A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Serum bactericidal activity against Neisseria meningitidis in patients with C3 nephritic factors is dependent on IgG allotypes.

The main mechanisms of immune defense against Neisseria meningitidis are serum bactericidal activity (SBA) and opsonophagocytosis. Many complement deficiencies, among them acquired partial C3 deficiency due to stabilizing autoantibodies against the alternative pathway C3 convertase (C3 nephritic factors, C3 NeF); increase the risk of meningococcal infection. SBA against meningococci in patients with C3 NeF was determined along with allelic variants (GM alleles) of the immunoglobulin constant heavy G chain (IGHG) genes. In patients with C3 NeF and in control children, individuals homozygous for G1M*f and G3M*b showed higher SBA against meningococci than heterozygous individuals. Partial complement deficiency in early childhood might explain the influence of GM variants on SBA in control children. These novel findings imply that the IGHG genotype is important in defense against meningococci in individuals with low complement function and possibly in combination with other immunodeficiencies.

Immunoglobulin G (IgG) subclasses and human disease.

The isotypes of IgG, IgG1, IgG2, IgG3, and IgG4 were determined in immunoglobulin preparations and the effect on serum levels of treated patients. Serum IgG subclass deficiencies were recorded in different patient groups: (1) IgG2-IgG4 deficiency was associated with IgA deficiency. (2) IgG2-IgG4 deficiency was found in patients with ataxia telangiectasia. (3) Low IgG2 levels were recorded in patients with SLE; one of these patients with recurrent pericarditis was treated with immunoglobulin with good results. (4) Low IgG2 and/or low IgG3 levels were found in patients with juvenile diabetes mellitus. (5) Mothers giving birth to severely group B streptococci infected infants showed low levels of IgG subclasses indicating that the newborns were IgG subclass deficient at birth. (6) In a prospective study of children with recurrent otitis media aged 12 and 32 months the IgG2 levels were significantly reduced in the group with considerably high otitis proneness. In patients with IgG2-IgG4 deficiency, absence of antibodies to polysaccharide antigen teichoic acid and the protein antigen alpha-toxin of staphylococci was demonstrated. Imbalanced IgG subclass pattern with increased IgG4 was recorded in patients with different diseases such as atopic diseases and also in combination with increased IgE, Henoch-Schönlein vasculitis, idiopathic pulmonary hemosiderosis, chronic mucocutaneous candidiasis, and in patients with Trichuris trichiura infection.

Pneumococcal serum antibody concentrations during the first three years of life: a study of otitis-prone and non-otitis-prone children.

One hundred and thirteen children were followed prospectively from birth until the age of 3, serum being obtained from cord blood, and at the ages of 3, 6, 12, 18, 24, 30 and 36 months. Thirteen children developed recurrent acute otitis media (rAOM), 29 remained very healthy and the remaining children formed an intermediate group. Cord serum concentrations were determined of total IgG class, of IgG1 and IgG2 subclasses, as well as of specific IgG antibodies against the pneumococcal capsular types, 3, 6A and 19F. The specific pneumococcal IgG as well as IgA and IgM antibodies were also followed in the sequential serum samples up to the age of 3 in the rAOM and healthy children. Despite total IgG class and IgG1 and IgG2 subclass concentrations being of the same magnitude in cord serum of rAOM (median: 11.15, 7.48 and 2.16 g/l for IgG, IgG1 and IgG2, respectively) as in that of healthy children (median: 10.21, 8.16 and 2.16 g/l, respectively), both in cord serum and in most serum samples drawn during the first year of life, specific IgG antibodies against types 6A and 19F, but not against type 3, were significantly lower in the rAOM group than in the healthy children. In the intermediate group, cord serum concentrations of specific IgG antibodies to type 6A were of the same magnitude as in the healthy children. The only significant difference in specific IgM and IgA antibody concentrations against types 3, 6A and 19F between the two groups was noted for type 6A antibodies at 36 months of age where rAOM children exhibited lower values. The results indicate an association between pre-existing low specific IgG antibody levels against AOM-associated pneumococcal types and the development of rAOM.

[Ataxia-telangiectasia surveyed in Sweden]. / Ataxia-telangiectasia kartlagd i Sverige.

Ataxia-telangiectasia (AT) is a rare autosomal recessive disease with a complex phenotype involving cerebellar degeneration, immunodeficiency, cancer risk and radiosensitivity. Our aim has been to identify Swedish AT patients in order to study the possible "Swedish phenotype" of the disease. In the 19 patients identified in Sweden we found a phenotype fairly similar to what has been described internationally, with the exception of some differences including lower cancer incidence in patients and their relatives and somewhat more pronounced immunodeficiency and concomitant susceptibility to infections.

Immunoglobulin constant heavy G chain genes as risk factors in childhood allergies.

BACKGROUND: Several candidate genes have been found to be associated with the inflammatory response of IgE-mediated allergy, so also the immunoglobulin constant heavy G chain (IGHG) genes. The IGHG genes are situated close to the IGHE gene on chromosome 14q32, 5'mu, delta, gamma3, gamma1, alpha1, gamma2, gamma4, epsilon, alpha2, 3'. They are inherited in a Mendelian fashion and expressed randomly in allelic exclusion. The alternative and functionally different gamma3, gamma1 and gamma2 gene variants are found in four IGHG haplotypes, coding four B cell variants. OBJECTIVE: The aim of this study was to assess the frequency of different IGHG genes in relation to phenotypes associated with allergy, in a case-control study. METHODS: We identified the constant heavy-chain genes of IgG in 198 allergic and non-allergic children participating in the Phase II of the International Study of Asthma and Allergy in Children. The IGHG genes were assessed by the alternative serum IgG subclass allotypes expressing the alternative alleles of gamma3, gamma1 and gamma2 genes, using ELISA and double immunodiffusion. RESULTS: The IGHG*bfn haplotype (=B1 cells) and IGHG2*n allele dominated (51% vs. 24%, P=0.002) and the IGHG*bf-n haplotype (=B2 cells) was infrequent (16% vs. 52%, P < 0.001) in allergic children with a family history of allergy, clinical manifest allergy and positive skin prick test (SPT). The frequency of IGHG genes was similar in children with maternal and paternal heredity and in children with wheezing, eczema or rhinitis, as well as in children with different positive SPT. The IGHG*bfn haplotype with the IGHG2*n allele was strongly associated with heredity for allergy. The IGHG*bf-n haplotype was inversely related to allergy. Conclusions IgG allotypes, immunochemical and functional variants of IgG molecules from IGHG genes are associated with atopy. The IGHG*bfn haplotype (=B1 cells) with the IGHG2*n allele dominates, associated with an increased risk for atopy. In contrast, the IGHG*bf-n haplotype (=B2 cells) with the IGHG2*-n allele is associated with low risk.

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens.

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.

Lack of the G2m(n) allotype in IgG subclass deficiency, in IgG2 deficiency together with lack of G1m(a) and G3m(g), and in IgG3 deficiency together with lack of G1m(f) and G3m(b).

Lack of G2m(n) was demonstrated in both IgG2-deficient and IgG3-deficient Caucasian patients. Lack of G2m(n) or G2m(",") was found together with homozygosity for both G1m and G3m allotypes as the dominant finding, i.e. for IgG2-deficient patients together with G1m (f,f) and G3m(b,b), constituting the Gm(f,",b) phenotype, and for IgG3-deficient patients together with G1m(a,a) and G3m(g,g), constituting the Gm(a,",g) phenotype. The group with IgG2 deficiency and the selected patients with the Gm(f,",b) phenotype expressed characteristically very low or undetectable IgG4, significantly increased IgG3, and normal IgG1. The group with IgG3 deficiency and the selected patients with the phenotype Gm(a,",g) expressed instead normal IgG4 and nearly normal IgG2 and IgG1 levels. The lack of G2m(n) together with lack of one or the other of the alternative G1m genes and corresponding G3m genes give different IgG2 levels and different IgG subclass patterns. The frequency of G1m allotypes and corresponding G3m allotypes also deviated significantly when the IgG2 deficiency and IgG3 deficiency groups were compared with each other. Most IgG subclass-deficient patients are homozygous in the Gm system and lack genetic variants in the three IgG subclasses, IgG1, IgG2, and IgG3.

Antigenic variants of human IgG subclasses. Restriction of murine monoclonal IgG subclass antisera.

A comparison of murine monoclonal IgG subclass antisera with rabbit polyclonal IgG subclass antisera restriction of the monoclonal antisera and further antigenic heterogeneity within the IgG subclasses of at least two antigenic variants of IgG1, two of IgG2, two of IgG3 and a third subtype of IgG4.

Crossed immunoelectrophoresis and electroimmunoassay of human IgG subclasses.

Human IgGl, IgG2, IgG3 and IgG4 in WHO pool 67/97, a normal serum pool, Cohn Fraction II and individual sera were examined by crossed immunoelectrophoresis and electroimmunoassay in agarose with IgG subclass specific rabbit antisera. In these methods the fact that IgG subclasses differ in the electrophoretic field is utilized: IgG4 is located anodically, IgG3 cathodically, and IgG2 and IgG1 both anodically and cathodically. The mean, S.D. and range of serum IgG1, IgG2, IgG3 and IgG4 in 20 normal adults found by the electroimmunoassay were given and related to the amount in the WHO pool 67/97. The IgG subclasses values obtained by electroimmunoassay agreed with the values obtained by single radial diffusion. The reproducibility of double determinations (interplate variations) was 1.5--5.5 per cent. Repeated freezing, thawing and storage of the serum at room temperature did not influence quantitation of IgG subclasses. Cohn Fraction II was found to contain smaller amounts of IgG1, IgG2, and IgG4 than those found in the normal serum pools. Crossed immunoelectrophoresis and electroimmunoassay also easily reveal failing quality of IgG subclass antisera. To obtain good antisera in rabbits against IgG subclasses immunization should be done with several myeloma proteins with different electrophoretic mobility within the same subclass.

Imbalanced switch of the IGHG (immunoglobulin constant heavy G chain) Gm(bfn) genes in atopic childhood asthma.

BACKGROUND: The IGHG genes on chromosome 14q32, 5'micron delta gamma3 gamma1alpha1 gamma2 gamma4 epsilon alpha2 3', as studied by Gm allotypes, are involved in the inheritance of atopy. The 5'micron delta b f alpha1 n gamma4 epsilon alpha2 3', Gm(bfn) haplotype of the genetic B1-cell variant has been found to be associated with the atopic phenotype of children with bronchial asthma. METHODS: An indirect competitive enzyme-linked immunosorbent assay for quantitation in serum of the alternative serum Gm allotypes from the gamma3-, gamma1-, and gamma2 loci and radial immunodiffusion for quantitation of IgG subclasses were used. Children with the genetic B1-cell variants B1/B1 (= Gm[bfn/bfn]), B1/B2 (=Gm[bfn/bf-n]), and B1/B4 (=Gm[bfn/ga-n]) and bronchial asthma were investigated and compared to healthy children of the same age and B-cell type. RESULTS: The three groups with B1/B1, B1/B2, and B1/B4 cells exhibited increased IgE. In both homozygous and heterozygous B1 or Gm(bfn), the serum G1m(f) levels from gamma1 loci were significantly downregulated to 75% of normal, while G2m(n) from gamma2 loci were significantly upregulated to about double the normal level. In heterozygous patients with additional B2 or B4 cells, the G2m(-n) levels from gamma2 loci were instead downregulated. G1m(a) from gamma1 of B4 cells was also downregulated. CONCLUSIONS: Children with atopic bronchial asthma demonstrated an imbalanced class switch in rearrangement of the genes for IgG. The activity of G1m(f) from the gamma1 locus was downregulated, but G2m(n) from gamma2 was upregulated together with the closely situated epsilon locus downstream of the IGH genes. Low levels of G1m(f), Glm(a), and G2m(-n) indicated a low pressure of infections. The imbalanced activation of the IGH genes in more hygienic environments might be one explanation of the increased prevalence of atopy in children in recent decades.

Correlation between atopy and Gm allotypes.

In 50 consecutive atopic Caucasian patients with increased IgE greater than 600 kU/l, the phenotypic Gm allotype constellation deviated from that to be expected, with significantly increased frequency of patients with the phenotype Gm(f,n,b). There was an increased frequency of the G2m(n) allotype, more frequent in patients with IgE greater than 1,000 kU/l, and in patients with IgG4 greater than 1 g/l. In patients with IgE greater than 1,000 kU/l the phenotype Gm(a,f,n,b) was significantly increased and in patients with IgG4 greater than 1 g/l the phenotype Gm(f,n,b) was significantly increased. Those atopic patients with increased IgE and increased IgG4, according to earlier studies known to have the most severe forms of the disease, were thus mainly found to have the m(f,n,b) phenotype.

Gm allotype genes and gene dosage affecting both IgG subclass and IgE levels in atopic patients.

The imbalanced IgG subclass levels of 50 atopic patients with IgE greater than 600 kU/l reflected the Gm expression of the patients. IgG1 was significantly increased but only in patients with the phenotypes Gm(f,f,n,n) and Gm(a,a,'',''). The typical IgG4 increase was found in the most frequent Gm(f,f,n,n) and Gm(a,f,n,'') phenotypes. Significant increase of IgG2, IgG3 and IgG4 was found in patients homozygous for G2m(n) compared to those lacking this allotype. IgG2 decrease was found in those with the Gm(f,f,'','') phenotype and IgG3 decrease in those with the Gm(a,a,'','') phenotype. Also the IgE levels were influenced by the Gm allotypes with increased IgE levels in the G1m(f,f) patients compared to the G1m(a,a) patients.

Suppressed G2m(n) levels from IGHCG2 in IgA deficiency.

The aim of the study was to investigate the production of IgG from alternative alleles of IGHCG1, IGHCG2 and IGHCG3, closely related to IGHCA1 and IGHCA2 on chromosome 14, in IgA deficient (IgAD; serum IgA levels < 0.05 g/l) for individuals. Sixty-two IgAD individuals were included in the study and sera were investigated with the sensitive competitive indirect ELISA for measuring serum concentrations of the Gm allotypes G1m(a), G1m(f), G2m(n) and G3m(b), performed with specific monoclonal antisera and purified myeloma proteins in combination with IgG subclass quantitation. The known 'compensatorily increased' serum levels of IgG1 and IgG3 were recognized with significantly increased G1m(a) and G1m(f) from IGHCG1 and significantly increased G1m(g) and G3m(b) from IGHCG3. The quotients of G1m(a)/G1m(f) from IGHCG1 and G3m(g)/G3m(b) from IGHCG3 were also normal. Instead, the levels of G2m(n) from IGHCG2 were selectively decreased in combination with normal or increased levels of G2m(") from the same IGHCG2. The quotient G2m(n)/G2m(") was also significantly decreased. As the selectively decreased G2m(n) allotype expression from IGHCG2 was situated close to the non-expressing IGHCA1, the origin of most serum IgA could be the result of a defective common regulating factor. The selectively decreased G2m(n) allotype levels from IGHCG2 must also be discussed with a view to Gm allotype suppression described in mice. The selectively decreased G2m(n) allotype levels in G2m(n,") heterozygous IgAD individuals could be the result of a preferential allelic exclusion of G2m(n) favoring G2m(") on IGHCG2 in many cells.

Serum IgG and IgG subclass contents in different Gm phenotypes.

Different serum IgG and IgG subclass levels were found among Gm phenotypes of a normal population. One hundred and fifty-seven Caucasian blood donors were investigated for the reciprocal Gm allotypes on IgG subclass loci namely: for IgG1, G1m(f) and G1m(a); for IgG2, G2m(n) and G2m("); and for IgG3, G3m(b) and G3m(g), and subgrouped in the seven most common Gm phenotypes. The frequencies of Gm phenotypes and haplotypes were given, including numbers of the previously little known G2m(n,") heterozygous individuals. Mean serum quantities +/- SD and range of IgG, IgG1, IgG2, IgG3 and IgG4 were given for different Gm phenotypes. The IgG content was significantly lower in the Gm(f,",b/f,",b) phenotype in which the IgG2 levels were also significantly lower, compared with values of the other phenotypes. IgG3 levels were significantly lower in the Gm(a,",g/a,",g) phenotype compared with other phenotypes. These data imply the importance of Gm(f,",b/f,",b) and Gm(a,",g/a,",g) phenotypes causing lower amounts of IgG antibodies. In evaluating IgG subclass deficiency, the range for the low responding Gm(f,",b/f,",b) and Gm(a,",g/a,",g) phenotypes should be considered.

IgG subclass levels in infancy and childhood.

The concentrations of IgG1, IgG2, IgG3 and IgG4 were determined by electroimmunoassay in 10 pairs of maternal and cord sera and in sera of 162 healthy children, aged 6 weeks to 15 years. Specific rabbit antisera against the IgG subclasses were used. The content of the normal serum pool WHO 67/97 was used as reference. The mean value, standard deviation and normal range of each IgG subclass were calculated for each age group and compared with the adult values. All IgG subclasses were present in cord serum except for IgG4 in those cases where also the maternal serum lacked demonstrable IgG4. The IgG subclasses followed the pattern of total IgG with a fall during the first 3--6 months and a subsequent gradual rise with age. The IgG1 and IgG3 levels rose faster with age than IgG2 and IgG4. Adult levels were not reached before puberty. No IgG4 was detectable in 12--21% of the children above 7 years of age.

Quantitative and qualitative investigations of serum IgG subclasses in immunodeficiency diseases.

Determinations of IgG subclasses were made by electroimmunoassay and crossed immunoelectrophoresis, and Gm markers were typed in sera from seventeen patients with well-defined immunodeficiency diseases. Certain IgG subclass and Gm patterns were recognized in various diseases: IgG2 deficiency and homozygosity of Gm (4,5) in the cartilage-hair-hypoplasia syndrome, in the ataxia telangiectasia syndrome and in selective IgG subclass deficiency; and IgG3 deficiency and homozygosity of Gm(1,-5) in the Wiskott-Aldrich syndrome. The findings suggest a common structural or regulator gene defect in some immunodeficiency diseases. In IgA deficiencies, the levels of IgG1 were raised. In patients with IgG subclass deficiencies there was sometimes a compensatory increase of the remaining IgG subclasses, with a preponderance of IgG1 and IgG3. The increased Ig1 showed restricted heterogeneity with only an increase of the electrophoretically cathodal part. This part contained both kappa and lambda chaings. IgG subclass deficiency indicates treatment with gammaglobulin even if the serum levels of IgG are normal or increased.

Preparation of IgG subclass allotypes from polyclonal IgG.

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(-n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion-exchange and protein A-Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(-n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(-n) molecules is of special interest as no genetic marker has been found to identify this allotype.

IgG subclass concentrations in preterm neonates.

Serum IgG subclass concentrations were measured in 158 sera from preterm, appropriate for gestational age, infants born between 27 and 37 weeks of gestation, pregnancy, delivery and neonatal period being uncomplicated in all cases. At birth the IgG subclass concentrations were inversely correlated to the degree of prematurity. The IgG subclass concentrations decreased mainly proportionately during the neonatal period. The most immature infants born before the 30th week of gestation had critically low concentrations of all IgG subclasses. All immature infants, already in the first week after birth, showed capacity for IgG1 and IgG3 synthesis.

Serum Gm allotype levels in common variable immunodeficiency: preponderance of homozygous G2m(",") on IGHCG2.

Common variable immunodeficiency (CVI) is one of the most frequent primary immunodeficiency diseases, characterized by defective antibody formation and associated with chronic sinopulmonary infections, autoimmunity and malignancies. The genes for the constant heavy chains of IgG are located on chromosome 14 and were further studied by identifying allelic, alternative Gm allotypes. These were defined by different epitopes for three of the IgG subclasses, G1m(a) and G1m(f) for IgG1, G2m(n) and G2m(") for IgG2 and G3m(g) and G3m(b) for IgG3. A sensitive competitive ELISA method for quantitation of the Gm allotypes G1m(a), G1m(f), G2m(n) and G3m(b) were used together with radial immunodiffusion IgG subclass quantitation. The dominating number of 25 of 33 patients (p < 0.001) expressed the homozygous G2m(",") allotype on IGHCG2 in combination with homozygous or heterozygous Gm allotypes on IGHCG1 and IGHCG3, namely Gm(f,f;",";b,b), Gm(a,a;",";g,g) and Gm(f,a;",";b,g). Studies of Gm allotype quantities revealed a progressive sequential impediment of the programmed cascade for downstream IGHCG gene rearrangements. According to the order of the IGHCG genes, the G3m allotype levels from the IGHCG3 were often normal, and G1m allotype levels from IGHCG1 were suppressed; G1m(a) was suppressed more than G1m(f), and most suppressed were G2m allotype levels from IGHCG2, both G2m(n) and G2m("). The susceptibility of CVI is associated to G2m(",") expression from the IGHCG2 locus on chromosome 14, which has also been found in IgA IgG subclass deficiency, conditions known among first-degree relatives.