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We have read the insightful comments on our article "RNA-Seq analysis reveals sex-dependent transcriptomic profiles of human subacromial bursa stratified by tear etiology" published in the Journal of Orthopaedic Research.1 We thank the authors of the I Letter to the Editor i 2 for their interest in our article and have decided to carefully respond to their concerns. The plot indicates moderate to highly up- and downregulated genes with log2 fold-changes >= |2| in females with traumatic injuries over females with degenerative injuries with modest levels of statistical significance p-value <= 0.05 are distinct from those genes with low to no statistical significance and log2 fold-changes <= |2|. Mean log2 counts-per-million to log2 fold-change amplitude plot illustrating the range of statistically significant genes with log2 fold-changes >= |2|. [Color figure can be viewed at wileyonlinelibrary.com] gl Second, the authors of letter noted that samples #15 (female degenerative) and #29 (female traumatic) expressed high levels of genes located on the Y chromosome which were expressed at the same level as their male counterparts. [Extracted from the article]

Rotator cuff tendinopathy, a major cause of shoulder disability, occurs due to trauma or degeneration. Our molecular understanding of traumatic and degenerative tears remains elusive. Here, we probed transcript level differences between traumatic and degenerative tears. Subacromial bursa tissues were collected from patients with traumatic or degenerative tears during arthroscopy (N = 32). Transcripts differentially expressed by tear etiology were detected by RNA‐seq. RNA‐seq results were validated by real‐time quantitative polymerase chain reaction. We identified 334 protein‐coding transcripts differentially expressed between traumatic and degenerative tears in females and 167 in males at a fold‐change greater than 2. In females, XIRP2, MYL1, MYBPC1, TNNT1, and LMOD2, were highly expressed in traumatic tears whereas TPSD1, CDSN, RCVRN, LTBP4, and PTGS1 were elevated in degen tears. Transcripts elevated in traumatic tears represented muscle cell differentiation and development, and muscle contraction whereas those elevated in degenerative tears represented cell activation and immune response. In males, AZGP1, CNTFR, COL9A1, ZNF98, and EREG were highly elevated in traumatic tears whereas MYL2, HOXD11, SLC6A7, CADM1, and MMP17 were highly expressed in degenerative tears. Transcripts elevated in traumatic tears represented metabolic/catabolic processes, and transmembrane protein transport while processes related to cell cycle were mainly enriched in degenerative tears. Numerous long noncoding RNAs were differentially expressed between traumatic and degenerative tears in both sexes. In summary, this study provides insights into molecular biology of bursa in patients with rotator cuff tendon disease based on tear acuity and novel sex‐based transcript differences that could inform clinical decision making in treating patients with traumatic or degenerative shoulder injuries. [ABSTRACT FROM AUTHOR]

Rotator cuff tears are a common pathology in the shoulder and generally have two underlying etiologies: traumatic and degenerative. Little is known about the molecular underpinning of these etiologies. Here we queried transcript level differences in tear etiology stratified by sex in 31 patients with rotator cuff tears. Tendon tissues were isolated from females (N = 16) and males (N = 15) with traumatic (N = 16) or degenerative (N = 15) tears during arthroscopy. Differentially expressed transcripts were identified by RNA‐seq and biological processes were probed computationally. Expression of some transcripts was validated by real‐time quantitative polymerase chain reaction (qPCR). We identified 339 and 336 transcripts differentially expressed by tear etiology in females and males, respectively, at a fold‐change greater than |2|. In females, GSTM1, MT1G, S1008A, ACSM3, DSC, FAM110C, and VNN2 were elevated in traumatic tears representing metabolic/catabolic processes, and immune response whereas CHAD, CLEC3A, IBSP, TNMD, APLNR, and CPA3 were elevated in degenerative tears representing tissue morphogenesis and developmental processes, angiogenesis, and extracellular matrix organization. In males, ELOA3B, CXCL8, ADM, TNS4, and SPOCK1 were elevated in traumatic tears representing localization of endoplasmic reticulum, chromosome organization, leukocyte/neutrophil degranulation, and protein transport whereas MYL2, TNNC1, MB, CPA3, APLNR, and CA3 were highly upregulated in degenerative tears representing muscle cell differentiation and development and angiogenesis. Numerous novel lncRNAs were identified to be differentially expressed by tear etiology in both sexes. Real‐time qPCR confirmed RNA‐seq data. This study improves our understanding of tendon biology based on underlying etiology (trauma or degeneration) in a sex‐specific manner. These findings may help drive clinical decision‐making in females and males with traumatic and degenerative shoulder injuries. [ABSTRACT FROM AUTHOR]

Anterior cruciate ligament (ACL) tears occur in isolation or in tandem with other intra‐articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of the injured ACL is unknown. Here, we tested the hypothesis that the biological response of the ACL to injury varies based on the presence or absence of concomitant meniscus tear. We performed RNA‐seq on 28 ACL tears remnants (12 isolated, 16 combined). In total, 16,654 transcripts were differentially expressed between isolated and combined injury groups at false discovery rate of 0.05. Due to the large number of differentially expressed transcripts, we undertook an Ensembl approach to discover features that acted as hub genes that did not necessarily have large fold changes or high statistical significance, but instead had high biological significance. Our data revealed a negatively correlated module containing 5,960 transcripts (down‐regulated in combined injury) and a positively correlated module containing 2,260 transcripts (up‐regulated in combined injury). TNS1, MEF2D, NOTCH3, SOGA1, and MLXIP were highly‐connected hub genes in the negatively correlated module and SCN2A, CSMD3, LRC44, USH2A, and LRP1B were critical hub genes in the positively correlated module. Transcripts in the negatively correlated module were associated with biological adhesion, actin‐filament organization, cell junction assembly, and cell matrix adhesion. The positively correlated module transcripts were enriched for neuron migration and exocytosis regulation. These findings indicate genes and pathways reflective of healing deficiency and gain of neurogenic signaling in combined ACL and meniscus tears, suggesting their diminished repair potential. The biological response of ACL to injury could have implications for healing potential of the ligament and the long term health of the knee. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2612–2621, 2018. Anterior cruciate ligament (ACL) tears occur in isolation or with other intra‐articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of ACL tears is unknown. RNA‐seq identifies genes and pathways demonstrating significant healing deficiency in combined ACL and meniscus tears compared to isolated ACL tears. The impact of injury pattern on the biological response of ACL to injury could have implications for healing potential of the ligament and long‐term knee health. [ABSTRACT FROM AUTHOR]

ABSTRACT: Microarrays and RNA‐seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA‐seq and microarrays for genome‐wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA‐seq and microarrays and to assess potential benefits of RNA‐seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time‐from‐injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA‐seq (N = 8). The correlation of biological replicates in RNA‐seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA‐seq data and the individual microarrays were low, but correlations between the RNA‐seq values and the geometric mean of the microarrays values were moderate. The cross‐platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA‐Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA‐seq and microarrays data. These findings demonstrate that RNA‐seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484–497, 2018. [ABSTRACT FROM AUTHOR]