Novel AIRE mutations and P450 cytochrome autoantibodies in Central and Eastern European patients with APECED

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare recessive disorder that results in several autoimmune diseases due to the mutations in the AIRE (autoimmune regulator) gene. APECED patients develop several autoimmune endocrine disorders and are characterized by the high titer autoantibodies to organ-specific antigens such as the steroidogenic P450 cytochromes. So far, 38 mutations have been identified in the AIRE gene. We report here the genetic and autoantibody analysis of 27 APECED patients of Eastern and Central European origins and one Egyptian patient. From 54 analyzed APECED chromosomes, eight mutations were detected, four of which (T16M, W78R, IVS1_IVS4, 30-53dup23bp) are novel. The most prevalent reason for APECED in these populations was the occurrence of R257X (36 chromosomes) that has been described earlier as a common and recurrent mutation in several other populations. The analysis of humoral immunity to steroidogenic P450 cytochromes by the immunoblotting of E. coli expressed antigens in the 18 APECED patients showed that 67%, 44%, and 61% of the Eastern and Central European APECED patients had autoantibodies to P450c17, P450c21, and P450scc, respectively.     

Molecular analysis of mucopolysaccharidosis type VI in Poland, Belarus, Lithuania and Estonia

Mucopolysaccharidosis VI (MPS VI) is a rare autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Over 130 ARSB gene mutations have been identified thus far and most mutations are unique to individual families. We aimed to analyze the spectrum of mutations in the ARSB gene responsible for the disorder in Poland, Belarus and Baltic States. Twenty one families with MPS VI patients, in whom diagnosis was confirmed biochemically and enzymatically, were studied. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, fourteen different disease-causing mutations were found. Three novel mutations included insertion c.375_376insT, a missense mutation c.499G>A (p.G167R) and deletion/insertion c.750_754delinsCCTGAAGTCAAG. We also report 11 previously described mutations (p.A33V, p.W57C, p.Q88X, p.T92K, p.Q97X, p.R152W, p.R160Q, p.R160X, p.Y210C, p.Y266S, p.G302R). The mutation p.R152W was present at a high prevalence of 50% (21/42) the mutated alleles in this group of patients. High prevalence of p.R152W mutation in Poland, Belarus and Baltic States indicates a possible founder effect and suggests that screening for this mutation may be appropriate in MPS VI patients from this region. Our study has also provided evidence to support genotype-phenotype correlation.     

Polymorphisms at +49A/G and CT60 sites in the 3' UTR of the CTLA-4 gene and APECED-related AIRE gene mutations analysis in sporadic idiopathic hypoparathyroidism

Autoimmune diseases such as Graves' disease and type 1 diabetes have been linked with +49A/G and CT60 single nucleotide polymorphisms (SNPs) in the 3' UTR of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene. Both these SNPs are functionally relevant and linked with T-lymphocyte activation. Hypoparathyroidism is seen in 70% of patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome (APECED). Although calcium sensing receptor autoantibodies (CaSRAb) and generalized activation of T lymphocytes are reported among patients with sporadic idiopathic hypoparathyroidism (SIH), CTLA-4 gene SNPs and APECED-related autoimmune regulator (AIRE) gene mutations have not been assessed in them. We studied lead CTLA-4 gene SNPs and APECED-related AIRE gene mutations in 73 patients with SIH and 114 healthy subjects. The CTLA-4 gene SNPs +49A/G in exon 1, CT60A/G in 3' UTR and -318C/T in the promoter region were genotyped by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) using BstEII, NcoI and MseI endonucleases, respectively. The APECED-related AIRE gene mutations, which is R257X (Finn-major) in exon 6, 4-bp insertion and 13-bp deletion in exon 8, and Iranian Jews population 'Y85C' mutation in exon 2, were studied by PCR-RFLP (Taq-I), PCR and nucleotide sequencing, respectively. CaSRAb were studied by immunoblotting. The frequencies of CTLA-4 A/A(49), A/G(49) and G/G(49) genotypes in the patients (47.9%, 38.4% and 13.7%) and controls (45.6%, 39.5% and 14.9%, respectively) and the frequencies of CT60 A/A, A/G, and G/G genotypes in the patient (42.4%, 37.0% and 20.6%) and the control (38.6%, 40.4% and 21.0%, respectively) groups were not significantly different. The frequencies of various haplotypes including genetic loci +49A/G and CT60 and frequencies of G alleles at these positions were comparable between patient and the control groups and its presence did not correlate with clinical and biochemical indices of the disease. None of the patients had APECED-related AIRE gene mutations. Lack of significant difference in the pattern of CTLA-4 A/G(49) and/or CT60A/G genotypes and absence of common APECED syndrome-related AIRE gene mutations among patients and controls suggest that these sites do not play a role in the development of the SIH.     

The molecular basis of UDP-galactose-4-epimerase (GALE) deficiency galactosemia in Korean patients.

PURPOSE: UDP-galactose-4-epimerase (GALE) deficiency galactosemia is an autosomal recessive disorder and the prevalence of the disease varies among ethnic groups. We aimed to investigate molecular characteristics of the Korean patients with attenuated GALE activity and elevated galactose-1-phosphate levels in blood. METHODS: In order to characterize the molecular defects underlying GALE deficiency, the GALE gene of 7 patients showing severe activity decreases was sequenced. PCR-RFLP was performed to confirm the presence of the mutations identified by sequencing. RESULTS: Nine mutations were identified: 8 missense mutations (p.A25V, p.R40C, p.D69E, p.E165K, p.R169W, p.R239W, p.G302D, and p.R335H) and one nonsense mutation (p.W336X). Except for p.R335H, all of these mutations are novel. Six patients were compound heterozygotes (p.D69E/p.G302D, p.R40C/p.R169W, p.D69E/p.E165K, p.R239W/p.R335H, p.A25V/p.R169W, and p.G302D/p.R335H) and the remaining patient had only one mutation (p.W336X/not detected). Thirty patients with moderately reduced GALE activity were also tested by PCR-RFLP for the presence of the above mutation, and mutations were detected in 17 of these 30 patients. The frequency of p.G302D (9/30), p.R239W (6/30) and p.R169W (5/30) in our Korean patients with GALE deficiency galactosemia was relatively high. CONCLUSIONS: We detected 9 mutations of the GALE gene in Korean galactosemia patients, and confirmed allelic heterogeneity in this disease.     

Two novel mutations of the AIRE protein affecting its homodimerization properties

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.     

Genetic analysis of autoimmune regulator haplotypes in alopecia areata

Alopecia areata is an immune-mediated disorder, occurring with the highest observed frequency in the rare recessive autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome caused by mutations of the autoimmune regulator (AIRE) gene on chromosome 21q22.3. We have previously detected association between alopecia areata and a single nucleotide polymorphism (SNP) in the AIRE gene in patients without APECED, and we now report the findings of an extended examination of the association of alopecia areata with haplotype analysis including six SNPs in the AIRE gene: C-103T, C4144G, T5238C, G6528A, T7215C and T11787C. In Caucasian groups of 295 patients and 363 controls, we found strong association between the AIRE 7215C allele and AA [P = 3.8 x 10(-8), OR (95% CI): 2.69 (1.8-4.0)]. The previously reported association between AA and the AIRE 4144G allele was no longer significant on correction for multiple testing. The AIRE haplotypes CCTGCT and CGTGCC showed a highly significant association with AA [P = 6.05 x 10(-6), 9.47 (2.91-30.8) and P = 0.001, 3.51 (1.55-7.95), respectively]. To select the haplotypes most informative for analysis, we tagged the polymorphisms using SNPTag software. Employing AIRE C-103T, G6528A, T7215C and T11787C as tag SNPs, two haplotypes were associated with AA; AIRE CGCT and AIRE CGCC [P = 3.84 x 10(-7), 11.40 (3.53-36.9) and P = 3.94 x 10(-4), 2.13 (1.39-3.24) respectively]. The AIRE risk haplotypes identified in this study potentially account for a major component of the genetic risk of developing alopecia areata.     

11例中国人粘多糖贮积症Ⅰ型轻型患者的基因突变检测

目的 对经酶学确诊的粘多糖贮积症Ⅰ型患者进行α-L-艾杜糖苷酸酶(α-L-iduronidase,IDUA)基因突变检测,了解中国北方地区粘多糖贮积症Ⅰ型轻型患者基因突变特点.方法 应用PCR扩增技术及直接测序技术对11例中国北方地区经酶学确诊的粘多糖贮积症Ⅰ型患者IDUA基因14个外显子及相邻区域进行突变筛查,同时进行亲本来源分析.结果 11例患者中检测出7种基因突变:A79V、R89Q、R89w、E178K、G197S、L346R和W626X.7种突变均为已报道过子,11例中6例患者为纯合改变,1例为无义突变杂合子;发现了9种已知多态性位点.结论 中国北方地区粘多糖贮积症Ⅰ型轻型患者IDUA基因的突变特点可能不同于其他国家和地区.

Abstract：

Objective Mucopolysaccharidosis type Ⅰ (MPS Ⅰ ) is an autosomal recessive diseaseresulting from the deficiency in the lysosomal enzyme α-L-iduronidase (IDUA). The present study was conducted to identify IDUA gene mutations in attenuated (MPS Ⅰ H/S and MPS Ⅰ S) patients with MPS Ⅰin northern China. Methods Fourteen exons with adjacent intronic sequences of the IDUA gene in 11 MPS Ⅰ patients were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced directly and origin analysis was conducted. Results Seven mutations were detected in the 11 MPS Ⅰ patients, i.e., c. 236 C＞T(p. A79V), c. 266 G＞A(p. R89Q), c. 265 C＞T(p. R89W), c. 532G＞A(p.E178K), c. 589G＞A(p. G197S), c. 1037T＞G(p. L346R), and c. 1877 G＞A(p. W626X). All of them were known mutations. Six patients were homozygotes and 1 was heterozygote with nonsense mutation. In addition, 9 reported single nucleotide polymorphism (SNP) were detected, i. e., p. A8, p. A20, p. H33Q,p. R105Q, p. A314, p. A361T, p. T388, p. T410 and p. V454I. Conclusion The mutation spectrum of the IDUA gene in attenuated MPS Ⅰ Chinese patients may be different from that in patients from other countries.     

Molecular background of polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome in a Polish population: novel AIRE mutations and an estimate of disease prevalence

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autosomal-recessive autoimmune disease caused by autoimmune regulator gene mutations. The aim of this study was to examine the mutation profile of Polish APECED patients, determine the carrier rate of the most frequent mutation(s) and estimate disease prevalence. While studying 14 unrelated patients, we identified three novel mutations (c.1A>T, affecting the start codon; [IVS1 + 1G>C; IVS1 + 5delG], a complex mutation affecting splice site; c. 908G>C, p.R303P, a missense mutation in plant homeodomain (PHD) and three previously reported mutations (c.769C>T, p.R257X; c.967_979del13bp, C322fsX372; c.931delT, p.C311fsX376). Eleven patients had mutations on both chromosomes, whereas in three patients only a single alteration with proven or likely pathogenic effect was detected. The most frequent was the p.R257X mutation (71% of chromosomes); its carriage rate was assessed in the background population. Analysis of 2008 samples showed eight heterozygotes, indicating the frequency of 0.40% (1:250) and the disease prevalence - 1:129,000 (95% confidence interval: 1:555,000 to 1:30,000). Comparison with an epidemiological estimate (1:619,000, derived for women) suggested that in Poland, APECED is underdiagnosed. Among the patients, no genotype/phenotype correlations were found, but we noted that women had earlier onset of hypoparathyroidism (p < 0.02) and were younger at diagnosis (p < 0.05) than men.     

Identification of four novel mutations of the XLRS1 gene in Japanese patients with X-linked juvenile retinoschisis. Mutation in brief no. 234. Online

The XLRS1 gene (HUGO-approved symbol, RS1) has been found to cause X-linked recessive retinoschisis (RS) which is characterized by splitting of the superficial layer of the retina. Recent mutation analysis of this gene revealed 82 different mutations in 214 patients with RS. We have now identified 10 mutations of the XLRS1 gene in 11 unrelated Japanese males with RS. Mutations found in these patients were; 1) a 20-kb deletion in exon 1 region; 2) mutations in the initiation sequence (M1V); 3) mutations in the splice donor site (IVS1 + 1 g-->a); 4) two nonsense mutations (Q88X, W163X); and 5) five missense mutations (E72K, Y89C, R182C, G109E, P203L). Four (M1V, Q88X, G109E, and W163X) of the 10 mutations were novel. The R182C mutation was identified in 2 unrelated patients. The 3 mutations found between exons 1 and 3 cause premature translation termination in the XLRS1 protein. The rest of the 7 mutations were clustered between exons 4 and 6. This region of the protein is homologous to the proteins implicated in cell-cell adhesion.     

Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment

Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing loss cases. Approximately half result from mutations in the connexin 26 (Cx26) gene, GJB2, in Caucasian populations. Heterozygous mutations in GJB2 occasionally co-occur with a deletion of part of GJB6 (connexin 30; Cx30). It is estimated that approximately 1% of deafness is maternally inherited, due to mutations in mitochondrial DNA (mtDNA). Few studies have focused on the frequency of mutations in connexins or mtDNA in African American (AA) and Caribbean Hispanic (CH) admixture populations. In this study, we performed bidirectional sequencing of the GJB2 gene and polymerase chain reaction (PCR) screening for the common GJB6 deletion, as well as PCR/RFLP analysis for three mutations in mtDNA (A1555G, A3243G, A7445G), in 109 predominantly simplex AA and CH individuals. Variations found were a 101T > C (M34T; 1/101 cases), 109G > A (V37I; 1/101), 35delG (mutation; 4/101, (3/4) of non-AA/CH ethnicity), 167delT (mutation; 1/101), 139G > T (mutation; E47X; 1/101 homozygote, consanguineous), -15C > T (1/101), 79G > A (V27I; 9/101), 380G > A (R127H; 4/101; Guyana, India, Pakistan ethnicity), 670A > C (Indeterminate; K224Q; 1/101), 503A > G (novel; K168R; 3/101) and 684C > A (novel; 1/101). All but one of the AA and CH patients had monoallelic variations. There were no hemizygous GJB6 deletions in those with monoallelic GJB2 variations. We also did not identify any patients with the three mutations in mtDNA. Bidirectional sequencing of the GJB2 gene was performed in 187 AA and Hispanic healthy individuals. Our results reveal that GJB2 mutations, GJB6 deletions, and mtDNA mutations may not be significant in these minority admixture populations.     

中国人自身免疫性多内分泌腺病综合征Ⅰ型AIRE基因突变

目的 研究一个中国自身免疫性多内分泌腺病综合征Ⅰ型(autoimmune polyendocrinopathy syndrome type Ⅰ,APS-Ⅰ)家系自身免疫调节因子(autoimmune regulator,AIRE)基因突变.方法 采用聚合酶链反应和DNA直接测序技术对该家系成员进行AIRE突变位点检测,应用限制性酶切分析的方法证实,并用生物信息学方法进行结构和功能预测.结果 患儿具有AIRE的A19T和R257X的复合杂合突变,患儿父亲仅有第1外显子的A19T突变,与本家系无亲缘关系的100名健康人不存在这一突变;患儿母亲仅有第6外显子的R257X突变.结论 发现中国APS-Ⅰ患儿存在AIRE突变,经检索人类基因突变数据库和最新文献,A19T尚未见报道,R257X尚未在亚洲人中报道.     

Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan

Mutations in OTOF , encoding otoferlin, cause non-syndromic recessive hearing loss. The goal of our study was to define the identities and frequencies of OTOF mutations in a model population. We screened a cohort of 557 large consanguineous Pakistani families segregating recessive, severe-to-profound, prelingual-onset deafness for linkage to DFNB9 . There were 13 families segregating deafness consistent with linkage to markers for DFNB9 . We analyzed the genomic nucleotide sequence of OTOF and detected probable pathogenic sequence variants among all 13 families. These include the previously reported nonsense mutation p.R708X and 10 novel variants: 3 nonsense mutations (p.R425X, p.W536X, and p.Y1603X), 1 frameshift (c.1103_1104delinsC), 1 single amino acid deletion (p.E766del) and 5 missense substitutions of conserved residues (p.L573R, p.A1090E, p.E1733K, p.R1856Q and p.R1939W). OTOF mutations thus account for deafness in 13 (2.3%) of 557 Pakistani families. This overall prevalence is similar, but the mutation spectrum is different from those for Western populations. In addition, we demonstrate the existence of an alternative splice isoform of OTOF expressed in the human cochlea. This isoform must be required for human hearing because it encodes a unique alternative C-terminus affected by some DFNB9 mutations.     

Mutation analysis and haplotype correlation for 139 cystic fibrosis patients from the Nebraska Regional Cystic Fibrosis Center

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian populations with an approximate frequency of one in 2,500 live births and a carrier frequency of one in 25. We studied 400 individuals seen at The Nebraska Regional Cystic Fibrosis Center that included 139 CF patients, 206 parents, and 55 unaffected siblings to determine the frequency of the ΔF508, R117H, G542X, S549R/N, G551D, R553X, R560T, and W1282X mutations. In addition, we determined haplotypes on each of these individual's chromosomes using four markers that included XV-2c, KM-19, pMP6d.9, and G2. Results from this study showed that the ΔF508 mutation was present in 70% of CF chromosomes. Of the 139 CF patients 74 (53%) were homozygous for the ΔF508 deletion, 47 (34%) were heterozygous for the ΔF508 deletion and an unknown mutation, and 18 (13%) carried two unknown mutations. Four additional-mutations were also found in our population and included G542X (6%), G551D (5%), R553X (4%), and R560T (1%). One patient was documented to be a compound heterozygote for G542X/G551D. A polymorphism, F508C, that has previously been reported in several families was also present in our study. The most common haplotype associated with the ΔF508 deletion in our CF patients was the E haplotype (CF Consortium B) while other mutations were associated with a variety of haplotypes. © 1993 Wiley-Liss, Inc.     

Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) occurs worldwide. The most common mutations in the CYP21A2 gene in 716 unrelated patients were analyzed and the mutations were grouped by ethnicity, as defined through self-declaration corroborated by review of pedigrees extending to two or three generations. Prevalent allelic mutations and genotypes were found to vary significantly among ethnic groups, and the predominance of the prevalent mutations and genotypes in several of these populations was significant. There are ethnic-specific mutations in the CYP21A2 gene. A large deletion is prevalent in the Anglo-Saxons; a V281L (1685 G to T) mutation is prevalent in Ashkenazi Jews; an R356W (2109 G to A) mutation is prevalent in the Croatians; an IVS2 AS -13 (A/C to G) mutation is prevalent in the Iranians and Yupik-speaking Eskimos of Western Alaska; and a Q318X (1994 C to T) mutation is prevalent in East Indians. Genotype/phenotype non-correlation was seen when at least one IVS2 AS -13 (A/C to G) mutation in the CYP21A2 gene was present.     

婴儿严重肌阵挛癫痫钠离子通道SCNIA基因突变分析

目的 研究婴儿严重肌阵挛癫痫(severe myoclonic epilepsy of infancy,SMEI)患儿钠离子通道a1亚单位基因(sodium channel al subunit gene,SCNIA)突变筛查及遗传特征.方法 收集SMEI患儿23例及其家系成员的临床资料及外周血DNA,采用PCR扩增和DNA直接测序的方法筛查SCNIA基因的26个外显子的突变.结果 23例SMEI患儿中17例有SCNlA基因突变.基因突变率约为73.9%(17/23),其中8例为错义突变(F90S、I91T、A239T、W952G、T1210K,V1335M、V1390M、G1433E),3例为无义突变(R612X、W768X、w1408X),3例为缺失突变(A395fsX400、L556fsX557、V1778fsX1800),1例为插入突变(Y1241fsX1270),1例为剪切部位突变(IVS10+3A>G),1例为同义突变(K1492K),截断突变约占总突变的47.1%(8/17).13个突变位点(F90S、I91T、T1210K、V1335M、G1433E、R612X、W768X,A395faX400、L556fsx557、V1778fsXl800、Y1241fsXl270、IVS10+3A>G、K1492K)经相关检索未见报道.14例突变已证实为新生突变,其余3例突变尚不能确定突变来源.结论 SCNIA基因是SMEI患儿的主要致病基因,约一半为截断突变.SMEI患儿的SCNIA基因突变无热点,且多为新生突变.