Hemoglobin F Only Syndrome at Birth: A Case of Maternal HbA2' Complicating the Diagnosis of ß-Thalassemia.

An asymptomatic infant of Ghanaian descent had hemoglobin F only detected on newborn screening. ß-globin gene sequencing identified the intervening sequence (IVS)-II-849 (A → G) mutation with no normal ß-globin gene. ß-globin/δ-globin gene sequencing showed that both parents were heterozygous for the IVS-II-849 (A → G) mutation. The mother was heterozygous for the HbA2' δ-globin mutation (δ16 (A13) Gly → Arg), thus ß-thalassemia trait was unrecognized due to coinheritance of HbA2'. The infant developed anemia, splenomegaly, and began transfusion therapy by the age 6 of months. This is the first report of ß-thalassemia major with homozygous IVS-II-849 (A → G) mutations. This case highlights the importance of δ-globin gene mutations in prenatal testing.

Thalassemia-like phenotype in a novel complex hemoglobinopathy with α, ß, δ globin chain abnormalities.

The occurrence of multiple abnormalities of α, ß, δ, and γ globin genes may lead to unusual and complex phenotypes when they arise simultaneously in the same individual. Here, we report the findings of an African American boy who coinherited 3 heterozygous globin gene abnormalities: the unstable ß-globin chain variant; hemoglobin (Hb) Showa-Yakushiji [ß110(G12) Leu→Pro], the δ-globin chain variant; HbB2 [δ16(A13) Gly→Arg] and α-thalassemia (α-thal); (α-/αα). Hb Showa-Yakushiji had been previously described in Japanese, Indian, and European populations. We report its first occurrence in a child of African ancestry who presented with anemia not responsive to iron and an incomplete ß-thalassemia minor phenotype. Although the clinical and laboratory features of Hb Showa-Yakushiji mimic those of a ß-thalassemia, the coinheritance of the δ-globin chain variant Hb B2 suppressed the relative increase in Hb A2 usually observed in heterozygotes for the Hb Showa-Yakushiji mutation. Protein-based methods detected only a trace amount of HbB2 and failed to reveal presence of Hb Showa-Yakushiji and α-thal. The latter were only identified through DNA analyses. The diagnostic difficulties, molecular characteristics, and genotype/phenotype correlations of this novel complex hemoglobinopathy syndrome are reviewed.

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: a RPE specific phenomenon.

SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.

Molecular characterization and expression pattern of taurine transporter in zebrafish during embryogenesis.

Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na(+)/Cl(-)-dependent manner with a Na(+):Cl(-):taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.

Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family.

SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296+/-88 microM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid.

Expression of slc5a8 in kidney and its role in Na(+)-coupled transport of lactate.

We report here on the expression of slc5a8 in kidney and its relevance to Na(+)-coupled reabsorption of lactate. slc5a8 is the murine ortholog of SLC5A8, a candidate tumor suppressor gene, which we recently cloned from human intestine and demonstrated its functional identity as a Na(+)-coupled transporter for short-chain fatty acids and lactate. The slc5a8 cDNA, cloned from mouse kidney, codes for a protein consisting of 611 amino acids. When expressed heterologously in mammalian cells or Xenopus oocytes, slc5a8 mediates Na(+)-coupled electrogenic transport of lactate/pyruvate as well as short-chain fatty acids (e.g. acetate, propionate, and butyrate). The Na+/fatty acid stoichiometry varies depending on the fatty acid substrate (2:1 for lactate and 4:1 for propionate). This phenomenon of variable Na+/substrate stoichiometry depending on the fatty acid substrate is also demonstrable with human SLC5A8. In situ hybridization with sagittal sections of mouse kidney demonstrates abundant expression of the transcripts in the cortex as well as the medulla. Brush border membrane vesicles prepared from rabbit kidney are able to transport lactate in a Na(+)-coupled manner. The transport process exhibits the overshoot phenomenon, indicating uphill lactate transport in response to the transmembrane Na+ gradient. The Na(+)-coupled lactate transport in these membrane vesicles is inhibitable by short-chain fatty acids. We conclude that slc5a8 is expressed abundantly in the kidney and that it plays a role in the active reabsorption of lactate. slc5a8 is the first transporter known to be expressed in mammalian kidney that has the ability to mediate the Na(+)-coupled reabsorption of lactate.

Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans.

We have cloned and functionally characterized an Na+-coupled citrate transporter from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size.

Human sodium-coupled citrate transporter, the orthologue of Drosophila Indy, as a novel target for lithium action.

NaCT (sodium-coupled citrate transporter) is an Na(+)-coupled citrate transporter identified recently in mammals that mediates the cellular uptake of citrate. It is expressed predominantly in the liver. NaCT is structurally and functionally related to the product of the Indy (I'm not dead yet) gene in Drosophila, the dysfunction of which leads to lifespan extension. Here, we show that NaCT mediates the utilization of extracellular citrate for fat synthesis in human liver cells, and that the process is stimulated by lithium. The transport function of NaCT is enhanced by lithium at concentrations found in humans treated with lithium for bipolar disorders. Valproate and carbamazepine, two other drugs that are used for the treatment of bipolar disorder, do not affect the function of NaCT. The stimulatory effect of Li+ is specific for human NaCT, since NaCTs from other animal species are either inhibited or unaffected by Li+. The data also suggest that two of the four Na(+)-binding sites in human NaCT may become occupied by Li+ to produce the stimulatory effect. The stimulation of NaCT in humans by lithium at therapeutically relevant concentrations has potential clinical implications. We also show here that a single base mutation in codon-500 (TTT-->CTT) in the human NaCT gene, leading to the replacement of phenylalanine with leucine, stimulates the transport function and abolishes the stimulatory effect of lithium. This raises the possibility that genetic mutations in humans may lead to alterations in the constitutive activity of the transporter, with associated clinical consequences.