Katarina Trebušak Podkrajšek

Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A–producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis.

Universal Screening for Familial Hypercholesterolemia in Children.

BACKGROUND Individuals with familial hypercholesterolemia (FH) who are untreated have up to 100-fold elevated risk for cardiovascular complications compared with those who are unaffected. Data for identification of FH with a universal screening for hypercholesterolemia in children are lacking. OBJECTIVES This study sought genetic identification of FH from a cohort of children with elevated serum total cholesterol (TC) concentration, detected in a national universal screening for hypercholesterolemia. METHODS Slovenian children born between 1989 and 2009 (n = 272) with TC >6 mmol/l (231.7 mg/dl) or >5 mmol/l (193.1 mg/dl) plus a family history positive for premature cardiovascular complications, identified in a national universal screening for hypercholesterolemia at 5 years of age were genotyped for variants in LDLR, PCSK9, APOB, and APOE. RESULTS Of the referred children, 57.0% carried disease-causing variants for FH: 38.6% in LDLR, 18.4% in APOB, and none in PCSK9. Nine novel disease-causing variants were identified, 8 in LDLR, and 1 in APOB. Of the remaining participants, 43.6% carried the APOE E4 isoform. Estimated detection rate of FH in the universal screening program from 2009 to 2013 was 53.6% (95% confidence interval [CI]: 34.5% to 72.8%), peaking in 2013 with an upper estimated detection rate of 96.3%. Variants in LDLR, APOB, or the APOE E4 isoform occurred in 48.6%, 60.0%, and 76.5%, respectively, of patients with a family history negative for cardiovascular complications. CONCLUSIONS Most participants who were referred from a national database of universal screening results for hypercholesterolemia had genetically confirmed FH. Data for family history may not suffice for reliable identification of patients through selective and cascade screening.

AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies

Summary APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Genetic Polymorphisms in Genes Encoding Antioxidant Enzymes Are Associated With Diabetic Retinopathy in Type 1 Diabetes

OBJECTIVE Oxidative stress plays an important role in the development of microangiopathic complications in type 1 diabetes. We investigated polymorphic markers in genes encoding enzymes regulating production of reactive oxygen species in association with diabetic retinopathy or diabetic nephropathy. RESEARCH DESIGN AND METHODS A total of 124 patients with type 1 diabetes were investigated in this case-control study. All subjects were matched for sex, age, and duration of diabetes. Genotyping was conducted using real-time PCR for p.Val16Ala polymorphism in the MnSOD gene and c.C−262T in the promoter region of the CAT gene. Multiplex PCR method was used for determination of GSTM1 and GSTT1 polymorphic deletions. Fluorescence-labeled PCR amplicons and fragment analysis was used for assessing the number of pentanucleotide (CCTTT)n repeats in inducible nitric oxide synthase. RESULTS A positive association of MnSOD genotype Val/Val (odds ratio [OR] 2.49, 95% CI 1.00–6.16, P = 0.045) and GSTM1–1 genotype (2.63, 1.07–6.47, P = 0.031) with diabetic retinopathy but not with diabetic nephropathy was demonstrated. Additionally, the combination of the two genotypes conveyed an even higher risk (4.24, 1.37–13.40, P = 0.009). No other investigated genetic polymorphisms were associated with either diabetic retinopathy or diabetic nephropathy. CONCLUSIONS Selected polymorphisms in genes encoding MnSOD and GSTM1 could be added to a panel of genetic markers for identification of individuals with type 1 diabetes at an increased risk for developing diabetic retinopathy.

Detection of a complete autoimmune regulator gene deletion and two additional novel mutations in a cohort of patients with atypical phenotypic variants of autoimmune polyglandular syndrome type 1

OBJECTIVE Autoimmune polyglandular syndrome type 1 (APS-1) is characterised by multiple autoimmune diseases. Detection of autoimmune regulator (AIRE) gene mutations facilitates timely and precise diagnosis. DESIGN AIRE mutation detection was performed in a cohort of 11 patients. Two did not meet clinical APS-1 criteria and several started with atypical presentation. METHODS Sequencing and TaqMan genotyping were used to identify AIRE mutations. Complete AIRE deletion was confirmed and framed by real-time PCR, long-range amplification and analysis of the microsatellite markers. RESULTS Seven different mutations were detected, three were novel: c.892G>A in exon 8, silent mutation c.462A>T in exon 3 most likely affecting splicing, and a complete deletion of a single AIRE allele ((?_68)_(1567-14_?)del). Novel (chronic otitis) and rare (systemic juvenile rheumatoid arthritis, autoimmune bronchiolitis, epilepsy) clinical presentations were observed. CONCLUSIONS AIRE mutation detection was valuable in the diagnostics of APS-1 in patients with atypical presentation. Chronic otitis media possibly broadened the cluster of APS-1 manifestations.

Severe progressive obstructive cardiomyopathy and renal tubular dysfunction in Donohue syndrome with decreased insulin receptor autophosphorylation due to a novel INSR mutation

Donohue syndrome (leprechaunism; OMIM *246200) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene (INSR) causing either defects in insulin binding or receptor autophosphorylation and tyrosine kinase activity. We report a patient with pronounced clinical picture of leprechaunism who developed severe progressive hypertrophic obstructive cardiomyopathy (HOCM) and renal tubular dysfunction which improved on continuous subcutaneous infusion of recombinant human insulin-like growth factor-1 (rhIGF-I). INSR gene molecular analysis and insulin receptor (IR) autophosphorylation on cultured fibroblasts were performed. A novel homozygous missense mutation p.Leu795Pro was found, located in the extracellular portion of the β subunit of the insulin receptor. The post-binding defect of the insulin receptor signaling in cultured fibroblasts demonstrated decreased insulin receptor autophosphorylation. Conclusion: Treatment with rhIGF-I partially reversed severe progressive HOCM and renal tubular dysfunction in a patient with Donohue syndrome associated with a novel p.Leu795Pro INSR gene mutation causing a severe decrease in IR autophosphorylation.

Craniofacial characteristics and genotypes of amelogenesis imperfecta patients

The aims of the study were to identify craniofacial characteristics in patients with the rough hypoplastic amelogenesis imperfecta (AI) phenotype and to evaluate whether craniofacial variables are related to a mutation in either of the two genes associated with AI, enamelin (ENAM) and amelogenin (AMGX). Eight children (five males and three females) with rough hypoplastic AI phenotype, aged 6.5-15 years, from three families and their parents (three males and three females) were examined clinically, radiographically, and genetically. Seventeen variables were measured on lateral cephalometric radiographs in AI affected (n = 11) and AI unaffected (n = 3) members. Craniofacial measurements were statistically analysed using a Students t-test. In all 14 individuals, mutation analysis of the ENAM and AMGX genes was performed by direct sequencing of the coding region. All AI affected patients had hypoplastic enamel with a rough surface and malocclusions. In the vertical plane, all AI children presented an anterior and/or posterior open bite (OB). Craniofacial analysis confirmed increased vertical relationships, with increased vertical jaw relationships and higher values for gonial angle. In two AI affected families, A and B, the same heterozygous ENAM g.8344delG mutation was confirmed, while in the third family, neither ENAM nor AMGX mutation was found. All patients with rough hypoplastic AI had a moderate to severe malocclusion with increased vertical dimensions regardless of the presence or absence of the ENAM g.8344delG mutation. As an OB requires appropriate timing of therapy, it is important to diagnose these patients as early as possible.

Cathepsin C Gene 5′-Untranslated Region Mutation in Papillon-Lefèvre Syndrome

Background: Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disorder characterized by palmoplantar keratoderma together with a severe form of generalized aggressive periodontitis and associated with mutations in cathepsin C gene (CTSC). Objective: To investigate the clinical and mutational characteristics of 6 PLS patients from 4 unrelated Slovenian families. Methods:CTSC mutational and functional analyses were performed. Results: In all patients, a novel homozygous substitution, c.-55C>A, in the CTSC 5′-untranslated region (UTR) was detected on genomic DNA level and confirmed by mRNA analysis, resulting in the almost complete loss of CTSC mRNA expression and CTSC activity. In silico analysis revealed the potential of the mutation to disrupt putative transcription factor binding sites (TFBSs) for AP-2 and Sp families of transcription factors. Conclusion: Identification of a novel CTSC 5′-UTR mutation together with a severe reduction of CTSC mRNA expression and virtually nonexistent CTSC activity was suggestive of a novel mechanism of TFBS dysfunction associated with PLS.

DEPTOR promoter genetic variants and insulin resistance in obese children and adolescents.

Insulin resistance (IR) is one of the major metabolic complications of obesity in children and adolescents. DEP domain‐containing mammalian target of rapamycin interacting protein (DEPTOR) is involved in downstream insulin signaling and DEPTORs effects are regulated by its level of expression.

GPR143 Gene Mutation Analysis In Pediatric Patients With Albinism

Background: X-linked ocular albinism type 1 is difficult to differentiate clinically from other forms of albinism in young patients. X-linked ocular albinism type 1 is caused by mutations in the GPR143 gene, encoding melanosome specific G-protein coupled receptor. Patients typically present with moderately to severely reduced visual acuity, nystagmus, strabismus, photophobia, iris translucency, hypopigmentation of the retina, foveal hypoplasia and misrouting of optic nerve fibers at the chiasm. Materials and Methods: Following clinical ophthalmological evaluation, GPR143 gene mutational analyses were performed in a cohort of 15 pediatric male patients with clinical signs of albinism. Results: Three different mutations in the GPR143 gene were identified in four patients, including a novel c.886G>A (p.Gly296Arg) mutation occurring “de novo” and a novel intronic c.360 + 5G>A mutation, identified in two related boys. Conclusions: Four patients with X-linked ocular albinism type 1 were identified from a cohort of 15 boys with clinical signs of albinism using mutation detection methods. Genetic analysis offers the possibility of early definitive diagnosis of ocular albinism type 1 in a significant portion of boys with clinical signs of albinism.