Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Allan-Herndon-Dudley syndrome and the monocarboxylate transporter 8 (MCT8) gene

Allan-Herndon-Dudley syndrome was among the first of the X-linked mental retardation syndromes to be described (in 1944) and among the first to be regionally mapped on the X chromosome (in 1990). Six large families with the syndrome have been identified, and linkage studies have placed the gene locus in Xq13.2. Mutations in the monocarboxylate transporter 8 gene (MCT8) have been found in each of the six families. One essential function of the protein encoded by this gene appears to be the transport of triiodothyronine into neurons. Abnormal transporter function is reflected in elevated free triiodothyronine and lowered free thyroxine levels in the blood. Infancy and childhood in the Allan-Herndon-Dudley syndrome are marked by hypotonia, weakness, reduced muscle mass, and delay of developmental milestones. Facial manifestations are not distinctive, but the face tends to be elongated with bifrontal narrowing, and the ears are often simply formed or cupped. Some patients have myopathic facies. Generalized weakness is manifested by excessive drooling, forward positioning of the head and neck, failure to ambulate independently, or ataxia in those who do ambulate. Speech is dysarthric or absent altogether. Hypotonia gives way in adult life to spasticity. The hands exhibit dystonic and athetoid posturing and fisting. Cognitive development is severely impaired. No major malformations occur, intrauterine growth is not impaired, and head circumference and genital development are usually normal. Behavior tends to be passive, with little evidence of aggressive or disruptive behavior. Although clinical signs of thyroid dysfunction are usually absent in affected males, the disturbances in blood levels of thyroid hormones suggest the possibility of systematic detection through screening of high-risk populations.     

Aminoaciduria, but normal thyroid hormone levels and signalling, in mice lacking the amino acid and thyroid hormone transporter Slc7a8

LAT2 (system L amino acid transporter 2) is composed of the subunits Slc7a8/Lat2 and Slc3a2/4F2hc. This transporter is highly expressed along the basolateral membranes of absorptive epithelia in kidney and small intestine, but is also abundant in the brain. Lat2 is an energy-independent exchanger of neutral amino acids, and was shown to transport thyroid hormones. We report in the present paper that targeted inactivation of Slc7a8 leads to increased urinary loss of small neutral amino acids. Development and growth of Slc7a8(-/-) mice appears normal, suggesting functional compensation of neutral amino acid transport by alternative transporters in kidney, intestine and placenta. Movement co-ordination is slightly impaired in mutant mice, although cerebellar development and structure remained inconspicuous. Circulating thyroid hormones, thyrotropin and thyroid hormone-responsive genes remained unchanged in Slc7a8(-/-) mice, possibly because of functional compensation by the thyroid hormone transporter Mct8 (monocarboxylate transporter 8), which is co-expressed in many cell types. The reason for the mild neurological phenotype remains unresolved.     

A novel mutation in the RPS6KA3 gene in a patient with Coffin-Lowry syndrome

Coffin-Lowry syndrome is an X-linked disorder characterized by mental retardation, characteristic facial features, skeletal abnormalities, and tapering fingers. Herein we report a novel missense mutation in exon 7 at codon 180 in the RPS6KA3 gene in a boy with Coffin-Lowry syndrome.     

Potent blockers of the monocarboxylate transporter MCT1: novel immunomodulatory compounds

A novel series of potent blockers of the monocarboxylate transporter, MCT1, is disclosed. From very potent but lipophilic lead compounds, systematic changes to all parts of the molecule, targeting reduction in log D, afforded compounds with significantly improved overall properties. These compounds show potent immunomodulatory activity.     

A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene

Thyroid hormones are iodothyronines that control growth and development, as well as brain function and metabolism. Although thyroid hormone deficiency can be caused by defects of hormone synthesis and action, it has not been linked to a defect in cellular hormone transport. In fact, the physiological role of the several classes of membrane transporters remains unknown. We now report, for the first time, mutations in the monocarboxylate transporter 8 (MCT8) gene, located on the X chromosome, that encodes a 613-amino acid protein with 12 predicted transmembrane domains. The propositi of two unrelated families are males with abnormal relative concentrations of three circulating iodothyronines, as well as neurological abnormalities, including global developmental delay, central hypotonia, spastic quadriplegia, dystonic movements, rotary nystagmus, and impaired gaze and hearing. Heterozygous females had a milder thyroid phenotype and no neurological defects. These findings establish the physiological importance of MCT8 as a thyroid hormone transporter.     

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.     

A novel thyroid hormone receptor-beta mutation,not anticipated to occur in resistance to thyroid hormone,causes variable phenotypes

Resistance to thyroid hormones (RTH) is a syndrome characterized by a variable tissue hyposensitivity to thyroid hormones and is linked to mutations in the thyroid hormone receptor-beta (TRbeta) gene. We report here for the first time in vivo the mutation R429W (CCG-->TCG) located in the exon 10. The artificial mutant obtained in vitro displayed a normal T(3)-binding affinity and transactivation function. Therefore, it was thought to produce little, if any, clinical effect and to escape to clinical detection. The present report is at least in part discordant with this prediction since the propositus and his grandmother had an authentic hyperthyroidism with high FT(4) plasma level in the presence of inappropriate TSH. On the other hand, spontaneous variations of clinical features and - interestingly - of plasma FT(4) concentrations with time in the propositus, and the phenotype observed in his mother who never complained with thyrotoxic symptoms, confirmed the in vitro binding and functional predictions. The most intriguing is the clinical course of the grandmother as she first presented with predominant pituitary RTH and a diffuse goiter and finally with a toxic multinodular goiter with normal T(3) and T(4) plasma concentrations and suppressed TSH. In conclusion, we report a novel mutation in the gene encoding the thyroid hormone receptor responsible for predominant pituitary RTH already described in vitro but not in vivo. The fluctuant phenotype of the propositus suggests that other factors modulate the degree of tissue resistance that is under genetic control. Toxic multinodular goiter, possibly due to chronic TSH stimulation during RTH, in addition to the phenotype variability, increases the difficulty to diagnose this thyroid disorder.     

A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance.

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.     

A novel COMP mutation in a Chinese patient with pseudoachondroplasia

A 2.75-year-old Chinese boy presented with typical clinical features of pseudoachondroplasia, including disproportionate short-limb short stature, brachydactyly, genu varus and waddling gait. Radiologically, tubular bones were short with widened metaphyses, irregular and small epiphyses; anterior tonguing or beaking of vertebral bodies were characteristic. DNA sequencing analysis of the COMP gene revealed a heterozygous mutation (c.1511G>A, p.Cys504Tyr) in the patient but his parents were unaffected without this genetic change. The missense mutation (c.1511G>A) was not found in 100 healthy controls and has not been reported previously. Our findings expand the spectrum of known mutations in COMP leading to pseudoachondroplasia.     

A novel missense mutation of the ATP2C1 gene in a Chinese patient with Hailey-Hailey disease

Benign familial chronic pemphigus (Hailey–Hailey disease, HHD; MIM 169600) is a rare autosomal dominant hereditary disorder characterized by pruritic vesicles, painful erosions and scaly erythematous plaques at the sites of friction and flexures. Mutations in ATP2C1, which encoding the human secretory pathway Ca²⁺/Mn²⁺-ATPase protein 1 (hSPCA1), have been identified as the pathogenic gene of HHD. We found a novel, distinct, heterozygous mutation during study of a Chinese patient with HHD. We identified a C→T transition at nucleotide 1235 (p.Thr352IIe), in exon 13 of ATP2C1. This observation would be useful for genetic counseling and prenatal diagnosis for affected families and in expanding the repertoire of ATP2C1 mutations underlying HHD.     

The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter

The late-infantile-onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile-onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide-polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518-amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs.     

A novel de novo PDE4D gene mutation identified in a Chinese patient with acrodysostosis

Acrodysostosis is an extremely rare disorder at birth, that is, characterized by skeletal dysplasia with short stature and midfacial hypoplasia, which has been reported to be caused by PDE4D and PRKAR1A gene mutations. Here, a Chinese boy with acrodysostosis, ventricular septal defect, and pulmonary hypertension was recruited for our study, and his clinical and biochemical characteristics were analyzed. A novel de novo heterozygous missense mutation (NM_001104631: c.2030A>C, p.Tyr677Ser) of the PDE4D gene was detected by whole exome sequencing and confirmed by Sanger sequencing. The c.2030A>C (p.Tyr677Ser) variant was located in exon 15 of the PDE4D gene, predicted to be damaging by a functional prediction program and shown to be highly conserved among many species. Further functional analysis showed that the p.Tyr677Ser substitution changes the function of the PDE4D protein, affects its subcellular localization in transfected cells, increases PDE4 activity in the regulation of cAMP signaling and affects cell proliferation. Our study identified a novel de novo PDE4D mutation in acrodysostosis of Chinese origin that not only contributes a deeper appreciation of the phenotypic characteristics of patients with PDE4D mutations but also expands the spectrum of PDE4D mutations.     

Single base mutation in the hormone binding domain of the thyroid hormone receptor beta gene in generalised thyroid hormone resistance demonstrated by single stranded conformation polymorphism analysis.

Thyroid hormone resistance is a syndrome of considerable clinical heterogeneity. Three mutations in the c-erb A beta gene encoding the human beta thyroid hormone receptor have been described in different kindreds. We report here, in a family affected with peripheral thyroid hormone resistance, a unique point mutation in the ligand binding domain of the c-erb A beta gene resulting in histidine replacement of an arginine residue at position 438. The region in which the mutation occurred was identified by single stranded conformation polymorphism analysis and confirmed by subcloning and sequencing of the mutant alleles from each of the affected members. Binding of tri-iodothyronine to isolated nuclei from family members was normal suggesting the mechanism of thyroid hormone resistance in this family is not mediated by abnormal binding of ligand and receptor.     

A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens

Background

Autosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA.

Results

The proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain.Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition.

Conclusions

A novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.

     

A novel RUNX2 mutation in exon 8, G462X,in a patient with Cleidocranial Dysplasia

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1‐8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real‐time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C‐terminus of proline/serine/threonine‐rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2‐G462X transfected cells. In the cyclohexamide chase assay, Runx2‐G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase‐13, bone sialoprotein, and osteopontin) decreased in G462X‐transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2‐G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.