Compound heterozygous deletion of the PROP-1 gene in children with combined pituitary hormone deficiency

Mutations in the prophet of Pit-1 gene (PROP1) have been shown to be responsible for combined pituitary hormone deficiency (CPHD) with deficiencies of growth hormone (GH), Prolactin (Prl), thyroid-stimulating hormone (TSH) and gonadotropins. We previously reported that homozygosity for a 2bp deletion in exon 2 (296delGA) accounted for CPHD in three patients from two Russian families. Here we report a second mutational hot spot in exon 2. This 2bp 149delGA deletion results in a frame shift that leads to the same serine to stop codon change at codon 109 (S109X). The predicted proteins are each truncated at residue 108 but diverge from the wild type sequence at different points in the homeodomain. Compound heterozygosity for the two mutations (149delGA/296delGA) was detected in 5 of 14 CPHD children from 4 families (36%). This provides the first evidence of heterozygosity for two common deletions as a cause of CPHD in Russian children.     

A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome

The human Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of non-bile salt organic anions. A highly similar phenotype has been described for a mutant Wistar rat strain, the transport-deficient (TR-) rat, which is defective in the canalicular multispecific organic anion transporter (cmoat). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The complementary DNA (cDNA) encoding rat cmoat has recently been cloned, and the mutation underlying the defect in TR- rats has been identified. In the present study, we have isolated the human homologue of rat cmoat, human cMOAT, and analyzed the corresponding cDNA from fibroblasts of a DJS patient for mutations. Our results show that a mutation in this gene is the cause of DJS.     

The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301-302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301-302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301-302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301-302delAG deletion suggests that rather than being inherited from a common founder, the 301-302delAG may be a recurring mutation.     

4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-3-deoxy-D-manno-2-octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus

A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C-->G point mutation in exon 8 causing a change from TAC (Tyr)-->TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C-->T mutation in exon 5 which gave a Ser-->Leu transition. The KpnI site introduced by the C-->G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C-->G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.     

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.     

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.     

Immunohistochemical evaluation of Bcl-2 gene family expression in liver of hepatitis C and cirrhotic patients: a novel mechanism to explain the high incidence of hepatocarcinoma in cirrhotics

Malonyl coenzyme A (CoA) decarboxylase (E.C.4. 1.1.9) catalyzes the conversion of malonyl CoA to acetyl CoA. The metabolic role of malonyl CoA decarboxylase has not been fully defined, but deficiency of the enzyme has been associated with mild mental retardation, seizures, hypotonia, cardiomyopathy, vomiting, hypoglycemia, metabolic acidosis, and malonic aciduria. Here we report the isolation and sequencing of the human gene encoding malonyl CoA decarboxylase, and the identification of a mutation causing malonyl CoA decarboxylase deficiency. Human malonyl CoA decarboxylase cDNA sequences were identified by homology to the goose gene, and the intron/exon boundaries were determined by direct sequencing of a PAC clone containing the entire human gene. The 1479 basepair human cDNA is 70 percent identical to the goose sequence, and the intron/exon boundaries are completely conserved between the two species. The genetic mutation underlying malonyl CoA decarboxylase deficiency was determined in a patient with clinical features of this defect, malonic aciduria, and markedly reduced malonyl CoA decarboxylase activity.     

RMA1, an Arabidopsis thaliana gene whose cDNA suppresses the yeast sec15 mutation, encodes a novel protein with a RING finger motif and a membrane anchor

To identify molecules that function in the plant secretory pathway, we screened for Arabidopsis thaliana cDNA clones that complement the temperature-sensitive (ts), secretion-deficient sec15 mutation of yeast Saccharomyces cerevisiae. RMA1, one of the genes obtained in this screening, suppressed not only the ts growth of sec15 but also its secretory defect. RMA1 is not a structural homologue of SEC15 but encodes a novel 28 kDa protein with a RING finger motif and a C-terminal membrane-anchoring domain. Mutational analysis indicates that the RING finger motif of RMA1 is important for its suppression activity. In Arabidopsis plant, RMA1 is ubiquitously expressed. A search for homologous proteins in the database revealed that Arabidopsis, nematode, mouse and human possess close homologues of RMA1.     

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax-3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.     

An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.     

Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.     

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1+ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron.     

The use of a highly informative CA repeat polymorphism within the abetalipoproteinaemia locus (4q22-24)

Abetalipoproteinaemia is a rare autosomal-recessive disorder caused by a defect in the large subunit of the microsomal triglyceride transfer protein (MTP) which is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. We report here the use of a polymorphic CA dinucleotide repeat in intron 10, MTPIVS10, of the large subunit of the human MTP protein in the analysis of a pregnancy in a consanguineous family, in which abetalipoproteinaemia was suspected, although prenatal diagnosis was subsequently refused. The mutation in the family has been identified as a novel four-nucleotide insertion/duplication of exon 17 between nucleotides 2349 and 2350 of the cDNA sequence of the MTP gene. However, the marker, MTPIVS10, can be used as an alternative to the time-consuming mutation detection techniques.     

The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoid element in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparent KD and only about a 2-fold reduction in the Bmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.     

Bright light: a countermeasure for jet lag?

We report the cDNA cloning, partial genomic organization, and expression pattern of Stra10, a novel retinoic acid-inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated, which are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx-related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH-2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence (Nakamura et al. [1996] Oncogene 13:2235-2242). The Meis2 homeodomain is identical to that of Meis1, and is most closely related to those of the Pbx/TGIF homeobox gene products. By in situ hybridization analysis, we show that the Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs.