Primary immunodeficiency disorders (PID), also termed as inborn errors of immunity (IEI), encompass a heterogeneous array of conditions characterized by compromised immune system function involving essential components, such as neutrophils, macrophages, dendritic cells (DC), complement proteins and natural killer cells, as well as T and B lymphocytes (1).

To date, a variety of >400 disorders have been listed under the term IEI and many of them have been described only in a small number of patients and families (2-4). While PIDs are primarily hereditary, acquired deficiencies have also been identified (5). It is crucial to differentiate IEI from secondary immunodeficiencies, which arise more frequently due to various factors such as infections, medications, metabolic disease and environmental conditions. By contrast, IEI stem from genetic anomalies resulting in impaired immune cell functionality (5).

Immunoglobulin A deficiency is the most common IEI, estimated to affect 1 in 300 to 1 in 500 individuals, whereas other IEI are rarer, with an approximate prevalence of 1 in 1,200 live births (6). The clinical indications of IEI are highly diverse and patients have a higher infection susceptibility. IEI may be diagnosed as a normal infection (ears, lungs and the sinuses), although they may be undetected if misdiagnosed (7). The diagnosis of such disorders requires specialized testing, accurate and timely diagnosis, as well as a relatively high suspicion (8). An experienced clinical immunologist in the evaluation of immunodeficiencies should be consulted for an accurate and early diagnosis, as well as precise treatment, which could prevent the rate of morbidity related to such disorders and it should improve the patient outcomes (9). Furthermore, Amaya-Uribe et al (10) found that there is a high prevalence of autoimmune disease (AD) among cases of IEI, indicating a link between the occurrence of both conditions. The authors found that a defect in autoimmune regulator and T-regulatory cells of the central and peripheral tolerance is associated with AD development. In addition, the study found that chronic and recurrent infection through molecular mimicry, as well as activation of super antigens, are vital for the progression of AD, which are characteristics of IEI (10).

The basic classification of IEI depends upon the type of immune system defects; they are broadly classified as adaptive or innate immunity disorders, respectively (1). The advent of whole-exome sequencing (WES) has significantly contributed to identifying novel causative genes in IEI cases (11). Particularly in regions with elevated rates of parental consanguinity, such as Jordan, where ~40% of marriages are consanguineous, WES has proven effective in identifying disease-associated genes and variants. The prevalence of consanguinity before 1980 in Amman was ~30%, leading to a heightened predisposition to IEI (according to www.consang.net). However, identifying new mutations requires robust evidence of their clinical relevance, necessitating functional genomic studies. These investigations delve into the impact of novel mutations on gene transcription, translation, transcriptional regulation and protein structure and function, ultimately shedding light on the precise effects of these mutations on proteins. Thus, genetic testing is a must when a family history of IEI has been detected in one of the family members (12). The present study, for the first time, investigated a cohort of Jordanian patients in whom signal transducer and activator of transcription 1 (STAT1), elastase, neutrophil expressed (ELANE) and interferon induced with helicase C domain1 (IFIH1) mutations were identified, and a new association with variants in NLR family, pyrin domain-containing 12 (NLRP12), G-protein-signaling modulator 1 (GPSM1) and TNFα-induced protein 3-interacting protein 1 (TNIP1) was discovered.

Primary immunodeficiencies encompass a diverse spectrum of disorders that necessitate precise diagnostic strategies. The emergence of NGS techniques has revolutionized clinical immunology. These methodologies not only aid in pinpointing the causative genetic variations but also inform therapeutic interventions, tailoring treatments to individual patient's genetic profiles (13,14).

A salient factor influencing the incidence of novel mutations is consanguinity. In Jordan, consanguinity is more prevalent in rural areas (~40%) than in its capital, Amman, where these levels are ~25.5%. The historical trend of consanguinity in Amman also seems to have influenced the prevalence of PID. Marriages before 1980 had a consanguinity rate of ~30%. (15)

Consanguinity does not seem to be a relevant bias in the present cohort of patients with PID. A novel homozygous mutation of uncertain significance was found in GPSM1, only in patient ID-32. In addition, the homozygous IFIH1 variant was found in a family where consanguinity was not previously reported. In all other patients, only heterozygous variants/mutations were found.

Arunachalam et al (14) elucidated that PIDs are typically monogenic, arising from an SNV. By contrast, polygenic diseases often manifest as autoimmune conditions such as rheumatoid arthritis or systemic lupus erythematosus. Of note, certain genetic variations that are associated with PIDs also appear in autoimmune diseases, underscoring the interwoven nature of immunological disorders (14,15).

It is true that several studies have shed light on the genetic landscape of PIDs, but with different results. For instance, Arunachalam et al (14) identified numerous genetic variants across different PID subtypes. In addition, studies from Aghamohammadi et al (16,17), Abolhasssani et al (18,19) and Kilic et al (20) provided valuable insight into the distribution of PID subtypes in Iranian and Turkish populations, respectively.

In the present study, the genetic underpinnings of PIDs in 9 Jordanian patients were explored, identifying mutations in key immunological genes, including STAT1/2, MBL2, TYK2, UNG, TNIP1, CARMIL2 and ELANE, as well as a 9-bp insertion in IL17RC, and one frameshift and nonsense mutation in the same exon in GPSM1. Of note, a STAT2 mutation was found in patient ID-26 suffering from hypogammaglobulinemia (after treatment with rituximab). However, STAT2 heterozygosity may have no impact on the patient; thus, it should be investigated along with PIK3R4 in order to determine their effect on the patient.

Furthermore, mutations in TYK2 and UNG were found in patient ID-34, who had been diagnosed with unclassified arthritis and esophageal candidiasis.

Mutations in STAT1 have been reported in different ethnicities, affecting critical cellular processes, such as STAT1-mediated responses to IFNG and IL27 (21,22) In the present cohort, patient ID-30 had a heterozygous SNV in the CARMIL2 and STAT1 genes. CARMIL2 is associated with immunodeficiency-58 (IMD58). This is an AR primary immunologic disorder distinguished by early-onset skin lesions, such as eczematous dermatitis, infectious abscesses and warts, as well as recurrent respiratory infections or allergies, among others. CARMIL2 deficiency, as described by Wang et al (23), presents with a unique set of clinical features, with compromised T-cell subsets and antibody responses. STAT1 deficiency, on the other hand, is associated with three distinct phenotypes depending on whether it's loss of function (LOF) AD/AR or gain of function (GOF) AD, while LOF mutations correlate with compromised Th1 immunity. GOF mutations are associated with impaired Th17 immunity-in the case of patient ID-30, GOF mutations were more likely suggested based on the patient's clinical presentation. IMD31C is an immunologic dysregulation disorder with highly variable manifestations caused by AD GOF mutations in STAT1. Most patients present with chronic mucocutaneous candidiasis in infancy or early childhood (24). Other highly variable characteristics include persistent infections, enteropathy with villous atrophy and autoimmune disorders such as hypothyroidism and diabetes mellitus. In addition, IMD31C is inherited in an AD manner (25-27). Both genes (STAT1, CARMIL2) could be relevant to the patient's disease, STAT1 function (e.g. phosphorylation) should be investigated, in addition to IL-17A production testing post-stimulation by phorbol 12-myristate 13-acetate and ionomycin, are useful to activate transcription factors for intracellular signaling and production of cytokines of many different immune cell types in the presence of Brefeldin, as well as the enumeration of Th17 lymphocytes, feasible through C-C motif chemokine receptor 6 and C-X-C motif chemokine receptor 3 markers, to ascertain the diagnosis. In addition, CARMIL2 expression should be detected in the patient. Similarly, mutations in IL17RA and TYK2 have been associated with characteristic clinical phenotypes and cellular defects (28-30). In the present study, a new IL17RA mutation was detected in a patient suffering from hypergammaglobulinemia and steroid-induced lymphopenia, namely ID-29. Two new mutations, one in STAT1 and another in CARMIL2, were also detected in a patient suffering from chronic mycosis of the skin, developmental delay and hypothyroidism, namely ID-30. In addition, a new TNIP1 mutation was found in ID-31, a patient diagnosed with biliary stasis at the age of 2 months, with are current history of fever since the early infantile period, and a history of short liver synovitis and microscopic hematuria. Furthermore, studies have emphasized the clinical significance of mutations in genes such as UNG, MBL2 and IL17RC (14,31-35).

Furthermore, studies reported the existence of NLRP12 deficiency in Chinese patients, being a compelling case of the diverse clinical manifestations associated with PID mutations, ranging from erythema nodosa to systemic symptoms such as fever and lymphadenopathy (36-38).

ELANE encodes leukocyte elastase, being produced by neutrophil precursors, and its mutations have been associated with different immunodeficiencies, such as congenital neutropenia (14,39,40). In the present study, ELANE mutation was found in patient ID-28, who suffered from severe neutropenia. The patient was diagnosed with no maturation arrest in the bone marrow study performed at the age of 3 months.

IFIH1 (IMD95) encodes an RIG-I-like receptor involved in the sensing of viral RNA (41,42). IFIH1 variants are also related to hepatitis C virus clearance (43). Heterozygous gain-of-function IFIH1 mutations have also been associated with autoimmune diseases (44). Furthermore, rare gain-of-function mutations in IFIH1 significantly increase the production of type I IFN, leading to Aicardi-Goutières or Singleton-Merten syndromes (45,46). Nonetheless, IMD95 is an AR disorder characterized primarily by recurrent and severe viral respiratory infections beginning in infancy or early childhood (47). Infections such as human rhinovirus which is the most common viral infectious agent in humans and is the predominant cause of the common cold and respiratory syncytial virus, a contagious virus that causes infections of the respiratory tract, frequently necessitate hospitalization or respiratory support. Immunologic testing is usually normal, though some minor abnormalities may be detected. The disorder is caused by a loss of the innate immune system's ability to sense viral genetic information, resulting in a lack of interferon production, poor response to viral and immunologic stimulation and failure to control viral replication (48). The patient in whom the IFIH1 mutation was detected had a low CD4 count. She was diagnosed with secondary alveolar proteinosis at the age of 8 months, had recurrent admissions with chest infection, as well as developmental delay and epilepsy disorder at the age of 2 years, which were associated with the patient's symptoms and initial diagnosis (15).

Familial cold autoinflammatory syndrome-2 is an AD autoinflammatory disorder that causes episodic and recurring rash, urticaria, arthralgia, myalgia and headaches. In most patients, these episodes are accompanied by fever and serologic signs of inflammation. The majority of patients, but not all, report that exposure to colds triggers their episodes. Other symptoms may include abdominal pain, thoracic pain and sensorineural deafness. The age of onset varies, ranging from infancy to middle age, and the severity and clinical manifestations are diverse (49). In the present study, patient ID-32 had a mutation in NLRP12 and according to various databases, the variant found is pathogenic. The patient had a history of chronic cough, failure to thrive and chronic diarrhea, which explains the symptoms. Furthermore, a mutation in GPSM1 was detected for the same patient; he had decreased naïve T cells and a reduced number of switched memory B cells.

Despite the high rate of consanguinity (4 out of 9 patients), only two homozygous variants were identified, with only one potentially linked to an IEI. This can be explained by considering several factors related to genetic diversity, the complexity of inheritance patterns and the nature of the diseases involved. IEIs are often genetically heterogeneous, meaning they can be caused by mutations in different genes. Despite the high rate of consanguinity, the patients may carry mutations in different genes that lead to similar clinical phenotypes. In addition, not all individuals with a pathogenic variant will necessarily manifest the disease (incomplete penetrance) and the severity of the disease can vary among those who do (variable expressivity) (38,50). This could result in only some of the patients showing symptoms of an IEI despite having related genetic backgrounds. Certain genetic conditions are caused by having two different mutations in a gene (one on each allele) rather than two identical mutations (homozygosity). In consanguineous populations, there may still be a presence of compound heterozygous variants that are not immediately apparent as homozygous but that lead to disease (51). Besides homozygous variants, other mechanisms, such as copy number variations, epigenetic changes or mutations in regulatory regions may be responsible for IEIs and may not be easily detected by standard genetic screening focused on homozygous variants (52-54). Finally, IEIs may be influenced by multiple genes and environmental factors. Therefore, not all individuals with consanguinity and clinical symptoms will have a clear, single-gene cause that can be identified as homozygous mutations.

The IL-17 family of cytokines plays crucial roles in both acute and chronic inflammatory responses and IL-17A, the hallmark cytokine of the Th17 cells, has important roles in protecting the host against extracellular pathogens, but also promotes inflammatory pathology in autoimmune disease (55).

The mutation in STAT1 affects the differentiation of Th17 cells, which are a subset of CD4+ T cells involved in epithelial cell- and neutrophil-mediated immune responses against extracellular microbes and in the pathogenesis of autoimmune diseases. The same mutation along with the c.1342C>T mutation in TYK2 also affects the JAK-STAT signaling pathway. This is the principal signaling mechanism for a wide array of cytokines and growth factors. Following the binding of cytokines to their cognate receptor, STATs are activated by members of the JAK family of tyrosine kinases. Once activated, they dimerize and translocate to the nucleus and modulate the expression of target genes. In addition to the activation of STATs, JAKs mediate the recruitment of other molecules such as the MAPKs and PI3K. These molecules process downstream signals via the Ras/Raf/MAPK and PI3K pathways, which results in the activation of additional transcription factors.

Our analysis also hints towards a deregulation in the NET formation through the mutation in ELANE. Neutrophils play a central role in innate immune defense. One of the mechanisms of neutrophil action is the formation of NETs, the extracellular structures composed of chromatin coated with histones, proteases and granular and cytosolic proteins that help catch and kill microorganisms.

Finally, two more pathways affected by mutations in GPSM1 and IL17RC were those of cocaine addiction and cytokine-cytokine receptor interaction, respectively.

The present study has certain limitations, which should be acknowledged. It focuses on the Jordanian population, thus limiting the generalizability of the present findings. Furthermore, the sample size was relatively small (nine families), potentially affecting the statistical power of the study. In addition, the present study lacks functional validation of the main findings. However, it still provides a direction for future research.

In conclusion, the intricate tapestry of primary immunodeficiencies is gradually being unraveled with the aid of advanced genetic techniques. Understanding the genetic basis of these disorders among consanguineous Jordanian families holds promise for the development of personalized therapeutic interventions and improved patient outcomes.