Identification of deletion and triple α-globin gene haplotypes in the montreal β-thalassemia screening program: Implications for genetic medicine

We obtained blood samples in a screening program designed to detect β-thalassemia heterozygotes in Montreal; additional samples were obtained from referred persons. We analyzed DNA for variant numbers of α-globin genes, notably the α-thalassemia₂ (- α/), α-thalassemia₁, (– –/), and triplicated ζ-globin gene (ααα/) haplotypes using restriction enzymes and probes for α-globin and α-globin gene sequences. We estimated the numbers of Montreal residents of Italian and Greek ethnic origin with –α/αα genotype. Thus, 4.3% of Italians and 1.5% of Greeks, or about 7,500 persons, are estimated to be α-thalassemia₂, trait (silent carriers), largely (80%) in the –α^3.7/type I form. The triplicated α-globin gene haplotype was also found. The risk of a severe (α-thalassemia₁) phenotype associated with inheritance of – –;/αα or –α/ –α genotypes was low and was found predominantly in this study, in persons of Asian ethnic origin. The sample of Asians was too small to estimate carrier frequencies; however, based on results from the β-thalassemia screening program, we estimated that about 4% of Asians (about 1,300 persons) in Montreal are α-thalassemia carriers. We identified persons heterozygous for both β-thalassemia and α-thalassemia mutations. In these double heterozygotes, the effect of the triplicated α-globin gene was to make the erythrocyte parameters used for screening (MCV and %HbA₂) more deviant from normal whereas deletion of 2 α-globin genes tended to normalize the erythrocyte values. These findings have implications for the screening program and reproductive counseling.     

α-thalassaemia due to a single codon deletion in the α-1-globin gene. Computational structural analysis of the new α-chain variant

A new unstable α-globin chain associated with α-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the α-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the α-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 or DK61 results in a structurally abnormal α-globin chain, not previously described, named Hb Clinic, which leads to the α-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this α-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer. Hum Mutat 11:412, 1998. © 1998 Wiley-Liss, Inc.     

Identification of two novel β°-thalassemia mutations in a filipino family: Frameshift codon 67 (− TG) and a β-globin gene deletion

The frequency of β-thalassemia alleles among Filipinos is estimated to be 0.02, although little is known about the actual mutations represented in this population. Here, we describe a Filipino family in which a child has severe β-thalassemia major. Molecular studies of the family revealed that the proband is a compound heterozygote for two previously unreported β°-thalassemia alleles: a frameshift mutation ( ? TG) at codon 67 and a deletion of the entire β-globin gene. The 5′ endpoint of this novel deletion is located ～3.9 kb to ～4.3 kb upstream of β-globin gene, and the deletion extends 3′ beyond the β-globin gene for an undetermined distance. The occurrence of two novel β-thalassemia alleles in a single family suggests that the Filipino population may have a unique spectrum of β-thalassemia alleles. © 1993 Wiley-Liss, Inc.     

Scanning method of identify the molecular heterogeneity of δ-globin gene especially in δ-thalassemias: Detection of three novel substitutions in the promoter region of the gene

A scanning strategy for the detection of δ-globin gene mutations and polymorphisms is presented. This procedure is based on the denaturing gradient gel electrophoresis (DGGE) of four different artificially amplified DNA fragments which cover the promoter, the exons, as well as IVS I of the reported gene. To estimate the efficiency and sensitivity of the proposed procedure, we analysed the appropriate controls of δ-thalassemic carriers, uncharacterised δ-thalassemias and cases with normal hematological phenotype, but slightly increased (up to 3.5%) HbA₂. DGGE results permitted the identification of δ-globin gene mutations and the polymorphism - 199 (T→C). Three novel base substitutions inside the promoter region of the gene [−65(A→G), −55(T→C), −36(C→A)], were also revealed. These changes are either linked in cis with other mutations or are responsible for thalassemias or for positive regulatory effect in δ-globin gene expression. The proposed experimental strategy consists of an accurate, rapid, safe and inexpensive screening procedure for establishing the molecular basis of δ-globin gene defects, suitable for the application for both research and diagnostics. Hum Mutat 9:465–472, 1997 © 1997 Wiley-Liss, Inc.     

Deletion mapping of the β-glucuronidase gene

GUSB, the gene for β-glucuronidase, has been localized to the proximal long arm of chromosome 7 between 7q11.2 and 7q22. Deficiency of β-glucuronidase results in mucopolysaccharidosis type VII (MPS VII, Sly syndrome). The enzymatic defect has been demonstrated in cultured skin fibroblasts, leukocytes and serum of affected patients. An 8-yr-old boy presented with manifestations similar to MPS VII (mental retardation, short stature, “coarse” facial appearance, mild skeletal involvement and recurrent lower respiratory tract infection) but other, discrepant abnormalities, e. g., bilateral iris colobomata and cleft palate. Normal activity of β-glucuronidase was found in the patient's leukocytes. Chromosome analysis disclosed an interstitial deletion of 7q with one breakpoint at the interface between bands 11.22 and 11.23 and the other breakpoint within band 21.1. DNA from this patient's leukocytes was analyzed for dosage of GUSB sequences. This locus appeared to be present at the normal diploid level. These findings suggest that GUSB is not in the portion of chromosome 7 deleted in our case, narrowing the smallest region of overlap to 7q21.1 → 7q22. We therefore assign the β-glucuronidase gene to 7q21.1 → 7q22.     

Novel single nucleotide polymorphisms in the 3′‐UTR of the TGFβRI and TGFβRIII genes

Transforming growth factor beta (TGFβ) family members are multifunctional cytokines that play a key role in cellular growth, proliferation and differentiation. Transmembrane signalling by TGFβ occurs via a complex of the serine/threonine kinases TGFβ type 1 (TGFβRI), type 2 (TGFβRII), and type 3 (TGFβRIII) receptors. Previous studies have implicated TGFβ receptors (TGFβR) in a variety of important hereditary clinical disorders. Mutations of the TGFβR genes have been observed in several human cancers. The aim of this study was to identify and confirm novel single nucleotide polymorphisms (SNPs) in TGFβRI and RIII and to determine the relative allele and genotype frequencies of these SNPs. SNPs were identified from the examination of sequence alignments held in databases and were confirmed by DNA sequencing. A polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) method was devised for genotyping TGFβ receptor polymorphisms. DNA samples from 91 controls were examined. The observed heterozygosities of TGFβRI and TGFβRIII gene polymorphisms in the control population were 43 and 33%, respectively, suggesting these SNPs could be useful markers in disease association studies.     

Role of the thymus in the generation of skin-homing αβ and γδ virgin T cells

Current models of lymphocyte traffic suggest that homing specificities of T cells to tissues such as skin are generated outside the thymus as a result of activation of naive T cells by antigen in lymph nodes. Virgin T cells are thought to home to high endothelial venules in lymph nodes, but are thought to be unable to home to extra-lymphoid tissues such as skin. We used the technique of in situ labeling of the thymus with fluorescein isothiocyanate to examine the homing specificities of authentically naive T cells in vivo, immediately after their export from the thymus. We report that homing specificities for skin as well as lymph node are imprinted on T cells inside the thymus, independent of antigen. We also show that both αβ and γδ emigrant T cells exhibit homing patterns to skin and lymph nodes which are identical to those of mature T cells. Our findings demonstrate a key role for the thymus in the induction of skin-homing specificities on T cells indicating that skin-homing specificities of T cells are not generated solely outside the thymus as a result of the activation of virgin T cells by antigen. The migration of thymic emigrants to extra-lymphoid tissues within a few hours of leaving the thymus may have implications for mechanisms of peripheral self-tolerance. This pathway provides an opportunity for direct virgin T cell interactions with self components only expressed in the periphery at a time when emigrants may be more susceptible to tolerance induction than mature circulating T cells.     

A Novel γ2-Herpesvirus of the Rhadinovirus 2 Lineage in Chimpanzees

Old World monkeys and, recently, African great apes have been shown, by serology and polymerase chain reaction (PCR), to harbor different gamma2-herpesviruses closely related to Kaposi's sarcoma-associated Herpesvirus (KSHV). Although the presence of two distinct lineages of KSHV-like rhadinoviruses, RV1 and RV2, has been revealed in Old World primates (including African green monkeys, macaques, and, recently, mandrills), viruses belonging to the RV2 genogroup have not yet been identified from great apes. Indeed, the three yet known gamma2-herpesviruses in chimpanzees (PanRHV1a/PtRV1, PanRHV1b) and gorillas (GorRHV1) belong to the RV1 group. To investigate the putative existence of a new RV2 Rhadinovirus in chimpanzees and gorillas we have used the degenerate consensus primer PCR strategy for the Herpesviral DNA polymerase gene on 40 wild-caught animals. This study led to the discovery, in common chimpanzees, of a novel gamma2-herpesvirus belonging to the RV2 genogroup, termed Pan Rhadino-herpesvirus 2 (PanRHV2). Use of specific primers and internal oligonucleotide probes demonstrated the presence of this novel gamma2-herpesvirus in three wild-caught animals. Comparison of a 1092-bp fragment of the DNA polymerase obtained from these three animals of the Pan troglodytes troglodytes subspecies, one from Gabon and the two others from Cameroon, revealed <1% of nucleotide divergence. The geographic colocalization as well as the phylogenetic "relationship" of the human and simian gamma2-herpesviruses support the model according to which herpesviruses have diversified from a common ancestor in a manner mediating cospeciation of herpesviruses with their host species. By demonstrating the existence of two distinct Rhadinovirus lineages in common chimpanzees, our finding indicates the possible existence of a novel human gamma2-herpesvirus belonging to the RV2 genogroup.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Functional characterization and exon 2 – intron 2 – exon 3 gene sequence of HLA-B*2712 as found in a British family

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR–SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712 ; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.