Background/Purpose:

To test the hypothesis that viruses are associated with Sjogren’s syndrome, salivary gland RNA extracts from Sjogren’s patients and non-Sjogren’s controls were screened for virus-associated sequences using a multiplexed NanoString nCounter chip designed to detect sequences from all known human viral and endogenous retroviral families.

Methods:

Samples were obtained following IRB approved protocols from study subjects recruited at the rheumatology clinics of The First Afflicted Hospital, Xiamen University, China who had minor salivary gland biopsies performed as part of routine clinical care.  Using the classification criteria from the American-European Consensus Group, subjects were classified as having (cases, N = 12) or not having Sjogren’s Syndrome.  Subjects who did not meet Sjogren’s classification criteria, and who did not have any salivary inflammatory process were designated the controls (N = 7). RNA was extracted from a 10 μm whole FFPE tissue section for each sample on a Siemens VERSANT robotic kPCR Molecular System. RNA was then directly assayed by Nanostring nCounter gene expression system for 334 viral, retroviral, and human immune gene sequences at once.

Results:

Cases (11 female, 8 primary SS, 4 in RA) were confirmed by serology, histopathology, and parotid sialography.  Controls were 7 patients (6 female) with normal histopathology, including 4 with negative ANA, SS-A, and SS-B. RNA extract quality and nCounter internal controls were robust for all tested samples.  Gene expression in all samples was normalized to GAPDH expression.  Cases and controls were compared for expression levels of the studied sequences.  The 45 genes with the highest fold difference between cases and controls were all increased in the Sjogren’s cases.  Genes represented in this group included primarily HPV and polyoma-derived viral sequences but not endogenous retroviruses.  The SS group had two distinct subsets, one of which (N = 6) showed increased expression of multiple viral genes compared to controls, and one of which (N = 6) showed reduced expression of multiple viral genes compared to controls.  Subgroup analysis of the cases with high viral expression revealed multiple HPV Type 35 and 56 genes that were at least 2 fold overexpressed compared to controls.  CD79a was the only tested gene at least 2 fold upregulated in the patients without high viral gene expression when compared to controls.  A trend toward more Primary SS was present in patients with high viral expression (5/6 vs 2/6 for other SS, Fisher’s Exact p = 0.2).

Conclusion:

Using a new highly sensitive and specific technique, we find evidence of overexpressed viral elements in salivary tissue in a subset of a Chinese Sjogren’s Syndrome cohort.  This data increases scrutiny of HPV and polyoma subtypes for potential pathogenic roles in SS, but does not support a role for endogenous retroviruses.  This work also for the first time identifies two distinct subsets of SS patients with regard to salivary viral expression.

 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multiplexed-nanostring-screening-for-salivary-gland-viral-elements-in-sjogrens-syndrome/