Report of two siblings with APECED in Serbia: is there a founder effect of c.769C>T AIRE genotype?

We discuss the features of the two new siblings by retrospectively evaluating the clinical and genetic characteristics of other 11 Serbian APECED patients (10 of which with available confirmatory AIRE genotype) reported in literature from 2001 till date (Table 1). The data are compared with those retrieved from North (Finland) [19] and South of Europe (Sardinia) [20], North and South America [4], Turkey [21] and India [22].

The details of the total cohort of 13 Serbian APECED patients, clinically diagnosed based on the classic criteria, are given in Table 1 [15‐18]. The female/male ratio was 8/4, median age at referral was 16.9 yr (range 3.5–33 yr) and early disease onset had a median age of 6.36 yr (range 1–15 yr), based on the appearance of the first component of the triad and the severity of the phenotype. At the end of the observation period, the classical triad was present in 9 of the 13 patients and the dyad in 12 of the 13 patients. A mean of 5.53 manifestations were reported per patient and the frequencies of the diseases are summarized in Fig. 1. Figure 2 refers to the mean age of appearance of clinical manifestations.

Epidemiological investigations conducted on different populations demonstrate that CMC is usually the first manifestation of APECED, often before 5 years of age [19, 23]. The frequency of CMC ranges between 17 and 100% in APECED patients in different series, with the lowest incidence in Iranian Jews [23]. CH is the second most common manifestation in order of appearance (rev in [3, 8, 11, 19, 23]) whilst AAD is usually the third manifestation, occurring in 22–95% of the patients (rev in [8, 11]). Concurrently, APECED patients suffer additional endocrine and non-endocrine autoimmune conditions [2, 3].

Among the 13 Serbian patients described, in 7/13 whose age of onset was known (53.8%) CMC was the first disease to manifest, isolated or in association with other symptoms, with a mean age of onset of 8.57 yr (range 1–16 yr) (Table 1). Of the 13, 10 (76.9%) exhibited CMC (2 males, 7 females based on available data) at some stage after the onset of the disease (Fig. 1). Among patients diagnosed before 30 yr of age and genetically confirmed by AIRE gene screening, CMC was present in 10/13 (76.9%) of Serbian patients compared to 81.48% of Indian patients [22], 70% Turkish [21], 95.5% Sardinian [20] and all Finnish patients [19] (Fig. 3).

Of the 13 patients, 84.6% (11/13) developed CH (3 males, 8 females) (Fig.1). CH, as the first disease to manifest, isolated or in association with other symptoms, had a mean age of onset of 8.85 yr (range 4–15 yr). According to published data, CH develops in 50–100% of APECED patients worldwide [8, 11]. Among patients diagnosed before the age of 30 (12/13 with clinical diagnosis confirmed by AIRE gene screening), CH was found in 11/13 (84.6%) of the Serbian patients, 23/27 (85.1%) Indian patients [22], 90% Turks [21] and 77.3% Sardinians [20] but only in 40% of Finnish patients [19] (Fig. 3).

In the Serbian APECED series, 100% of the patients were affected by AAD; this occurred as first manifestation (isolated or with other symptoms) in 11/13 (84.6%) patients at a mean age of 10.6 for those whose age of disease onset was available (range 3.5–16 yr). On the whole, 100% (13/13) of Serbian, 44.44% Indian [22], 60% Turkish [21], 68.2% Sardinian [20] and 70% Finnish [19] patients with genetic diagnosis suffered from AAD (Fig. 3).

Other endocrinopathies include POF that manifests as primary or secondary amenorrhea with hypergonadotropic hypogonadism in 0–71% of cases according to national surveys (rev in [8, 11]). Among Serbians, OF was present in 5/8 of the evaluated females (62.5%), 28.5% Indians [22], 20% Turks [21], but nearly two thirds (61.5%) of Sardinians [20] (Fig. 3). Autoimmune thyroid disease (AITD), mostly Hashimoto’s thyroiditis and rarely Graves’ disease, was diagnosed in 29% of all reported APECED patients at a median age of 19 yr (rev in [8, 11]). Among Serbians AITD was reported in 3/13 (23%) (Hashimoto’s thyroiditis in 2 patients and Graves’ disease in one patient), 18.5% (5/27) Indians [22], 30% Turks [21], 20% Finns [19] but none of the Sardinians [20] (Fig. 3).

A higher incidence (40–80%) of non-endocrine manifestations was reported in North American APECED patients compared to < 5–20% in some other cohorts [4]. Non-endocrine manifestations encountered in the Ferre/Lionakis criteria help accelerate early diagnosis of APECED in the American cohort [4] compared to the classic criteria. These expanded criteria for clinical diagnosis are fulfilled if patients develop 2 out of 6 manifestations, which include non-endocrine urticarial eruption, intestinal dysfunction and enamel hypoplasia, along with the classic components CMC, AAD and CH [4]. In the Serbian series, a high incidence of non-endocrine manifestations was reported in 12 out of 13 patients with a mean of 2 manifestations per patient. These included ectodermal dystrophy (n = 3, patient 2, 5 and 7, Table 1), alopecia (n = 6, patient 5, 7, 11 and 12, diffuse alopecia with madarosis in patient 2, alopecia universalis in patient 13), pure red cell aplasia (patient 3), renal dysfunction (patient 3), exocrine pancreas insufficiency (patient 3), pernicious anemia (n = 4, patient 4, 6, 9, 12), Lichen ruber planus (patient 4), chronic otitis media with effusion (patient 6), autoimmune bronchiolitis manifested as asthma-like dyspnea (patient 6), Systemic Juvenile Rheumatoid Arthritis (patient 6), vitiligo (n = 4, patient 5, 7, 8 and 13), chronic active hepatitis (patient 5 and 9) and malabsorption (n = 4, patient 3, 7, 10 and 12) [15‐17]. Based on the limited retrospective information available, the expanded criteria [4] allowed an early diagnosis in only 3 patients (patients 7, 10, 12 in Table 1) out of 9 whose information was sufficient for evaluation. Prospective studies conducted on a larger number of patients will be helpful to compare the diagnostic efficacy of these two sets of criteria.

Among the non-endocrine manifestations, ectodermal dystrophy was present in 3 out of 13 (23%) patients from the Serbian cohort (1 male, 1 female based on the available information) (Fig. 1), 12/27 (44.4%) Indian [22], 4/10 (40%) Turkish [21], 81.8% Sardinian [20] and absent in Finnish patients [19] (Fig. 3). Alopecia was present in 6/13 Serbian patients (46%), progressing to alopecia universalis in one female. Vitiligo has been reported in 6–50% and alopecia areata in 7–52% APECED patients in different studies [8, 11]. Vitiligo was observed in 4/13 (30%) Serbian patients (one male, 2 females based on the available information) (Fig. 1), 6/27 (22.2%) Indian [22], 2/10 (20%) Turkish [21], 22.7% Sardinian [20] and 40% Finnish patients [19] (Fig. 3).

Autoimmune gastritis, manifesting as mucosal atrophy with submucosal lymphocytic infiltration and loss of parietal cells, is reported in 4–32% APECED patients worldwide, either in isolation or in association with pernicious anemia (rev in [23]). Gastric parietal cell failure was not documented in the Serbian series (Table 1), though pernicious anemia affected 4 out of the 13 (30%) (patient 4, 6, 9, 12 in Table 1) APECED patients (Fig. 1). For comparison, pernicious anemia was reported in 11.1% Indian, 20% Turkish, 18.2% Sardinians, and 30% Finnish patients [19‐22] (Fig. 3).

Intestinal dysfunction in APECED can be due to rare conditions like celiac disease, exocrine pancreas insufficiency, intestinal lymphangiectasia, or intestinal infections such as Candida albicans, Giardia lamblia, Clostridium difficile etc. There is also evidence of an autoimmune origin associated with the presence of circulating antibodies against tryptophan hydroxylase (TPHAbs), histidine hydroxylase (HD) or L-amino acid decarboxylase (AADC) Abs [1, 24, 25]. Its incidence varies between 15 and 22% in different series (rev in [23]). Intestinal dysfunction appeared in 4/13 (30.7%) Serbian patients, 25.9% Indian [22], 10% Turkish [21], 50% Sardinian [20] and 40% Finnish [19] patients (Fig. 3).

Additional rare manifestations reported in previous studies (rev in [23]) were present in a few Serbian APECED patients. Among these, pure red cell aplasia manifested in patient 3 (see above); it is a rare syndrome characterized by severe normocytic anemia, reticulocytopenia, and the absence of detectable erythroid precursors in the bone marrow [26]. Few APECED patients have been reported in literature with this associated autoimmune disorder due to the presence of autoantibodies against erythroid cells or erythropoietin [15, 26]. Hyperactivity of T cells, especially of large granular subsets, or NK cells was also considered a possible pathogenic mechanism [15]. Another rare condition represented in the Serbian series is the Vogt-Koyanagi-Harada syndrome manifesting with uveitis, right hypoacusia and right hemiparesis (patient 13) (Table 1) [18].

APECED is a monogenic recessive syndrome with 100% penetrance. The inheritance of two variant AIRE alleles predicts progression to APECED. The AIRE gene maps on chromosome 21q22.3 with 14 exons; the encoded protein is 545 aminoacids in length and weighs 58 kDa [2]. More than 100 AIRE mutations have been identified to date (Human Gene Mutation Database-http://​www.​hgmd.​cf.​ac.​uk; http://​bioinf.​uta.​fi/​AIRE-base/​) within the exon/intronic sequence of the gene including single nucleotide substitutions or large deletions [23]. As highlighted above, some AIRE mutations that cause APECED are prevalent in certain populations [8, 10, 11, 20, 27‐30]. Indeed, AIRE mutation c.769C > T (R257X) is prevalent in the Finnish population [19], c.415C > T (R139X) in the Sardinian [20] and c.254A > G (Y85C) in the Iranian Jew populations, suggesting a founder effect for these variants [12]. The R257X genotype was also found at high frequency in the Turkish population [21] and is overall responsible for 75% of alleles in patients with APECED in Central and Eastern Europe [16]. In particular, regarding the Italian population, 3 hot spots of incidence were identified, the first in Sardinia, the second in Apulia and the third in Veneto. In Sardinia, the presence of a peculiar AIRE gene mutation was identified and defined as R139X [20]. W78R was the prevalent mutation in Apulia and R203X in Sicily [28]. In Northern Italian populations, R257X mutation was reported frequently and often in association with mutation 1094-1106del13 [10]. These 2 mutations have been described in European populations [31].

On general ground, a genotype-phenotype correlation was observed only in a few populations. As regard to Iranian Jews, the nonsense Y85C mutation, CMC and AAD had low incidence and keratopathy was not observed [20]. Furthermore, the presence of the R257X mutation in Finnish patients was correlated with CMC [12, 20]. Of note, the autosomic dominant G228W mutation was discovered in a patient from Tuscany affected by CH and autoimmune thyroiditis with the presence of circulating 21OH antibodies [13].

In the Serbian APECED series of the present investigation, all genetically confirmed APECED patients (12 out of 13) harbored the homozygous R257X (p.Arg257X, c.769C > T) mutation in 10 out of 12 patients. Two patients presented the R257X mutation in heterozygosity with the c.462A > T (p.Glu298Lys) or the c.462A > T (p. (=)) mutation. These data suggest the presence of a founder effect for the R257X mutation in the Serbian population. No genotype/phenotype correlation was observed although all patients were affected by adrenal insufficiency and 10 out of 13 patients presented CMC. A high incidence of pernicious anemia among non-endocrine manifestations was also evidenced (Fig. 3).