GWAS meta‑analysis of axial spondyloarthritis and Behçet’s disease identifies CXCR6 as a novel MHC‑I‑opathy gene in a Turkish population

Author: Dr. Mohammad Saeed

Dr. Mohammad Saeed is a member of the EULAR MHC-I-opathy group.

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“MHC-I-opathy” is a family of inflammatory disorders with a strong genetic link to HLA-I. These include Axial spondyloarthritis (AxSpA; previously Ankylosing spondylitis (AS) associated with HLA-B27), Behçet’s disease (BD; associated with HLA-B51) and psoriasis. Though the MHC-I genetic associations were discovered about 50 years ago, the pathogenesis of these disorders is not fully understood. To identify immunogenetic pathways for MHC-I-opathies, a cluster-based meta-analysis of AxSpA and BD Genome-wide association studies (GWAS) using OASIS was conducted. Both selected GWAS were from Turkiÿe for homogenous population effect.

OASIS (Objective Assimilation of SNPs Interacting in Synchrony) is a GWAS clustering algorithm, developed by Dr. Mohammad Saeed to identify genes of modest significance. These are often missed in classic analysis that use Manhattan plots to identify significant associations in GWAS. Clustering or binning methods, such as OASIS, increase power by decreasing the number of tests per study.

HLA-I was the only locus that was highly significant (P<1× ­10–8) in both GWAS datasets. OASIS identified 234 independent modestly significant non-HLA loci in the two GWAS datasets (Bonferroni correction: P≤2.14×10⁻4; −log10 P≥3.67). The most signficant genes that this study identified were IL23R (1p31.3), LACC1 (13q14.11) and the novel CXCR6 (3p21.31). These are significantly associated with both AxSpA and BD. All three genes are down-regulated in these MHC-I-opathies. Protein interaction network showed that CXCR6 is part of the interactome of IL23R and LACC1 (P=0.00243). The novel gene, CXCR6, identified here codes for a G-protein coupled receptor expressed in several T-cell subsets and regulate their migration to tissues. A previous biological study showed that CXCR6+ CD8+ T-cells were actively recruited to psoriasis-affected skin. It also showed that TNF-α inhibition, which is part of the therapeutics (such as the biologic medications Eternacept, Adalimumab and Infliximab) for AxSpA and BD, reduced the CXCR6-mediated T-cell recruitment.

In summary, this study identified 15 loci common to both AxSpA and BD that may provide diagnostic and therapeutic targets for MHC-I-opathy. Major candidate genes for MHC-I-opathy identified here are IL23R, LACC1, and CXCR6. By integrating locus-level significance with functional validation in independent expression datasets, we ensured that only statistically robust and biologically relevant associations were highlighted, thereby strengthening the reliability and interpretability of our findings.

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Cross-Reactivity of SARS-CoV-2 Antibodies With Typhoid Flagellar-H Protein.

Aneela Pasha, Mohammad Saeed

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Figure 2A of the article (Copyright with TMIH Journal) and depicts the molecular overlap between Salmonella typhi Flagellar-H antigen and SARS-CoV-2 RBM antigen. This overlap was eliminated due to the Delta mutation (Figure 2B). This, we hypothesize underlies the antibody cross-reactivity between the two organisms.

COVID-19 affected different regions of the world with varying intensity leading to high mortality in some regions and low in others. This proof-of-concept study presents evidence that one potential infection that may have provided natural herd immunity against COVID-19, due to adaptive heterologous immunity, is Typhoid fever.

During the COVID-19 pandemic several patients presented to ImmunoCure from interior Sindh with COVID-19 symptoms and a positive Typhidot test. They had been treated with antibiotics for Typhoid fever. However, their symptoms and clinical exam was suggestive of mild to moderate COVID-19. After informed consents patients were tested for both SARS-COV-2 antibodies and Typhidot (without charge). We found high correlation between the two tests (~72%).

Dr. Mohammad Saeed suspected antibody cross-reactivity, which occurs when antibody against one antigen (usually a protein) reacts against a different antigen / protein. Usual matching analysis (using the BLAST software) did not show any overlap. However, with specialized analysis involving cutting the protein sequences into small pieces and matching them (k-mer analysis) showed that a 5-amino acid sequence overlapped between SARS-CoV-2 (that causes COVID-19) and Salmonella typhi (that causes Typhoid fever).

This 5-amino acid sequence, NGVEG, was found in the Flagellar-H protein of Salmonella typhi and very interestingly in the critical region of SARS-CoV-2 called the Spike protein. The Spike protein is responsible for the entry of the SARS-CoV-2 virus in the cells and vaccines are also made against it to protect from COVID-19. Moreover, NGVEG was found in the special region of the Spike protein called the Receptor Binding Motif (RBM) which is the critical most region of SARS-CoV-2. The Delta variant of SARS-CoV-2 emerged in India after a super-spreader event and the mortality rate in South Asia and around the world from COVID-19 skyrocketed. This Delta variant contained a mutation E484Q. This mutation targeted the very sequence that overlapped between Salmonella typhi and SARS-CoV-2 i.e. NGVEG, turning it into NGVQG. This single amino acid change, completely altered the 3-dimensional structure of the SARS-CoV-2 RBM as shown in the Figure 2 of the article (above).

We next looked at whether COVID-19 mortality was impacted by Typhoid prevalence. Epidemiological data showed that Typhoid endemic regions, such as Pakistan, Bangladesh and others, had lower COVID-19 mortality than Typhoid non-endemic regions such as Europe and United States.

After the COVID-19 pandemic, the Government of Pakistan permanently banned the use of the Typhidot test due to widespread concerns over false-positive results. This policy action, while driven by diagnostic concerns indirectly supports the idea that cross-reactivity between SARS-CoV-2 and Salmonella typhi was of real-world relevance. Our study brings to light this possibility and attempts to highlight the science-policy interface and the public health implications of diagnostic cross-reactivity in endemic settings.

@AneelaPasha and @DrMSaeed_pk

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Dr. Mohammad Saeed was invited to present his research on the ‘Genetics of MHC-I-Opathy’ at the British Society for Rheumatology (BSR) (April 24, 2024). The summary of the research was published in the official BSR Journal, Rheumatology.

The presentation was well-received by the august audience of Rheumatologists and Immunologists at the BSR 2024. A lengthy follow-up meeting was held at the University of Leeds and a collaboration for data and analysis exchange was established with Dr. Mohammad Saeed. University of Leeds has been recognized as a center of excellence in Rheumatology by EULAR (European League against Rheumatism) since 2005.

This research received no funding to perform. However, participation at the BSR 2024 Meeting was sponsored by High-Q and KIAZEN Pharmaceuticals, Pakistan. This was considered Advertising campaign for the Sponsors and declared at the BSR Meeting Oral Presentation. 

Python – The Logo Way (Book)

The book is written with the child in mind to teach programming by drawing using “Logo” concepts. I hope to spark greater creativity in children, make education fun and fulfill the need to learn today’s essential skill, i.e, being able to program and ‘talk to computers’. Therefore, a simple text for an accessible programming language such as Python was much needed.

Dr. Mohammad Saeed

Consultant Rheumatology and Immunogenetics

Twitter: @DrMSaeed_pk

Lecture #3: “How to choose a Mentor?” by Prof. Anwar Siddiqui

Friday, October 16, 2020. Timing: 3 – 4 pm (Tweet)

Zoom Online Meeting Recorded Video

Language requirement: Both English and Urdu

Lecture #2: “Why do Research?” by Prof. Anwar Siddiqui

Friday, September 25, 2020. Timing: 3 – 4 pm (Tweet)

Recorded Video of Lecture:

Lecture #1: “Research Integrity” by Prof. Anwar Siddiqui

🇵🇰 Happy Independence Day

At ImmunoCure we celebrated this important day with the first Seminar. Prof. Anwar Siddiqui enlightened us with his rich experience and concrete examples on "Research Integrity", a theme that blends with our desire to rise as a nation on solid footings. pic.twitter.com/YcrV6ZV64K

— ImmunoCure (@ImmunoCure_pk) August 14, 2020

Dr. Mohammad Saeed
Founder & CEO
Consultant Rheumatology and Immunogenetics

Rheumatology Clinic: Mon to Thu and Sat
Timings: 10 am to 5 pm
Appointments: +92 308 282 2712

Dr. Mohammad Saeed, M.D.
Consultant Rheumatology and Immunogenetics
Fellowships (USA): Rheumatology, Musculoskeletal Ultrasound, Genomics, Immunology

Dr. Mohammad Saeed graduated from Aga Khan University Medical College and has Fellowships in Genomics from Northwestern University, Chicago, and Immunology from the University of Arkansas for Medical Sciences (UAMS). He trained in Internal Medicine and Rheumatology at UAMS and has further specialization in Musculoskeletal Ultrasound from Boston University (USSONAR program). He has been faculty at UAMS and Staff Scientist at Nephropath, USA.

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He is an active researcher working on the genetics of lupus and other autoimmune disorders and has previously contributed substantially to Neurogenetics. He has several publications in international journals and has been a member of professional organizations including the American College of Rheumatology, European Lupus Society (SLEuro) and American Society of Human Genetics. He is a member of the Pakistan Society for Rheumatology and Health Research Advisory Board, Pakistan.

Rheumatology deals with inflammation anywhere in the body (kidneys, lungs, brain, and nerves, or even eyes) as well as disorders of joints and bones. These conditions are often difficult to diagnose due to the variety of symptoms they produce in different organ systems. Dr. Saeed has a special interest in patients who have not had a diagnosis despite visiting several doctors. Other common Rheumatologic diseases Dr. Saeed manages, include Osteoporosis and Osteoarthritis, Rheumatoid arthritis, Gout, Systemic lupus erythematosus, Myositis, Reactive arthritis, Psoriasis, and Vasculitis. These conditions can vary from mild to severe disease but have good treatments available especially if diagnosed early.

Research Publications

Curriculum vitae

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Two Abstracts were accepted from ImmunoCure at EULAR 2019 Meeting in Madrid, Spain. 

Abstract AB1101 is about the small subset of Rheumatoid arthritis patients in whom steroids cannot be withdrawn – an interesting rheumatologic dilemma. We showed that these patients have high frequency of Mi2 and Ku antibodies and are in fact Overlap syndromes. 

Abstract AB0016 identifies new genes for Psoriasis using Dr. Saeed’s GWAS analytic method, OASIS, in combination with gene expression analysis.

AB1101 (2019)
ANA SUBSETS ANTIBODY PROFILE OF EARLY UNDIFFERENTIATED ARTHRITIS (UA) IN PAKISTAN
Syed Hussain Azhar Rizvi, Tariq Gazdar1, Aneela Pasha, Mohammad Saeed
ImmunoCure, Karachi, Pakistan
Background: Undifferentiated Arthritis (UA) is a group of inflammatory disorders where early synovitis (duration less than 12 months) is present, however patients do not meet criteria for established rheumatologic disorders such as Rheumatoid arthritis, Lupus or Spondyloarthritis (1). On long term follow up, a subpopulation of UA patients converge into definitive Rheumatologic disorders. Up to 50% of early arthritis in European cohorts has been reported to be UA (1, 2).

Objectives: Identify Anti-nuclear antibody (ANA) Subset antibodies in patients presenting with early UA.

Methods: Over a 1-year period (2017-18) patients with early UA were prospectively evaluated. This included clinical exam, routine laboratory investigations including ESR and CRP, RF and anti-CCP as well as Musculoskeletal ultrasound (MSKUS) (3). These patients underwent further testing for ANA Subset antibodies. ANA subsets (17 antibodies) were performed using standard immunoblot assays (EUROIMMUN) at our clinical laboratory (4).

Results: 110 UA patients were found to have positive ANA subsets. The most frequent antibodies were Ku (77.3%) and Mi2 (73.6%) followed by dsDNA (30%) and Nucleosome antibody (29%) ( Figure 1 ). Low frequency antibodies included Sm (n=1), Ribosomal-P and PCNA (n=2 each), Jo-1 (n=3), PM-Scl100 (n=4) and SSB (n=5), whereas Scl70 antibodies were absent. SSA (n=9) and Ro-52 (n=14) together constituted 21% frequency. These antibodies had high signal intensities (mean ± SD SSA = 52±35 and Ro-52 = 46±25). Ku (17±11) and Mi2 (22±15) had modest signal intensities ( Figure 2 ). Clinically 42 patients were classified as RA (RF+ = 33%; anti-CCP+ = 14%) and 41 patients met ACR criteria for SLE while 2 patients met criteria for both and were classified as Rhupus. ANA subset antibody frequencies did not statistically differ between the RA and SLE groups signifying that the clinically classified RA patients in fact had early Rhupus which did not meet ACR criteria for SLE.

Conclusion: Early UA is difficult to categorize clinically, sometimes even after long term follow up, though a substantial portion converge into RA. This study shows that there is a significant portion of early Rhupus in the RA group whose steroid discontinuation leads to a disease flare. ANA Subset antibody profile in such patients may help in their more accurate diagnoses and treatment.

REFERENCES:

[1] van Aken J, van Bilsen JH, Allaart CF, Huizinga TW, Breedveld FC. The Leiden Early Arthritis Clinic. Clin Exp Rheumatol. 2003 Sep-Oct;21(5 Suppl 31):S100-5. PMID: 14969059.

[2] Hülsemann JL, Zeidler H. Undifferentiated arthritis in an early synovitis out-patient clinic. Clin Exp Rheumatol. 1995 Jan-Feb;13(1):37-43. PubMed PMID: 7774101.

[3] Horton SC, Tan AL, Wakefield RJ, Freeston JE, Buch MH, Emery P. Ultrasound-detectable grey scale synovitis predicts future fulfilment of the 2010 ACR/EULAR RA classification criteria in patients with new-onset undifferentiated arthritis. RMD Open. 2017;3(1):e000394. PMID: 28469936.

[4] Op De Beéck K, Vermeersch P, Verschueren P, Westhovens R, Mariën G, Blockmans D, Bossuyt X. Antinuclear antibody detection by automated multiplex immunoassay in untreated patients at the time of diagnosis. Autoimmun Rev. 2012;12(2):137-43. PMID: 22387973.5.

ANA Subsets Antibody Frequency

ANA Subsets Antibody Intensity

Disclosure of Interests: Syed Hussain Azhar Rizvi Grant/research support from: Participation in EULAR 2019 Meeting is sponsored by Searle Pak, Tariq Gazdar Shareholder of: Partner at ImmunoCure which performs Autoimmune Disease testing, Aneela Pasha: None declared, Mohammad Saeed Shareholder of: Partner at ImmunoCure – laboratory, which performs Autoimmune antibody testing, Grant/research support from: Participation in EULAR 2019 Meeting is sponsored by High-Q Pharma Pak

Citation: Ann Rheum Dis, volume 78, supplement 2, year 2019, page A2014
Session: Other orphan diseases (Scientific Abstracts)

DOI: http://dx.doi.org/10.1136/annrheumdis-2019-eular.126

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AB0016 (2019)
COMBINED GENOME-WIDE ASSOCIATION AND GENE-EXPRESSION META-ANALYSIS IDENTIFIES NOVEL PSORIASIS GENES
Mohammad Saeed
ImmunoCure, Karachi, Pakistan
Background: Psoriasis (Ps) is a common, inflammatory disorder affecting skin, joints and associated connective tissue, with significant genetic underpinnings. Multiple genome-wide association studies (GWAS) have been conducted with identification of several Ps loci. However, these only explain about a third of Ps genetic risk indicating that additional loci of modest effect remain to be discovered (1).

Objectives: Identify novel psoriasis genes that have functional consequences for psoriasis by altered gene-expression

Methods: Association clustering methods such as gene- and locus-based tests are more powerful than single variant analysis for identifying modest genetic effects (2, 3). Here, a dbGAP GWAS dataset (4) for Ps (pha002855: 1348 Ps cases and 1368 controls, genotyped for 448K SNPs) was analyzed using the locus-based algorithm, OASIS (3), to identify 13 highly significant loci other than the HLA-C locus. In these loci 50 genes were identified using SNIPPER, which were then subjected to gene expression analysis in three Ps GEO datasets.

Results: This genetic and functional analysis identified a total of 18 genes that were significantly associated and had altered expression in psoriasis skin. The most significant of these were two genes that were two-fold upregulated in Ps, IL12B ( P = 9×10 -11 ) and TTC39B ( P = 3×10 -12 ) and two genes that were repressed <50%, MAML2 ( P = 8×10 -19 ) and EBF1 ( P = 1×10 -12 ). Interestingly, the expression of IL23R remained unaltered. Other genes that were significantly upregulated (1.5 – 2.0 fold) were IL12RB2 , TTC1 and PSMD6 . Genes that were also significantly repressed (0.5 – 0.8 fold) were SERBP1 , ATXN7 , PSIP1 , ZNF385D , SIPA1L1 .

Conclusion: This combined genetic and functional meta-analysis elucidated novel genes, functional networks and pathways for psoriasis. These results will lead to important insights into the immunopathogenesis and treatment of psoriasis.

REFERENCES:

[1] Tsoi LC, Stuart PE, Tian C, et al. Large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants. Nat Commun. 2017May24;8:15382. PMID: 28537254.

[2] Luo L, Peng G, Zhu Y, et al. Genome-wide gene and pathway analysis. Eur J Hum Genet. 2010Sep;18(9):1045-53. PMID: 20442747.

[3] Saeed M. Novel linkage disequilibrium clustering algorithm identifies new lupus genes on meta-analysis of GWAS datasets. Immunogenetics. 2017May;69(5):295-302. PMID: 28246883.

[4] Nair RP, Duffin KC, Helms C, et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet. 2009Feb;41(2):199-204. PMID: 19169254.

Disclosure of Interests: Mohammad Saeed Shareholder of: Partner at ImmunoCure – laboratory, which performs Autoimmune antibody testing, Grant/research support from: Participation in EULAR 2019 Meeting is sponsored by High-Q Pharma Pak

Citation: Ann Rheum Dis, volume 78, supplement 2, year 2019, page A1474
Session: Genomics, genetic basis of disease and antigen presentation (Scientific Abstracts)

DOI: http://dx.doi.org/10.1136/annrheumdis-2019-eular.306

#EULAR2019

Full Text Article

ImmunoCure is pleased to announce its publication in Immunogenetics identifying novel genes for Parkinson’s disease based on Dr. Mohammad Saeed’s statistical genetic algorithm, OASIS. This study provides further evidence that Parkinson’s disease has significant links with inflammation. It also strengthens the field of Neuroimmunology, for which ImmunoCure provides a range of special tests.

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Dr Mohammad Saeed
ImmunoCure

Dr. Mohammad Saeed’s Review on Lupus published in the journal “Immunogenetics”

‘The challenge will be more robust clinical classification of SLE. As more antibodies are discovered, such as MDA5 and PLA2R, and genetic tests seep into clinical practice, it is likely that SLE classification may change further. Perhaps, SLE clinical definition may incorporate a mandatory presence of nuclear antibodies (ANA and its subsets) and the nuclear-antibody negative SLE may be classified according to the specific antibodies or mutation findings.’…..’On the other hand, expert panels may take a more inclusive approach by subclassifying SLE into more refined phenotypes. Whatever the course SLE classification takes in the future, genetic exploration of this complex disease has not only enhanced its own pathobiology but opened doors to understanding autoimmune disease in general as well.’

Lay Summary of the Lupus Review

1. Lupus is a syndrome rather than a single disease. 330 types of lupus are possible. Therefore, every lupus patient is unique.
2. Lupus is initiated with aberrant processing of DNA (and RNA) during apoptosis. This means the clearance system of the body is defective and unable to remove properly the debris of dying cells.
3. This nuclear material is picked up by cells of the immune system specifically plasmacytoid dendritic cells (pDCs) and B-cells which react to it (through intricate cellular pathways) by releasing high levels of interferon-alpha.
4. These cells may be further activated by viral and bacterial infections or conditions like pregnancy.
5. Both pDCs and B-cells present the nuclear material in immunoreactive forms (antigens) to T-cells which coordinate the formation of antibodies by the B-cells.
6. The cells and antibodies attack the ‘self’-antigens in tissues such as the kidney and brain.
7. This sets up a perpetual cycle of immunologic abnormalities that defines lupus – aberrant apoptosis followed by immune complex formation and deposition.
8. Antibodies are often present in lupus but do not necessarily cause lupus. They however do indicate a dysfunctional immune system. SLE can result even without any detectable antibodies.
9. SLE treatment has so far largely been guided by clinical experience. With the recent advent of targeted biologic therapies, this paradigm has started to change.
10. Molecular analysis has shown the similaries and differences between various SLE drugs.
11. Biologic medications such as Rituximab, Belimumab (both anti-B cell) and the upcoming Anifrolumab (designed against the interferon alpha receptor – anti-pDC and B-cell) are powerful lupus treatment medications.
12. Given more than 100 genes now known for lupus and a plethora of antibodies, it is expected that the clinical disease definition of lupus will change as well. The new definition may be more clinically robust.

‘Lupus pathobiology based on genomics’