The Clinical, Pathological And Genetic Characteristics Of Oculopharyngeal Myopathy Syndrome

Oculopharyngeal myopathy syndrome is a series of muscular diseases, mainly involving ocular and pharyngeal muscles. It’s characterized by ptosis, ophthalmoplegia, dysphagia, with or without limb muscle weaknesss, including oculopharyngeal muscular dystrophy (OPMD) and oculopharyngodistal myopathy (OPDM). Though chronic progressive external ophthalmoplegia (CPEO) may share similar phenotype, it’s excluded owing to specific mitochondrial changes in myopathology.OPMD is a late-onset neuromuscular disorder characterized by progressive ptosis, dysphagia and proximal muscle weakness. It’s inherited usually in an autosomal dominant pattern, and rarely in an autosomal recessive pattern. Myopathology shows myopathic changes with rimmed vacuoles. In 1980, Tome et al. revealed unique nuclear tubular filamentous inclusions in skeletal muscle fibers, which were considered as its pathological golden criteria. In 1998, Brais et al. identified that the underlying cause was the abnormal (GCN) expansion in exon 1 of the polyadenylate binding protein nuclear 1 gene (PABPN1). OPMD has a worldwide distribution, and at least 300 papers from 35 countries have been published. The incidence of OPMD in Europe is about 1:100000 and cases are distributed evenly. In America, most cases are in USA and Canada. It appears to be sparse among East Asians, with most families being reported in Japan. Only rare cases have been reported in Chinese population.OPDM has been delineated as a rare adult-onset muscular disorder with putative autosomal dominant or recessive inheritance. It is characterized by ptosis, ophthalmoplegia, dysphagia and distal limb muscle weakness. In 1998, Umaya et al. manifested that the cardinal pathologic finding was muscle fibers containing rimmed vacuoles. By electron microscope,15-18nm sarcoplasmic tubular filamentous inclusions could be found, which is non-specific and could also be seen in inclusion body myositis and hereditary inclusion body myopathy. The causative gene has not been identified yet. Hence, the diagnosis of OPDM has to rely on the clinical manifestation and exclusion of other diseases. The lack of causative gene limits the recognition of OPDM. In 2011, Durmus et al. reported the largest series of 47 patients from 9 Turkish families. Besides, only several families scattered in Japan, England, Italy and China with OPMD being excluded genetically.In this study, we summarized detailed clinical, pathological and genetic findings for OPMD and OPDM. These findings prompted us to gain more insight into the diagnosis and differential diagnosis.PART Ⅰ Clinical Features of Oculopharyngeal Myopathy SyndromeAll 18 unrelated families were recruited through the Neurology Department of Qilu Hospital of Shandong University and Peking University First Hospital from 1995 to 2013. Muscle biopsy and PABPN1 gene screening were performed on all probands. Thirteen families with (GCN) expansion in exon 1 of PABPN1 gene were diagnosed as OPMD, including 34 patients. The other 5 families without PABPN1 mutations were diagnosed as OPDM, including 21 patients.For our OPMD cohort, the mean age at onset was 47.2±11.2 years (range,27-67 years). Dysphagia was the most common initial symptom, accounting for 53%(18/34), with the mean age at onset of 44.8±10.7 years. Ptosis was the initial symptom in 9 out of 34 cases, with the mean age at onset of 57.8±7.7 years. There was a significant difference between the age at onset in the dysphagia group with that in the ptosis group as studied by Mann-Whitney U test (P=0.0036).The mean disease duration was 15.5±12.6 years (range,1-53 years) at the time of the last examination. Twenty-seven of 34 cases had ptosis, with visual impairment caused by the ptosis being the main complaint in our cohort. Dysphagia occurred in 30 out of 34 cases. Ophthalmoplegia was present in 18 out of 34 cases. Fourteen out of 34 patients had predominantly proximal limb weakness. Our cohort showed intrafamilial homogeneity on initial symptom and occurrence of limb muscle weakness.For our OPDM cohort, the mean age at onset was 26.4 years (range,22-39 years). Limb muscle weakness was the most common initial symptom, accounting for 38% (8/21). The mean disease duration was 15.6±8.99 years (range,1-34 years) at the time of the last examination. Ptosis and ophthamoplegia presented in 20 out of 21 cases. Dysphagia occurred in 11 out of 21 cases,4 of which had to feed on semi-liquid diets in advanced stage. Predominantly distal limb weakness was present in 12 out of 21 cases,5 of which were wheelchair-bound 12 to 15 years after onset. Patients with limb muscle weakness at onset showed a more rapid progress. Two patients died at the age of 40 due to pneumonia and malnutrition,17 years after onset. Intrafamilial homogeneity was found in our OPDM cohort on age at onset, initial symptom and/or progress.PART Ⅱ Pathological Features of Oculopharyngeal Myopathy SyndromeAll probands form 18 families had muscle biopsy. For histological and enzyme histochemistry examination, serial sections were stained with hematoxylin-eosin (H&E), modified Gomori trichrome (MGT), periodic acid Schiff (PAS), oil red O (ORO), nicotinamide adenine dinucleotide tetrazolium dehydrogenase (NADH-TR), succinate dehydrogenase (SDH), cytochrome c oxidase (COX), and adenosine triphosphatase (ATPase; pH 4.3,4.6 and 10.8). Immunohistochemistry study was performed according to standard protocol, with antibodies against dystrophin-R/N/C terminal, dysferlin, α/β/γ/δ-sarcoglycan and caveolin-3. Samples from 7 OPMD probands and 3 OPDM probands were examined by transmission electron microscopy. Muscle samples with normal histology were used as controls in above studies.Muscle biopsies revealed myopathic changes with rimmed vacuoles in both OPMD and OPDM under light microscope. The mean frequencies of rimmed vacuoles were 1.1 ±0.9% in OPMD and 4.2±2.1% in OPDM. There was a significant difference between the mean frequencies of rimmed vacuoles in OPMD group with that in the OPDM group as studied by Mann-Whitney U test (P=0.0159). Compared to OPMD, the myopathic damages were more prominent in OPDM, with atrophic fibers in small groups, necrotic fibers with phagocytosis and significant interstitial hyperplasia in some cases. There was no remarkable finding for immunohistochemistry study. By electron microscopy, intranuclear inclusions formed by tubular filaments were found in 5 of 7 OPMD probands, with the outer diameter being around 8.5nm. We failed to find intranuclear or sarcoplasmic inclusions in OPDM probands.PART Ⅲ Genetic Analysis of Oculopharyngeal Muscular DystrophyGenetic analysis of 13 unrelated families revealed 7 genotypes, of which pure (GCG) expansions of the PABPN1 gene in 10 families; specifically, we saw (GCG)8 in 1 family, (GCG)9 in 6 families, (GCG)10 in 2 families, and (GCG)11 in 1 family. We detected (GCA) triplet interspersions in 3 families. The (GCG)6(GCA)1(GCG)3(GCA)3(GCG)1 allele was found in 2 families. In family 1, the proband was a compound heterozygote with (GCG)6(GCA)1 (GCA)3(GCG),/(GCG)6(GCA)1(GCG)1 (GCA)3(GCG),. Her elder sister had one normal allele and the mutated allele of (GCG)6(GCA)1(GCG)1(GCA)3(GCG)1. Her younger brother, who carried the (GCG)6(GCA)1(GCA)3(GCG)i allele, was asymptomatic.Although cryptic (GCN)11 has been widely referred to as the polymorphic allele, to the best of our knowledge, (GCG)6(GCA)1(GCA)3(GCG)1 was first described here.There was no difference in phenotype between patients with pure (GCG) expansions and those with (GCA) interspersions. As for the correlation between severity of the disease and the length of the repeats, no conclusion could be drawn based on our patients. In family 1, the proband with (GCN)11/(GCN)12 had a more severe phenotype than her sister with (GCN)10/(GCN)12, which suggests (GCN)11 polymorphism may act as a modifier.PART Ⅳ Study of the Causative Gene for Ocuolopharyngodistal MyopathyAlthough exome only account for 1% of the genome sequences, but it contains the main information for encoding and regulating protein synthesis. For diseases inherited in a Mendelian genetic model, more than 85% of the causative mutations are located in exome. Up to now, whole exome sequencing technology (WES) has successfully identified more than 100 previously unknown causative genes for rare diseases, complex diseases, cancer and so on. The causative gene of OPDM is still unknown and rarely studied, which may be limited by the lack of samples. Compared with linkage analysis and whole genome sequencing, the outstanding advantage of WES is that it may identify the causative gene based on small families, or even sporadic cases. Therefore, we chose WES to explore the causative gene in OPDM.Two patients (Ⅲ7, Ⅲ9) and 1 normal control (the father of Ⅲ9) from family 2 were selected for whole exome sequencing, with genomic DNA being extracted from their whole peripheral blood. Then we construct a dominant model in statistical analysis to identify possible causative gene. Other family members and family 4 were used for further confirmation.An average of 105641 genetic variations per subject were generated, including non-synonymous mutations, splice site variations, and insertions or deletions (indels). The list of candidate variants was reduced to 52 non-synonymous mutations and 3 indels after subsequent data analysis and functional prediction. Unfortunately, we failed to identify the causative gene of OPDM.Conclusions1. OPMD and OPDM are two different disease entities. Dysphagia was the most common initial symptom in our OPMD cohort. The patients with dysphagia as the initial symptom had a much earlier onset compared with those with ptosis as the initial symptom (P=0.0036). In our OPDM cohort, patients with limb muscle weakness as the initial symptom may indicate a bad prognosis. Phenotypic intrafamilial homogeneity exists in both OPMD and OPDM cohort, which suggests that other genetic factors may contribute to modification of the phenotype.2. Compared to OPMD, rimmed vacuoles were more common in OPDM (F=0.0159).3. The heterogeneous genetic basis of OPMD implies that there is no single founder effect in Chinese population. Our data also lend support to the point that (GCN)11 polymorphism may have a disease-modifying effect.4. We failed to identity the causative gene for OPDM by WES. Short tendem repeat and copy number variation, which are beyond the capacity of exome sequencing, need to be considered. Besides, the causative gene may lie in the non-coding sequences.