Enrichment of Immune Dysregulation Disorders in Adult Patients with Human Inborn Errors of Immunity

Inborn errors of immunity (IEI), previously known as primary immunodeficiencies, are a large group of heterogeneous diseases originating from molecular alterations in genes related to the innate or adaptive immune system. Patients with these alterations develop an increased susceptibility to infectious diseases (mediated by viruses, bacteria, or fungi) and/or a growing diversity of autoimmune, autoinflammatory, allergic, bone marrow failure, or malignant phenotypes. Although IEI are considered rare diseases, the number of this type of disorders has been growing year by year, and more than 485 genetic defects related to IEI are currently described. The International Union of Immunological Societies (IUIS) categorizes IEI into nine main groups and an extra 10th group for IEI-phenocopies. These groups are based on overlapping phenotypes shown by different patients [1, 2].

Due to the genetic origin of most of these diseases, symptoms usually appear at the first stages of life and diagnosis is commonly made during childhood. This early diagnosis allows the establishment of a tailored treatment and appropriate genetic counseling to achieve considerable improvement and even cure these patients. Nevertheless, some IEI patients with pathologic variants are diagnosed in adulthood because of a late onset of symptoms [3] or nonspecific multisystemic presentations that finally give rise to a delay in ordering specific molecular studies for the diagnosis of IEI. There are different hypotheses to explain the symptomatic delay of these diseases, such as contact with a specific environmental trigger, the low penetrance of some hypomorphic variants, the development of epigenetic modifications of specific genes, or the presence of somatic variants [3].

An immunophenotype-based approach followed by Sanger sequencing was the most common strategy to reach a genetic diagnosis of IEI until approximately a decade ago. However, there were difficulties in finding an accurate diagnosis of patients using this strategy, such as the large number of IEI-related genes, high variability in the genotype–phenotype relationship, or similar phenotypes shared by different genotypes. Furthermore, Sanger sequencing of multiple candidate genes is a time-consuming, inefficient, and expensive methodology [4]. The growing incorporation of next-generation sequencing (NGS) in hospital environments has allowed a better recognition of previously undiagnosed IEI, not only in infants but also in adult patients [3, 4]. NGS is a sensitive and cost-effective sequencing technology that can be used as a first-line molecular study to identify IEI [5]. The improvement in diagnostic efficiency is clearly demonstrated in some patients with atypical presentations of known IEI [6]. In spite of the benefits achieved by NGS, the identification of disease-causing genetic defects in IEI patients is still a challenge and the diagnostic yield barely reaches 50% in the best of circumstances [5, 7, 8]. Additionally, IEI could fit with other more complex genetic scenarios such as polygenic, epigenetic, or even somatic; not only monogenic models should be considered [9].

In this work, we show the compiled experience in molecular IEI-diagnosis of adult patients between 2005 and 2023. We report on our performance ability to identify IEI in adults, the different types of IEI identified, and the most frequent ages of onset of these pathologies. In addition, we show the most frequent clinical features associated with the detection of IEI in adult patients. We also relate the advantages of a modified list of warning signs for adult patients based on the Jeffrey Model Foundation 10 warning signs to anticipate the presence of an IEI- associated molecular defect.

This work is a retrospective study conducted in the University Hospital 12 de Octubre between 2005 and 2023. Inclusion criteria were adult patients older than 18 years old with suspected IEI that presented clinically as increased susceptibility to infections, autoimmunity, autoinflammatory diseases, allergy, bone marrow failure, and/or malignancy for which their physicians required a molecular study. All patients provided written informed consent in accordance with the Declaration of Helsinki and as approved by the Institutional Research Ethics Committees of University Hospital 12 de Octubre.

Primary clinical manifestations and genetic analysis results were collected and analyzed. Molecular diagnosis was made through Sanger sequencing from 2005 to 2015 (11 years). PCR was used to amplify the coding sequence and the flanking regions of the candidate genes using specific primers. Double-strand DNA templates were sequenced using the dideoxy chain-terminator method of Sanger using an ABI PRISM 3130 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA). NGS was performed from 2016 to 2023 (7 ½ years), and two sequencing approaches were used. In the first approach, an in-house-designed targeted 192-gene panel (from January, 2016, to December, 2021) or 434-gene panel (from January, 2022, until June, 2023) (Tables S1 and S2) involved in IEI was sequenced in an Ion Torrent PGM (Thermo Fisher Scientific, Waltham, MA, USA) or MiSeq platform (Illumina, San Diego, CA, USA), respectively. In the second approach, whole exome sequencing (WES) was carried out in cases with atypical clinical and laboratory phenotypes or a negative result in a previous molecular study by Sanger sequencing or the 192/434 gene panels (Table S3). The sequencing workflow followed by our center is shown in Fig. S1. An xGen Exome Panel v1.0 kit (Integrated DNA Technologies, Coralville, IO, USA) and paired-end sequencing (2 × 75 bp) was conducted on a NextSeq 550 (Illumina, San Diego, CA, USA). Bioinformatic analysis was performed using the Karma tool, an in-house pipeline integrating BWA (v0.7.17) and Bowtie2 (v2.4.1) for sequence alignment to the reference genome (hg19 assembly), GATK (v4.1), and VarDict (v1.7.0) for genotyping, ExomeDepth (v.1.10) for CNV detection, AnnotSv (v2.4) for CNV annotation, and ANNOVAR (v2018Apr16) for variant annotation. Karma follows the validation recommendations of the Association for Molecular Pathology (AMP) [10]. Genetic variants were filtered according to alignment and genotyping quality metrics. Variants identified from alignments with low mapping quality, variants with strand bias, variants with a frequency greater than 3% in the gnomAD population database (v2.1.1) [11], and variants classified as benign and probably benign according to the ClinVar database were not evaluated. Clinical interpretation and final classification of the identified variants were performed using information extracted from the gnomAD population database; the genetic variant databases ClinVar, Leiden Open Variant Database (LOVD), and Human Gene Mutation Database (HGMD); the protein domain and structure databases Uniprot, PFAM, and Prosite; and the hereditary disease databases OMIM, Orphanet, and GeneReviews. Variants were classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS) according to the recommendations of the American College of Medical Genetics and Genomic (ACMG) [12]. Variant filtering and prioritization were performed based on a 455-gene custom panel targeted (Table S3) at the clinical suspicion of IEI. The human genomics community VarSome [13] was used for a comprehensive interpretation of the variants. Candidate gene variants were confirmed by PCR and Sanger sequencing using the same strategy described previously.

Furthermore, copy number variants (CNVs), a 60K chromosomal microarray (CMA) (60K KaryoNIM®, NIMGenetics, Madrid, Spain), were carried out in cases where a heterozygous variant was detected and the form of the disease was autosomal recessive (AR). Analysis and interpretation of the results were performed using Cytogenomics (v.4.0.3.12, Agilent) software. A threshold of ≥ 5 consecutive probes was established to consider a CNV. CNVs detected were classified following previous CMA recommendations for clinical practice. For fluorescence in situ hybridization (FISH), metaphase chromosome spreads were prepared from peripheral blood lymphocytes harvested routinely, and FISH was performed using a commercially available probe, LSI Tuple 1 (Vysis, Abbott), according to the manufacturer’s protocols.

Statistical tests were performed using GraphPad Prism version 7 (GraphPad Prism Software, La Jolla, CA, USA) and RStudio (Posit team (2022); RStudio: Integrated Development Environment for R. Posit Software, PBC, Boston, MA. URL http://www.posit.co/). Statistical significance was calculated by the chi-square test. Sensitivity and specificity were compared by the exact binomial test. Predictive values were compared using the weighted generalized score statistic proposed by Kosinski [14]. Significance was considered only when P values were less than 0.05 (*p < 0.05; **p < 0.001).

One-fifth of the patients (20.45%, 9/44) in the cohort w_MolDx received allogeneic hematopoietic stem cell transplantation (HSCT) (Fig. 2E). Only the patient with the biallelic form of TET2 died after HSCT [24]. Although HSCT is considered the main therapeutic and curative approach for children with IEI, it has been relatively unusual among IEI-adults due to difficulties related to the selection of adequate candidates and donors, optimal timing, conditioning regimens, and specific management after transplantation. The decision to transplant remains a complex clinical decision [55]. Nevertheless, negative HSCT outcomes have been mostly related to complications arising from the specific IEI of the patient, more than with the age of the candidates [56]. Therefore, an early molecular diagnosis is imperative to allow IEI adults to benefit from HSCT and avoid the occurrence of adverse effects that would contraindicate it.

The aim of this work is to show that IEI-related genetic alterations are not infrequent findings in adult patients. As a rule, clinical manifestations due to genetic alterations usually appear in childhood and suspicion of IEI-causing variants in adult patients is less frequent. The delay from initial manifestations to diagnosis of IEI for index cases frequently takes several years. Sometimes, this molecular diagnostic delay can be critical and often involves frequent, costly, and unhelpful specialist visits and unnecessary laboratory tests, which represents a diagnostic odyssey. It should be necessary to expand access to screening of adult patients for IEI, especially when disease onset occurs in childhood [8]. In this work, patients with initial symptoms before 18 years of age should have been studied as soon as possible, but several reason could have influenced the delay of a molecular diagnosis: a. symptomatic delay of the disease with a poor specificity of presenting symptoms, as in the case of some CVID patients; b. multisystemic manifestations without a multidisciplinary evaluation (TET2 deficiency, STAT3 LoF, 22q11.2del patients); c. limited access to NGS techniques in the healthcare field when the patients were under 18 years of age (PGM3, PIK3R1, RAB27A patients); d. incomplete penetrance with mild clinical phenotypes where the diagnosis was carried out through the patients’ offspring (ELANE patients).

Unfortunately, the resulting diagnostic delay can interfere with properly tailored treatment, causing unnecessary distress, diminished quality of life, and sometimes death. A point to take into account is the case of patients with CVID, where approximately 50% of the patients’ onset is in the second decade of life [15], and in some cases, molecular diagnosis represents a challenge.

In conclusion, NGS is a first-tier diagnostic test for monogenic adult IEI that can be used in patients with typical and atypical presentation. Therefore, adult patients with immune dysregulation in the form of autoimmunity, cytopenias, lymphoproliferation, and neoplasia should be considered for NGS screening.

The ever-changing clinical and genetic landscape of IEI and the advances of the last decade have shown how clinical, translational, and basic science networks have enhanced the field of immunodeficiencies [57]. The growing number of genetic discoveries has increased clinical awareness, and targeted biologics and definitive treatments such as gene therapy will be important in the next decade.