Clinical Manifestations, Immunological Characteristics and Genetic Analysis of Patients with Hyper-Immunoglobulin M Syndrome in Iran
Mitra Tafakori Delbari, Taher Cheraghi, Reza Yazdani, Saba Fekrvand, Samaneh Delavari, Gholamreza Azizi, Zahra Chavoshzadeh, Seyed Alireza Mahdaviani, Hamid Ahanchian, Maryam Khoshkhui, Fatemeh Behmanesh, Soheila Aleyasin, Hossein Esmaeilzadeh, Farahzad Jabbari-Azad, Morteza Fallahpour, Mohammadali Zamani, Seyedeh Panid Madani, Bobak Moazzami, Sima Habibi, Arezou Rezaei, Azadeh Lotfalikhani, Masoud Movahed, Mansoureh Shariat, Arash Kalantari, Delara Babaei, Mahshid Darabi, Nima Parvaneh, Nima Rezaei, Hassan Abolhassani Asghar Aghamohammadi
a Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Science, Tehran, Iran;
b Department of Pediatrics, 17th Shahrivar Children’s Hospital, Guilan University of Medical Sciences, Rasht, Iran;
c Non-Communicable Diseases Research Center, Alborz University of Medical Sciences, Karaj, Iran;
d Pediatric Infections Research Center, Mofid Children’s Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
e Pediatric Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
f Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran;
g Allergy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran;
h Department of Allergy and Clinical Immunology, Rasool e Akram Hospital, Iran University of Medical Sciences, Tehran, Iran;
i Department of Immunology and Allergy, Shahrekord University of Medical Sciences, Shahrekord, Iran;
j Division of Allergy and Clinical Immunology, Department of Pediatrics, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran;
k Department of Allergy and Clinical Immunology, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran;
l Department of Allergy and Clinical Immunology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran;
m Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institute at Karolinska University Hospital Huddinge, Stockholm, Sweden
Abstract
Background: Hyper-immunoglobulin M (HIGM) syndrome is a rare heterogeneous group of primary immunodeficiency disorders characterized by low or absent serum levels of IgG and IgA along with normal or elevated serum levels of IgM. Methods: Clinical and immunological data were collected from the 75 patients’ medical records diagnosed in Chil- dren’s Medical Center affiliated to Tehran University Medical Sciences and other Universities of Medical Sciences in Iran. Among 75 selected patients, 48 patients (64%) were ana- lyzed genetically using targeted and whole-exome sequenc- ing. Results: The ratio of male to female was 2.9:1. The me- dian age at the onset of the disease, time of diagnosis, and diagnostic delay were 10.5, 50, and 24 months, respectively. Pneumonia and lower respiratory tract infections (61.3%) were the most common complications. Responsible genes were identified in 35 patients (72.9%) out 48 genetically ana- lyzed patients. Cluster of differentiation 40 ligand gene was the most mutated gene observed in 24 patients (68.5%) fol- lowed by activation-induced cytidine deaminase gene in 7 pa- tients, lipopolysaccharide-responsive and beige-like anchor (1 patient), nuclear factor-kappa-B essential modulator (1 pa- tient), phosphoinositide-3-kinase regulatory subunit 1 (1 pa- tient), and nuclear factor kappa B subunit 1 (1 patient) genes. Nineteen (25.3%) patients died during the study period, and pneumonia was the major cause of death occurred in 6 (31.6%) patients. Conclusion: Physicians in our country should carefully pay attention to respiratory tract infections and pneumonia, particularly in patients with a positive fam- ily history. Further investigations are required for detection of new genes and pathways resulting in HIGM phenotype.
Introduction
Hyper-immunoglobulin M (HIGM) syndrome is a rare heterogeneous group of primary immunodeficiency disorders (PIDs) characterized by low or absent serum levels of IgG and IgA along with normal or elevated se- rum levels of IgM, accounting for approximately 0.3– 2.9% of all primary immunodeficient patients [1, 2]. To date, defects in B cell signaling, class switch recombina- tion, somatic hyper mutation, and DNA repair mecha- nisms have been identified to be associated with HIGM phenotype. Cluster of differentiation 40 ligand (CD40L) and nuclear factor-kappa-B essential modulator (NEMO) genes with X-linked inheritance, as well as CD40 and ac- tivation-induced cytidine deaminase (AICDA) genes with autosomal inheritance, are the most frequently re-ported mutations among HIGM patients [3, 4]. However in Western cohorts, CD40L is the most prevalent genetic defect, making up nearly 65–70% of all HIGM cases and resulting in a more severe form of the disease affecting both the humoral and the cellular immunity [5].
In accordance to the genetic underpinning, there is a heterogeneous spectrum of clinical manifestations among the affected patients including severe recurrent common and opportunistic infections; pulmonary complications; gastrointestinal involvement such as oral ulcers, protract- ed diarrhea, sclerosing cholangitis, and liver cirrhosis;, autoimmune and inflammatory disorders; lymphoprolif- eration; and malignancy [6–11]. Defective CD40L:CD40 interaction and nuclear factor-kappa-B pathway in HIGM patients are associated with combined cellular (T-cell) and humeral (B-cell) immunodeficiency, while AID-de- ficient patients have intact cellular immunity. Moreover, CD40L- and CD40-deficient HIGM patients are reported to have reduced numbers of CD27+ switched memory B cells along with normal numbers of total peripheral blood B lymphocytes, and patients with AID deficiency are re- ported to have normal amounts of both peripheral and CD27+ memory B lymphocytes [6, 12, 13].
In regard to the clinical and genetic diversities and a scarce number of publications concerning HIGM pa- tients [14, 15], in the present study, we aimed to describe an update report on demographic characteristics and clinical, laboratory, and molecular features in a relatively large cohort of Iranian patients with HIGM syndromes.
Material and Methods
Study Population
During 1993–2018, a total of 75 patients diagnosed with HIGM syndrome and followed up at Children’s Medical Center (Pediat- rics Center of Excellence affiliated to Tehran University of Medical Sciences, Tehran, Iran) and other Universities of Medical Sciences in Iran (Guilan, Shahid Beheshti, Mashhad, Shiraz, Iran, and Shahrekord) were enrolled in this study [16, 17]. Based on the di- agnostic criteria for PID recommended by European Society for Immunodeficiencies, the inclusion criteria for diagnosis of HIGM were low serum IgG (2 SD below age-related normal values in at least twice measurement) and normal or elevated serum IgM, no evidence of profound T-cell deficiency along with at least one of the followings: increased susceptibility to infections, immune dys- regulation, cytopenias, malignancy, and affected family member (https://esid.org/Working-Parties/Registry-Working-Party/Di- agnosis-criteria). Patients with secondary cause of immunodefi- ciency and incomplete diagnostic criteria were excluded. This study was approved by the Ethics Committee of the Tehran Uni- versity of Medical Sciences, and written informed consents were obtained from all patients and/or their legal guardians.
Data Collection
Detailed clinical and immunological information were retro- spectively collected from the patients’ medical records. These data consisted of demographic information, age at onset of symptoms, age at diagnosis, delay in diagnosis, parental consanguinity, family history of immunodeficiency, clinical manifestations, and immu- nologic data that were performed at the time of diagnosis of the disease, using standard techniques administered by highly author- itative technicians as explained previously [18, 19]. Diagnostic de- lay was defined as the time period between the onset of symptoms and the time of clinical diagnosis of HIGM.
Genetic Analysis
Genomic DNA was extracted from whole blood of all available patients by the previously described procedure [20]. Targeted Sanger sequencing for the most probable mutated genes including CD40L, CD40, NEMO, AICDA and UNG was carried out in the mentioned patients [3]. As a result of the failure of targeted se- quencing in the detection of underlying genetic mutations, whole- exome sequencing was performed and analyzed in unsolved pa- tients as explained in details in previous studies [3, 21].
Statistical Analysis
Statistical analysis was accomplished using SPSS (version 24) software (SPSS Inc., Chicago, IL, USA). Kolmogorov-Smirnov and Shapiro-Wilk tests were used to test for the normality of data. Cen- tral and descriptive statistics were reported for quantitative data. For variables with skewed distribution, median and interquartile range (IQR) were reported as the index of data dispersion. Ana- lytical analyzes were performed using Mann-Whitney and chi- square and Fisher’s exact tests.
Results
Demographic Characteristics
A total of 75 patients (19 female and 56 male) with median (IQR) age at the time of the study of 11.0 (6.0–20.5) years were enrolled in the study. Median (IQR) age at the onset of the disease, time of diagnosis, and delay in diagnosis was 10.50 (6.0–33.0), 50.0 (19.5– 74.0), and 24.0 (7.0–53.0) months, respectively. Forty five (60%) of the patients were born to consanguineous families. At the time of the study, 45 (60%) patients were alive while 19 (25.3%) of them were dead, and the current vital status of 11 (14.7%) patients was not clear. Pneumonia was the most common cause of death among the deceased patients, accounting for 31.6% (6 patients) of the cases. The positive family history of im- munodeficiency and/or early death of a family member was observed in 18 (24%) patients (Table 1). As depict- ed in Figure 1, respiratory tract infection (59%) was the most common first presentation among the patients, followed by skin infection/abscess (10%) and diarrhea (9%).
Clinical Manifestations
Clinical presentations of the study population are rep- resented in Figure 2. Infectious manifestations were the most prevalent feature among the study population. Pneumonia and lower respiratory tract infections were recorded in 46 (61.3%) of the patients followed by upper respiratory tract infections (44 patients, 58.7%), otitis media (41 patients, 54.7%), and sinusitis (23 patients, 30.7%).
Among the noninfectious complications, lymphopro- liferative disorders (30 patients, 40%), recurrent diarrhea (23 patients, 30.7%), splenomegaly (20 patients, 26.7%), and failure to thrive (20 patients, 26.7%) were the com- monest. The mean episodes/person-years of pneumonia (0.730 vs. 0.134, p < 0.001), otitis media (0.537 vs. 0.008, p < 0.001), and sinusitis (0.203 vs. 0.083, p = 0.008) were significantly higher before the diagnosis of HIGM syn- drome in comparison to after the diagnosis. Table 2 pre- cisely demonstrates the complicated organs among the study population. Respiratory tract (60 patients, 80%) was the most involved organ followed by hematologic system (34 patients, 45.3%) including anemia (30 patients, 40%), neutropenia (21 patients, 28%), thrombocy-topenia (9 patients, 12%), and leucopenia (2 patients, 2.7%). Multiple organs complication was also reported in 50 patients (79.7%).
Laboratory Features
In comparison to the age-matched normal immuno- logical values, 7.1% of the patients had decreased T cell count, while 12% and the remaining 80.9% had increased and normal counts, respectively. CD4+ T cell count was decreased in 21.1% of the patients, while CD8+ count was increased in 34.5% of the study population. B cell count was found to be decreased by 3.8% of the patients. Other detailed information about laboratory features is provid- ed in Table 3.
Mutation Analysis
Genetic analyses were performed in 48 patients from which molecular diagnosis was confirmed in 35 patients (72.9%). CD40L gene was the most mutated gene (24 pa- tients, 68.5%) followed by AICDA (7 patients, 20%) and NEMO in 1 patient. Of note, we also identified mutations in lipopolysaccharide-responsive and beige-like anchor protein, phosphoinositide-3-kinase regulatory subunit 1, and nuclear factor kappa B subunit 1 genes each in 1 pa- tient (Fig. 3). Missense mutations were the most preva- lent, making up 62.8% (22 patients) of all mutations, fol- lowed by frameshift nonsense (5 patients, 14.2%), splice- site (4 patients, 11.4%), and stop gain (3 patients, 8.5%) mutations. In CD40L gene, being the most affected, the most common mutations were missense (16 patients, 66.6%), affecting the tumor necrosis factor homologous domain (17 patients, 70.8%) of the CD40L protein. More- over, in AID-deficient patients, missense mutations were predominant (4 patients, 57.1%), and the cytidine mono- phosphate deaminase domain residues were the main af- fected domain of the protein (6 patients, 85.7%). Detailed information about amino acid and DNA sequence change, zygosity, a method of genetic analysis, affected domain, type of mutation, predicted severity, and medical severity for each of the 35 genetically evaluated patients is pro- vided in Table 4.
Discussion
In the current cohort study, we presented the recent findings in regard to demographic data, and clinical and laboratory data of 75 Iranian HIGM patients, 35 of whom were also genetically diagnosed (only 27% unsolved pa- tients indicating the best diagnostic yield worldwide). This study includes the largest number of HIGM patients among the previously published cohort studies in the country.
Median diagnostic delay age of HIGM syndrome in Iran has decreased from 31 months in 2009 [14] to 24 months in the present study in 2019; this improvement in earlier diagnosis is partly due to the recent advances in research and diagnostic procedures and increased aware- ness of physicians about PIDs in the country [16, 22]. In comparison to other countries, this series’ median diag- nostic delay was lower than reports from Turkey [23], India [24], China [25, 26], and Tunisia [27] and higher than those of previously reported retrospective studies from Brazil [28] and Latin America [29]. High rates of consanguineous marriage in our country along with rela- tively high rate of positive family history for a PID and/or early death of a family member in the present study high- light the necessity of increasing public awareness about possible unfavorable consequences of consanguineous marriage and importance of genetic counseling, prenatal diagnosis, and neonatal screening for PIDs specially in susceptible families for earlier diagnosis and manage- ment of the probable underlying PID.
HIGM syndrome has wide clinical diversities and in- volvement of various organs with respiratory tract infec- tions being the most prominent feature in almost all the previous retrospective studies [4, 8, 23–25, 27–39]. Also in our cohort study, lower respiratory tract infections were the most frequent presentation. Identification of pathogenic microorganisms responsible for infections was not performed for all patients in this study as some of them were lost to follow-up together with lack of sen- sitive and cost-effective microbiologic methods and false negative results due to empiric antibiotic interventions. However, new policies and protocol should be made considering detection of microorganisms as an important and requisite step in health-care setting of HIGM patients since it could help to a relevant management and decreased infectious morbidity of the patients. In the present survey, among the cases for whom microor- ganism led to severe complications, Pneumocystis jirove- cii in 3 cases, Klebsiella pneumonia, Giardia lamblia, Cryptosporidium parvum, Blastocystis hominis, Aspergil- lus, and cytomegalovirus each in one case were isolated as previously reported [4, 8, 23–39]. Among the patho- genic agents isolated in some of the previous studies, Pneumocystis jirovecii has been reported as the most common opportunistic microorganism responsible for pneumonia among CD40L-deficient HIGM patients [4, 8, 29, 30, 34, 36, 37, 39–41], which should be paid atten- tion particularly in developing countries, where it is en- demic.
Autoimmune disorders are another feature of HIGM syndrome presented mostly among CD40L-and AID-de- ficient patients [4, 8, 24, 26, 27, 29, 30, 32, 34, 39]. In our study, autoimmunity was observed in 25.3% of the pa- tients and slightly higher in CD40L-deficient patients. Autoimmune hemolytic anemia was the most frequent followed by rheumatoid arthritis, idiopathic thrombocy- topenic purpura, Gullain-Barré syndrome, diabetes mel- litus, Evans syndrome, reactive arthritis, and alopecia. Probable underlying mechanisms responsible for occur- rence of autoimmunity among HIGM patients include role of CD40:CD40L interactions in the selection of T-cell repertoire as well as development of regulatory T cells as- sociated with decreased T-cell autoreactivity and coun- terselection of transgenic autoreactive B cells, skew of IgM reactivity repertoire toward self-antigens, role of reg- ulatory T-cells in the elimination of peripheral autoreac- tive B cells, defective peripheral B cell tolerance mecha- nism, increased serum B-cell activating factor levels and the CD40 molecule itself [42].
Among the hematologic manifestations, neutropenia was reported in 21 of our study population mainly in CD40L-deficient cases, most of whom had concomitant oral ulcers. The accompaniment of neutropenia to gas- trointestinal mucosal involvement such as oral, rectal, and esophageal ulcers, gingivitis, and proctitis has previ- ously been reported [4, 8, 25, 26, 29, 36, 43]. Production of granulopoiesis-promoting factors is facilitated by CD40:CD40L interactions [44]; therefore, defective CD40L signaling is believed to be the underlying cause of neutropenia and subsequent lesions among CD40L- deficient HIGM patients [45]. Five of our patients had atrophic tonsils, in 3 of whom the genetic evaluation in- dicated CD40L gene mutations, while AID-deficient HIGM patients generally present with lymphoid hyper- plasia [29, 32, 35]. Taken together, patients with HIGM phenotype, atrophic tonsils, and neutropenia are sug- gested to be evaluated for mutations in the CD40L gene, as these manifestations are almost characteristic of CD40L-deficient HIGM patients.
To prevent severe forms of extrapulmonary TB, Bacil- lus Calmette-Guérin (BCG) vaccination should be per- formed in endemic countries such as Iran; however, se- vere disseminated and lethal complications may occur following vaccination in immunocompromised patients [46]. In the present study, 2 of the patients manifested adverse reactions to BCG vaccination, one of whom was previously reported [14] and the other one had axillary lymphadenitis positive for Mycobacterium bovis. These findings suggest that similar to severe forms of PID, HIGM patients are also prone to infection with Mycobac- terial species and complications following BCG vaccina- tion, thus postponing BCG vaccination to a few months later after ruling out possible PIDs including HIGM es- pecially in those families with positive history of PID, could prevent adverse and in some cases, the lethal out- come of vaccination in the affected immunodeficient pa- tients.
CD40L gene is the most common affected gene among HIGM patients accounting for approximately 65–70% of all cases [35]. Similarly, 68% of our molecu- larly defined patients were CD40L-deficient with AID gene mutations taking the second place. By findings of NEMO, lipopolysaccharide-responsive and beige-like anchor, phosphoinositide-3-Kkinase Rregulatory Ssub- unit 1 and nuclear factor kappa B subunit 1 deficiencies, and presence of heterogeneous underlying genetic de- fects, we strongly recommend the need for an unbiased molecular evaluation of all HIGM patients. For finding out the underpinning genetic mutations and possible hot-spots among Iranian descent and even new genes responsible for HIGM phenotype, the whole-genome sequencing should be considered for unsolved patients. Although the involvement of nongenetic markers, in- cluding epigenetic and environmental modifying fac- tors, is also possible.
In our study, missense mutations were the most prev- alent among the CD40L-deficient patients followed by frameshift nonsense mutations, which is similar to find- ings of Winkelstein et al. [36] and Cabral-Marques et al. [28], but in contrast to that of previous studies reporting deletions as the commonest mutations among X-linked CD40L-deficiency [4, 24–26, 41, 47]. In our study, mis- sense mutations were also the commonest among AID-deficient patients same as the cohort study by Quartier et al. [32].
Pneumonia was the leading cause of death in our se- ries, which is in compatible with that in some previously reported surveys [24, 36, 41]. High frequency of pneumo- nia and its severe and lethal outcome in our country may partly be due to environmental factors and been given less attention, pointing out the essentiality for increasing the awareness of physicians of its significance and impor- tance of earlier detection and management before irre- versible complication by means of prophylactic antibi- otic therapy and immunoglobulin replacement treat- ment. Earlier diagnosis may also provide insider for correct management by hematopoietic stem cell trans- plantation in those with cellular immunodeficiency, which could lead to patients’ survival and better quality of life.
Advanced next generation sequencing for detection of possible new genes responsible for Cytidine phenotype is strongly suggested as identification of defects in new genes and pathways could lead to a better understanding of features observed among HIGM patients along with the application of new and coherent therapeutic proce- dures.