Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity

Parvovirus B19 (B19) can cause chronic anemia due to persistent infection in immunocompromised hosts who cannot produce neutralizing antibody necessary for clearing B19. Three patients with X-linked hyper-IgM syndrome (XHIM), who were all asymptomatic until they developed B19-induced chronic anemia at the ages of 8, 14, and 17 years, respectively, were found to have mutations of the CD40L gene, including a missense mutation (T254M), a nonsense mutation resulting in a new initiation codon and loss of the intracellular domain (R11X), and a splice site mutation (nt 309+2t-->a). Antibody responses to the T cell-dependent antigen, bacteriophage phiX174, were impaired, but neutralizing antibody titers were higher than in XHIM patients with classic phenotype. All 3 patients responded to intravenous immune globulin (IVIG) treatment. Certain mutations of the CD40L gene result in a mild XHIM phenotype that may become apparent following B19 infection in patients not on IVIG therapy and therefore not protected from B19 infection.     

Characterization of nine novel mutations in the CD40 ligand gene in patients with X-linked hyper IgM syndrome of various ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome-specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates

We determined full-length nucleotide sequence of hepatitis B virus (HBV) genome in sera from 40 Japanese patients with HBsAg-positive hepatocellular carcinoma (HCC), in order to obtain information on HCC-specific characteristics, if any, of the HBV genome. Direct sequencing of the long distance PCR products starting from 50 microliters of serum samples revealed that 95% of our isolates were of genotype C, and that mutations and deletions/insertions were very common. With respect to envelope protein genes, deletions and missense mutations were frequent in preS2, and the determinant a domain of HBsAg was rich in "antibody-escape" mutations. Within the precore/core region, the most remarkable mutation was the replacement of proline of wild type by other amino acids at codon 130 of the core gene, which was found in 58% of our isolates, while precore-stop mutation was found in 45%. Most interestingly, however, about 90% of our isolates had mutations at nt positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter, which had been implicated in "e-suppressive" phenotype of HBV genome. G-to-A at nt 1613 and C-to-T at nt 1653 within enhancer II and T-to-C/A at nt 1753 within core promoter were also evident: 38%, 53%, and 40%, respectively. It was interesting that some of the characteristics observed in our isolates form HCC patients had been previously implicated in fulminant hepatitis and/or acute exacerbation of chronic hepatitis.     

Molecular and structural characterization of five novel mutations in the Bruton's tyrosine kinase gene from patients with X-linked agammaglobulinemia

BACKGROUND: The Btk (Bruton's tyrosine kinase) gene has been shown to be mutated in the human immunodeficiency disease, XLA (X-linked agammaglobulinemia). Btk is a member of the Tec family of cytosolic protein tyrosine kinases with distinct functional domains PH, TH, SH3, SH2, and kinase. Mutations have been observed in each of the Btk subdomains in XLA. We have analyzed the Btk gene in six XLA patients from five unrelated families. MATERIALS AND METHODS: DNA was prepared from the patients peripheral blood. The Btk exons including the junctional sequences were analyzed by single-strand conformation polymorphism (SSCP) followed by direct nucleotide sequencing after PCR-amplification. For structural analysis, the missense mutations were introduced into three-dimensional models of the PH and kinase domains of Btk and the outcome was predicted based on the knowledge of the protein function. RESULTS: Five novel mutations and two novel polymorphisms, all of which resulted from single-base alterations, were identified. Three of the five mutations were in the PH domain and two were in the kinase domain of Btk. Three of these mutations were of the missense type, two of which altered the same codon in the PH domain; the third one was located in the kinase domain. The fourth mutation was a point deletion in the PH domain causing a frameshift followed by premature termination. The fifth mutation was a splice donor-site mutation within the kinase domain which could result in an exon skipping. In four of the five instances, mothers of the patients were shown to be obligate carriers. In one instance, a sibling sister was identified as a heterozygote establishing her as a carrier. CONCLUSIONS: Functional consequences of the mutations causing frameshifts and altered splicing can be inferred directly. Functional consequences of the missense mutations were interpreted by 3-dimensional structural modeling of Btk domains. It is proposed that the two PH domain mutations will interfere with membrane localization while the kinase domain mutation will interfere with the enzymatic function of Btk. This study provides further insight into the role of Btk in XLA.     

IL-2 and IL-7 induce heterodimerization of STAT5 isoforms in human peripheral blood T lymphoblasts

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface of in vitro activated CD4+ T cells. Approximately 90% of in vitro activated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% of in vitro activated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of the in vitro activated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.     

Missense mutations in the PAX6 gene in aniridia

PURPOSE: Aniridia is caused by a mutation of the PAX6 gene. Haploinsufficiency of the gene product is thought to result in the aniridia phenotype, because most mutations thus far detected have been large deletions encompassing the entire gene and nonsense, frameshift, or splice errors that result in premature translational termination on one of the alleles. Only two missense mutations have been detected in aniridia pedigrees, each of which occurs in its paired domain or homeodomain. In this study, four novel missense mutations were found in three aniridia pedigrees. METHODS: Polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing of the PAX6 gene were performed using genomic DNA of three aniridia pedigrees and more than 100 healthy control subjects. RESULTS: Three mutations occurred in the N-terminal subdomain of the paired domain, namely N17S, I29V, and R44Q, the first two of which were detected on the same allele of one patient. The other mutation (Q178H) was in the linking portion of the paired domain and homeodomain. CONCLUSIONS: These missense mutations give rise to haploinsufficiency by another route, because the missense mutations presented here resulted in an aniridia phenotype indistinguishable from that caused by a heterozygous deletion of the entire PAX6 gene.     

Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy

A candidate gene, myotubularin, involved in the pathogenesis of X-linked myotubular myopathy (MTM1) was isolated recently. Mutations originally were identified in 12% of patients examined for 40% of the coding sequence, raising the possibility that additional genes could be responsible for a proportion of X-linked cases. We report here the identification of mutations in 26 of 41 independent male patients with muscle biopsy-proven MTM, by direct genomic sequencing of 92% of the known coding sequence of the myotubularin gene. Eighteen patients had point mutations, including one A/G transition found in four patients which alters a splice acceptor site in exon 12 and leads to a three amino acid insertion. Six patients had small deletions involving <6 bp, while two larger deletions encompassed two or six exons, respectively. No differences were noted among the types of mutations between familial and sporadic cases. However, all of the five patients with a mild phenotype had missense mutations. While 50% of the mutations were found in exons 4 and 12, and three distinct mutations were found in more than one patient, no single mutation accounted for more than 10% of the cases. The low frequency of large deletions and the varied mutations identified suggest that direct mutation screening for molecular diagnosis may require gene sequencing.     

Leukocyte transfusion-associated granulocyte responses in a patient with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a severe congenital immunodeficiency caused by mutations in CD154 (CD40 ligand, gp39), the T cell ligand for CD40 on B cells. Chronic or cyclic neutropenia is a frequent complicating feature that heightens susceptibility to severe infections. We describe a patient with a variant of XHIM who produced elevated levels of serum IgA as well as IgM and suffered from chronic severe neutropenia. Eight of ten leukocyte transfusions with cells from a maternal aunt, performed because of mucosal infections, resulted in similar episodes of endogenous granulocyte production. Transfection studies with the mutant CD154 protein indicate that the protein is expressed at the cell surface and forms an aberrant trimer that does not interact with CD40. The data suggest that allogeneic cells from the patient's aunt, probably activated T cells bearing functional CD154, may interact with CD40+ recipient cells to produce maturation of myeloid precursors in the bone marrow.     

Caudal appendage in a full-term infant

We report studies of two unrelated Japanese patients with 17alpha-hydroxylase deficiency caused by mutations of the 17alpha-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient's mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17alpha-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.     

Thin foil preparation of metal particles in brittle ceramic matrices

Predisposing germline mutations in the BRCA1 gene were identified recently in families with 17 q-linked breast and ovarian cancers. Using single-strand conformation polymorphism (SSCP) analysis, we examined primary breast cancers for mutations in coding exons of BRCA1 in a panel of 103 patients, of whom all either represented early-onset cases (< 35 of age), were members of multiply-affected families, and/or had developed bilateral breast cancers. Mutations were detected in tumors from four patients, all of whom had developed breast cancers bilaterally: a frame-shift due to a 2-bp deletion at codon 797; a nonsense mutation at codon 1214; and two missense mutations, one at codon 271 leading to Val-->Met substitution, and the other at codon 1150 leading to Pro-->Ser substitution. In each case the same mutation was present in constitutional DNA. The mean age of onset was 49 years among the Japanese carriers of BRCA1 mutations identified in this study, in contrast to the mean age of 35 observed among carriers of BRCA1 mutations in a similar U.S. study (Futreal et al., 1994). The evidence reported here supports a rather limited role of BRCA1 in breast carcinogenesis.     

Mutations in haemophilia A

In the present study DNA from 281 unrelated haemophilia A patients including 15 inhibitor patients has been analysed by Southern blotting technique. Using various restriction enzymes, cloned factor VIII cDNA probes and genomic fragments we have identified 14 mutations. Six of the mutations are novel partial factor VIII gene deletions. One deletion affects exon 1, two deletions concern exon 6, another deletion, of which breakpoints are sequenced, takes part of exon 16 and two deletions affect exon 26. Besides the deletions, eight point mutations have been found at the TaqI restriction sites of exons 18, 24 and 26. Five C-->T mutations resulted in nonsense mutations, one in exon 18, one in exon 26 and three in exon 24. Two G-->A mutations caused a missense mutation in exon 24 leading to an arginine/glutamine exchange. Although two patients showed this mutation, their clinical phenotypes were different, possibly due to an additional unidentified sequence polymorphism. A G-->T mutation in exon 26 substituted the arginine with leucine. All deletions and seven of the point mutations are associated with severe disease with a detectable inhibitor in the patient with the TaqI-point mutation in exon 18. One of the G-->A mutations is associated with mild haemophilia but the patient also has developed an inhibitor. Amongst these mutations the origin of the mutation could be determined in four kindred, one of which showed maternal mosaicism.     

Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.     

Interpersonal relations in nursing: a philosophical-ethical analysis of the work of Hildegard E. Peplau

We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing. The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA. Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin. Carrier frequency may be as high as 1/10 in some groupings. The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme. These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.     

Spectrum of mutations in alpha-mannosidosis

alpha-Mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). The resulting intracellular accumulation of mannose-containing oligosaccharides leads to mental retardation, hearing impairment, skeletal changes, and immunodeficiency. Recently, we reported the first alpha-mannosidosis-causing mutation affecting two Palestinian siblings. In the present study 21 novel mutations and four polymorphic amino acid positions were identified by the screening of 43 patients, from 39 families, mainly of European origin. Disease-causing mutations were identified in 72% of the alleles and included eight splicing, six missense, and three nonsense mutations, as well as two small insertions and two small deletions. In addition, Southern blot analysis indicated rearrangements in some alleles. Most mutations were private or occurred in two or three families, except for a missense mutation resulting in an R750W substitution. This mutation was found in 13 patients, from different European countries, and accounted for 21% of the disease alleles. Although there were clinical variations among the patients, no significant LAMAN activity could be detected in any of the fibroblast cultures. In addition, no correlation between the types of mutations and the clinical manifestations was evident.     

Correction of neutropenia and hypogammaglobulinemia in X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation

X-linked hyper-IgM (X-HIM) syndrome is a primary immunodeficiency disease characterized by defects in both cellular and humoral immunity. X-HIM is caused by mutations in the gene for CD40 ligand (CD40L), a T cell membrane protein that mediates T cell-dependent immune functions. We report the case of a 6-year-old male with X-HIM due to an intronic mutation resulting in aberrant CD40L RNA splicing and absence of detectable CD40L protein. The patient had a history of multiple infectious complications and chronic neutropenia requiring treatment with recombinant granulocyte colony-stimulating factor, and underwent allogeneic bone marrow transplantation from an HLA-matched sibling donor. Following successful engraftment, T cell CD40L expression and immunoglobulin isotype switching were reconstituted and neutropenia resolved. Allogeneic bone marrow transplantation can correct neutropenia and reconstitute immune function in X-HIM.     

CD40/CD40 ligand interactions in normal, reactive and malignant lympho-hematopoietic tissues

CD40 is a 48 Kd integral membrane protein expressed by cells of B cells, origin, dentritic cells, monocytes, epithelial cells, endothelial cells and tumor cells including carcinomas, B cell lymphomas/leukemias and Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin's disease (HD). CD40 has been clustered as a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily with the corresponding counterstructure, the CD40 ligand (L) being mainly expressed by activated CD4+ T cells, but also some activated CD8+ T cells, basophils, eosinophils, mast cells and stromal cells. CD40L shares significant amino acid homology with TNF particularly in its extracellular domain ("TNF homology region") and is therefore viewed as a member of the TNF ligand superfamily. Binding of CD40L+ T cells to CD40+ B cells is thought to play a major role in T cell-dependent B cell activation, B cell proliferation, Ig isotype switching, memory B cell formation and rescue of B cells from apoptotic death in germinal centers. Mutations of the CD40L gene have been associated with the X-linked hyper-IgM immunodeficiency syndrome, pointing to the critical role of the CD40/CD40L interaction in the T cell-B cell interplay. Accordingly, expression of CD40 by human lympho-hematopoietic tumors has been shown in most of the B cell neoplasias, H-RS cells and HD and some carcinomas. In contrast, CD40L+ tumor cells are almost invariably restricted to CD4+/CD8- T cell lymphomas. Overall, functional CD40/CD40L interactions appear to be critical for cellular activation signals during immune responses and neoplastic tumor cell growth. The understanding of the biology of CD40L has improved our diagnostic and therapeutic repertoire in the management of several human diseases, including CD40+ tumors.     

Effect of contrast medium on corpus cavernosum smooth muscle

OBJECTIVE: To study the molecular basis of complete androgen insensitivity syndrome (AIS). STUDY DESIGN: The coding region of the human androgen receptor (hAR) gene in two women with AIS was amplified with polymerase chain reaction using 12 pairs of oligonucleotide primers and then sequenced with a dye terminator method. RESULTS: Both patients had mutation in exon E of the androgen-binding domain. In one patient, codon 732 GAC (aspartic acid) was changed to ACC (asparagine), and her CAG polyglutamine tract had 27 repeats. In the other patient, codon 765 GCC (alanine) was changed to ACC (threonine), and her CAG polyglutamine tract in exon A had 19 repeats. CONCLUSION: Except for CAG polyglutamine polymorphism, these two missense mutations were the only differences detected in the coding region of the hAR gene. Both mutations involved the CpG sequence, which has been regarded as a mutation hotspot. To the best of our knowledge, these two mutations have not been observed before in Chinese women. Elucidation of the molecular defects of AIS patients would be very helpful for genetic counseling and prenatal diagnosis.