Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease

Mutation scanning and direct DNA sequencing of all 50 exons of ABCR were completed for 150 families segregating recessive Stargardt disease (STGD1). ABCR variations were identified in 173 (57%) disease chromosomes, the majority of which represent missense amino acid substitutions. These ABCR variants were not found in 220 unaffected control individuals (440 chromosomes) but do cosegregate with the disease in these families with STGD1, and many occur in conserved functional domains. Missense amino acid substitutions located in the amino terminal one-third of the protein appear to be associated with earlier onset of the disease and may represent misfolding alleles. The two most common mutant alleles, G1961E and A1038V, each identified in 16 of 173 disease chromosomes, composed 18.5% of mutations identified. G1961E has been associated previously, at a statistically significant level in the heterozygous state, with age-related macular degeneration (AMD). Clinical evaluation of these 150 families with STGD1 revealed a high frequency of AMD in first- and second-degree relatives. These findings support the hypothesis that compound heterozygous ABCR mutations are responsible for STGD1 and that some heterozygous ABCR mutations may enhance susceptibility to AMD.     

Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele

We have encountered an allele which seems to be another type of O allele at the human histo-blood group ABO locus. We have determined the nucleotide sequence of this allele over the coding region in the last two coding exons. This allele does not possess the single-nucleotide deletion found common among all the O alleles previously analyzed. Compared with A1 allele, this allele has three nucleotide substitutions resulting in two amino acid substitutions. The introduction of these amino acid substitutions into the A1 transferase expression construct apparently abolished the enzymatic activity of A1 transferase.     

A small influence of HSP90 levels on the trehalose and heat shock element inductions of the yeast heat shock response

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.     

T-cell receptor V region beta-chain gene expression in the autoimmune thyroiditis of non-obese diabetic mice

The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.     

Residues near the amino and carboxyl termini of staphylococcal enterotoxin E independently mediate TCR V beta-specific interactions

Previous studies identified three COOH-terminal residues in staphylococcal enterotoxin E (SEE; Asp200, Pro206, and Asp207) that in part mediate TCR V beta recognition. We have identified an additional three residues near the NH2-terminus of SEE (Arg20, Asn21, and Ser24 that are needed in conjunction with these COOH-terminal residues to fully restore native levels of V beta-specific T cell proliferation. A staphylococcal enterotoxin A SEA-SEE hybrid molecule containing the NH2-terminal V beta determinants of SEE to activate alone exhibited V beta specificities of both SEA and SEE, indicating that these residues of SEE independently contribute to V beta recognition and do not obscure the native V beta determinants of SEA. These findings suggest that the ability of SEE to activate certain V beta-specific T cell subsets may result from multiple interactions with a single TCR beta-chain or perhaps by cross-linking two TCR. High affinity binding to HLA-DR1, a property of native SEA, was not altered in the SEA-SEE hybrid enterotoxins containing amino acid substitutions in regions 20 to 24 and 200 to 207, indicating that residues comprising the V beta determinants of SEE are separate from residues that contribute to HLA-DR1 binding affinity. Computer models of the predicted structure of SEE revealed that the V beta determinants of SEE are located on two adjacent solvent-exposed loops. Thus, the residues of SEE that mediate V beta recognition may coalesce to form a TCR binding site with specificities for multiple TCR beta-chains.     

Caring for patients with HIV disease: the experience of a generic hospice

Following the recent realization that TCR beta transgenes can severely inhibit the rearrangement of endogenous Vbeta gene segments in the absence of pre-TCR alpha (pT alpha) chains, we tested whether the pre-TCR has an essential role in TCR beta allelic exclusion under more physiological conditions by analyzing TCR rearrangement in immature thymocytes by single-cell PCR. Our results in pT alpha+ mice are consistent with an ordered model of TCR beta rearrangement beginning on one allele and continuing on the other only when the first attempt is unsuccessful. By contrast, a higher proportion of thymocytes from pT alpha-/- mice exhibited two productive TCR beta alleles. Thus, the pre-TCR-independent suppression of rearrangement by TCR beta transgenes represents a transgene artifact, whereas under physiological conditions the pre-TCR is essential for allelic exclusion.     

The Gln-Arg191 polymorphism of the human paraoxonase gene (HUMPONA) is not associated with the risk of coronary artery disease in Finns

The human paraoxonase gene (HUMPONA) is codominantly expressed as alleles A and G. The A allele codes for glutamine (A genotype) and the G allele for arginine (B genotype) at position 191 of the paraoxonase enzyme. This genetic polymorphism has been suggested to be associated with the predisposition to coronary artery disease (CAD). We investigated the frequency of paraoxonase A and G alleles in 380 well-characterized CAD patients and in 169 controls. The most common genotype in both the patients with CAD (211/380) and in healthy Finnish individuals (87/169) was AA (Gln/Gln). The heterozygous AM (Gln/Arg) genotype was present in 140 of the patients and in 75 controls. The frequency of the A allele was 0.74 in both patients and controls. The genotype distribution between the two groups did not differ (P = 0.12, chi2 test). The genotype distributions were also similar to those reported earlier in other caucasoid populations. In conclusion, we found no association between the Gln-Arg 191 polymorphism of the human paraoxonase gene and coronary artery disease in Finns.     

Polymorphism of the thiopurine S-methyltransferase gene in African-Americans

The molecular basis for the genetic polymorphism of thiopurine S -methyltransferase (TPMT) has been estab-lished for Caucasians, but it remains to be elucidated in African populations. In the current study, we determined TPMT genotypes in a population of 248 African-Americans and compared it with allele frequencies in 282 Caucasian Americans. TPMT genotype was determined in all individuals with TPMT activity indicative of a heterozygous genotype (10.2 U/ml pRBC, n = 23 African-Americans, n = 21 Caucasians). No mutant alleles were found in the high activity control groups. The overall mutant allele frequencies were similar in African-Americans and Caucasians (4.6 and 3.7% of alleles, respectively). However, while TPMT*3C was the most prevalent mutant allele in African-Americans (52.2% of mutant alleles), it represented only 4.8% of mutant alleles in Caucasians ( P < 0.001). In contrast, TPMT*3A and TPMT*2 were less common in African-Americans (17.4 and 8.7% of mutant alleles), whereas TPMT*3A was the most prevalent mutant allele in Caucasians (85.7% of mutant alleles). A novel allele ( TPMT*8 ), containing a single nucleotide transition (G644A), leading to an amino acid change at codon 215 (Arg-->His), was found in one African-American with intermediate activity. These data indicate that the same TPMT mutant alleles are found in American black and white populations, but that the predominant mutant alleles differ in these two ethnic groups.     

Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins

The TCR confers immunity by the specific recognition of foreign Ag peptides in the context of self-MHC molecules. The mechanisms controlling TCR selection and repertoire generation are not clearly understood and seem to occur in an apparently random, (self) Ag-driven manner. To address the question to what extent the TCR repertoire is randomly shaped or genetically predetermined, we have analyzed the alpha beta TCR repertoire of the CD4+ and CD8+ subsets of peripheral blood lymphocyte cultures of monozygotic twins by using the polymerase chain reaction technique with TCR V region gene family-specific oligonucleotide primers. Our studies demonstrate that there is high concordance in the overall patterns of V gene usage within a pair of twins, particularly in V beta usage (mean V beta CD4+ R2 = 0.869 and CD8+ R2 = 0.833) and to a lesser extent V alpha usage (mean V alpha CD4+ R2 = 0.621 and CD8+ R2 = 0.627); whereas the patterns between unrelated individuals show more variability. This study has also demonstrated that the V alpha and V beta genes are not randomly used within the CD4+ and CD8+ subsets. We observed significant preferential skewing of several V alpha or V beta gene families to either the CD4+ or CD8+ subset in the majority of individuals analyzed (p-value range = 0.0476 to < 0.001). In particular, V alpha 11, 17, 22, and V beta 3, 9, 12, 18 were skewed to the CD4+ subset; whereas V alpha 2, 6, 12, 15, 20 and V beta 7, 14, 17 were skewed to the CD8+ subset. Furthermore, a number of the V genes showed patterns of skewing consistent only within a pair of twins. In three pairs of twins, V beta 2 was skewed to the CD4+ subset, whereas the fourth pair used almost equal frequencies of V beta 2 in both subsets. This observation was made for the V beta 2, 4, 5, 6, 8, 19 and V alpha 7, 16, 18, 21 families. Finally, the ratio of the relative V gene usage frequency that could be observed within an individual was conserved within the sets of twins; for instance, the relative amount of V beta 2 to that of V beta 3 was higher in both individuals of one set of twins, whereas it was lower in all of the other three sets. Together these observations suggest that the predominant influence shaping the TCR repertoire is genetically predetermined, of which, HLA-predicted selection mechanisms exerted during thymic maturation might be contributing factors.     

Identification of an endogenous mammary tumor virus involved in the clonal deletion of V beta 2 T cells

Expression of V beta (beta-chain variable region) gene segments was investigated in the Mus m. domesticus DDO strain, which possesses a large genomic deletion encompassing 20 of the 29 V beta gene segments known in BALB/c. Stainings using V beta-specific monoclonal antibodies revealed that up to 60% of the peripheral T cells use 3 V beta gene segments. Variable frequencies of V beta 2 T cells were observed among DDO individuals. Segregation analyses of F2 crosses between V beta 2-deletor mice and mammary tumor virus (Mtv)-free mice led to the identification of a new endogenous Mtv, named Mtv-DDO, mediating V beta 2 T cell clonal deletion. Mtv-DDO structure is conserved with the exception of the carboxy-terminal region as compared to other Mtv. Comparison between Mtv sharing the same V beta specificity and isolated from laboratory or wild mice confirms that a stretch of 11 amino acids, defined as the V beta-specific region, is required for the V beta-specific interaction. Limited substitutions in this region account for the shift of the Mtv specificity towards different V beta.     

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency: structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.     

Reactions of anti-immunoglobulin sera with synthetic T cell receptor peptides: implications for the three-dimensional structure and function of the TCR beta chain

The derived amino acid sequence of the human TCR beta chain shows considerable homology to lg lambda light chains in its variable (V) and constant (C) domains, and in its joining segment (J). We assessed the cross-reactivity between TCR beta chains and lg light chains by synthesizing a set of nested, overlapping 16-mer peptides that duplicated the sequence that corresponds to the continuous VDJC sequence of TCR beta chain and determining the capacity of rabbit antisera to human or murine lgs to react with these peptides. The reactivities we observed were consistent with homologies to lambda and kappa light chains. The strongest reactivity in ELISA binding and competitive inhibition was with a peptide that corresponds to the 'switch peptide' of light chains. The sequence is encoded by the C-terminal region of the J segment (Fr4) and the N-terminus of the C region. Other regions reactive with anti-light chain sera corresponded respectively to CDR1 and Fr3 segments of the V region, and a segment of the constant region predicted to loop out of the tight globular structure. The peptide immunochemical results, coupled with the identification of specific regions of sequence correspondence between TCR beta and the characterized lambda light chain Mcg, allowed us to develop a three-dimensional model of the beta chain consistent with its role in antigen recognition and response to superantigens.     

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.     

Molecular genetics of metachromatic leukodystrophy

Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disease. It can be caused by mutations in two different genes, the arylsulfatase A and the prosaposin gene. These genes encode two proteins that are needed for the proper degradation of cerebroside sulfate, a glycolipid mainly found in the myelin membranes. Deficiency of arylsulfatase A or of a proteolytic product of prosaposin leads to the accumulation of cerebroside sulfate, which causes a lethal progressive demyelination. Mutations in the arylsulfatase A gene are far more frequent than those of the prosaposin gene. So far 31 amino acid substitutions, one nonsense mutation, three small deletions, three splice donor site mutations, and one combined missense/splice donor site mutation have been identified in the arylsulfatase A gene. Two of these mutant alleles are frequent, accounting for about one-half of all mutant alleles, whereas the remainder are heterogeneous. Amino acid substitutions cluster in exons 2 and 3, a region that shows a high degree of conservation among sulfatases of different function and origin. Different mutations are associated with phenotypes of different severity, but there is a remarkable variability of severity when patients with identical genotypes are compared. Demonstration of an arylsulfatase A deficiency is not a proof of metachromatic leukodystrophy, since a substantial deficiency without any clinical consequences is frequent in the general population. This deficiency is caused by an arylsulfatase A allele, which due to certain mutations encodes greatly reduced amounts of functional enzyme. However, these amounts are sufficient to sustain a normal phenotype. In the diagnosis and genetic counseling, these deficiencies must be differentiated from those causing metachromatic leukodystrophy. So far only six patients with mutations in the prosaposin gene have been described, in which three defective alleles two with amino acid substitutions and one with a 33-bp insertion have been identified.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Polymorphism detection and sequence analysis of human T-cell receptor V alpha-chain-encoding gene segments

The T-cell receptor (Tcr) provides specificity for antigen recognition by its variable domain, primarily consisting of two germline encoded variable (V) region gene segments. Thus it has been suggested that inherited polymorphisms in the TCRV gene segments could contribute to differential immune responsiveness (e.g., autoimmunity) in human populations. In the present study, we have sought potentially functional polymorphisms in the germline TCRAV gene segments. Using denaturing gradient gel electrophoresis on polymerase chain reaction (PCR)-amplified products from the pooled DNA of many individuals, we identified polymorphisms in the TCRAV2S1, AV4S1, AV7S1, and AV8S1 gene segments. A complete DNA sequence analysis of these PCR products identified polymorphisms that affected amino acids in the predicted antigen-binding regions of the Tcr alpha chain, as well as polymorphisms in the introns. Genotype analysis of all nine DNA point mutations showed a 5%-50% range (averaging 35%) of minor allele frequencies, often resulting in individuals homozygous for the alternate allele forms. All possible haplotype combinations of the amino acid-affecting polymorphisms were found, indicating that in human populations there are a large number of different germline haplotypes encoding V gene segment alleles. These TCRAV coding region polymorphisms provide the rationale for, and allow the direct testing of, hypotheses concerning inherited polymorphisms within the T-cell receptor genes that may contribute to autoimmune susceptibility.     

Specific V beta T cell subsets are associated with cat and birch pollen allergy in humans

Cognate interaction between TCRs and MHC class II molecules plays an important role in initiating the allergen-specific immune response. Therefore, we analyzed the TCR distribution of human PBLs of 56 atopic and nonatopic (NA) individuals, including 4 monozygotic twin pairs, from two extended and four nuclear families. The expression of 23 V beta and 3 V alpha elements was analyzed. The blood samples of symptomatic birch pollen-sensitized individuals that were taken < or = 6 wk after the birch pollen season (n = 8) showed a significantly higher frequency of V beta 16.1+ and V beta 20.1+ T cells compared with the blood samples of birch pollen-sensitized individuals that were obtained out of allergen season (n = 10) or from NA individuals (p < 0.0005 and p < 0.0001, respectively). Allergen-specific lymphocyte proliferation was detected in the allergic individuals, and the distribution of V beta 16.1+ and V beta 20.1+ T cells returned to normal levels after the pollen season. The frequency of these V beta-expressing T cells correlated with the levels of allergen-specific IgE Abs. In addition, cat-sensitized individuals (n = 8) showed a significantly higher frequency of V beta 17.1-expressing T cells than did NA individuals (p < 0.005). Our results indicate restricted TCR-V beta gene usage in cat and birch pollen allergies; we suggest that both genetic and environmental factors contribute to TCR-V beta gene expression and to the development of a specific T cell response.     

Conserved sequence motifs of CDR3 loops of TCR specific for two major epitopes of the grass pollen allergen PhI p 1

We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped alphabeta TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3beta and most of the CDR3alpha of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR.