Technological readiness and implementation of genomic-driven precision medicine for complex diseases.

The fields of human genetics and genomics have generated considerable knowledge about the mechanistic basis of many diseases. Genomic approaches to diagnosis, prognostication, prevention and treatment - genomic-driven precision medicine (GDPM) - may help optimize medical practice. Here, we provide a comprehensive review of GDPM of complex diseases across major medical specialties. We focus on technological readiness: how rapidly a test can be implemented into health care. Although these areas of medicine are diverse, key similarities exist across almost all areas. Many medical areas have, within their standards of care, at least one GDPM test for a genetic variant of strong effect that aids the identification/diagnosis of a more homogeneous subset within a larger disease group or identifies a subset with different therapeutic requirements. However, for almost all complex diseases, the majority of patients do not carry established single-gene mutations with large effects. Thus, research is underway that seeks to determine the polygenic basis of many complex diseases. Nevertheless, most complex diseases are caused by the interplay of genetic, behavioural and environmental risk factors, which will likely necessitate models for prediction and diagnosis that incorporate genetic and non-genetic data.

Population-based study of patients with primary Sjögren's syndrome and lymphoma: lymphoma subtypes, clinical characteristics, and gender differences.

Objective: To examine lymphoma subtypes, clinical characteristics, and gender differences in patients with primary Sjögren's syndrome (pSS) and lymphoma in a population-based setting.Method: Patients with Sjögren's syndrome and lymphoma diagnoses were identified by linkage of the Swedish Patient Register 1964-2007 with the Cancer Register 1990-2007. Clinical data were collected from medical records and lymphoma tissues were re-examined. The lymphoma subtype distribution was compared with the Swedish Lymphoma Register.Results: We identified 105 pSS patients with lymphoma. Diffuse large B-cell lymphoma (DLBCL) (32%) and marginal zone lymphoma [MZL including mucosa-associated lymphoid tissue (MALT) lymphoma] (31%) were the most common lymphoma subtypes. The proportion of DLBCL was not increased compared to the general population reference (32%, p = 1), in contrast to MZL (general population 5%, p < 0.0001). Compared to DLBCL, MALT lymphoma was diagnosed at a younger age (55 vs 67 years, p = 0.0001), and earlier after patient-reported sicca onset (7 vs 18 years, p = 0.0001) and pSS diagnosis (2 vs 9 years, p = 0.0005). Sixteen of the pSS-lymphoma cases were men (15%), twice the proportion in general pSS populations. Compared to women, men had a shorter median time from pSS diagnosis to lymphoma diagnosis (1 vs 8 years, p = 0.0003) and more often had lymphoma in the salivary glands (56% vs 29%, p = 0.04).Conclusion: DLBCL and MZL are common in pSS patients, but only MZL/MALT lymphoma occurs at an increased relative frequency in pSS compared to the general population. The study supports increased awareness of signs of lymphoma in men in the first years after pSS diagnosis.

Concomitant Ro/SSA and La/SSB antibodies are biomarkers for the risk of venous thromboembolism and cerebral infarction in primary Sjögren's syndrome.

BACKGROUND: To assess the risk of incident cardiovascular disease in patients with primary Sjögren's syndrome, overall and stratified by Ro/SSA and La/SSB autoantibody status. METHODS: A cohort of patients with primary Sjögren's syndrome in Sweden (n = 960) and matched controls from the general population (n = 9035) were included, and data extracted from the National Patient Register to identify events of myocardial infarction, cerebral infarction and venous thromboembolism. Hazard ratios were estimated using cox proportional hazard regressions. RESULTS: During a median follow-up of 9.5 years, the overall hazard ratio (HR) was 1.6 (95% CI 1.2-2.1) for myocardial infarction, 1.2 (95% CI 0.9-1.7) for cerebral infarction and 2.1 (95% CI 1.6-2.9) for venous thromboembolism. Patients positive for both Ro/SSA and La/SSB autoantibodies had a substantially higher risk of cerebral infarction (HR 1.7, 95% CI 1.0-2.9) and venous thromboembolism (HR 3.1, 95% CI 1.9-4.8) than the general population. These risks were not significantly increased in Ro/SSA- and La/SSB-negative patients. Among autoantibody-positive patients, the highest HR of cerebral infarction was seen after ≥10 years disease duration (HR 2.8, 95% CI 1.4-5.4), while the HR for venous thromboembolism was highest 0-5 years after disease diagnosis (HR 4.7, 95% CI 2.3-9.3) and remained high throughout disease duration. CONCLUSIONS: Primary Sjögren's syndrome is associated with a markedly increased risk of cardiovascular disease and the presence of Ro/SSA and La/SSB autoantibodies identify the subgroup of patients carrying the highest risk. These findings suggest that monitoring and prevention of cardiovascular disease in this patient group should be considered.

Infections increase the risk of developing Sjögren's syndrome.

OBJECTIVE: Environmental factors have been suggested in the pathogenesis of rheumatic diseases. We here investigated whether infections increase the risk of developing primary Sjögren's syndrome (pSS). METHODS: Patients with pSS in Sweden (n = 945) and matched controls from the general population (n = 9048) were included, and data extracted from the National Patient Register to identify infections occurring before pSS diagnosis during a mean observational time of 16.0 years. Data were analysed using conditional logistic regression models. Sensitivity analyses were performed by varying exposure definition and adjusting for previous health care consumption. RESULTS: A history of infection associated with an increased risk of pSS (OR 1.9, 95% CI 1.6-2.3). Infections were more prominently associated with the development of SSA/SSB autoantibody-positive pSS (OR 2.7, 95% CI 2.0-3.5). When stratifying the analysis by organ system infected, respiratory infections increased the risk of developing pSS, both in patients with (OR 2.9, 95% CI 1.8-4.7) and without autoantibodies (OR 2.1, 95% CI 1.1-3.8), whilst skin and urogenital infections only significantly associated with the development of autoantibody-positive pSS (OR 3.2, 95% CI 1.8-5.5 and OR 2.7, 95% CI 1.7-4.2). Furthermore, a dose-response relationship was observed for infections and a risk to develop pSS with Ro/SSA and La/SSB antibodies. Gastrointestinal infections were not significantly associated with a risk of pSS. CONCLUSIONS: Infections increase the risk of developing pSS, most prominently SSA/SSB autoantibody-positive disease, suggesting that microbial triggers of immunity may partake in the pathogenetic process of pSS.

Comparison of patients with and without pre-existing lymphoma at diagnosis of primary Sjögren's syndrome.

OBJECTIVE: In the 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren's syndrome (pSS), pre-existing lymphoma is not an exclusion criterion for pSS diagnosis, as in earlier criteria. We aimed to explore whether there are differences between pSS patients with and without pre-existing lymphoma at pSS diagnosis. METHOD: Patients with ICD-7-10 codes for Sjögren's syndrome (SS) and a diagnosis of malignant lymphoma before or after SS diagnosis were identified by linking the Swedish Patient Register 1964-2007 with the Cancer Register 1990-2007 (n = 224). Clinical data were collected from medical records. Lymphoma diagnoses were evaluated by tissue review. Characteristics of pSS patients with and without pre-existing lymphoma were compared. RESULTS: We identified 107 patients with pSS as the reason for an SS diagnosis code and a verified lymphoma. Of these, 18 (17%) had a pre-existing lymphoma at pSS diagnosis, defined as lymphoma diagnosed before or within 6 months of pSS diagnosis. Male gender (39% vs 10%, p = 0.006), enlarged lymph nodes during the pSS disease (61% vs 27%, p = 0.01), mucosa-associated lymphoid tissue (MALT) lymphoma (50% vs 22%, p = 0.02), and salivary gland lymphoma (61% vs 26%, p = 0.006) were more common in patients with a pre-existing lymphoma at pSS diagnosis. Other pSS characteristics were similar. CONCLUSION: In a substantial proportion of patients, particularly in men, pSS remains undiagnosed until after lymphoma diagnosis. The study highlights the importance of pSS investigation in patients with lymphoma, especially MALT lymphoma, in the salivary glands.

Transcription profiling of peripheral B cells in antibody-positive primary Sjögren's syndrome reveals upregulated expression of CX3CR1 and a type I and type II interferon signature.

B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS). The aim of this study was to analyse the transcriptome of CD19+ B cells from patients with pSS and healthy controls to decipher the B cell-specific contribution to pSS. RNA from purified CD19+ B cells from 12 anti-SSA antibody-positive untreated female patients with pSS and 20 healthy blood donors was subjected to whole transcriptome sequencing. A false discovery rate corrected significance threshold of α < 0.05 was applied to define differential gene expression. As validation, gene expression in B cells from 17 patients with pSS and 16 healthy controls was analysed using a targeted gene panel. RNA-sequencing identified 4047 differentially expressed autosomal genes in pSS B cells. Upregulated expression of type I and type II interferon (IFN)-induced genes was observed, establishing an IFN signature in pSS B cells. Among the top upregulated and validated genes were CX3CR1, encoding the fractalkine receptor involved in regulation of B-cell malignancies, CCL5/RANTES and CCR1. Increased expression of several members of the TNF superfamily was also identified; TNFSF4/Ox40L, TNFSF10/TRAIL, TNFSF13B/BAFF, TNFRSF17/BCMA as well as S100A8 and -A9/calprotectin, TLR7, STAT1 and STAT2. Among genes with downregulated expression in pSS B cells were SOCS1 and SOCS3, CD70 and TNFAIP3/A20. We conclude that B cells from patients with anti-SSA antibody-positive pSS display immune activation with upregulated expression of chemokines, chemokine receptors and a prominent type I and type II IFN signature, while suppressors of cytokine signalling are downregulated. This adds insight into the autoimmune process and suggests potential targets for future functional studies.

Diminished CXCR5 expression in peripheral blood of patients with Sjögren's syndrome may relate to both genotype and salivary gland homing.

Genetic investigations of Sjögren's syndrome (SS) have identified a susceptibility locus at p23.3 of chromosome 11, which contains the CXCR5 gene. C-X-C motif chemokine receptor 5 (CXCR5) is a chemokine receptor expressed on B and T cell subsets, and binds the chemotactic ligand C-X-C motif chemokine ligand 13 (CXCL13). In this study we aimed to link the genetic association with functional effects and explore the CXCR5/CXCL13 axis in SS. Expression quantitative trait loci analysis of the 11q23.3 locus was performed using B cell mRNA expression data from genotyped individuals. Lymphocyte surface markers were assessed by flow cytometry, and CXCL13 levels by a proximity extension assay. CXCR5+ and CXCL13+ cells in minor salivary glands were detected using immunohistochemistry. Our results demonstrated that SS-associated genetic polymorphisms affected the expression of CXCR5 (P < 0·01). Notably, a decreased percentage of CXCR5+ cells, with lower CXCR5 expression, was observed for most circulating B and T cell subsets in SS patients, reaching statistical significance in CD19+ CD27+ immunoglobulin (Ig)D+ marginal zone (P < 0·001), CD19+ CD27+ IgD- memory (P < 0·05) and CD27-IgD double-negative (P < 0·01) B cells and CD4+ CXCR3- CCR6+ Th17 cells (P < 0·05). CXCL13 levels were increased in patient plasma (P < 0·001), and immunohistochemical staining revealed expression of CXCL13 and higher numbers of CXCR5+ cells (P < 0·0001) within focal infiltrates and interstitially in salivary glands of SS patients. In conclusion, we link a genetic susceptibility allele for SS to a functional phenotype in terms of decreased CXCR5 expression. The decrease of CXCR5+ cells in circulation was also related to homing of B and T cells to the autoimmune target organ. Therapeutic drugs targeting the CXCR5/CXCL13 axis may be useful in SS.

No association of primary Sjögren's syndrome with Fcγ receptor gene variants.

The genetic background of primary Sjögren's syndrome (pSS) is partly shared with systemic lupus erythematosus (SLE). Immunoglobulin G Fc receptors are important for clearance of immune complexes. Fcγ receptor variants and gene deletion have been found to confer SLE risk. In this study, four Fcγ receptor single-nucleotide polymorphisms (SNPs) and one copy number variation (CNV) were studied. Swedish and Norwegian pSS patients (N=527) and controls (N=528) were genotyped for the Fcγ receptor gene variant FCGR2A H131R (rs1801274) by the Illumina GoldenGate assay. FCGR3A F158V (rs396991) was analysed in 488 patients and 485 controls, FCGR3B rs447536 was analysed in 471 patients and 467 controls, and FCGR3B rs448740 was analysed in 478 cases and 455 controls, using TaqMan SNP genotyping assays. FCGR3B CNV was analysed in 124 patients and 139 controls using a TaqMan copy number assay. None of the SNPs showed any association with pSS. Also, no FCGR3B CNV association was detected. The lack of association of pSS with Fcγ receptor gene variants indicates that defective immune complex clearance may not be as important in pSS pathogenesis as in SLE, and may point to important differences between SLE and pSS.

Contribution of IKBKE and IFIH1 gene variants to SLE susceptibility.

The type I interferon system genes IKBKE and IFIH1 are associated with the risk of systemic lupus erythematosus (SLE). To identify the sequence variants that are able to account for the disease association, we resequenced the genes IKBKE and IFIH1. Eighty-six single-nucleotide variants (SNVs) with potentially functional effect or differences in allele frequencies between patients and controls determined by sequencing were further genotyped in 1140 SLE patients and 2060 controls. In addition, 108 imputed sequence variants in IKBKE and IFIH1 were included in the association analysis. Ten IKBKE SNVs and three IFIH1 SNVs were associated with SLE. The SNVs rs1539241 and rs12142086 tagged two independent association signals in IKBKE, and the haplotype carrying their risk alleles showed an odds ratio of 1.68 (P-value=1.0 × 10(-5)). The risk allele of rs12142086 affects the binding of splicing factor 1 in vitro and could thus influence its transcriptional regulatory function. Two independent association signals were also detected in IFIH1, which were tagged by a low-frequency SNV rs78456138 and a missense SNV rs3747517. Their joint effect is protective against SLE (odds ratio=0.56; P-value=6.6 × 10(-3)). In conclusion, we have identified new SLE-associated sequence variants in IKBKE and IFIH1, and proposed functional hypotheses for the association signals.

Increased carotid intima thickness and decreased media thickness in premenopausal women with systemic lupus erythematosus: an investigation by non-invasive high-frequency ultrasound.

OBJECTIVE: To determine whether high-frequency ultrasound (US) yielding separate assessments of intima and media thickness gives additional information about the vascular morphology compared with the total common carotid artery intima-media thickness (CCA-IMT). METHODS: Using a 22 MHz US instrument, we determined the near-wall CCA-IMT, the intima and media layers, and the intima/media (I/M) ratio in 47 premenopausal women with systemic lupus erythematosus (SLE), 20 healthy women, and 17 postmenopausal women (mean ages 37, 40, and 69 years, respectively). RESULTS: In SLE, the carotid intima was thicker (0.19 ± 0.04 vs. 0.12 ± 0.02 mm), the media thinner (0.45 ± 0.12 vs. 0.68 ± 0.24 mm), the I/M ratio higher (0.45 ± 0.17 vs. 0.20 ± 0.07) (all p < 0.0001), and the CCA-IMT lower (0.64 ± 0.13 vs. 0.80 ± 0.25 mm, p < 0.01) compared to age-matched controls. The SLE patients had a thicker carotid intima compared to the postmenopausal women (0.19 ± 0.04 vs. 0.14 ± 0.03 mm, p < 0.0001) and a similar I/M ratio. CONCLUSION: Separate assessment of carotid artery wall layers demonstrated a thicker intima, thinner media, and a higher I/M ratio in women with SLE compared to healthy controls and indicated an artery wall status in SLE comparable to 30-years-older healthy women. Separate estimates of carotid intima and media layers may be preferable to CCA-IMT in SLE patients.

TSGA10 - A target for autoantibodies in autoimmune polyendocrine syndrome type 1 and systemic lupus erythematosus.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High-titre autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in approximately 7% of APS1 patients, with immunoreactivity to pituitary tissue frequently described. Using APS1 patient serum to immunoscreen a pituitary cDNA expression library, testis specific, 10 (TSGA10) was isolated. Immunoreactivity against TSGA10 was detected in 5/99 (5.05%) patients with APS1, but also in 5/135 (3.70%) systemic lupus erythematosus (SLE) patients and 1/188 (0.53%) healthy controls. TSGA10 autoantibodies were not detected in the serum from patients with any other autoimmune disease. Autoantibodies against TSGA10 were detectable from a young age in 4/5 positive APS1 patients with autoantibody titres remaining relatively constant over time. Furthermore, real-time PCR confirmed TSGA10 mRNA to be most abundantly expressed in the testis and also showed moderate and low expression levels throughout the entire body. TSGA10 should be considered as an autoantigen in a subset of APS1 patients and also in a minority of SLE patients. No recognizable clinical phenotype could be found to correlate with positive autoantibody reactivity.

Association of EBF1, FAM167A(C8orf13)-BLK and TNFSF4 gene variants with primary Sjögren's syndrome.

We performed a candidate gene association study in 540 patients with primary Sjögren's Syndrome (SS) from Sweden (n=344) and Norway (n=196) and 532 controls (n=319 Swedish, n=213 Norwegian). A total of 1139 single-nucleotide polymorphisms (SNPs) in 84 genes were analyzed. In the meta-analysis of the Swedish and Norwegian cohorts, we found high signals for association between primary SS and SNPs in three gene loci, not previously associated with primary SS. These are the early B-cell factor 1 (EBF1) gene, P=9.9 × 10(-5), OR 1.68, the family with sequence similarity 167 member A-B-lymphoid tyrosine kinase (FAM167A-BLK) locus, P=4.7 × 10(-4), OR 1.37 and the tumor necrosis factor superfamily (TNFSF4=Ox40L) gene, P=7.4 × 10(-4), OR 1.34. We also confirmed the association between primary SS and the IRF5/TNPO3 locus and the STAT4 gene. We found no association between the SNPs in these five genes and the presence of anti-SSA/anti-SSB antibodies. EBF1, BLK and TNFSF4 are all involved in B-cell differentiation and activation, and we conclude that polymorphisms in several susceptibility genes in the immune system contribute to the pathogenesis of primary SS.

Additive effects of the major risk alleles of IRF5 and STAT4 in primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) shares many features with systemic lupus erythematosus (SLE). Here we investigated the association of the three major polymorphisms in IRF5 and STAT4 found to be associated with SLE, in patients from Sweden and Norway with primary SS. These polymorphisms are a 5-bp CGGGG indel in the promoter of IRF5, the single nucleotide polymorphism (SNP) rs10488631 downstream of IRF5 and the STAT4 SNP rs7582694, which tags the major risk haplotype of STAT4. We observed strong signals for association between all three polymorphisms and primary SS, with odds ratios (ORs) >1.4 and P-values <0.01. We also found a strong additive effect of the three risk alleles of IRF5 and STAT4 with an overall significance between the number of risk alleles and primary SS of P=2.5 x 10(-9). The OR for primary SS increased in an additive manner, with an average increase in OR of 1.78. For carriers of two risk alleles, the OR for primary SS is 1.43, whereas carriers of five risk alleles have an OR of 6.78. IRF5 and STAT4 are components of the type I IFN system, and our findings emphasize the importance of this system in the etiopathogenesis of primary SS.

A polymorphic variant in the MHC2TA gene is not associated with systemic lupus erythematosus.

Single-nucleotide polymorphisms (SNPs) in the major histocompatibility complex class II transactivator (MHC2TA) gene encoding the class II transactivator have been associated with multiple sclerosis, rheumatoid arthritis, and myocardial infarction in the Swedish population. We used a case-control approach to investigate the prevalence of a relevant variant in Swedish systemic lupus erythematosus (SLE) cohorts to determine whether SLE shares the same MHC2TA susceptibility allele as the other diseases. No differences were observed between cases and control subjects at either the allele or genotype levels. Furthermore, no significant correlations were found when comparing different clinical and serological SLE phenotypes. This particular polymorphism rs3087456 of the MHC2TA gene does not appear to influence genetic susceptibility to SLE in the Swedish population. We conclude that our data support neither allelic nor genotype association between the MHC2TA SNP and SLE.

Autoantibodies to alpha-fodrin in primary Sjögren's syndrome and SLE detected by an in vitro transcription and translation assay.

OBJECTIVE: To investigate the prevalence of alpha-fodrin autoantibodies in primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) with and without secondary SS, using an in vitro transcription and translation assay (ITT). METHODS: cDNA encoding JS-1, the amino-terminal portion of alpha-fodrin, was used for ITT. Immunoprecipitation was performed with sera from 56 primary SS patients and 67 SLE patients, 14 with and 53 without secondary SS. Correlations to RF, ANA, anti-dsDNA, anti-SS-A and anti-SS-B antibodies, hypergammaglobulinemia, labial salivary gland biopsy grade, extraglandular manifestations and a modified SLE disease activity index (mSLEDAI) were made. RESULTS: Autoantibodies against alpha-fodrin were detected in 16/56 (29%) of primary SS patients and in 25/53 (47%) of sera from SLE patients without secondary SS. In SLE patients with secondary SS the prevalence was 3/14 (21%). None of the blood donors showed alpha-fodrin reactivity. Correlations were found to RF, ANA, anti-dsDNA antibodies and a positive mSLEDAI score. CONCLUSION: The frequency of alpha-fodrin autoantibodies detected by this method is similar in sera from primary SS patients and SLE patients with or without secondary SS. The presence of alpha-fodrin autoantibodies seems to reflect non-organ-specific autoimmunity in primary SS and SLE and to be of limited discriminating value.

Limited Wegener's granulomatosis with central nervous system involvement and fatal outcome.

A 74-year-old woman was admitted to the medical department with vertigo and confusion, and suffered seizures three days later. Clinical, radiological and histopathological examination revealed limited Wegener's granulomatosis (WG) with central nervous system (CNS) involvement. Initially she was treated successfully with prednisolone and cyclophosphamide, but she relapsed after 25 months and died four weeks later. Autopsy showed widespread granulomas with vasculitis in the cerebrum. These findings emphasize the importance of considering WG in CNS disease of unclear origin, and demonstrate the aggressive nature of the disease.