment size, which suggest that female patients more easily suffered from the(epi)genetic or unkn own synergistic factors. Compared to families’ cases, the more strong correlatio n between Eco RI fragment size and clinical severity was observed in sporadic cases.It is suggest that the intrafamilial clinical variability is higher than interfa milial.3. The haplotypes distribution of 4q/10 q were relatively single, indicated that Chinese han population was less influenced by ethnic migration in human evol ution; Although it was shown that frequent rearrangements between chromosome s 4q and 10 q, the SSLP haplotypes has unique distribution on 4q and 10 q alle les;the sequence variants in different haplotypes of 4q/10 q may contribute to th e molecular basis of FSHD associated with 4q35 region.4. Compared to the haplotypes distributions in healthy control, all of patients with FSHD carried alleles with contraction D4Z4 repeats associated with 4A161 PAS. The evidences supported the hypothesis that the unifying genetic model in FSHD is closely related to DUX4 expression contributed to the development of FSHD.5、There is no difference of haplotypes distributions between asymptomatic carr iers and FSHD patients, which suggested that the current unifying genetic sign ature cannot explain moderate or no phenotypes in asymptomatic carriers and u nkown epigenetic factors/mechanisms modulate the disease. |
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