Molecular genetics of septo-optic dysplasia

Septo-optic dysplasia (SOD) is a highly variable condition characterized by midline neurological abnormalities associated with pituitary hypoplasia and optic nerve hypoplasia. The aetiology is unknown. Mutant mice, in which a novel homeobox gene, Hesx1, has been disrupted, exhibit a phenotype that resembles the phenotype of SOD. We therefore wished to explore the possibility that this gene is implicated in SOD. We cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis, followed by cloning and sequencing of any exons which showed a band shift. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain, leading to a loss of in vitro DNA binding. Subsequently, we have identified heterozygous mutations in HESX1 that are associated with milder pituitary phenotypes. Our studies indicate a vital role for Hesx1/HESX1 in forebrain and pituitary development in mouse and man, and hence in some cases of SOD.     

A novel SNP of the <Emphasis Type="Italic">Hesx1</Emphasis> gene in bovine and its associations with average daily gain

As an essential repressor, the homeobox gene Hesx1/HESX1 is required within the anterior neural plate for normal forebrain development. Mutations within the Hesx1 gene have been associated with GH deficiency or combined pituitary hormone deficiency. We detected the polymorphism of Hesx1 gene by PCR-SSCP and DNA sequencing methods in 702 individuals from four Chinese cattle breeds. A novel single nucleotide polymorphism (SNP) (IVS1 + 382T > C) was detected. The frequencies of genotype TC in four breeds were 0.000–0.222. Polymorphism of the Hesx1 gene was shown to be associated with growth in the Nanyang breed. Individuals with genotype TC was significantly lower average daily gain than TT at 18 months (P < 0.05).     

Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression

Pituitary tumor-transforming gene (PTTG) originally isolated from GH-secreting pituitary adenoma cells causes in vitro cell transformation, in vivo tumorigenesis, and induces basic fibroblast growth factor. These functions require an intact C-terminal proline-proline-serine-proline motif. PTTG1 is abundantly expressed in human pituitary tumors and plays a role in the early stages of experimental prolactinoma formation. We now determined direct effects of PTTG1 on hormonal phenotypes of functional pituitary tumor cells. Overexpression of PTTG1 C terminus (amino acids 147-202) containing intact proline-proline-serine-proline motifs in rat prolactin (PRL)- and GH-secreting GH3 cells markedly abrogates PRL mRNA expression by more than 90% (P < 0.001) and hormone levels (P < 0.001) and PRL promoter activity (P < 0.01) compared with control vector cells or to a PTTG1 C terminus mutant (P163A, S165Q, P166L, P170L, P172A, and P173L). Wild-type PTTG1 C-terminal transfectants formed smaller (P < 0.05) sc tumors in rats compared with control or mutated PTTG1 C-terminal transfectants. Estrogen (10 nm) treatment for 48 h partially restored PRL expression in stable wild-type PTTG1 C-terminal transfectants. These results indicate that targeting PTTG1-mediated signaling alters the hormonal phenotype in pituitary cells and disrupted PTTG1 action may be a potential subcellular therapeutic tool for repressing PRL hypersecretion.     

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.     

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.     

Intellectual-disability-associated mutations in the ceramide transport protein gene CERT1 lead to aberrant function and subcellular distribution

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT’s serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.     

Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating

Mutations in the gene encoding the voltage-gated sodium channel of skeletal muscle (SkMl) have been identified in a group of autosomal dominant diseases, characterized by abnormalities of the sarcolemmal excitability, that include paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP). We previously reported that PC mutations cause in common a slowing of inactivation in the human SkMl sodium channel. In this investigation, we examined the molecular mechanisms responsible for the effects of L1433R, located in D4/S3, on channel gating by creating a series of additional mutations at the 1433 site. Unlike the R1448C mutation, found in D4/S4, which produces its effects largely due to the loss of the positive charge, change of the hydropathy of the side chain rather than charge is the primary factor mediating the effects of L1433R. These two mutations also differ in their effects on recovery from inactivation, conditioned inactivation, and steady state inactivation of the hSkMl channels. We constructed a double mutation containing both L1433R and R1448C. The double mutation closely resembled R1448C with respect to alterations in the kinetics of inactivation during depolarization and voltage dependence, but was indistinguishable from L1433R in the kinetics of recovery from inactivation and steady state inactivation. No additive effects were seen, suggesting that these two segments interact during gating. In addition, we found that these mutations have different effects on the delay of recovery from inactivation and the kinetics of the tail currents, raising a question whether this delay is a reflection of the deactivation process. These results suggest that the S3 and S4 segments play distinct roles in different processes of hSkM1 channel gating: D4/S4 is critical for the deactivation and inactivation of the open channel while D4/S3 has a dominant role in the recovery of inactivated channels. However, these two segments interact during the entry to, and exit from, inactivation states.     

Prediction of the Damage-Associated Non-Synonymous Single Nucleotide Polymorphisms in the Human MC1R Gene

The melanocortin 1 receptor (MC1R) is involved in the control of melanogenesis. Polymorphisms in this gene have been associated with variation in skin and hair color and with elevated risk for the development of melanoma. Here we used 11 computational tools based on different approaches to predict the damage-associated non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region of the human MC1R gene. Among the 92 nsSNPs arranged according to the predictions 62% were classified as damaging in more than five tools. The classification was significantly correlated with the scores of two consensus programs. Alleles associated with the red hair color (RHC) phenotype and with the risk of melanoma were examined. The R variants D84E, R142H, R151C, I155T, R160W and D294H were classified as damaging by the majority of the tools while the r variants V60L, V92M and R163Q have been predicted as neutral in most of the programs The combination of the prediction tools results in 14 nsSNPs indicated as the most damaging mutations in MC1R (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H); C273Y showed to be highly damaging in SIFT, Polyphen-2, MutPred, PANTHER and PROVEAN scores. The computational analysis proved capable of identifying the potentially damaging nsSNPs in MC1R, which are candidates for further laboratory studies of the functional and pharmacological significance of the alterations in the receptor and the phenotypic outcomes.     

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin

In this report, methods are described to isolate milligram quantities of a mutant apolipoprotein A-I (apoA-I) protein for use in structure-function studies. Expression of the L159R apoA-I mutation in humans reduces the concentration of plasma wild-type apoA-I, thus displaying a dominant negative phenotype in vivo. Earlier attempts to express and isolate this mutant protein resulted in extensive degradation and protein misfolding. Using an Escherichia coli expression system used predominantly for the isolation of soluble apoA-I mutant proteins, we describe the expression and purification of L159R apoA-I (apoA-I(Fin)) from inclusion bodies. In addition, we describe a mass spectrometric method for measuring the L159R-to-wild-type apoA-I ratio in a 1 microl plasma sample. These new methods will facilitate further studies into the mechanism behind the dominant negative phenotype associated with the expression of the L159R apoA-I protein in humans.     

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.     

Mitochondrial DNA defects in Saccharomyces cerevisiae caused by functional interactions between DNA polymerase gamma mutations associated with disease in human

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.     

Novel insights into the aetiology and pathogenesis of hypopituitarism

Recent advances in our knowledge of pituitary development, acquired mainly from animal models, have enhanced our understanding of the aetiology of isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD), as well as several syndromic forms of growth hormone deficiency (GHD). A number of developmental genes known to be important for organ commitment and cell differentiation and proliferation (HESX1, LHX3, LHX4, PROP1 and PIT1) have been implicated in CPHD with or without other syndromic features. Phenotypes associated with these genetic mutations and their inheritance may be highly variable. Functional analyses of these mutations reveal valuable insights into the function of the proteins and hence into the effect of these mutations on phenotype. Novel insights have been gained into the mechanisms whereby these genes are associated with particular phenotypes as a result of murine transgenesis, e.g. type II autosomal dominant GHD. Mutations within known genes account for a small proportion of cases of IGHD and CPHD, suggesting the role of other as yet unidentified genetic and environmental factors. Hence, genetic testing will in the future have a greater role to play in understanding the mechanisms leading to particular hypopituitary phenotypes and also in predicting the evolution of these disorders. There is, however, no substitute for careful delineation of the phenotype prior to undertaking genetic studies.