INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 27, no. 3, 0-0 (2014)

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1 INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 27, no. 3, 0-0 (2014) LETTER TO THE EDITOR DO ELEVATED SERUM IgM LEVELS HAVE TO BE INCLUDED IN PROBABLE DIAGNOSIS CRITERIA OF PATIENTS WITH ATAXIA-TELANGIECTASIA? E. AZARSİZ 1, N.E. KARACA 1, N.C. GUNAYDİN 1, N. GULEZ 2, C. OZTURK 3, G. AKSU 1, F. GENEL 2 and N. KUTUKCULER 1 1 Ege University, Faculty of Medicine, Department of Pediatric Immunology, Izmir, Turkey; 2 Dr Behcet Uz Children s Hospital, Department of Pediatric Immunology, Izmir, Turkey; 3 Tepecik Training Hospital, Department of Pediatrics, Izmir, Turkey Received May 2, 2014 Accepted June 17, 2014 Ataxia-telangiectasia (AT) is a rare multisystem, neurodegenerative genetic disorder that is characterised by progressive neurological abnormalities, oculocutaneous telangiectasias and immunodeficiency. Delay in diagnosis or misdiagnosis is probable due to its wide clinical heterogeneity in infancy. Recurrent sinopulmonary infections are often the only presenting symptom and usually patients have decreased immunoglobulins. A total 10% of patients who present with decreased serum immunoglobulin G and A and with normal or elevated immunoglobulin M levels are often misdiagnosed as hyperimmunoglobulin M syndrome. Definitive diagnosis is made if a patient with progressive cerebellar ataxia has a disease causing mutation on the ATM gene. Ataxia-telangiectasia guideline of the European Society for Immunodeficiencies defines the probable diagnosis criteria. We evaluated twenty ataxia-telangiectasia patients (mean age 13.8±4.1 years) retrospectively who were followed-up for a mean of 38.6±27.0 months. Twelve patients had a family history of consanguinity. A total of 80% patients suffered from various infections. Neoplasms occurred in three of them. Patients showed immunological abnormalities as low IgG (45%), low IgA (65%) and elevated IgM (60%) levels. CD3 ± CD4 ± T lymphocyte frequency was low in 45% patients. The mean AFP concentration at the diagnosis was 191.9±140.1 ng/ ml and the raised IgM values did not show any statistically significant relationship with high AFP concentrations. Frequency of the elevated IgM concentrations in (60%) patients raises the concerns about thinking this finding has to be accepted as a probable diagnosis criterium. A description of patients with ataxiatelangiectasia (AT) was first published in 1926 by Syllaba and Henner (1). AT is a rare multisystem, neurodegenerative genetic disorder that is characterised by progressive neurological abnormalities, oculocutaneous telangiectasias and immunodeficiency. The prevalence of AT is estimated to be 1/ to 1/ (2). Delay in diagnosis or misdiagnosis is probably due to its wide clinical heterogeneity. Affected children are normal at birth, but by the age of 2-3 they lose muscle coordination and by the age of 10 they are usually confined to a wheelchair (3). Recognization of cerebellar ataxia and ocular telangiectasia are difficult in infancy and at this age recurrent sinopulmonary infections are often the only presenting symptom. The immunodeficiency phenotype in AT is Key words: ataxia-telangiectasia, serum IgM Mailing adderess: Elif Azarsiz, MD Ege University Faculty of Medicine, Department of Pediatric Immunology, Bornova- Izmir, Türkiye Tel.: elif.azarsiz@ege.edu.tr (2014) Copyright by BIOLIFE, s.a.s. This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.

2 342 E. AZARSİZ ET AL. variable and usually manifests as decreased or absent IgA, IgE and IgG2 (4). A total 10% of AT patients present with decreased serum IgG and IgA with normal or elevated IgM levels. These patients are often misdiagnosed as hyper-igm syndrome (HIGM) which is an immunoglobulin class switch recombination (CSR) deficiency and is generally associated with reduced somatic hypermutation. The mismatch repair system in which the ATM (ataxia telangiectasia mutated) gene is included is known to play a role in CSR (5). The gene responsible for AT was first localized to chromosome 11q by Gatti et al. (6) and then identified as the ATM gene by Savitsky et al. (7). ATM maintains genomic stability by coordinating the DNA-damage response, and in the absence of ATM, the cell cycle does not stop for repair of double-stranded DNA breaks caused by ionizing radiation and V(D)J recombination of immunoglobulin (Ig) and T-cell receptor genes. Missense mutations may partially present the classic immunological features of AT, but complete absence of enzyme activity would more likely present immunological abnormalities (8). Patients are considered to have ataxiatelangiectasia according to AT guidelines of the European Society for Immunodeficiencies (ESID). Definitive diagnosis is made if a patient with progressive cerebellar ataxia has a disease causing mutation on the ATM gene. Presence of ocular or facial telangiectasia, serum IgA concentration at least 2SD below normal for age and α-fetoprotein (AFP) concentration at least 2SD above normal for age in addition to progressive cerebellar ataxia are probable diagnosis criteria. The aim of this retrospective study is to search whether there is any other clinical or laboratory finding, such as elevated serum IgM level,s for the probable diagnosis criteria of AT patients. MATERIALS AND METHODS The clinical and laboratory data of twenty ataxia telangiectasia patients were evaluated retrospectively. The patients were all diagnosed according to ESID criteria. An evaluation sheet was used to summarize the patients demographic information including name, gender, date of birth, age at onset of symptoms, age at admission, age at diagnosis and clinical characteristics, such as frequency and type of infections, presence of ataxia-telangiectasia, neoplasms and autoimmunity, IVIg therapy, familial history and laboratory data. Serum immunoglobulins (IgG, IgA, IgM), absolute counts and percentages Table I. Demographical and laboratory data of patients with ataxia telangiectasia. mean±sd Age at the time of study (year) 13.8 ± 4.11 Age of symptoms (year) 4.86 ± 2.63 Age at diagnosis (year) 7.86 ± 3.55 Initial IgG (mg/dl) ± ±236 # Initial IgA (mg/dl) 56.3 ± ±47.4 # age-related normal values Initial IgM (mg/dl) ± ± 43.8 # CD19 ± B cells % 10.9 ± ± 4.2 CD3 ± T cells % 58.2 ± ± 6.3 CD3 ± CD4 ± T helper cells % 28.8 ± ± 5.4 CD3 ± CD8 ± T cytotoxic cells % 33.0 ± ± 3.3 CD3 - CD16 ± CD56 ± Natural killer cells % 31.4 ± ± 2.2 CD3 ± HLA DR ± Active T cells % 16.5 ± ± 3.2 α-fetoprotein (ng/ml) ± <10 # reference no 9, reference no 10

3 Int. J. Immunopathol. Pharmacol. 343 of lymphocyte subsets (CD3 ± T, CD19 ± B, CD3 - CD16 ± CD56 ± natural killer, CD3 ± CD4 ± T helper, CD3 ± CD8 ± T cytotoxic and CD3 ± HLA-DR ± active T cells) and α-fetoprotein concentrations were evaluated as laboratory findings. Statistical analysis Statistical analyses were performed using SPSS (Windows version 17.0, SPSS Inc, Chicago, IL). A p value <0.05 was considered to be significant. RESULTS The data of twenty patients (10 female and 10 male) were evaluated retrospectively. Demographical data and laboratory findings of the study group are summarized in Table I. The mean age of patients was 13.8±4.1 years (min: 9.5, max: 22 years) at the time of the study, 4.8±2.6 years (min: 1, max: 12 years) at the onset of symptoms and 7.8±3.5 years (min: 4, max: 17 years) at the time of diagnosis, respectively. Delay in diagnosis was about 4.8±3.3 years. The mean follow-up period was 38.6±27.0 months with a range of months. Four patients died after fourteen to forty-eight months of follow-up period. Twelve patients (60%) had a family history of consanguinity, and nine of them were first- degree and three of them were second-degree cousins. Two patients (10%) had a family history of malign breast neoplasm. On physical examination, all patients had presented ataxia and eighteen patients had oculocutaneous telangiectasia (90%). None of them had autoimmune findings. Sixteen patients (80%) suffered from infections. Infection spectrum showed a broad range from upper respiratory tract infection to mix type infection accompanied with sepsis. Neoplasms occurred in three (15%) patients. In one female case, mixed cellular type Hodgkin lymphoma had developed and this patient died after haemophagocytosis and septic shock. The second case was a male who had large B cell non- Hodgkin lymphoma, and who had undergone bone marrow stem cell transplantation at the age of eleven years. The third case had also Hodgkin lymphoma at the age of eight and died one year later because of massive pulmonary infection. Fourteen (70%) patients received IVIg replacement therapy which was started at the mean age of 102.6±42.3 months (min: 49, max: 191). Immunological abnormalities were present in all of our patients. Mean initial IgG, IgA and IgM concentrations were 774.2±466, 56.3±68.4 and 169.7±91.2 mg/dl, respectively. Of all the patients, nine had low IgG (45%), thirteen had low IgA (65%) and twelve had elevated IgM (60%), and three had low IgM (15%) values on admission. Mean initial IgG and IgA concentrations were low compared to age-related normal levels (Table I). In five patients with initial high IgM levels, IgM concentrations showed descending acceleration during followup while the others continued to rise or stayed at similar levels. Initial IgM concentrations showed no statistically significant relationship with presence of infection (p: 0.626) or IVIg therapy (p: 0.853). Regarding cellular immunity, a total of 45% patients (n: 9) had decreased levels of CD3 ± CD4 ± T lymphocytes in flow cytometric lymphocyte subset analysis. CD19 ± B lymphocytes were in the normal range in 80% of patients. Percentages of CD3 - Table II. Mean initial serum AFP and follow-up IgM concentrations of patients (* pearson correlation). follow-up period mean±sd p value* AFP (ng/ml) initial ± IgM (mg/dl) 0-6 months ± months-1 year ± years 95.3 ± years ± years ± >4 years ±

4 344 E. AZARSİZ ET AL. CD16 ± CD56 ± natural killer cells were elevated in nine (45%) and active T cells were elevated in seven patients (35%). Serum concentrations of AFP were elevated in all except one patient in whom the diagnosis was established by the presence of typical neurologic features. The mean AFP concentration at the diagnosis was 191.9±140.1 ng/ml. Initial serum IgG concentrations did not show any relationship with high AFP concentrations (p: 0.940), but IgA concentrations were significantly lower in thirteen of nineteen patients who had elevated AFP (p: 0.044). The raised IgM values in eleven patients did not show any statistically significant relationship with high AFP concentrations (p: 0.653). In addition, no positive correlation was found between serum IgM levels obtained during followup years and AFP concentrations (Table II). Genetic analyses for ATM gene were carried out only on six patients, therefore statistical evaluation was not made. Genetic mutations were as follows: pro1054arg heterozygous/ Asn19385 homozygous compound mutation; 3 UTR5486>T heterozygous, 3 UTR55IT>C heterozygous mutation and c del AAGAT (p.lys1754.aspfsx13) in exon 13 -c6047a/g (p.asp2016 Gly) in exon 43, double heterozygous c6047a>g, c8207dupa and c dubt heterozygous, c.3102t>g heterozygous mutation in two siblings. our patients, mean age at the time of ataxia onset was 4.86±2.63 years but 7.86±3.55 years at the time of diagnosis. So, the relatively high delay in diagnosis of patients might be due to absence of specific features at early stages of life, clinical heterogeneity and low awareness about ataxia-telangiectasia. Delay in diagnosis was 58 months, similar to us, in Iranian patients (12). Oculocutaneous telangiectasia was also seen in 90% and 83.8% patients in both groups, respectively. We followed-up patients for a mean period of 38.6 months and four of them died during this period. In patients with AT, early death is frequently due to pulmonary disease, but malignancies are also a common cause (4). The common cause of mortality was severe infections in our patients and two of them had also malignity. The incidence of malignancy is higher than in healthy persons (13). The risk of cancer in these patients is estimated to be 200 times higher than in the general population (3) and lymphoreticular malignancies are the most common tumors (14). Micol et al. (15) reported that cancer was a major risk factor for mortality among patients with null mutations, whereas respiratory tract infections were the leading cause of death among patients with hypomorphic mutations. Neoplasms occurred in three of our (15%) patients. Two of them who had Hodgkin lymphoma died after sepsis and massive pulmonary infection and the third patient had B cell non-hodgkin lymphoma and recovered after stem cell transplantation. Close relatives of AT patients should also be controlled regularly, as malignancies in parents of AT patients are common (16) and there is a higher risk for female breast cancer (17). In our group, two patients (10%) had a family history of malign breast neoplasm and one patient s brother sibling had acute lymphoblastic leukemia. Similarly, 16% patients had history of malignancy in their families in the Iranian group. There is a wide range of immunologic abnormalities in AT that affects both humoral and cell- mediated immunity. A total of 60-80% patients have immunodeficiency which raises the susceptibility for recurrent infections (18). A total of 80% patients suffered from infection in our group and the infection spectrum showed a broad range from upper respiratory tract infection to mixed type infection accompanied with sepsis. DISCUSSION The estimated cumulative prevalence rate for all forms of primary immunodeficiencies is 30.5/ in the general population and it is 18.8/ in Ege University (11). AT is classified in other well defined immunodeficiency syndromes and shows a frequency of 1.5% in our PID patients (11). In Turkey, consanguineous marriages are more common (21%) compared to European countries and this has a great effect on prevalance. Consanguinity increases the risk, given its transmission by autosomal recessive inheritance (4). A total of 60% patients had a family history of consanguinity in this study group while it was 76% in an Iranian group which had similar geographical features (12). Ataxia is usually the first diagnostic symptom in AT, having its onset in the first years of life. Among

5 Int. J. Immunopathol. Pharmacol. 345 The immunodeficiency phenotype in AT is variable and decrease or absence of IgA, IgE and IgG2 is frequently seen (4). On the other hand, case reports with normal immunoglobulin levels are also reported (19). In our group, mean initial IgG and IgA concentrations were low in a total of 45% and 65% patients, respectively. IgG deficiencies are reported as 18% (4), 12% (12) and Ig A deficiencies as 58.3% (20), 63% (4), 75.4% (12) and 79.3% (15) in the literature. Recently, case reports presenting with an unexplained immunodeficiency syndrome with decreased immunoglobulin levels in the presence of B cells and high IgM concentrations (21-23) have been published in the literature. In our group, the raised initial IgM values in 60% patients were noteworthy although concentrations showed no statistically significant relationship with the presence of infection (p: 0.626) or IVIg therapy (p: 0.853). Descending IgM acceleration in five patients may be explained by regular IVIg treatment. Novak- Wegrzyn et al. (4) reported that frequency of patients with high IgM was 26% and patients who had more than one measurement manifested no trend to decreasing levels with age. Occasionally, AT patients have been described with high IgM levels, associated with splenomegaly, lymphadenopathy, lymphocytic interstitial pneumonitis and neutropenia, and it was suggested that hyper-igm in subjects with AT might represent an inefficient immunological response to infection (21, 24, 25). As none of the patients in our study group presented with hepatosplenomegaly, lymphadenopathy or lymphocytic interstitial pneumonitis, we could not make a correlation between the high IgM levels and such clinical findings. Patients with AT generally have a progressive T cell defect which causes viral and opportunistic infections but some patients present with a typical CSR deficiency with elevated IgM, and a contrastingly strong decrease in IgG and IgA levels which requires Ig replacement therapy (21, 26, 27). The failure of DNA repair during CSR suggests a role for ATM gene in CSR-induced double-stranded DNA break repair, so patients B cells fail to CSR, leading to inadequate antibody production (28). Patients with complete absence of enzyme activity are much more likely to show clinical and/or immunological features of immunodeficiency than those with residual activity. Regarding cellular immunity, T lymphocyte deficiency is the most common deficiency in AT patients. Approximately one-third of the patients display mild lymphocytopenia (29). Frequency of T cell deficiency was about 75% in Teruel et al. s series (20) while Nowak-Wegrzyn et al. (4) reported CD3 ± deficiency in 57% and CD3 ± CD4 ± T lymphocytes deficiency in 69% patients. A total of 45% patients had decreased levels of CD3 ± and CD3 ± CD4 ± T lymphocytes in our group. Moin et al. (12) also showed severe lack of CD4 ± T cells in 44.4% patients. B lymphocytes were in normal range (80%) except four patients, in contrast to the study by Nowak-Wegrzyn et al. (4) in which B cells were low in 75% patients and natural killer cell frequency was high in 45% patients similar to 66% in that study. An elevated serum AFP level is a useful screening test for ataxia-telangiectasia (30, 31). AT patients with a normal serum AFP have also previously been described (32). In this study, serum AFP concentrations were high in all except one patient who did not have an AFP measurement. The mean value was 191.9±140.1 ng/ml in our and 133 ng/ ml in Novak- Wegrzyn et al. s study (4). Serum initial IgA concentrations were significantly lower in patients with elevated AFP (p: 0.044) but, on the other hand, neither IgG nor IgM values showed any statistically significant relationship with high AFP concentrations. Patients with truncating mutations of the ATM gene and an absence of ATM kinase activity present with the severe classic form of AT. Conversely, missense or splice-site mutations of the ATM gene tend to present clinically milder forms of AT (33). Staples et al. (8) speculate that AT patients with mutations leading to no expression of ATM kinase activity have a more severe immunological phenotype. In our group, genetic analyses were performed only on six patients, so we did not have enough data to make an explanation for clinical heterogeneity. In conclusion, AT is a rare primary immunodeficiency disease in which high IgM concentrations may be the only presenting feature and may be misdiagnosed as hyper-igm syndrome. It is thought that, in case of missense mutation or complete absence of ATM gene, defective

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