Clinical Study And Investigation Of Molecular Diagnosis Of Myotonic Dystrophy Type 1

Objective1. To analyze and summarize clinical features of DM1, further understand DM1 and improve DM1 diagnosis.2. To investigate the methods of molecular diagnosis of Myotonic Dystrophy Type 1.Methods1. The clinical data of 26 DM1 cases confirmed by molecular diagnosis were studied retrospectively.2. Routine PCR and southern blot analysis were performed for molecular diagnosis in 29 clinically diagnosed DM cases.3. TP-PCR analysis was performed for molecular diagnosis in 29 clinically diagnosed DM cases.Results1. There were 14 familial and 12 sporadic cases in the 26 DM1 cases. The clinical manifestations of these cases included muscle weakness, amyotrophy, myotonia and involvement of multisystem.2. Molecular diagnosis of DM1(1) CTG repeats on DMPK were amplified by PCR. Electrophoretogram of PCR products showed two normal size stripes in 1 cases of the 29 suspected cases, one normal size stripe in the rest and two normal size stripes in one congenital myotonia patient. One normal size stripe was found in 2 normal controls and two normal size stripes in the other 28 normal controls.(2) Southern blot was performed with digoxin labelled probe. A 3400bp stripe was found in all tested samples. One 4000bp stripe was found in one sample and a smear larger than 3400bp was found in another 3 samples. The results of the 4 cases were positive which were in agreement with those provided by TP-PCR. (3) Southern blot was performed withα-32P labelled probe. A normal size stripe with 3400bp and abnormal amplified stripe between 3400bp and 4400bp were found in 4 DM1 cases which had been confirmed by TP-PCR. Only one normal size stripe with 3400bp was found in one clinically diagnosed DM case and his parents. TP-PCR results also denied DM1 in the 3 cases. (4) DMPK gene was analyzed through TP-PCR. There existed 26 positive results in all 29 clinically suspected DM cases and 30 negative results in all normal controls. (5)CCTG repeated sequence on ZNF9 was amplified by PCR. Diagnosis of DM2 was ruled out in consideration of DM1 molecular diagnosis results in all cases.Conclusions1. There are currently two clinically and molecularly defined forms of DM:DM1 and DM2. Complicated clinical manifestations and differences of phenotype make it difficult to confirm the diagnosis of DM. Characteristics of adult onset DM1 includes insidious onset in young and middle-aged, slowing course, positive family history. Amyotrophy, weakness and myotonia are showed in multiunit musculature, particularly affecting muscles of face, neck and distal extremities. Extensors are more severely involved than flexors. Multisystemic involvements are showed. Premature alopecia in male patient is an outstanding signs of DM1. EMG shows myotonia potentials and myogenic damage in DM1. The characteristic of biopsy of DM1 are the findings of ingression of nuclear, centronucleus, sarcoplasmic masses, atrophy of fiber type 1 and hypertrophy of fiber type 2.2. Abnormal amplified simple tandem repeats with huge length contain high GC level. Most DM 1 patients can not been confirmed by PCR because of preponderant amplification. In our study routine PCR combined with genomic DNA southern blot and TP-PCR had been used and 26 DM1 were confirmed. This demonstrates that TP-PCR is a robust, reliable, easily performed and qualitative molecular diagnosis. The successful southern blot showed that the probe we prepared through PCR was effective. It merits more evaluation.