INTRODUCTION:

Recent progress in genome sequencing has greatly broadened our understanding of disease including rare autoimmune and autoinflammatory conditions by making genetic analysis more accessible within clinical settings. This shift is made possible by advancements since the Human Genome Project, which first mapped the human genome over a decade and at a cost of about $5 billion (when adjusted for inflation). Today, entire genomes can be sequenced for under $1,000. Integration of these advanced sequencing technologies with meticulous clinical assessment has revolutionized the identification of rare immune-mediated diseases and uncovered their specific genetic origins. Such discoveries not only provide deeper insights into these disorders but also pave the way for tailored, patient-specific therapies¹.

Historically, diagnosing rheumatic diseases has depended on the interplay of laboratory test results, clinical expertise, and established diagnostic criteria. Taking systemic lupus erythematosus (SLE) as an example, while agreed-upon classification standards exist, seasoned rheumatologists emphasize the pivotal role of clinical experience and positive anti-nuclear antibody results in making the final diagnosis. Laboratory findings, though crucial, must be interpreted alongside patient history and distinctive clinical clues. Yet, some individuals display unusual patterns or symptoms that escape traditional classifications. In such cases, integrating genomic data into the practice of rheumatology offers tremendous potential for more accurate diagnoses and the design of individualized treatment strategies.

Although the investigation of extremely rare autoimmune and autoinflammatory syndromes is often specialized, research into these disorders is yielding profound insights into the underlying mechanisms of broader rheumatic diseases. Unlike infectious diseases, where the cause is usually a specific pathogen, the triggers for most rheumatic conditions are still elusive. In this evolving landscape, the study of monogenic autoimmune and autoinflammatory diseases specially those involving the TREX1 cGAS STING pathway has transformed current perspectives on the origins, diagnosis, and management of rheumatic disorders. This focus is particularly important as the TREX1 cGAS STING pathway is implicated in several monogenic inflammatory syndromes and inherited vasculopathies, offering new pathways for understanding disease biology and therapeutic intervention².

Advent of Affordable Genome Sequencing:

Traditionally, DNA sequencing relied on the classical chain-termination technique, known as Sanger sequencing. This process uses fluorescently labeled dideoxynucleotides modified bases that permanently stop DNA strand extension during the PCR extension step. When one of these terminators is incorporated, it produces a DNA fragment whose length depends on where the termination occurred.

These fragments, each labeled with a distinct fluorescent dye corresponding to the specific base, are then separated by size through capillary electrophoresis. By detecting the fluorescence pattern, the exact DNA sequence can be reconstructed. While Sanger sequencing delivers short yet highly accurate reads, it is more expensive and slower compared to today’s advanced high-throughput sequencing methods².

Advances in Sequencing Technology and Clinical Accessibility:

Recent advances in technology have greatly enhanced the affordability and accessibility of genome and exome sequencing for clinical use. Unlike traditional methods, next-generation sequencing (NGS) employs a fundamentally different process one that is more error-prone but significantly cheaper. In a widely used NGS method, DNA fragments are randomly fixed in clusters on a flow cell, a specialized surface that captures them. Nucleotides are then added one at a time to each cluster, with only a single base attaching to the 3′ hydroxyl group in each cycle. After incorporating a fluorescently labeled nucleotide, the flow cell is imaged, and the emitted color reveals which base was added.

A major cost-reduction method in genomics is sequencing only the exome the genome’s protein-coding regions rather than the entire genome, dramatically lowering the number of bases needing analysis, since much of human DNA is non-coding³.

A distinctive aspect of NGS is that, after imaging, the fluorescent label can be chemically removed while retaining the incorporated base. The 3′ hydroxyl is then restored, enabling addition of the next base and another imaging step. Repeating this cycle produces long, continuous sequences from individual DNA molecules. Despite a higher error rate compared with Sanger sequencing, NGS offers major advantages in cost and throughput, as it can process millions of DNA strands simultaneously. Because errors are more common, multiple reads of the same DNA segment described as sequencing “depth” or “fold coverage” are essential for reliability. For example, 50-fold coverage implies most bases are read at least 30 times, boosting accuracy, while higher coverage further strengthens confidence in results. However, exome sequencing may introduce biases, such as those from PCR amplification or binding kinetics, which must be addressed during analysis.

Interpreting sequencing results is complex because every person carries a combination of unique mutations, harmless variants, and common genetic polymorphisms. The American College of Medical Genetics and Genomics (ACMG) has established standardized terms for classification: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign, based on factors like population frequency and functional evidence. Laboratory experiments, including cell-based or animal models, are often necessary to confirm the biological effects of suspected mutations⁴.

Because of these complexities, genetic counseling is essential both before and after testing. Counselors guide patients and families especially when rare, disease-causing mutations are found through potential implications, including those related to life insurance and reproductive planning. Expert input also helps prevent misinterpretation of results and promotes informed choices regarding genetic testing.

Rare diseases and the impact of genetic insights:

The life stories of people with rare rheumatic diseases illustrate the critical role of genetic insights in guiding patient care. In 1988, doctors encountered a case involving a patient with a mysterious, multi-organ disorder affecting the kidneys, liver, brain, and eyes. Standard immunosuppressive therapies failed, and the patient along with several relatives succumbed to severe systemic damage. Autopsy revealed widespread blood vessel disease, especially in the brain and eyes, along with brain lesions resembling radiation injury.

Family history revealed that nearly half the patient’s relatives had experienced similar health problems but were incorrectly diagnosed with conditions like multiple sclerosis or systemic lupus erythematosus (SLE). Roughly twenty years later, the true cause was discovered retinal vasculopathy with cerebral leukoencephalopathy (RVCL, also known as RVCL-S or HERNS) a fatal disorder caused by autosomal-dominant, C-terminal frameshift mutations in the TREX1 gene⁵.

In RVCL, all affected individuals inherit the same pattern of mutation in the C-terminal portion of TREX1, leading to progressive multi-organ failure that usually starts around age 40, with most patients dying within 5–10 years of symptom onset. While RVCL shares the TREX1 mutation link with Aicardi Goutières syndrome (AGS), the two conditions differ markedly: AGS can be caused by mutations in multiple genes, follows an autosomal-recessive inheritance pattern, and is the result of complete loss of TREX1 function. RVCL, by contrast, is triggered by a truncating mutation in just one allele of TREX1.

TREX1 encodes a DNA exonuclease that normally acts as a brake on type I interferon activity by preventing cytosolic double-stranded DNA (dsDNA) from persistently activating the cGAS–STING pathway. The amino-terminal domain drives exonuclease activity, while the C-terminal domain ensures proper localization near the nucleus. In AGS, the absence of functioning TREX1 leads to dsDNA buildup and chronic interferon activation. For RVCL, the mechanism is less well understood but may involve mis localization of an otherwise functional enzyme or abnormal activation of cGAS–STING signalling caused by the C-terminal mutation⁶.

RVCL poses one of the biggest hurdles for patients and families because there is currently no effective treatment. The disease progressively leads to vision loss, kidney and liver damage, strokes, dementia, osteonecrosis, thyroid and gastrointestinal disorders, chronic pain, and ultimately early death. While the biology behind RVCL remains partially unclear, the discovery of TREX1 mutations has transformed clinical awareness. Genetic testing is now available even before symptoms appear, allowing affected families to plan for the future and consider reproductive options such as in vitro fertilization with preimplantation genetic testing. This also enables patient enrolment in clinical research and potential access to emerging therapies⁷.

Finding TREX1 as the culprit has provided a clear therapeutic target. Research is now centered on deciphering the molecular mechanisms of RVCL, developing gene therapy approaches to correct the mutation, and exploring small-molecule drugs to counteract the abnormal protein function.

In 2014, another interferonopathy—STING-associated vasculopathy with onset in infancy (SAVI)—was described by Raphaela Goldbach-Mansky and colleagues at the NIH. Presenting within the first year of life, SAVI causes severe Raynaud phenomenon, digital tissue loss from vasculopathy, rashes, and progressive lung fibrosis. Its root cause is gain-of-function mutations in STING, which keep the protein permanently active and drive ongoing immune activation. Laboratory models introducing SAVI mutations via CRISPR confirmed their pathogenicity, and patient immune cells show chronic overexpression of type I interferon stimulated genes. JAK inhibitors, which block interferon receptor signaling, can reduce inflammation but often fail to halt disease progression, implying that interferon-independent processes are also at play. Current research aims to pinpoint these alternative mechanisms and the initiating cell types⁸.

Fig.2 The role of the TREX1–cGAS–STING pathway in rare rheumatic diseases

A related STING-linked disease, COPA syndrome, was identified in 2015 and traced to mutations in the COPA gene, which codes for the α-COP subunit of the coatomer complex I (COPI) essential for transporting proteins from the Golgi back to the ER. Mutations disrupt this trafficking, causing STING to remain trapped in the Golgi and remain persistently active. Clinically, COPA syndrome shares features with SAVI but tends to cause pulmonary haemorrhage, kidney inflammation, and ANCA-associated vasculitis rather than severe peripheral vascular disease or autoamputation. In some cases, SAVI mutations have also been linked to ANCA-associated vasculitis, indicating possible overlap in disease pathways.

Together, these findings reinforce that continuous activation of STING underlies a spectrum of rare autoinflammatory and autoimmune diseases, making this pathway a prime target for precision therapies ranging from small molecules that modulate signalling to advanced genetic correction techniques. The most remarkable takeaway is that, despite their varying symptoms, these rare conditions converge on the same TREX1–cGAS–STING pathway—a central immune pathway also implicated in more common disorders. As research advances, additional mutations in this pathway or related signaling networks are likely to be uncovered, offering fresh opportunities for targeted treatments that may benefit both rare disease patients and those with more widespread autoimmune or autoinflammatory conditions⁹.

Somatic Mutations:

One of the most significant recent breakthroughs linking genetics to rheumatology was the discovery of VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) in 2020. Prior to this finding, affected patients often received varied and sometimes unrelated diagnoses because of their diverse clinical symptoms some were classified as having classic polyarteritis nodosa, others giant cell arteritis, and others relapsing polychondritis. In reality, these disparate conditions were different manifestations of the same underlying genetic defect.

Researchers at the U.S. National Human Genome Research Institute, working with other NIH experts, analysed genomic data from over 2,500 individuals with unexplained inflammatory disorders, focusing on about 800 genes linked to ubiquitylation a protein modification process that controls protein activity and degradation. Their investigation pinpointed the UBA1 gene as the cause. Every patient diagnosed with VEXAS carried a somatic mutation in UBA1. Blood cell analysis demonstrated reduced ubiquitylation and abnormally increased activation of innate immune pathways in myeloid cells¹⁰.

This discovery represents a step toward redefining diseases based on molecular mechanisms rather than solely on clinical signs, paving the way for more targeted and personalized treatment strategies.

The finding also supports a broader idea that many rheumatic conditions could stem from rare somatic or inherited mutations. Some inherited mutations may show incomplete penetrance, making it harder to confirm them as causative, while somatic mutations could act as disease triggers in more common conditions similar to cancer¹¹.

Detecting these mutations, however, can be challenging. The pathogenic variants might occur in only a small fraction of a patient’s cells, making it essential to identify and sample the right tissue for sequencing. Moreover, they may be so rare within that tissue that they evade detection by standard next-generation sequencing. Unlike cancer, where tumour DNA is readily compared to healthy DNA from the same patient, rheumatic disease research often demands deep sequencing repeatedly reading the same gene hundreds or thousands of times alongside careful tissue selection to locate these variants. Once found, in-depth mechanistic studies are necessary to clarify how such mutations contribute to disease development¹².

Route to Personalized Medicine:

A confirmed molecular diagnosis can pave the way for precisely targeted and personalized treatment strategies. In some autoinflammatory diseases such as TNF receptor associated periodic syndrome, neonatal-onset multisystem inflammatory disease, Muckle-Wells syndrome, and familial cold autoinflammatory syndrome therapies directed at specific cytokines (like IL‑1β or TNF inhibitors) have proven effective in controlling inflammation. Research on these conditions also led to the discovery of the NLRP3 inflammasome, now known to be a central regulator of innate immunity, spurring the development of NLRP3 inhibitors for clinical use¹³.

However, many other autoinflammatory and related disorders remain difficult to treat. For instance:

· VEXAS often proves fatal despite immunosuppressive therapy.

· SAVI can continue to progress even when treated with JAK inhibitors.

· RVCL does not respond to existing immunomodulatory therapies.

A major challenge in developing effective treatments for such rare diseases is the limited patient population, which makes large, well‑powered clinical trials nearly impossible. In this context, animal models are crucial for studying disease mechanisms and testing new experimental approaches¹⁴.

RVCL, emerging personalized strategies include:

· Proteolysis-targeting chimeras (PROTACs) to bind and tag mutant proteins for ubiquitylation and proteasomal destruction.

· Genome editing using CRISPR/Cas9 or TALENs to directly repair harmful mutations.

Before pursuing such advanced therapies, researchers must identify the specific cellular compartments driving disease a question that remains unresolved for SAVI, COPA syndrome, and RVCL. Bone marrow chimera experiments in SAVI mouse models indicate that radioresistant parenchymal/stromal cells expressing mutant STING play a key role in lung inflammation by recruiting pathogenic lymphocytes, with type II interferon (IFNγ) signaling also contributing.

Developing effective personalized treatments will require a combined approach involving:

· Detailed clinical phenotyping of patients

· Studies using patient-derived cells

· Robust animal models expressing human disease‑associated proteins

These preclinical systems will be vital for testing candidate therapies such as siRNA, small‑molecule inhibitors, gene‑editing tools, and PROTACs before moving into phase I human trials¹⁵.

Significant progress is also being made in designing drugs to target the cGAS STING pathway. Advances in structural biology have enabled the design of:

· Molecules that block DNA binding or catalytic activity of cGAS, preventing cGAMP synthesis

· Agents that compete with cGAMP for STING binding

· H‑151, an inhibitor that prevents STING activation by blocking its palmitoylation, a crucial step for its ER-to-Golgi trafficking and signaling

· STING‑targeting PROTACs to degrade STING, potentially relevant for AGS, SAVI, and COPA syndrome

By directly modulating the TREX1 cGAS STING axis, these therapies aim to stop all downstream inflammatory processes whether driven by T-cell activation, type I interferon signaling, or other pathways offering promise not only for rare interferonopathies but also for more common autoimmune and autoinflammatory diseases¹⁶.

CONCLUSIONS:

For most common autoimmune and autoinflammatory diseases, the precise molecular mechanisms remain poorly defined, and diagnoses are still largely based on patterns of clinical symptoms. Treatment decisions in rheumatology typically follow the established clinical diagnosis. However, in certain cases, patients with apparently typical rheumatic syndromes fail to respond to standard therapies, indicating that there may be considerable molecular and immunological diversity among individuals grouped under the same diagnostic label.

Advances in next-generation sequencing, supported by bioinformatics, have significantly enhanced our understanding of these diseases, revealing shared molecular features among conditions that previously seemed unrelated. Traditionally, it was assumed that similar clinical presentations implied similar molecular mechanisms, but genomic insights have challenged this view, reshaping disease classification in rheumatology. The current approach, which relies heavily on clinical categorization, may contribute to the difficulty of addressing genetic and molecular heterogeneity in research.

In the future, moving away from a broad division into “autoimmune” versus “autoinflammatory” conditions toward a molecular systems-based classification could greatly improve clinical understanding. The creation of large-scale genotype–phenotype databases will be an essential step toward enabling precise molecular diagnoses and developing personalized therapies for both rare and prevalent forms of these disorders.

REFERENCES:

1. Tan EM, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982; 25:1271-7.

2. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997; 40:1725.

3. Slatko BE, Gardner AF, Ausubel FM. Overview of next-generation sequencing technologies. Curr Protoc Mol Biol. 2018;122: e59.

4. Richards S, et al. Standards and guidelines for the interpretation of sequence variants. Genet Med. 2015; 17:405-24.

5. Grand MG, et al. Cerebroretinal vasculopathy: a new hereditary syndrome. Ophthalmology. 1988; 95:649-59.

6. Stam AH, et al. Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations. Brain. 2016; 139:2909-22.

7. Kothari PH, et al. TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia. Brain Pathol. 2018; 28:806-21.

8. Richards A, et al. C-terminal truncations in human 3’-5’ DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy. Nat Genet. 2007; 39:1068-70.

9. Crow YJ, et al. Mutations in the gene encoding the 3’-5’ DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus. Nat Genet. 2006; 38:917-20.

10. Rice G, et al. Clinical and molecular phenotype of Aicardi-Goutières syndrome. Am J Hum Genet. 2007; 81:713-25.

11. Liu Y, et al. Activated STING in a vascular and pulmonary syndrome. N Engl J Med. 2014; 371:507-18.

12. Watkin LB, et al. COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis. Nat Genet. 2015; 47:654-60.

13. Beck DB, et al. Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease. N Engl J Med. 2020; 383:2628-38.

14. Jesus AA, Goldbach-Mansky R. IL-1 blockade in autoinflammatory syndromes. Annu Rev Med. 2014; 65:223-44.

15. Lama L, et al. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat Commun. 2019; 10:2261.

16. Hong Z, et al. STING inhibitors target the cyclic dinucleotide binding pocket. Proc Natl Acad Sci USA. 2021;118: e2105465118.

Received on 25.08.2025 Revised on 05.11.2025

Accepted on 30.12.2025 Published on 27.01.2026

Available online from February 02, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(1):34-40.

DOI: 10.52711/2231-5675.2026.00006

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.