Multiorgan autoimmunity in a Turner syndrome patient with partial monosomy 2q and trisomy 10p

Gene 515 (2013) 439–443

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Short Communication

Multiorgan autoimmunity in a Turner syndrome patient with partial monosomy 2q and trisomy 10p Armando Grossi a, Alessia Palma b, Ginevra Zanni c, Antonio Novelli d, Sara Loddo d, Marco Cappa a, Alessandra Fierabracci b,⁎ a

Division of Endocrinology, Bambino Gesù Children's Hospital IRCCS, Rome, Italy Immunology Area, Bambino Gesù Children's Hospital IRCCS, Rome, Italy Unit of Molecular Medicine, Bambino Gesù Children's Hospital IRCCS, Rome, Italy d Cytogenetic Laboratory, Institute CSS Mendel, Rome, Italy b c

a r t i c l e

i n f o

Article history: Accepted 4 December 2012 Available online 20 December 2012 Keywords: X chromosome condition Autoimmune polyglandular syndrome Autoantibodies AIRE polymorphism PTPN22 polymorphism SNP-array analysis

a b s t r a c t Turner syndrome is a condition caused by numeric and structural abnormalities of the X chromosome, and is characterized by a series of clinical features, the most common being short stature and gonadal dysgenesis. An increased frequency of autoimmune diseases as well as an elevated incidence of autoantibodies has been observed in Turner patients. We present a unique case of mosaic Turner syndrome with a complex rearrangement consisting of a partial deletion of chromosome 2q and duplication of chromosome 10p {[46],XX,der(2)t(2;10)(2pter→2q37::10p13→ 10pter)[127]/45,X,der(2)t(2;10)(2pter→ 2q37::10p13→ 10pter)[23]}. The patient is affected by partial empty sella, in association with a group of multiorgan autoimmunity-related manifestations including Hashimoto's thyroiditis, celiac disease, insulin-dependent diabetes mellitus (Type 1 diabetes, T1D), possible autoimmune inner ear disease with sensorineural deficit, preclinical Addison disease and alopecia universalis. The patient was previously described at the age of 2.4 years and now re-evaluated at the age of 14 years after she developed autoimmune conditions. AIRE gene screening revealed heterozygous c.834 C>G polymorphism (p.Ser278Arg) and IVS9+6G>A variation, thus likely excluding autoimmune polyendocrine syndrome Type 1 (APECED). Heterozygous R620W polymorphism of the protein tyrosine phosphatase non receptor type 22 (PTPN22) gene was detected in patient's DNA. SNP-array analysis revealed that autoimmunity-related genes could be affected by the partial monosomy 2q and trisomy 10p. These data suggest that early genetic analysis in TS patients with complex associations of multiorgan autoimmune manifestations would permit a precise diagnostic classification and also be an indicator for undiscovered pathogenetic mechanisms. © 2012 Elsevier B.V. All rights reserved.

Abbreviations: AIED, autoimmune inner ear disease; ANA, antinuclear antibodies; AIRE, autoimmune regulator gene; Abs, antibodies; ACTH, adrenocorticotropic hormone; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome; arr, rearrangement; AT, annealing temperature; AU, alopecia universalis; B, basophils (white blood cells); BMD, body mineral density; bp, base pair(s); °C, degree Celsius; CD, celiac disease; CD25, cluster differentiation 25; ChAS, Chromosome Analysis Suite software; CXCR7, chemokine orphan receptor 1 gene; der, derivative chromosome; DEXA, dual energy X-ray absorptiometry; dl, deciliter; DNA, deoxyribonucleic acid; E, eosinophils (white blood cells); FSH, follicle-stimulating hormone; FOXP3, forkhead box P3; FT4, free thyroxine; GADA, anti-glutamic acid decarboxylase isoform 65 autoantibodies; g/dl, gram in deciliter; GH, growth hormone; Hb, hemoglobin; HbA1c, glycated hemoglobin; hg, human genome; HT, Hashimoto's thyroiditis; ICA-IgG, islet cell antibodies (immunoglobulin G); ID, identification; IFN ω, interferon ω; IGF1, insulin-like growth factor 1; IL-2, interleukin 2; IL2RA, interleukin 2 receptor, alpha chain precursor/CD25; IL15RA, interleukin 15 receptor, alpha isoform 2; IVGTT, intravenous glucose tolerance test; L, lymphocytes (white blood cells); LH, luteinizing hormone; M, monocytes (white blood cells); Mb, megabytes; mcU/ml, microunits per milliliter; mg/24 h, milligrams in 24 hours; ml, milliliter; MRI, magnetic resonance imaging; μl, microliter; N, neutrophils (white blood cells); neg, negative; ng/ml, nanograms per milliliter; nr, normal range; OMIM, Online Mendelian Inheritance in Man; p, short arm of chromosome; PCR, polymerase chain reaction; PDCD1, programmed cell death 1 precursor; pg, picogram; pg/ml, picograms per milliliter; pter, terminal part of chromosome short arm; PTH, parathyroid hormone; PLT, platelets; PTPN22, protein tyrosine phosphatase non receptor type 22; POF, premature ovarian failure; pos, positive; q, longer arm of chromosome; RBC, red blood cells; rs, single reference SNP; SD, standard deviation; SNHL, sensorineural hearing loss; SNP, single nucleotide polymorphism; T1D, Type 1 diabetes; TG, thyroglobulin; TPO, thyroperoxidase; TRAF3IP1, TNF (tumor necrosis factor) receptor-associated factor 3 interacting; Tregs, T regulatory cells; TRG, transglutaminase; TS, Turner syndrome; TSH, thyroid stimulating hormone; UCSC, University of California Santa Cruz Genome Browser; UrCa, urinary calcium; UrCr, urinary creatinine; UrCr/UrCa, urinary creatinine/calcium ratio; WBC, white blood cells. ⁎ Corresponding author at: Immunology Area, Children's Hospital Bambino Gesù IRCCS, Piazza S. Onofrio, 4, 00165 Rome, Italy. Tel.: +39 06 6859 2656; fax: +39 06 6859 2904. E-mail address: fi[email protected] (A. Fierabracci). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.007

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1. Introduction Turner syndrome (TS) is a condition caused by numeric and/or structural abnormalities of the X chromosome, with a series of clinical features, the most common being short stature and gonadal dysgenesis (Larizza et al., 2009). The birth prevalence of TS is about 1 in 2500 female live births (McCarthy and Bondy, 2008). Approximately 1–2% of all conceptuses have a 45,X chromosome constitution. An increased frequency of autoimmunity as well as an elevated incidence of autoantibodies is observed in TS (Gawlik et al., 2011; Lleo et al., 2012; Mortensen et al., 2009). The underlying cause possibly derives from the interaction of genetic and environmental factors and may be due in part to the haploinsufficiency of genes on the X chromosome (Bianchi et al., 2012). Since autoimmunity is frequently diagnosed in families of TS patients, abnormal gametogenesis and non-dysjunctional events may be a consequence of altered autoimmune responsiveness. The prevalence of autoimmune disease increases with age. It has been estimated that 50% of TS patients suffer from Hashimoto's thyroiditis (HT). Other associated conditions are celiac disease (CD), ulcerative colitis, Crohn's disease, juvenile rheumatoid arthritis, psoriasis, vitiligo and idiopathic thrombocytopenic purpura (Jørgensen et al., 2010). In a Danish registry, when compared to Danish women in general, TS subjects exhibited a 4-fold increased risk of developing male-predominant autoimmune diseases such as insulin dependent diabetes mellitus (Type 1 diabetes, T1D). Other male-predominant autoimmune diseases which could affect TS patients include amyotrophic lateral sclerosis, ankylosing spondylitis, reactive arthritis and Dupuytren's contracture. This phenomenon may be interpreted as indicating that while in women with a normal karyotype there is compensation by the normally functioning copy on the other X chromosome, a harmful allele will occur in TS females and in males due to X monosomy (Jørgensen et al., 2010). Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) was also reported in a TS patient with premature ovarian failure (POF) and primary amenorrhea (Reato et al., 2011). We describe a case of mosaic TS with unique complex karyotype (Grammatico, et al., 1999), underlining the importance of identifying immune gene defects for early diagnostic classification of associated multiorgan autoimmune manifestations as well for identifying their pathogenetic mechanisms. 2. Materials and methods 2.1. Molecular analysis of the AIRE gene Genomic leukocyte DNA was extracted from whole blood of the patient by QIAamp DNA blood mini kit (Qiagen, Hilden Germany). PCR (polymerase chain reaction) was carried out with specific primers for all 14 exons of the gene (GenBank ID: AJ009610). Primer sequences were selected as follows (Fierabracci et al., 2012): exon 1 forward 5′-CGTGCCAGTGTCCCGGGACCCACC-3′ and reverse 5′-GGGCGGGGTT CCTCCTGGAACTTCC-3′ [annealing temperature (AT) 55 °C] identified a product of 276 bp (base pair) (Cervato et al., 2009); exon 2 forward 5′-AGTCATGATGGAGATGGGC-3′ and reverse 5′-GAGCAGGTGACAGCA GC-3′ (AT 62.5 °C) identified a product of 330 bp; exon 3 forward 5′-GTCTGGCCAAGGTGTCC-3′ and reverse 5′-GCAGTGGGTGGGAGC-3′ (AT 58.5 °C) identified a product of 350 bp; exon 4 forward 5′-GGCACTCACCCCCACT-3′ and reverse 5′-ACACCAGGCCAGCACG-3′ (AT 62.5 °C) identified a product of 280 bp; exon 5 forward 5′-GCA TAGAGTATGTGCTTGG-3′ and reverse 5′-TCCGGTCTGCTGTGG-3′ (AT 58.5 °C) identified a product of 330 bp; exon 6/7 forward 5′-CTGGGG CCTACACGACTGC-3′ and reverse 5′-TGCCCAGGTAAAGGCAGAGG-3′ (AT 67 °C) identified a product of 650 bp; exon 8 forward 5′-AAGGA GGTGGCTCTCAGGA-3′ and reverse 5′-CTTCCCTTCAGGGTCAGTGG-3′ (AT 58.5 °C) identified a product of 310 bp (Cervato et al., 2009); exon 9 forward 5′-CGCTGTCTTGTTCTGCATGT-3′ and reverse 5′-ACA GGACTCCAGGGGACAG-3′ (AT 58.5 °C) identified a product of 251 bp (Cervato et al., 2009); exon 10 forward 5′-CCTGGGTTTCAGGGTCCC-3′

and reverse 5′-CCCCAGGCCCTGTGC-3′ (AT 65.8 °C) identified a product of 500 bp; exon 11 forward 5′-TCGGGTTGAGCTACATTTCC-3′ and reverse 5′-GTGTGGTTGTGGGCTGTATG-3′ (AT 58.5 °C) identified a product of 279 bp (Cervato et al., 2009); exon 12 forward 5′-GAGGT GGCACTCCTGCTC-3′ and reverse 5′-TCTGCCCTGAGATGTGCTC-3′ (AT 58.5 °C) identified a product of 247 bp (Cervato et al., 2009); and exon 13 forward 5′-GAGCTGGGTGTAAGAATTCCC-3′ and reverse 5′-ACGGCTCAAGAGCAGTGG-3′ (AT 58.5 °C) identified a product of 270 bp. Two couples of primers were used to amplify exon 14 (Cervato et al., 2009): forward 5′-GGAGGTTCTCACCGTCACTC-3′ and reverse 5′-AGTAGGTCACCAGGCAAGGA-3′ (AT 58.5 °C) with a product of 344 bp and forward 5′-AATTAAACCCTGCCCCACTT-3′ and reverse 5′-TCCATTCAGGAAGCTGGAAC-3′ (AT 58.5 °C) with a product of 364 bp. Molecular analysis of the AIRE gene was conducted by direct sequencing. PCR sequencing was carried out with the BigDye Terminator v.3.1 Cycle sequencing protocol (LifeTechnologies, Applied Biosystem, Paisley, Scotland, UK). Products were then purified and sequenced with the Genetic Analyzer 3500 (Applied Biosystem HITACHI system). 2.2. Molecular analysis of the PTPN22 gene Molecular analysis of the PTPN22 R620W (C1878T) polymorphism of the autoimmunity predisposing gene protein tyrosine phosphatase non receptor Type 22 (PTPN22) (Bianco et al., 2010) was evaluated using a XcmI restriction fragment length polymorphism-PCR method. 2.3. SNP array analysis Whole genome SNP-array analysis of patient's DNA was performed using GeneChip 6.0 platform (Affymetrix, Santa Clara, CA), consisting of about 906,600 SNP sequences and about 900,000 non-polymorphic oligonucleotides. 3. Results 3.1. Case history We report a TS patient, previously described at the age of 2.4 years (Grammatico et al., 1999), who subsequently developed a complex association of clinical autoimmune manifestations (Table 1). The patient, delivered at term by cesarean section, is the second daughter of non-consanguineous parents, with no family history of autoimmune disorders. The patient and her family are from the Lazio region. She was referred to our hospital in September 2003 at the age of 3.5 years with a history of dysmorphic features including pterygium, short neck, and short broad fingers. Standard karyotype analysis had shown mosaicism for X monosomy and a complex rearrangement involving chromosome 2 and chromosome 10 (Grammatico et al., 1999), characterized by SNP-array as arr 2q37.1q37.3 (233,426,419-243,046,383)x1, 10p15.3p12.32 (72,797-18,674,330)x3, Xp22.33q28 (152,439-155, 182,342)x1~2. MRI investigation of the pituitary region revealed ‘partial empty sella’ with biventricular dilatation (Laube et al., 2002). On first observation we diagnosed autoimmune thyroiditis with subclinical hypothyroidism and celiac disease. Levothyroxine treatment was initiated. Additional classical clinical features included sensorineural deficit (Stenberg et al., 2007), cutaneous dystrophy of the legs with muscle weakness and hypotrophy (Larizza et al., 2009). Adaptive behavior scored very low on Vineland Adaptive Behavior Scales in the domains of communication, motor ability, socialization and daily activity skills. At the age of 7.9 years there was persistent alopecia universalis (AU) (Larizza et al., 2009). Severe intellectual disability (according to the Wechsler Intelligence Scale for Children) was diagnosed. The patient was not able to perform any task due to her speech delay, psychomotor retardation and interaction difficulties. High titres of antithyroperoxidase (TPO), anti-thyroglobulin (TG) and anti-glutamic acid decarboxylase isoform 65 autoantibodies (GADA) were detected.

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Table 1 Clinical presentation, laboratory and instrumental parameters of the patient. Age

Clinical presentation

Laboratory and instrumental parameters

Birth to 2.4 years

Dysmorphic features in Turner syndrome Psychomotor retardation Facial eczema, alopecia universalis Intellectual disability, sensorineural deficit CD, alopecia universalis HT with subclinical hypothyroidism Leg lipodystrophy Muscle weakness and hypotrophy All above features Height at the 25th centile Subclinical hyposurrenalism

Karyotype 46,XX,der(2)t(2;10)(2pter→2q37::10p13→10pter) [127]/45,X,der(2)t(2;10)(2pter→2q37::10p13→10pter)[23] Thyroid ultrasound: thyroiditis TSH 6.81 (nr 3.8–5.2) mcU/ml; FT4 1.09 (nr 0.7–1.8) ng/ml MRI: partial empty sella with biventricular dilatation TRG Abs pos

3.5 years

7.9–8.3 years

8.7–10.2 years

10.8–11.8 years

13 years 14:04 years

All above features Low weight gain Height at the 25th centile Above clinical features Weight loss Type 1 diabetes and above features Spontaneous thelarche and above clinical manifestations Height −1.1 SD

DEXA scan: BMD −3.9 SD; PTH b5 (10–65) pg/ml UrCa: 74.94 mg/24 h UrCr: 254.5 mg/24 h; UrCr/UrCa = 0.29 IVGTT: normal insulin response Normal liver enzymes ACTH 110 pg/ml; cortisol in the normal range IGF-1 36 ng/ml (nr 50–158) GADA and ANA pos Anti-thyroid Abs pos IGF-1 63 ng/ml; PTH normal values DEXA scan: BMD −3.7 SD Anti-21OH hydroxylase Abs pos ACTH 117 pg/ml; IVGTT: impaired insulin response ACTH test: altered cortisol response PTH 43.8 pg/ml RBC 5.69; WBC 8.67 (L 34.7%, N 54.4%, E 5.1%, B 0%, M 5.8%); PLT 230; Hb 12.8 g/dl FSH, LH, estradiol: near puberty normal values ICA-IgG pos; anti-TG, anti-TPO pos Anti-ovary Abs neg RBC 5.53; WBC 8.96 (L 19.9%, N 73.5%, E 2.9%, B 0.1%, M 3.6%); PLT 245; Hb 11.4 g/dl

pos = positive; neg = negative. TRG = transglutaminase. nr = normal range; ANA = anti-nuclear antibodies. ICA-IgG = islet cell antibodies. Altered parameters in bold.

Low body mineral density (BMD) was associated with low PTH level, for which calcium and vitamin D therapy was started. Height was at the 25th centile according to TS growth charts. At the age of 8.3 years ACTH levels were elevated and cortisol was tested within the normal range (Table 1) in the presence of anti-21OH hydroxylase autoantibodies, indicative of subclinical Addison disease (Reato et al., 2011). In view of the high titers of GADA, an intravenous glucose tolerance test (IVGTT) was performed and found normal. PTH values were repeatedly tested in the normal range, thus excluding primary hypoparathyroidism. Insulin-like growth factor 1 (IGF1) levels at 8.3 and 10.2 years were in the lower range of normal values. Height at 8.3 and 10.2 years was stable at the 25th centile. Taken together, these results (borderline IGF1, BMD) were interpreted as being due to malabsorption and malnutrition in CD, rather than to GH deficiency. At the age of 10.8 years the patient presented reduction of adipose tissue, decreasing muscular strength and hypotrophy, as well as neuromuscular deficit. Subclinical impairment of glucose metabolism was diagnosed at this time by IVGTT. High ACTH levels persisted with reduced cortisol response to ACTH stimulation. At the age of 13 years the diagnosis of T1D was made and insulin substitutive treatment was started with rapid and persistent amelioration of blood glucose profile. At the age of 14.4 years initial spontaneous thelarche (B2) was observed at physical examination which could be explained by the X/XX mosaicism. Spontaneous menarche did not occur. Testing for FSH, LH, and estradiol showed near-puberty normal values. Testing for antiovary autoantibodies was negative. Glycated hemoglobin (HbA1c) was in the normal range for T1D affected patients under intensive treatment. Over the years worsening of muscle weakness and hypotrophy was observed. AU persisted as did leg cutaneous dystrophy. Over the years blood cell counts were always tested within the normal range for sex and age. At the last examination red blood

cells (RBC) counted 5.53 × 10 6/μl [normal range (nr) 3–6], hemoglobin (Hb) 11.4 g/dl (nr 9–16), white blood cells (WBC) 8.96 × 10 3/μl (nr 4–14) [neutrophils 73.5% (nr 10–74), eosinophils 2.9% (nr 0–7), basophils 0.1% (nr 0–1.5), lymphocytes 19.9% (nr 20–65), monocytes 3.6% (nr 3.4–11)], and platelets (PLT) 245 × 10 3/μl (nr 150–450) (Table 1). 3.2. AIRE gene screening and search for PTPN22 R620W polymorphism Due to the presence of multiple confirmed or suspected autoimmune clinical manifestations, subclinical Addison disease and ectodermal dystrophy, molecular analysis of the AIRE (Fierabracci, 2011) and PTPN22 genes was conducted. Within the AIRE gene heterozygous polymorphism c.834 C>G (961 C>G, p.Ser278Arg, rs 1800520) in exon 7 (Faiyaz-Ul-Haque et al., 2009; Tazi-Ahnini et al., 2002; Tóth et al., 2010) and intronic variation c.1095 + 6G>A (IVS9+6G>A, rs 1800525) (Ferrera et al., 2007) were detected (not shown). Anti-IFN ω antibodies were negative. These findings likely excluded the diagnosis of APECED. Screening of the PTPN22 gene detected the heterozygous polymorphism R620W (Bianco et al., 2010) (not shown). 3.3. SNP array analysis SNP-array analysis was performed to identify well known autoimmune-related genes that could have been affected by the TS karyotype 46,XX,der(2)t(2;10)(2pter→ 2q37::10p13→ 10pter)[127]/ 45,X,der(2)t(2;10)(2pter → 2q37::10p13 → 10pter)[23] and genotype/ phenotype correlations. SNP-array analysis revealed partial deletion of 2q, about 10 Mb in size. The deletion spanned from 233,426,419 bp to 243,046,383 bp (USCS Genome Browser; http://genome.ucsc.edu; hg18 release), at 2q37.1 → q37.3. The genes located at the 2q deleted region included immune-function genes such as CXCR7 (OMIM* 610376;

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chemokine orphan receptor 1), PDCD1 (OMIM* 600244; programmed cell death 1 precursor), and TRAF3IP1 (OMIM* 610376; TNF receptorassociated factor 3 interacting). The analysis also showed the partial duplication of the short arm of chromosome 10 of about 18 Mb, mapping to 10pter → p12.32, from 72.797 bp to 18,674,330 bp. Duplication region included immunological related genes IL2RA (OMIM* 147730; interleukin 2 receptor, alpha chain precursor/CD25), IL-15 RA (OMIM* 601070; interleukin 15 receptor, alpha isoform 2) (Fig. 1). SNP-array analysis confirmed mosaicism for X monosomy, but the percentage was higher (40%) than the standard karyotype (15%), corresponding to possible changes in percent of mosaicism over time and within tissues. 4. Discussion Our patient is affected by some of the most frequent autoimmune disorders associated with TS, including HT, CD, and T1D (Larizza et al., 2009). Notably, the patient had concomitant manifestations of HT, intellectual disability and cognitive defect with sensorineural hearing loss (SNHL) (Grammatico et al., 1999; Ross et al., 2006; Stenberg et al., 2007). Ear and hearing problems in TS have been correlated to the degree of X chromosome loss, leading to growth disturbances

during fetal life (Stenberg et al., 2007). Furthermore, autoimmune mechanisms may be causative of neurological conditions associated with the autoimmune thyroid disorder known as Hashimoto's encephalopathy (Tamagno et al., 2010). The presence of circulating anti-thyroid antibodies and GADA leads to speculation that immunemediated mechanisms may contribute to the patient's various neural symptoms associated with deafness. Autoimmune inner ear disease (AIED) is rare (Bovo et al., 2009; Stenberg et al., 2007), accounting for less than 1% of all cases of hearing impairment or dizziness. The profound psychomotor retardation also reflects the cumulative effect of the two chromosomal aberrations (Grammatico et al., 1999). Non pathogenic AIRE polymorphisms were detected in the patient's DNA (Faiyaz-Ul-Haque et al., 2009; Ferrera et al., 2007; Tazi-Ahnini et al., 2002; Tóth et al., 2010), thus excluding APECED and the PTPN22 R620W variant, known to be related to several autoimmune conditions and to autoimmune disease risk in TS (Bianco et al., 2010). High resolution SNP-array analysis of deleted 2q and duplicated 10p regions revealed that some immune genes could be functionally affected in particular those coding for IL-2RA (Brand et al., 2007), and PDCD1 which is involved in the regulation of T cell function during immunity and tolerance (Francisco et al., 2010). On a speculative

Fig. 1. SNP-array analysis of patient's DNA. Chromosome 2 profile generated by Chromosome Analysis Suite software (ChAS; Affymetrix) and detail of deletion (red bar and red triangle) at 2q37.1 → q37.3, spanning about 10 Mb (a) and chromosome 10 profile and detail, showing a duplication (blue bar and blue triangle), of about 18 Mb within 10pter →p12.32 region (b).

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basis, their duplication or deletion could be responsible for subtle changes in T regulatory cells (Tregs) (Geiger and Tauro, 2012), although their frequency and ability to suppress effector T cell function of T cell subsets expressing X-linked FOXP3 was previously found unaltered in TS patients with autoimmunity (Su et al., 2009). In support of a role of the IL-2 receptor pathway, linkage disequilibrium mapping has already identified the CD25 region as a general susceptibility locus for autoimmune disease (Brand et al., 2007). In this TS patient the complex assortment of autoimmune manifestations possibly derives from the complicity of AIRE/PTPN22 variants with chromosome abnormalities affecting intrinsic immune response genes, not being of the same origin as APECED. Interestingly, chromosome abnormalities relative to chromosome 2q and 10p, have been associated with acute myelocytic leukemia (Christiansen et al., 2005; Stegmann et al., 1995). Over the years blood counts have always been in the normal range for sex and age in our patient, thus excluding the association or the possible development of hematological malignancies. In this regard, data from the Danish Cancer registry did not observe hematological malignancies in 597 women with the syndrome (Hasle et al., 1996), although a variety of hematological malignancies were reported in TS patients (45,X) (Caballe et al., 2000; Welborn, 2004). Thus very little is known about the risk of these disorders in TS syndrome. 4.1. Conclusion Genetic investigations aimed at defining autoimmune phenotypic/ genotypic correlation in TS should be considered a useful tool for early diagnostic classification of associated multiorgan autoimmune manifestations, as well as for indicating as yet unknown pathogenetic mechanisms. Conflict of interest The authors declare no conflict of interest in the conduction of this study. Acknowledgements This work was supported by the Italian Ministry of Health. We thank Dr J. Furmaniak and Dr S. Chen of FIRS Laboratories, RSR Ltd, Cardiff, UK for measuring the IFN ω autoantibodies and Professor C. Betterle, Medical and Surgical Sciences Department, Padua University, Italy for testing the anti-adrenal and anti-ovary autoantibodies. References Bianchi, I., Lleo, A., Gershwin, M.E., Invernizzi, P., 2012. The X chromosome and immune associated genes. J. Autoimmun. 38, J187–J192. Bianco, B., et al., 2010. PTPN22 polymorphism is related to autoimmune disease risk in patients with Turner syndrome. Scand. J. Immunol. 72, 256–259. Bovo, R., Ciorba, A., Martini, A., 2009. The diagnosis of autoimmune inner ear disease: evidence and critical pitfalls. Eur. Arch. Otorhinolaryngol. 266, 37–40. Brand, O.J., et al., 2007. Association of the interleukin-2 receptor alpha (IL-2Ralpha)/ CD25 gene region with Graves' disease using a multilocus test and tag SNPs. Clin. Endocrinol. 66, 508–512.

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