Mutational spectrum of Chinese LGMD patients by targeted next-generation sequencing

<div><p>This study aimed to study the diagnostic value of targeted next-generation sequencing (NGS) in limb-girdle muscular dystrophies (LGMDs), and investigate the mutational spectrum of Chinese LGMD patients. We performed targeted NGS covering 420 genes in 180 patients who were consecutively suspected of LGMDs and underwent muscle biopsies from January 2013 to May 2015. The association between genotype and myopathological profiles was analyzed in the genetically confirmed LGMD patients. With targeted NGS, one or more rare variants were detected in 138 patients, of whom 113 had causative mutations, 10 sporadic patients had one pathogenic heterozygous mutation related to a recessive pattern of LGMDs, and 15 had variants of uncertain significance. No disease-causing mutation was found in the remaining 42 patients. Combined with the myopathological findings, we achieved a positive genetic diagnostic rate as 68.3% (123/180). Totally 105 patients were diagnosed as LGMDs with genetic basis. Among these 105 patients, the most common subtypes were LGMD2B in 52 (49.5%), LGMD2A in 26 (24.8%) and LGMD 2D in eight (7.6%), followed by LGMD1B in seven (6.7%), LGMD1E in four (3.8%), LGMD2I in three (2.9%), and LGMD2E, 2F, 2H, 2K, 2L in one patient (1.0%), respectively. Although some characteristic pathological changes may suggest certain LGMD subtypes, both heterogeneous findings in a certain subtype and overlapping presentations among different subtypes were not uncommon. The application of NGS, together with thorough clinical and myopathological evaluation, can substantially improve the molecular diagnostic rate in LGMDs. Confirming the genetic diagnosis in LGMD patients can help improve our understanding of their myopathological changes.</p></div

Histopathological abnormalities in different LGMD subtypes^a.

<p>Histopathological abnormalities in different LGMD subtypes<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175343#t001fn001" target="_blank">^a</a>.</p

Immunohistochemical findings in different LGMD subtypes^a.

<p>Immunohistochemical findings in different LGMD subtypes<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175343#t002fn001" target="_blank">^a</a>.</p

Flowchart of patients’ enrollment.

<p>Flowchart of patients’ enrollment.</p

Myopathological changes of LGMD patients.

<p>A. NADH-TR staining shows lobulated fibers (arrow) in a LGMD2B patient. B. MGT staining shows red-ragged fibers (arrow) in a LGMD2B patient. C. MGT staining shows nemaline bodies (arrow) in a LGMD2A patient. D. NADH-TR staining shows multi-minicores (arrow) in a LGMD2A patient. E. Immunohistochemical labelling of N-terminal dystrophin shows decreased or absent expression (asterisk) in a LGMD2A patient. F. Immunohistochemical labelling of dysferlin shows decreased expression (asterisk) in a LGMD2A patient. Abbreviations: LGMD = limb-girdle muscular dystrophy; NADH-TR = nicotinamide adenine dinucleotide-tetrazolium reductase; MGT = modified Gomori trichrome.</p

Image_1_Comparison of cytokine/chemokine profiles between dermatomyositis and anti-synthetase syndrome.TIF

ObjectivesDermatomyositis (DM) and anti-synthetase syndrome (ASS) are autoimmune diseases with multisystem involvement. Despite sharing some clinical and myopathological features, these are two diseases with different pathogeneses and prognoses. We aimed to clarify and compare cytokine/chemokine profiles in both disorders, which may help in the differential diagnosis.Materials and methodsWe collected clinical data and serum samples of consecutive patients with DM and ASS. Quantibody® Human Inflammation Array 3 for cytokines/chemokines was performed in the serum of all participants. Receiver operating characteristic analysis with the area under the curve and Youden's index were performed.ResultsEight newly diagnosed and treatment-naïve patients with DM, nine newly diagnosed and treatment-naïve patients with ASS, and 14 healthy controls were enrolled. Serum C-C motif chemokine ligand (CCL) 2, CCL4, C-X-C motif chemokine ligand (CXCL) 13, and tumor necrosis factor receptor 2 (TNFR2) were increased in patients with both DM and ASS. Serum interleukin (IL)-1 receptor type 1 (IL-1ra), IL-1b, CCL1, CXCL11, and CCL3 were modulated in patients with DM only, and IL-8, CXCL9, and tissue inhibitors of metalloproteinases-1 (TIMP-1) in patients with ASS only. Serum CCL2, CXCL13, and TNFR2 accurately distinguished patients with DM and ASS from healthy controls, as shown by the area under the curve >0.80. Moreover, receiver operating characteristic analysis showed that, as biomarkers for discrimination between DM and ASS, the combination of IL-1ra and TIMP-1, had an area under the curve of 0.944, a sensitivity of 87.5%, and a specificity of 88.9%.ConclusionOur study demonstrated that serum levels of cytokines/chemokines showed a different pattern in newly diagnosed patients with DM and ASS, in which serum IL-1ra and TIMP-1 could be used to distinguish between the two diseases.</p

Image_2_Comparison of cytokine/chemokine profiles between dermatomyositis and anti-synthetase syndrome.TIF

ObjectivesDermatomyositis (DM) and anti-synthetase syndrome (ASS) are autoimmune diseases with multisystem involvement. Despite sharing some clinical and myopathological features, these are two diseases with different pathogeneses and prognoses. We aimed to clarify and compare cytokine/chemokine profiles in both disorders, which may help in the differential diagnosis.Materials and methodsWe collected clinical data and serum samples of consecutive patients with DM and ASS. Quantibody® Human Inflammation Array 3 for cytokines/chemokines was performed in the serum of all participants. Receiver operating characteristic analysis with the area under the curve and Youden's index were performed.ResultsEight newly diagnosed and treatment-naïve patients with DM, nine newly diagnosed and treatment-naïve patients with ASS, and 14 healthy controls were enrolled. Serum C-C motif chemokine ligand (CCL) 2, CCL4, C-X-C motif chemokine ligand (CXCL) 13, and tumor necrosis factor receptor 2 (TNFR2) were increased in patients with both DM and ASS. Serum interleukin (IL)-1 receptor type 1 (IL-1ra), IL-1b, CCL1, CXCL11, and CCL3 were modulated in patients with DM only, and IL-8, CXCL9, and tissue inhibitors of metalloproteinases-1 (TIMP-1) in patients with ASS only. Serum CCL2, CXCL13, and TNFR2 accurately distinguished patients with DM and ASS from healthy controls, as shown by the area under the curve >0.80. Moreover, receiver operating characteristic analysis showed that, as biomarkers for discrimination between DM and ASS, the combination of IL-1ra and TIMP-1, had an area under the curve of 0.944, a sensitivity of 87.5%, and a specificity of 88.9%.ConclusionOur study demonstrated that serum levels of cytokines/chemokines showed a different pattern in newly diagnosed patients with DM and ASS, in which serum IL-1ra and TIMP-1 could be used to distinguish between the two diseases.</p

Table_1_Comparison of cytokine/chemokine profiles between dermatomyositis and anti-synthetase syndrome.DOCX

ObjectivesDermatomyositis (DM) and anti-synthetase syndrome (ASS) are autoimmune diseases with multisystem involvement. Despite sharing some clinical and myopathological features, these are two diseases with different pathogeneses and prognoses. We aimed to clarify and compare cytokine/chemokine profiles in both disorders, which may help in the differential diagnosis.Materials and methodsWe collected clinical data and serum samples of consecutive patients with DM and ASS. Quantibody® Human Inflammation Array 3 for cytokines/chemokines was performed in the serum of all participants. Receiver operating characteristic analysis with the area under the curve and Youden's index were performed.ResultsEight newly diagnosed and treatment-naïve patients with DM, nine newly diagnosed and treatment-naïve patients with ASS, and 14 healthy controls were enrolled. Serum C-C motif chemokine ligand (CCL) 2, CCL4, C-X-C motif chemokine ligand (CXCL) 13, and tumor necrosis factor receptor 2 (TNFR2) were increased in patients with both DM and ASS. Serum interleukin (IL)-1 receptor type 1 (IL-1ra), IL-1b, CCL1, CXCL11, and CCL3 were modulated in patients with DM only, and IL-8, CXCL9, and tissue inhibitors of metalloproteinases-1 (TIMP-1) in patients with ASS only. Serum CCL2, CXCL13, and TNFR2 accurately distinguished patients with DM and ASS from healthy controls, as shown by the area under the curve >0.80. Moreover, receiver operating characteristic analysis showed that, as biomarkers for discrimination between DM and ASS, the combination of IL-1ra and TIMP-1, had an area under the curve of 0.944, a sensitivity of 87.5%, and a specificity of 88.9%.ConclusionOur study demonstrated that serum levels of cytokines/chemokines showed a different pattern in newly diagnosed patients with DM and ASS, in which serum IL-1ra and TIMP-1 could be used to distinguish between the two diseases.</p

Table_2_Comparison of cytokine/chemokine profiles between dermatomyositis and anti-synthetase syndrome.docx

ObjectivesDermatomyositis (DM) and anti-synthetase syndrome (ASS) are autoimmune diseases with multisystem involvement. Despite sharing some clinical and myopathological features, these are two diseases with different pathogeneses and prognoses. We aimed to clarify and compare cytokine/chemokine profiles in both disorders, which may help in the differential diagnosis.Materials and methodsWe collected clinical data and serum samples of consecutive patients with DM and ASS. Quantibody® Human Inflammation Array 3 for cytokines/chemokines was performed in the serum of all participants. Receiver operating characteristic analysis with the area under the curve and Youden's index were performed.ResultsEight newly diagnosed and treatment-naïve patients with DM, nine newly diagnosed and treatment-naïve patients with ASS, and 14 healthy controls were enrolled. Serum C-C motif chemokine ligand (CCL) 2, CCL4, C-X-C motif chemokine ligand (CXCL) 13, and tumor necrosis factor receptor 2 (TNFR2) were increased in patients with both DM and ASS. Serum interleukin (IL)-1 receptor type 1 (IL-1ra), IL-1b, CCL1, CXCL11, and CCL3 were modulated in patients with DM only, and IL-8, CXCL9, and tissue inhibitors of metalloproteinases-1 (TIMP-1) in patients with ASS only. Serum CCL2, CXCL13, and TNFR2 accurately distinguished patients with DM and ASS from healthy controls, as shown by the area under the curve >0.80. Moreover, receiver operating characteristic analysis showed that, as biomarkers for discrimination between DM and ASS, the combination of IL-1ra and TIMP-1, had an area under the curve of 0.944, a sensitivity of 87.5%, and a specificity of 88.9%.ConclusionOur study demonstrated that serum levels of cytokines/chemokines showed a different pattern in newly diagnosed patients with DM and ASS, in which serum IL-1ra and TIMP-1 could be used to distinguish between the two diseases.</p

Table_3_Comparison of cytokine/chemokine profiles between dermatomyositis and anti-synthetase syndrome.pdf

ObjectivesDermatomyositis (DM) and anti-synthetase syndrome (ASS) are autoimmune diseases with multisystem involvement. Despite sharing some clinical and myopathological features, these are two diseases with different pathogeneses and prognoses. We aimed to clarify and compare cytokine/chemokine profiles in both disorders, which may help in the differential diagnosis.Materials and methodsWe collected clinical data and serum samples of consecutive patients with DM and ASS. Quantibody® Human Inflammation Array 3 for cytokines/chemokines was performed in the serum of all participants. Receiver operating characteristic analysis with the area under the curve and Youden's index were performed.ResultsEight newly diagnosed and treatment-naïve patients with DM, nine newly diagnosed and treatment-naïve patients with ASS, and 14 healthy controls were enrolled. Serum C-C motif chemokine ligand (CCL) 2, CCL4, C-X-C motif chemokine ligand (CXCL) 13, and tumor necrosis factor receptor 2 (TNFR2) were increased in patients with both DM and ASS. Serum interleukin (IL)-1 receptor type 1 (IL-1ra), IL-1b, CCL1, CXCL11, and CCL3 were modulated in patients with DM only, and IL-8, CXCL9, and tissue inhibitors of metalloproteinases-1 (TIMP-1) in patients with ASS only. Serum CCL2, CXCL13, and TNFR2 accurately distinguished patients with DM and ASS from healthy controls, as shown by the area under the curve >0.80. Moreover, receiver operating characteristic analysis showed that, as biomarkers for discrimination between DM and ASS, the combination of IL-1ra and TIMP-1, had an area under the curve of 0.944, a sensitivity of 87.5%, and a specificity of 88.9%.ConclusionOur study demonstrated that serum levels of cytokines/chemokines showed a different pattern in newly diagnosed patients with DM and ASS, in which serum IL-1ra and TIMP-1 could be used to distinguish between the two diseases.</p