Γ-chain Gene Research Articles

Mammalian gene targeting with designed zinc finger nucleases

Gene targeting by homologous recombination is a powerful method to manipulate the genome precisely and could be exploited to correct genetic defects. Zinc finger nucleases are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease. Zinc finger nucleases were generated that stimulated gene targeting from half-site sequences from the human β-globin gene and the human common γ-chain gene. Zinc finger nucleases were also generated that stimulated gene targeting at full sites from the green fluorescent protein gene and the human CD8α gene. This work built on the prior zinc finger design work of others and in targeting these four genes had a 100% success rate at designing nucleases to the consensus half-site 5′-GNNGNNGNN-3′ and the consensus full site 5′-NNCNNCNNCNNNNNNGNNGNNGNN-3′, suggesting that zinc finger nucleases can be empirically designed to stimulate gene targeting in a large portion of the mammalian genome.

Durable Clinical, Cytogenetic, and Molecular Remissions After Allogeneic Hematopoietic Cell Transplantation for Refractory Sezary Syndrome and Mycosis Fungoides

Sezary syndrome (SS) and tumor-stage mycosis fungoides (MF) are generally incurable with currently available treatments. We conducted a retrospective study to evaluate the outcome of allogeneic hematopoietic stem-cell transplantation (HSCT) in this patient population. From August 1996 through October 2002, eight patients with advanced MF/SS underwent allogeneic HSCT at our institution. All patients were heavily pretreated, having failed a median number of seven prior therapies (range, five to 12). Clonal T-cell populations in peripheral blood or bone marrow were detectable by polymerase chain reaction analyses of T-cell receptor gamma-chain gene rearrangements in six patients and cytogenetics in three patients. The conditioning regimen included total-body irradiation and cyclophosphamide (n = 3), busulfan and cyclophosphamide (n = 1), and the reduced-intensity regimen of fludarabine and melphalan (n = 4). Allogeneic hematopoietic stem cells were obtained from HLA-matched siblings (n = 4) and unrelated donors (n = 4). All patients achieved complete clinical remission and resolution of molecular and cytogenetic markers of disease within 30 to 60 days after HSCT. Two patients died from transplantation-related complications; graft-versus-host disease (GVHD; n = 1) and respiratory syncytial virus pneumonia (n = 1). With a median follow-up of 56 months, six patients remain alive and without evidence of lymphoma. Our results suggest that allogeneic HSCT from both HLA-matched sibling and unrelated donors can induce durable clinical, molecular, and cytogenetic remissions in patients with advanced cutaneous T-cell lymphoma that is refractory to standard therapies.

Structure of the Angiopoietin-2 Receptor Binding Domain and Identification of Surfaces Involved in Tie2 Recognition

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.

Diagnostic validation of capillary electrophoresis analysis of T-cell receptor gamma-chain gene rearrangements: prediction of malignant transformation of cutaneous T-cell lymphoproliferative disorders.

T-Cell lymphoproliferative disorders of the skin constitute in themselves a unique area of diagnostic difficulty for the clinician and the pathologist. Hence, although T-cell clonality analysis by PCR and capillary electrophoresis (CE) has been used extensively in lymphoproliferative disorders in general, there are very few reports describing their application to the differential diagnosis of cutaneous T-cell lymphoma (1)(2) or to cases of cutaneous lymphoproliferative disorders with less conclusive histology, where their superior resolution properties have provided strong confirmatory evidence of evolving lymphoma development (3). We present here our validation data of the use of CE in the analysis of T-cell receptor γ-chain (TCRγ) gene rearrangements for clinical use. We also present a report of a case that was judged to be benign based on clinical and pathologic grounds but was found to have the clonal molecular fingerprint that could have predicted malignant transformation at an early stage. This predictive value, in our opinion, enhances the diagnostic value of this test in the clinical setting. Pathologists are aware that distinction between benign and malignant T-cell lymphoproliferative disease by use of morphologic criteria can often be difficult, even with the aid of immunotyping. The best marker of T-cell malignancy is T-lymphocyte monoclonality, which is rarely encountered in nonneoplastic disease. Rearrangements of TCR genes in a rapidly proliferating neoplastic lymphoid cell population derived from a single progenitor cell are identical, involving unique combinations of specific variable (V), joining (J), diversity (D), and constant (C) gene segments, and thus serve as unique clonal markers (3)(4). In contrast, nonneoplastic and reactively proliferating T-cells yield different, polyclonal TCR gene rearrangements. TCR gene rearrangements can be …

Immune consequences of mutations in the human common γ-chain gene

A mutation (c.878T > A) in the common gamma chain (γ c) causes an X-linked combined immunodeficiency (XCID) in a large kindred of British origin. In the disease, γ c is expressed, but its binding to Jak3 is reduced. The immune deficiencies and clinical course were less marked in toddlers and school age children with XCID L293Q than in severe combined immunodeficiency (SCID). However, affected newborns were profoundly deficient in thymic size and T cells. In some affected infants, thymic size and numbers of T cells gradually increased during the first year. Their clinical course was relatively benign. In affected infants of one lineage, the number of blood T cells failed to increase substantially. They succumbed to opportunistic infections. T cell deficiencies in XCID L293Q progressively worsened during adolescence. Decreased thymic function, failure to rescue T cells from apoptosis, and replication senescence were possible causes. Blood T cells with the phenotype CD45RA +CD62L + (unstimulated T cells) were most depressed. CD4 + T cells were also deficient in a specific marker of recent thymic emigrants, episomal DNA deletion circles created during TcR gene rearrangements. Apoptosis of T cells was increased, but neither apoptosis nor cell death was age-related. In contrast, telomere shortening in T cells increased with age. Unlike murine γ c gene deletions, γδ T cells were prominent in affected adolescents and young adults. Furthermore, T cells with a Vδ2/Vγ9 specificity declined with age and were replaced in the oldest male with a Vδ1 specificity. Thus, the mutation provides many insights concerning the role of γ c in the biology of T cells.

T Cell Receptor γ-Chain Gene Polymerase Chain Reaction to Diagnose Central Nervous System Involvement by Cutaneous T Cell Lymphoma

The authors describe a patient who was suspected of having cutaneous T cell lymphoma involvement of the brain despite repeatedly negative cerebrospinal fluid (CSF) cytology, inconclusive flow cytometry, and no discrete lesion for brain biopsy. The diagnosis was made by polymerase chain reaction (PCR) analysis that showed a monoclonal T cell receptor gamma-chain gene rearrangement in the CSF, identically sized to that present in a skin biopsy specimen. Thus, PCR could be used early and routinely to diagnose central nervous system spread of T cell lymphomas, because of its potentially superior sensitivity and specificity to CSF cytology.

Establishment of a Monosomy 7 Leukemia Cell Line, MONO-7, With aras Gene Mutation.

A monosomy 7 leukemia cell line, designated MONO-7, was established from the peripheral blood of a patient with monosomy 7 acute myelocytic leukemia (French-American-British classification M0). The cells were cultured continuously for more than 24 months in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum. The cell line exhibits an unclassified appearance. Cytochemically, α-naphthol-acetate esterase and myeloperoxidase are negative. Immunophenotypi-cally, the cell line expresses CD33, CD13, CD56, CD34, CD38, HLA-DR, and CD45, but lacks T and B cell-associated antigens. Karyotypic analysis of the cell line showed only 45,XY,-7. Analysis of the N-ras gene mutation demonstrated identical mutations in fresh leukemic cells and the MONO-7 cell line. Clonal rearrangements of the immunoglobulin heavy-chain gene, T-cell receptor β-chain gene, or T-cell receptor γ-chain gene were not found in DNA extracted from MONO-7 cells. The growth of MONO-7 cells in vitro was stimulated by recombinant human granulocyte-macrophage colony-stimulating factor or interleukin 3. To our knowledge, this is the first report of the establishment of a cell line with the karyotype 45,XY,-7 with-out any other abnormality and with a ras gene mutation.

Interleukin 6 and Haemostasis

Interleukin 6 and Haemostasis

Identification of Four Novel Polymorphisms in the Aα and γ Fibrinogen Genes and Analysis of Association with Plasma Levels of the Protein

Four novel polymorphisms were identified in the fibrinogen gene cluster. Three of them were localized in the promoter regions of the Aα-chain (α −128 C/G, α −58 G/A) or the γ-chain (γ −239 A/G) gene, while the remaining one was identified in intron 9 of the γ-chain gene (γ 7792 C/T). Genotype distributions for these polymorphisms were analyzed in 200 healthy Italian individuals and were in Hardy–Weinberg equilibrium. Since high levels of plasma fibrinogen have been associated with an increased risk of cardiovascular disease and genetic variations have been evaluated as thrombotic risk predictors, we analyzed their role in determining the plasma levels of this protein. Owing to the low frequency of the rare allele of α −128 C/G and γ −239 A/G polymorphisms, association with plasma fibrinogen levels was investigated for only α −58 G/A and γ 7792 C/T. We also investigated in the same population two previously identified polymorphisms in the fibrinogen gene cluster (α TaqI and β −455 G/A) chosen for their widely studied association with plasma fibrinogen levels. In the multivariate linear regression analysis, no statistically significant association with plasma fibrinogen levels was found.

Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR gamma-chain gene sequencing.

Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30+ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30+ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR gamma-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells.

Assignment of T cell receptor (TCR) alpha-chain gene (A), beta-chain gene (B), gamma-chain gene (G), and delta-chain gene (D) loci on swine chromosomes by in situ hybridization and radiation hybrid mapping

Using the cDNA sequence of porcine T-cell receptor (TCR) α-, β-, γ-, and δ-chain genes, we screened a porcine BAC library to isolate clones containing these genes. The isolated BAC clones were confirmed to carry these TCR genes by partial nucleotide sequencing. Among the clones obtained in the present screening, two clones carried both the TCR α-chain gene (TRA) and the TCR δ-chain gene (TRD) while one clone each carried only the sequence of either TRA or TRD. This observation demonstrated that TRA and TRD are localized in close proximity on a swine chromosome. Also two clones contained the sequence of the TCR β-chain gene (TRB), and two clones contained the sequence of TCR γ-chain gene (TRG). Fluorescence in situ hybridization using the above BAC clones as probes revealed that TRA and TRD, TRB, and TRG loci reside on swine chromosomes 7q15.3→q21, 18q11.3→q12, and 9q21→q22, respectively. The chromosome positions of TRA and TRB are consistent with those determined by somatic cell hybrid analysis (Rettenberger et al., 1996). In addition, RH mapping of these genes was performed using the INRA-University of Minnesota RH panel DNA. The result confirmed the position of TRA and TRB reported earlier (http://imprh.toulouse.inra.fr/), and further demonstrated that TRG was located 11 cR away from genetic marker SW989 toward the marker S0019.

Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aα-chain gene, only 2 missense mutations in the C-terminal domain of the Bβ-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G→A transition at position +5 of intron 1 of the γ-chain gene. The predicted mutant fibrinogen γ-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen γ-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen γ-chain gene causing afibrinogenemia and indicates that, in addition to the Aα and Bβ-chain genes, the γ-chain gene must also be considered in mutation screening for afibrinogenemia.

Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation

AbstractCongenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aα-chain gene, only 2 missense mutations in the C-terminal domain of the Bβ-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G→A transition at position +5 of intron 1 of the γ-chain gene. The predicted mutant fibrinogen γ-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen γ-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen γ-chain gene causing afibrinogenemia and indicates that, in addition to the Aα and Bβ-chain genes, the γ-chain gene must also be considered in mutation screening for afibrinogenemia.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

AbstractCongenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

Large plaque parapsoriasis: clinical and genotypic correlations.

Twelve patients with large plaque parapsoriasis (LPP) were investigated for the presence of predominant T-cell clones, analyzing the T-cell receptor (TCR) gamma-chain gene. The diagnostic and prognostic significance of TCR gene rearrangement status was assessed by a correlation with the long-term clinical follow-up. Six out of 12 patients showed a clonal T-cell population. Clinically, among the patients with clonal disease one developed clearcut mycosis fungoides (MF) after a follow-up of 8 years, in the other 5 patients no such diagnosis could be made after follow-up of 2-21 years (median: 9 years). In patients with polyclonal infiltrates the lesions remained virtually unchanged. These findings indicate that in LPP TCR gene rearrangement status has no prognostic significance and does not allow distinction of LPP and early MF. Both conditions show a clonal T-cell infiltrate with similar frequency, are very similar in clinical and histologic presentation and according to recent studies share the same low risk to develop overt MF. Therefore both terms refer to the identical clinical situation. This should be designated as early MF and efforts should concentrate on identifying those patients that are at risk to develop aggressive disease.

A Case of Congenital Afibrinogenemia: Fibrinogen Hakata, a Novel Nonsense Mutation of the Fibrinogen γ-Chain Gene

Congenital afibrinogenemia due to a novel homozygous nonsense mutation of the fibrinogen gamma-chain gene, fibrinogen Hakata, was found in an 18-year-old Japanese girl who had received supplemental fibrinogen therapy since she was 4 months old. The plasma fibrinogen concentrations of the proband were measured as less than 10 mg/dl by a functional method and less than 17 mg/dl by an immunological method. Fibrinogen concentrations of her family were in the range of 94-164 mg/dl. The proband and her family had no other clinical symptoms. Genomic DNA of the proband and her family was isolated from leukocytes, and all exons of fibrinogen subunits and their intron/exon boundaries were analyzed. A genetic mutation, a guanine-to-thymine (G-to-T) transversion at the nucleotide position of 5860, was identified on exon 7 of the gamma-chain gene. This mutation changed the codon for the 231st residue of the gamma-chain from GAG (Glu) to TAG (stop). No other mutation was observed. Aalpha, Bbeta and gamma chains were observed in plasma of the heterozygous family members. However, only a trace amount of Aalpha chain and no gamma chain was detected in the plasma of the proband.

Hypofibrinogenemia Associated With a Heterozygous Missense Mutation γ153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates Defective Secretion of the Variant Fibrinogen

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of γ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.

Hypofibrinogenemia Associated With a Heterozygous Missense Mutation γ153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates Defective Secretion of the Variant Fibrinogen

AbstractWe genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of αγ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.

Severe combined immunodeficiency in an infant with multiple congenital abnormalities

Immunologic evaluations revealed low total lymphocytes, low T cells, and elevated B cells (Table I). IgG (101 mg/dL; normal range, 172 to 1069 mg/dL), IgA (undetectable), and IgM (63 mg/dL; normal range, 17 to 149 mg/dL); ADA, purine nucleoside phosphorylase, and thymulin levels were normal. The level of antibodies to diphtheria was 0.041 IU/mL (normal level, >0.01 IU/mL), and the level of antibodies to tetanus was 0.1 IU/mL (normal level, >0.1 IU/mL) after 3 doses of vaccine. Jak3 protein was analyzed by using polyclonal antibodies (C-20, N-15, and H-20; Santa Cruz Biotechnology, Santa Cruz, Calif) and SupraSignal ULTRA (Pierce, Rockford, Ill). EBV-transformed B cells were measured by immunofluorescence with anti-γ-chain mAb (TUGh4; Pharmingen, San Diego, Calif), and sequencing analysis of coding and splice regions of IL2R γ-chain gene in both patient and mother were normal. Cytogenetic analysis of peripheral blood failed because of nonproliferation after mitogen treatment. On the EBV-transformed cell line, a highresolution Giemsa-banded karyotype and fluorescence in situ hybridization were performed. The karyotype was normal male, 46XY, with no deletion of either allele at the chromosome 22q11.2 DiGeorge locus (TUPLE1 probe from Vysis, Downer’s Grove, Ill)3 and also no deletion at the chromosome 11p13-p14 region associated with a minority of cases of DiGeorge and velocardiofacial syndrome.4 The latter region was probed with YAC10p1 and YAC10p47 (kindly provided by Peter Scambler, Institute of Child Health, London, UK).4