Beta+-thalassemia in cis of a sickle cell gene: occurrence of a promoter mutation on a beta s chromosome

An atypical sickle cell trait with a very low level of hemoglobin S and features of heterozygous beta-thalassemia was recently described. In vitro globin chain synthesis strongly suggested the presence of the two abnormalities on the same chromosome. We report the corresponding beta S-thal gene. DNA sequence revealed a C----T base substitution in the distal promoter element CACCC, at position-88 from the cap site, in addition to the expected GAG----GTG mutation responsible for the structural variant (beta 6 Glu----Val). Reticulocyte mRNA titration and transient assay of the mutant gene in COS cells showed a defect in beta- mRNA production. Restriction haplotype and DNA sequence analyses revealed that the doubly mutated gene is associated with haplotype 19 (or Benin/Algeria haplotype). In particular, we found the (AT)9(T)4 repeated sequences specifically encountered 5' to the beta S gene of Benin Algeria type. These results support the view that the beta S-thal gene resulted from an independent thalassemic mutation having occurred on a beta S chromosome rather than (a) from a beta S mutation having altered a beta-thalassemic gene or (b) from a recombination event between two chromosomes, each carrying one of the mutations.     

A frameshift mutation of beta subunit of epithelial sodium channel in a case of isolated Liddle syndrome

BACKGROUND: Liddle syndrome is an autosomal dominant form of salt-sensitive hypertension caused by mutations in the epithelial sodium channel expressed in the distal nephron playing an essential role in Na absorption. All reported mutations in Liddle syndrome are either missense mutations or frameshift mutations destroying the PY motif closer to the C-terminus of the beta or gamma subunits causing the situation that the epithelial sodium channels are not degraded and sodium is pooled and thus hypertension and hypokalemia are caused. METHODS: We sequenced the C-terminus of the beta or gamma subunits of the epithelial sodium channel in a Japanese family of a patient clinically diagnosed as having Liddle syndrome. RESULTS: As a result, we found in the proband, a frameshift mutation of the beta subunit caused by a single cytosine insertion at the codon 595, introducing a new stop codon at 605 and deleting the last 34 amino acids from the normally encoded protein. CONCLUSION: This mutation is carried by neither parent (with paternity proven) and hence confirms this has occurred as a event within this family.     

A family with liddle's syndrome caused by a mutation in the beta subunit of the epithelial sodium channel

Liddle's syndrome is a rare form of autosomal-dominant salt-sensitive hypertension. Constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel (ENaC) is essential for salt-sensitive hypertension. Recently, several DNA analysis studies have indicated that there is a mutation of C-terminus of either the beta or y subunit. We sequenced the C-termini of the beta and -gamma subunits of the ENaC in a Japanese family with hypertension and hypopotassemia without excess minerarocorticoids, clinically diagnosed as Liddle's syndrome. The mutation of the ENaC of this family was beta R564X. Since such case seem to be rare in the literature, detailed data are shown in this report.     

A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase.

The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.     

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.     

beta 0 thalassemia, a nonsense mutation in man.

We determined the complete nucleotide sequence of the 5' noncoding region and the first 74 amino acids of the nonfunctional beta-globin mRNA in a patient with homozygous beta 0 thalassemia. We identified the molecular defect as a single nucleotide substitution in the coding region of the mRNA. At the position corresponding to amino acid 17, replacement of an adenine by a uracil changes the triplet AAG, which codes for lysine in the normal beta chain, to an amber termination codon, UAG. This type of beta 0 thalassemia represents an example of a nonsense mutation in man.     

A new mutation,R563Q,of the beta subunit of the epithelial sodium channel associated with low-renin,low-aldosterone hypertension

OBJECTIVE: To determine the relationship between R563Q, a mutation of the renal epithelial sodium channel, and hypertension. METHODS: Hypertensive patients with low renin and aldosterone, hypokalemia or resistant hypertension were selected for DNA analysis. Genomic DNA encoding the C-terminal domain of the epithelial sodium channel beta subunit from hypertensives and controls was amplified by polymerase chain reaction and screened for the R563Q mutation by digestion with Sfc1 restriction enzyme, or sequenced. RESULTS: A previously undescribed mutation, R563Q, of the beta epithelial sodium channel was found in 10 of 139 black hypertensives, but was not present in any of 103 black normotensives, a significant (P = 0.0058) difference in frequency. The frequency of the mutation in the subgroup of black low-renin, low-aldosterone hypertensives (four of 14) was significantly (P = 0.0001) greater than in normotensives, and was also greater (P = 0.041) than in normal-high renin hypertensives, suggesting that R563Q is an activating mutation of the epithelial sodium channel. R563Q was also found in seven out of 250 mixed ancestry hypertensives, and was significantly (P = 0.017) associated with low-renin, low-aldosterone hypertension in this population group. The mutation was found in one of 100 mixed ancestry normotensives but not in any of 136 white hypertensives. Of the 18 R563Q patients, 11 had severe hypertension, leading to renal failure in two cases, while only two had hypokalaemia. CONCLUSIONS: R563Q, a new variant of the beta epithelial sodium channel, is associated with low-renin, low-aldosterone hypertension, in South African black and mixed-ancestry patients. Only a minority of individuals with the R563Q allelle fully express the Liddle's syndrome phenotype.     

G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin.     

Identification of a point mutation in factor XIII A subunit deficiency.

Oligonucleotide primers have been designed for the amplification of all 15 exons of the human coagulation factor XIII A subunit gene. Each exon and its intron flanking regions has been amplified and sequenced from a patient with severe A subunit deficiency. A single G to A transition in the last base of exon 14 has been identified in the homozygous proband and in his heterozygous parents. The mutation would result in the substitution 681 Arg to His in the mature protein product. However, because the mutation is at a splice junction, the deficiency may result from a defect in pre-messenger RNA splicing.     

Workshop on programming beta cell development, impairment and regeneration

Helsing?r, the city of Hamlet in Denmark, provided the site for the workshop "Programming Beta Cell Development, Impairment and Regeneration" on October 23-26th, 2011. The same location has held two EASD Islet study group meetings, while the previous three workshops were held in Helsinki, Finland (2003), El Perello, Spain (2006) and Peebles, Scotland (2009). The meeting drew 190 attendees from 12 different countries. There were 37 main oral presentations, and 68 posters covered virtually all aspects of the pancreas and provided a dynamic snapshot of the most interesting areas of current investigation. In addition, six parallel workshops on stem cells, epigenetics, autoimmunity, β-cell imaging, β-cell identity, omics in β-cell research and a panel discussion on "to be or not to be a beta cell" were held. Here, we will review some of the newest highlights and still unanswered questions in the field.     

A novel beta-globin mutation, beta Durham-NC [beta 114 Leu-->Pro], produces a dominant thalassemia-like phenotype

Mutations within exon 3 of the beta-globin gene are relatively uncommon, and many of these mutations produce a dominant thalassemia- like phenotype. We describe a novel thalassemic hemoglobinopathy caused by a single nucleotide substitution (CTG-->CCG) at codon 114 resulting in a leucine to proline substitution and designate it beta Durham-NC [beta 114 Leu-->Pro]. The mutation producing this thalassemic hemoglobinopathy is located near to the beta Showa-Yakushiji mutation (beta 110 Leu-->Pro). Both of these hemoglobinopathies share similar phenotypic features with moderately severe microcytic anemia. Using computer imaging of the hemoglobin molecule, we examined several reported point mutations within exon 3 of the beta-globin gene. These point mutations cause a single amino acid substitution in the G helix, and result in a thalassemic and/or hemolytic phenotype. Computer imaging of nine separate examples suggests that amino acid substitutions affecting side chains that project into the heme pocket may destabilize the heme moiety within the beta-globin chain, resulting in a thalassemic phenotype. Hemolytic phenotypes may be the result of decreased alpha 1 beta 1 interactions. The beta Durham-NC mutation further characterizes a novel group of thalassemias/hemoglobinopathies that are clinically difficult to identify and require accessory laboratory testing.     

Null mutation in transforming growth factor beta1 disrupts ovarian function and causes oocyte incompetence and early embryo arrest

TGFbeta1 is implicated in regulation of ovarian function and the events of early pregnancy. We have investigated the effect of null mutation in the Tgfbeta1 gene on reproductive function in female mice. The reproductive capacity of TGFbeta1 null mutant females was severely impaired, leading to almost complete infertility. Onset of sexual maturity was delayed, after which ovarian function was disrupted, with extended ovarian cycles, irregular ovulation, and a 40% reduction in oocytes ovulated. Serum FSH and estrogen content were normal, but TGFbeta1 null mutant mice failed to display the characteristic proestrus surge in circulating LH. Ovarian hyperstimulation with exogenous gonadotropins elicited normal ovulation rates in TGFbeta1 null mutant mice. After mating with wild-type stud males, serum progesterone content was reduced by 75% associated with altered ovarian expression of mRNAs encoding steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase-1 and P450 17 alpha-hydroxylase/C17-20-lyase. Embryos recovered from TGFbeta1 null mutant females were developmentally arrested in the morula stage and rarely progressed to blastocysts. Attempts to rescue embryos by exogenous progesterone administration and in vitro culture were unsuccessful, and in vitro fertilization and culture experiments demonstrated that impaired development is unlikely to result from lack of maternal tract TGFbeta1. We conclude that embryo arrest is due to developmental incompetence in oocytes developed in a TGFbeta1-deficient follicular environment. This study demonstrates that TGFbeta1 is a critical determinant of normal ovarian function, operating through regulation of LH activity and generation of oocytes competent for embryonic development and successful initiation of pregnancy.     

Integrin beta1 subunit controls mural cell adhesion, spreading, and blood vessel wall stability

Growth, maturation, and integrity of the blood vessel network require extensive communication between the endothelial cells, which line the vascular lumen, and associated mural cells, namely vascular smooth muscle cells and pericytes. Pericytes extend long processes, make direct contact with the capillary endothelium, and promote vascular quiescence by suppressing angiogenic sprouting. Vascular smooth muscle cells are highly contractile, extracellular matrix-secreting cells that cover arteries and veins and provide them with mechanical stability and elasticity. In the damaged blood vessel wall, for example in atherosclerotic lesions, vascular smooth muscle cells lose their differentiated state and acquire a highly mitotic, so-called "synthetic" phenotype, which is thought to promote pathogenesis. Among other factors, extracellular matrix molecules and integrin family cell-matrix receptors may regulate this phenotypic transition. Here we show that the inactivation of the gene encoding the integrin beta1 subunit (Itgb1) with a Cre-loxP approach in mice leads to mural cell defects and postnatal lethality. Integrin beta1-deficient vascular smooth muscle cells display several hallmarks of the synthetic phenotype: Cell proliferation is enhanced, whereas differentiation and their ability to support blood vessels are compromised. Similarly, mutant pericytes are poorly spread but present in larger numbers. Our analysis of this mutant model shows that integrin beta1-mediated cell-matrix adhesion is a major determinant of the mural cell phenotype.     

A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.     

A mutation in segment IVS6 disrupts fast inactivation of sodium channels.

Na(+)-channel inactivation is proposed to occur by binding of an intracellular inactivation gate to a hydrophobic inactivation gate receptor in the intracellular mouth of the pore. Amino acid residues in transmembrane segment S6 of domain IV (IVS6) that are critical for fast inactivation were identified by alanine-scanning mutagenesis. Mutant VIL1774-6AAA, in which three adjacent residues (Val-Ile-Leu) at the intracellular end of segment IVS6 were converted to alanine, had substantial (> 85%) sustained Na+ currents remaining 15 ms after depolarization, while a nearby mutation of three residues to alanine had no effect. Single-channel analysis revealed continued reopenings late in 40-ms depolarizing pulses indicating that inactivation was substantially impaired compared to wild type. The mean open time for VIL1774-6AAA was longer than wild type, suggesting that this mutation also decreases the rate of entry into the fast inactivated state. These results suggest that residues near the intracellular end of segment IVS6 are critical for fast Na(+)-channel inactivation and may form part of the hydrophobic receptor site for the fast inactivation gate.     

T cell receptors recognizing type II collagen in HLA-DR-transgenic mice characterized by highly restricted V beta usage

OBJECTIVE: To determine the T cell receptor (TCR) structure recognizing type II collagen (CII) in HLA-DR-transgenic mice, and to examine the role of T cells with certain V(beta)-chains in collagen-induced arthritis (CIA). METHODS: T cell hybridomas were established from DR1- and DR4-transgenic mice and selected for their responses to CII and CII peptide containing the T cell determinants. RNA was extracted and reverse transcribed into complementary DNA, which was then amplified using appropriate V(beta)- and V(alpha)-subfamily-specific primers. The polymerase chain reaction products were purified and directly sequenced. To determine the role of T cells with certain V(beta)-chains in CIA, V(beta)-subfamily-specific antibodies were administered and the development and characteristics of arthritis were determined. RESULTS: TCRs of 23 clonally distinct T cell hybridomas that were derived from DR1-transgenic mice and that were reactive to the CII peptide containing the immunodominant determinant were analyzed. These hybridomas predominantly used the TCR V(beta)14 and V(beta)8 gene segments (70% and 30%, respectively). The same restriction in V(beta) usage was also found in CII-reactive T cell hybridomas from DR4-transgenic mice. There was also restricted use of V(alpha) genes, although this was less marked than that of V(beta). In contrast, the hybridomas expressed a diverse third complementarity-determining region. Deletion of both V(beta)14-bearing and V(beta)8-bearing T cells significantly reduced the incidence and severity of CIA. CONCLUSION: These data demonstrate that DR1 and DR4 not only bind and present the same CII immunodominant peptide, but also stimulate a highly restricted subset of T cells.     

Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo.The seminiferous epithelium is separated by tight junctions between Sertoli cells into a luminal compartment containing spermatocytes and spermatids and a basal compartment containing spermatogonial stem cells (SSCs) and spermatogonia. The basal compartment is bounded above and on the sides by Sertoli cells and below by the basement membrane of the seminiferous tubule and a layer of peritubular myoid (PM) cells. SSCs are thought to reside in a microenvironmental niche in the basal compartment, where extrinsic cues influence their decision to either self-renew or enter the pathway of spermatogonial development (1, 2). They are a minor fraction of the undifferentiated spermatogonia in the basal compartment. The other undifferentiated spermatogonia (progenitors) give rise to differentiating spermatogonia that proliferate mitotically to progress on a developmental pathway toward becoming spermatocytes (3, 4). Our current understanding of the progression of SSCs to differentiating spermatogonia comes mainly from cell kinetic studies, germ cell transplantation assays, and the use of molecular markers that identify different populations of spermatogonia.The leading model for spermatogonial development specifies that when SSCs divide, they either self-renew by becoming two type A-single (A_s) spermatogonia or give rise to type A-paired (A_pr) spermatogonia connected by an intercellular bridge to become undifferentiated spermatogonia (5–7). The pairs continue to divide to form short chains of bridge-connected undifferentiated type A-aligned (A_al) spermatogonia, and these in turn divide to form longer chains of differentiating (type A₁, A₂, A₃, intermediate, and B) spermatogonia.Although SSCs are single cells, not all A_s spermatogonia are likely to be SSCs. There are ∼35,000 A_s spermatogonia in the testes of adult mice (8), but only about 3,000 of these have the ability to regenerate spermatogenesis when transplanted to germ cell-depleted testes (9). Although there are no generally accepted molecular markers specific for SSCs, potential candidates are inhibitor of DNA binding 4 (ID4) and paired box 7 (PAX7), which are expressed in minor subsets of A_s spermatogonia (10–12). However, it remains to be reported if ID4 and PAX7 are coexpressed in the same subset of A_s spermatogonia. SSCs also share molecular markers with undifferentiated spermatogonia, including Nanos2, Gfra1, Zbtb16, Bcl6b, and THY1(13–17). In addition, differentiating spermatogonia have characteristic molecular markers, including Ngn3, Nanos3, Spo11, and KIT (18–22). These molecular markers have been proven to be valuable tools for monitoring the presence or absence of different populations of spermatogonia.A conditional knockout (cKO) of the androgen receptor (Ar) gene in PM cells resulted in progressive loss of spermatogonia beginning at postnatal d 21, leading to disorganization of the seminiferous epithelium and infertility (23). This strongly suggested that androgens regulate genes in PM cells whose products are essential for SSC maintenance. In other studies, mice heterozygous for a global mutation in the gene for glial cell-derived neurotrophic factor (Gdnf) had reduced stem cell reserves, whereas mice with a transgene overexpressing GDNF experienced an overaccumulation of undifferentiated spermatogonia (24). GDNF also was reported to be critical for development of SSCs in vitro and their ability to restore spermatogenesis after transplantation to the testes of germ-cell-depleted mice (25, 26). This led us to hypothesize that regulation of GDNF production in PM cells by testosterone (T) is essential for SSC maintenance. In studies to test this hypothesis, we determined that PM cells isolated from adult mice and treated in vitro with T produce GDNF but not when untreated. We also found that SSCs from neonatal mice co-cultured with T-treated PM cells and transplanted to testes of germ cell-depleted mice restored spermatogenesis but not when they were co-cultured with untreated PM cells (10). These results supported the hypothesis but did not rule out the possibilities that PM cells or Sertoli cells produce other factors in vivo in addition to GDNF that are responsible for SSC self-renewal and differentiation. In these studies, we generated mice with a cKO of the Gdnf gene in PM cells to test the hypothesis that the production of GDNF by PM cells is essential for the in vivo development of undifferentiated spermatogonia.