Inherited chronic obstructive pulmonary disease: new selective-sequencing workup for alpha1-antitrypsin deficiency identifies 2 previously unidentified null alleles

BACKGROUND: alpha(1)-Antitrypsin (alpha(1)AT) deficiency predisposes individuals to chronic obstructive pulmonary disease (COPD) and/or liver disease. Phenotyping of the protein by isoelectric focusing is often used to characterize alpha(1)AT deficiency, but this method may lead to misdiagnosis (e.g., by missing null alleles). We evaluated a workup that included direct sequencing of the relevant parts of the gene encoding alpha(1)AT, SERPINA1 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1], for patients with alpha(1)AT concentrations < or =1.0 g/L. METHODS: During a 5-year period, we identified 66 patients with alpha(1)AT concentrations < or =1.0 g/L and amplified and sequenced exons 2, 3, and 5 of the alpha(1)AT gene in these patients. To ensure that no relevant genotypes were missed, we sequenced the same exons in 48 individuals with alpha(1)AT concentrations between 1.0 and 1.5 g/L. RESULTS: Sequence analysis revealed 18 patients with combinations of disease-associated alpha(1)AT alleles: 8 homozygous for the deficient Z allele and 10 compound heterozygotes for various deficient or null alleles. We identified and named 2 new null alleles, Q0(soest) (Thr(102)-->delA, which produces a TGA stop signal at codon 112) and Q0(amersfoort) (Tyr(160)-->stop). No relevant disease-associated allele combinations were missed at a 1.0-g/L threshold. CONCLUSIONS: Up to 22% of the alleles in disease-associated alpha(1)AT allele combinations may be missed by conventional methods. Genotyping by direct sequencing of samples from patients with alpha(1)AT concentrations < or =1.0 g/L detected these alleles and identified 2 new null alleles.     

Use of two reporter dyes without interference in a single-tube rapid-cycle PCR: alpha(1)-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler

BACKGROUND: alpha(1)-Antitrypsin is the major plasma serine protease inhibitor. Its deficiency is mainly associated with the alleles PI*S and PI*Z and can lead to obstructive lung disease in adults and to liver cirrhosis during childhood. METHODS: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705. RESULTS: Unequivocal genotyping results were obtained for all investigated samples in an assay time of approximately 30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves. CONCLUSIONS: To our knowledge, this is the first report of simultaneous detection of two mutations in a single tube by PCR of genomic DNA and the use of two different reporter dyes with the LightCycler color compensation feature. This approach is a rapid, convenient, and economic alternative to other methods described to date for the detection of alpha(1)-antitrypsin deficiency alleles.     

Diagnosis of alpha-1-antitrypsin deficiency: An algorithm of quantification, genotyping, and phenotyping

BACKGROUND: Laboratory testing in suspected alpha-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. METHODS: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. RESULTS: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. CONCLUSIONS: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.     

The microheterogeneities of thyroxine-binding globulin and alpha 1 protease inhibitor (alpha 1-antitrypsin) are interrelated

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the alpha 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.     

Polymerase chain reaction-mediated site-directed mutagenesis detection of Z and S alpha-1-antitrypsin alleles in family members

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder with a reduction in serum A1AT levels. In a large family, we used a polymerase chain reaction (PCR)-mediated, site-directed mutagenesis assay to detect the two most common A1AT deficient variants, Z and S. By coamplification, using primers for both the Z and S mutations, we were able to detect heterozygous and homozygous genotypes for both mutations in a single reaction. We compared our results with phenotype studies obtained by standard immunofixation and isoelectric focusing techniques at two reference laboratories. Whereas PCR and isoelectric focusing agreed completely, there were five discrepancies in the results obtained by the immunofixation procedure. The reference laboratory that provided these discrepant results later informed us of a quality control problem that accounted for their error. The family study included 12 individuals representing three generations. Two individuals were MM homozygotes, three were MZ heterozygotes, four were MS heterozygotes, and three were SZ heterozygotes. A thirteenth family member was diagnosed as a ZZ homozygote at another institution. We have shown that this PCR coamplification technique provides accurate information about the M, S, and Z alleles that is at least as useful as current reference laboratory methodologies. © 1996 Wiley-Liss, Inc.     

Adenine phosphoribosyltransferase deficiency and its purine metabolism

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder that causes 2,8-dihydroxyadenine (DHA) urolithiasis. Based on the level of residual enzyme activity in cell extracts, two types of APRT deficiency have been identified. Type I is complete enzyme deficiency. Type II shows residual activity in cell lysates, but enzyme activity is not demonstrable in intact cells. Patients with type II have at least one M136T allele and have been identified mainly in Japanese. Thus, in Japanese, patients with type I deficiency are homozygous for APRT*Q0 (null alleles) and patients with type II deficiency are either homozygotes with APRT*J (M136T allele) or compound heterozygotes with APRT*J/APRT*Q0.     

Ethanol dependence of alpha 1-antitrypsin C-terminal Lys truncation mediated by basic carboxypeptidases.

BACKGROUND: Patients with hereditary emphysema are treated with alpha 1-antitrypsin (alpha 1-proteinase inhibitor [A1PI]) concentrates. High-resolution isoelectric focusing (IEF) analysis of A1PI shows that commercial A1PI products have different glycoisoform band patterns predominantly caused by varying degrees of C-terminal Lys truncation at position 394 from the A1PI molecule. Basic carboxypeptidases (CPs) are a group of enzymes that specifically cleave C-terminal basic amino acids (Arg or Lys) from peptides and proteins. STUDY DESIGN AND METHODS: In this study, whether A1PI is a substrate for basic CPs was investigated. CPN and CPU, two CPs present in plasma, and CPM, a GPI-anchored membrane protein highly expressed in lung tissues, were included. RESULTS: Basic CPs are able to mediate the C-terminal Lys truncation of A1PI although with a very low efficiency. However, presence of ethanol, for example, during Cohn fractionation, renders A1PI highly susceptible to cleavage by CP with the extent of Lys truncation depending on the ethanol concentration. This ethanol concentration dependence elegantly explains the varying amounts of des-Lys A1PI present in commercial preparations purified from different Cohn fractions. CONCLUSIONS: The cause of C-terminal truncation of A1PI present in products used for augmentation therapy has been identified, and it has been shown that A1PI becomes a substrate for CPs, specifically CPN, because of the presence of ethanol during Cohn fractionation.     

中国北方汉族急性白血病和慢性粒细胞白血病患者HLA等位基因及单倍型研究

目的 研究中国北方汉族急性白血病(AL)和慢性粒细胞白血病(CML)患者人群中人类白细胞抗原HLA-A、B、DRB1等位基因和单倍型的分布差异.方法 根据1 270例中国北方汉族AL病患者和803例中国北方汉族CML患者的HLA-A、B、DRB1表型数据,采用最大似然性方法分别计算两个群体的HLA-A、B、DRB1等位基因和单倍型频率,并采用(2检验方法比较其分布差异.结果 两个患者群体中,A位点的A*02、A*03、A*24,B位点的B*37、B*38、B*44、B*45、B*46、B*50、B*51、B*52、B*54、B*60、B*65,DR位点的DRB1*03、DRB1*09、DRB1*10、DRB1*15等位基因的分布有统计学差异.在两个患者群体中有326条A-B单倍型,357条B-DRB1单倍型,1 278条A-B-DRB1单倍型为共有单倍型,其中7.1%(23/326)A-B单倍型,6.2%(22/357)B-DRB1单倍型,4.0%(51/1278)条A-B-DRB1单倍型有统计学差异(x2>3.84,P<0.05).结论 中国北方汉族AL和CML两个患者群体的HLA-A、B、DRB1等位基因和单倍型均具有高度遗传多态性,并有其自身遗传特征.     

江苏骨髓分库汉族造血干细胞捐献志愿者HLA-A、B、DRB1基因多态性和单倍型研究

目的分析中国造血干细胞捐献者资料库(简称江苏分库)汉族人群中HLA-A,B,DRB1基因多态性和单倍型的分布特征。方法用PCR-SSP和PCR-SSOP基因分型技术对江苏分库20 248名无关志愿者作HLA-A、B、DRB1低分辨基因分型,以直接计数法计算等位基因频率,应用Arlequin 3.01软件以最大似然法分析单倍型频率。结果江苏分库汉族志愿者HLA抗原频率分布符合Hardy-Weinberg平衡;共检出HLA-A位点等位基因18个,B位点等位基因34个,DRB1位点等位基因13个;A位点频率最高的是A*02(29.6%),B位点频率最高的是B*15(14.4%),DRB1位点频率最高的是DRB1*09(16.2%);频率最高的HLA-A,-B,-DRB1单倍型是A*30-B*13-DRB1*07(6.92%),频率最高的HLA-A、B单倍型是A*30-B*13(8.04%),频率最高的HLA-B,DRB1单倍型是B*13-DRB1*07(8.07%),频率最高的HLA-A、DRB1单倍型是A*02-DRB1*09(7.70%)。结论对江苏分库汉族人群HLA的分布状况的了解,有助于指导临床寻找HLA匹配的无关骨髓供者,为HLA与疾病相关研究和我国人群群体遗传学研究等提供了有意义的基础性资料。     

Erythroid urea transporter deficiency due to novel JKnull alleles

BACKGROUND: The Kidd blood group antigens Jk^a and Jk^b are encoded by the red blood cell (RBC) urea transporter gene. Homozygosity for silent JK alleles results in the rare Jk(a–b–) phenotype. To date, seven JK ^null alleles have been identified, and of these, two are more frequent in the Polynesians and Finns. This study reports the identification of other JK ^null alleles in Jk(a–b–) individuals of different ethnic or geographic origins.
STUDY DESIGN AND METHODS: Nine Jk(a–b–) samples and a sample from a Jk(a–b+) mother of a Jk(a+b–) baby were investigated. Polymerase chain reaction amplification and sequence analysis of the JK gene was performed. Western blotting and urea lysis were used to confirm Jk(a–b–) RBCs.
RESULTS: Four novel alleles were identified: two different nonsense mutations, 202C>T (Gln68Stop) and 723delA (Ile262Stop) were identified on otherwise consensus JK * 1 and JK * 2 alleles, respectively. A missense mutation, 956C>T (Thr319Met), was identified in a JK * 1 allele from an African-American and a JK * 2 allele in two people of subcontinental Indian descent. Immunoblotting and urea lysis confirmed absence of JK glycoprotein in RBC membranes from a sample carrying the 956C>T mutation. Other previously described JK ^null mutations were found in samples of origins other than in which they were first identified.
CONCLUSION: The molecular bases of the Jk(a–b–) phenotype are diverse and this is the first report of JK ^null alleles in individuals of African and subcontinental Indian descent. Although rare, these alleles should be taken into consideration when planning genotyping strategies for blood donors and patients.     

Rapid and inexpensive detection of alpha1-antitrypsin deficiency-related alleles S and Z by a real-time polymerase chain reaction suitable for a large-scale population-based screening

alpha(1)-Antitrypsin (AAT) deficiency is one of the most common genetic disorders in Caucasians, leading to early onset pulmonary emphysema and/or liver disorders. Accumulating data suggest that AAT deficiency is commonly under-recognized or misdiagnosed by physicians. The need for a rapid, timesaving, and relatively inexpensive but reliable detection method for the two most common deficiency alleles was developed using real-time polymerase chain reaction (PCR) genotyping. We designed and validated a 5'-nuclease assay for typing of the PI*S and PI*Z alleles using dual-labeled target-specific fluorescent probes. As a reference method, we used restriction fragment length polymorphism. The real-time PCR method was tested on a large, cross-sectional epidemiological trial. Overall, we genotyped about 1200 samples and found a very good concordance with AAT serum levels and restriction fragment length polymorphism results. In addition, external interlaboratory validation confirmed the accuracy of the real-time PCR method. In our experience, the real-time qualitative PCR using 5'-nuclease assay is suitable as a genetic test for AAT deficiency. This method offers an acceptable balance between reliability and expenses. It seems appropriate for both population-based screening and clinical diagnosis of the deficiency.     

山东半岛地区汉族人群HLA-A、B、DRB1高分辨等位基因及单体型多态性的研究

目的研究山东半岛地区汉族人群HLA-A、B、DRB1等位基因多态性的分布特征。方法应用聚合酶链反应-直接测序分型(polymerase chain reaction sequence-based typing,PCR-SBT)法和序列特异性寡核苷酸探针杂交技术(Polymerase chain reaction and sequence-specific olignucleotide probe hybridizations,PCR-SSOP)高分辨试剂,对山东半岛地区865名无血缘关系的汉族健康人群HLA-A、B、DRB1进行基因分型。结果检出HLA-A、B、DRB1等位基因分别有33、73、43种,经统计分析这3个基因座分布均符合Hardy-Weinberg平衡定律(P>0.05),A*3001-B*1302-DRB1*0701(7.61%)和A*3303-B*4403-DRB1*1302(1.674%)单体型是山东半岛地区汉族人群最常见单体型。结论山东半岛地区汉族人群HLA-A、B、DRB1基因座单体型分布具有高度的遗传多态性且有其自身分布特点。本研究获得的的HLA-A、B、DRB1基因座单体型分布数据及相关遗传参数资料,为HLA在人类学、免疫遗传学、法医学和其它的组织器官移植方面的应用和科学研究提供和积累了基础资料。     

Genetic control of the eighth component of complement.

Using isoelectric focusing in polyacrylamide gel and a hemolytic assay for development of patterns, extensive, structural polymorphism in human C8 has been delineated. Two alleles, C8A and C8B, have been identified in orientals, with gene frequencies of 0.655 and 0.345. In blacks, what appears to be a third common allele was found, so that frequencies were 0.692, 0.259, and 0.049 for C8A, C8B, and C8A1. In whites, C8A1 was rare with a frequency of 0.003, and frequencies for C8A and C8B were 0.649 and 0.349. Inheritance was autosomal codominant in family studies and the distribution of types in random unrelated populations fit the Hardy-Weinberg equilibrium in all groups. C8 allotypes have been determined for two previously studied families, each with a homozygous C8-deficient propositus. This study suggests that C8 deficiency is a silent or null allele of the C8 structural locus, and that half normal levels of C8 cannot be used as a single criterion for the establishment of heterozygous C8 deficiency. C8 allotypes, as well as 18 other autosomal markers, were also determined for 24 families. The C8 structural locus is not closely linked to these markers, including the human histocompatibility loci complex.     

Cutoff level to detect heterozygous alpha 1 antitrypsin deficiency in Turkish population

Background: Alpha 1 antitrypsin (AT) deficiency is a hereditary disorder leading to the defective defence system against neutrophil elastasis in lung and accumulation of insoluble heterodimer AT molecules in hepatocytes. Knowledge of the prevalence of AT deficiency in each country is important to organize the public health policy. The aim of this study is to determine the prevalence of AT deficiency in Turkish population and to define the cutoff value of AT level in serum to detect heterozygous AT deficient subjects. Materials and Methods: Serum samples from 1,203 healthy blood donors were used, attending the Blood Bank of Hacettepe Medical Faculty. Isoelectric focusing method for determining PIM, PIS, and PIZ alleles and rate immune nephelometry for measuring the level of AT in serum were used. Results: Out of 1,203 healthy blood donors enrolled, 1,164 (%96.8) had normal variant PI MM allelee, 9 (%0.7) PI MZ, 7 (%0.6) PI MS, 6 (%0.5) MF, and 17 (%1.4) PI M? (unidentified variants with existing standards). Most individuals (89.6%) with low AT level (cutoff <100 mg/dl) in serum were positive for PI MM allele. The cutoff value to investigate PI MZ was 100.5 mg/dl, which had PPV and NPV of 5.0 and 99.9%, respectively. AT deficiency is a rare hereditary disorder in asymptomatic healthy Turkish blood donors. Although the cutoff value of 100.5 mg/dl for AT level in serum was able to detect heterozygous AT deficiency in the healthy population, this finding should be conformed to case‐control studies. J. Clin. Lab. Anal. 25:296–299, 2011. © 2011 Wiley‐Liss, Inc.     

中国满族人群HLA-A、B、DRB1基因和单倍型的多态性分析

目的研究中国满族人群HLA-A、B、DRB1等位基因和单倍型的多态性。方法根据2 183名满族骨髓捐献者的HLA-A、B、DRB1基因分型数据,采用最大似然性方法计算HLA-A、B、DRB1等位基因和单倍型频率。结果满族人群中共检出18个HLA-A、44个HLA-B和15个HLA-DRB1等位基因,其常见等位基因为A*02、A*11、A*24、A*30、A*33、B*13、B*35、B*46、B*51、B*40(B60)、B*40(B61)、B*15(B62)、DRB1*04、DRB1*07、DRB1*08、DRB1*09、DRB1*11、DRB1*12、DRB1*13、DRB1*14和DRB1*15。A*30-B*13、A*02-DRB1*15、B*13-DRB1*07和A*30-B*13-DRB1*07分别为其最常见A-B、A-DRB1B-DRB1和A-B-DRB1单倍型,31条A-B、24条A-DRB1和27条B-DRB1单倍型频率≥0.01,32条A-B-DRB1单倍型频率≥0.005。14条A-B、3条A-DRB1、14条B-DRB1和38条A-B-DRB1单倍型呈现强连锁不平衡格局(ALD≥0.40)。结论中国满族人群HLA-A、B、DRB1等位基因和单倍型的分布比较接近北方汉族人群并具有其自身分布特点。     

4082名上海骨髓库汉族无关供者HLA-A、B、DRB1高分辨等位基因及单体型多态性研究

目的 了解上海地区汉族人群HLA-A、B、DRB1在高分辨分型水平上的等位基因及单体型频率分布特征.方法 采用PCR-测序分型技术(Sequence-based typing,SBT)对4 082名随机、无血缘关系、健康的中华骨髓库上海分库汉族造血干细胞捐献者进行HLA-A、B、DRB1高分辨基因分型,利用计数法和最大似然性原理方法分别计算HLA-A、B 、DRB1等位基因及单体型频率.结果 4082例样本中,共观察到204个HL4等位基因,其中频率＞0.1的HLA-A、B、DRB1等位基因分别有A*11:01、A*24:02、A*02:01、B*46:01、DRB1* 09:01、DRB1* 15:01.841条A-B单体型中,70条单体型呈现显著的连锁不平衡(ALD ＞0,HF≥3.67 ×10-4,x2 ＞3.84),8条表现为强连锁不平衡(RLD ＞0.40),17条单体型的频率高于0.01;1 027条B-DRB1单体型中,99条单体型呈现显著的连锁不平衡(ALD＞0,HF≥3.67×10-4,x2＞3.84),12条表现为强连锁不平衡(RLD＞0.40),12条单体型的频率高于0.01;3 344条A-B-DRB1单体型中,681条单体型是“可靠的”(频率≥3.67×10-4),单体型频率高于0.001有135条.结论 获得上海地区汉族人群HLA-A 、B、DRB1高分辨等位基因频率和单体型分布数据及相关遗传参数,为临床移植供者的选择、中国人群CWD表的制订、骨髓库最适库容的评估及人类遗传学研究提供参考数据.     

NullCanada: A novel α1-antitrypsin allele with in cis variants Glu366Lys and Ile100Asn

Backgroundα₁-Antitrypsin (A1AT) deficiency predisposes patients to pulmonary disease due to inadequate protection against human neutrophil elastase released during inflammatory responses. A1AT deficiency is caused by homozygosity or compound heterozygosity for A1AT variants; individuals with A1AT deficiency most commonly have at least one Z variant allele (c.1096G > A (Glu366Lys)). Null variants that result in complete absence of A1AT in the plasma are much rarer. With one recent exception, all reported A1AT variants are characterized by a single pathogenic variant.CaseAn 8 years old patient from Edmonton, Alberta, Canada, was investigated for A1AT deficiency. His A1AT phenotype was determined to be M (wild type)/Null by isoelectric focusing (IEF) but M/Z by targeted genotyping. Gene sequencing revealed two heterozygous variants: Z and Ile100Asn (c.299 T > A). The Ile100Asn substitution is predicted to disrupt the secondary structure of an α-helix in which it resides and the neighbouring tertiary structure, resulting in intracellular degradation of A1AT prior to hepatocyte secretion.MethodsFamily testing was conducted to verify potential inheritance of an A1AT allele carrying the two mutations in cis, as this arrangement of the mutations would explain “Z” detection by genotyping but not by IEF. Molecular modeling was used to assess the effect of the variants on A1AT structure and stability.DiscussionCarrier status for a novel variant Null_Canada with in cis mutations (c.[299 T > A;1096G > A], p.[(Ileu100Asn;Glu366Lys)]) was confirmed. A sibling was identified as having A1AT deficiency on the basis of compound heterozygosity for two alleles: Null_Canada and the common Z allele. A separate pedigree from the Maritimes was subsequently recognized as carrying Null_Canada.ConclusionIn cis mutations such as Null_Canada may be more common than previously described due to failure to detect such mutations using historical testing methods. Combined approaches that include gene sequencing and segregation studies allow recognition of rare A1AT variants, including in cis mutations.     

Two subsets of HLA-DQA1 alleles mark phenotypic variation in levels of insulin autoantibodies in first degree relatives at risk for insulin-dependent diabetes.

Levels of insulin autoantibodies (IAA) vary among different first degree relatives of insulin-dependent diabetes mellitus patients, suggesting genetic regulation. We previously reported elevated IAA among DR4-positive at risk relatives. In this study, 72/82 at risk relatives were IAA positive, of whom 75% (54/72) carried DR4 versus 20% (2/10) of IAA-negative relatives (P = 0.0004). However, 69% (18/26) of DR4-negative relatives were IAA positive. Since DR4 did not account for all IAA positivity, we analyzed DQA1 and DQB1 alleles. Homozygosity for DQA1 alleles deriving from the evolutionary lineage 4 (*0401, *0501, *0601) was associated with low IAA levels, while lineage 1-3 alleles (*0101, *0102, *0103, *0201, *0301) correlated with higher levels. Most (93%, 65/70) relatives with lineage 1-3 alleles were IAA positive (mean = 360 +/- 63 SEM nU/ml). Only 7/12 relatives homozygous for lineage 4 alleles were IAA-positive, with lower levels than relatives with lineage 1-3 alleles (mean = 55 +/- 15 SEM nU/ml, P < 0.0001; 7/12 vs 65/70, P = 0.004). The amino acid sequences of lineage 1-3 alleles uniquely share glutamic acid (E) and phenylalanine (F) at positions 40 and 51 (EF alleles). Lineage 4 alleles have glycine (G) and leucine (L) at those positions (GL alleles). 90% (65/72) of IAA-positive relatives had an EF allele, while only 75% (54/72) had DR4 (P = 0.01). Homozygosity for GL alleles (often DQA1 *0501 on DR3 haplotypes) correlated with little or no humoral response to insulin. Thus, HLA-DQB1 GL alleles, or other genes on haplotypes (e.g., DR3) that carry these DQA1 alleles, may confer recessive low responsiveness to insulin.