Saethre-Chotzen Syndrome, Pro136His TWIST Mutation, Hearing Loss, and External and Middle Ear Structural Anomalies: Report on a Brazilian Family

Objective: To describe the clinical, speech, hearing, and imaging findings in three members of a Brazilian family with Saethre-Chotzen syndrome (SCS) who presented some unusual characteristics within the spectrum of the syndrome. Design: Clinical evaluation was performed by a multidisciplinary team. Direct sequencing of the polymerase chain reaction amplified coding region of the TWIST1 gene, routine and electrophysiological hearing evaluation, speech evaluation, and imaging studies through computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed. Results: TWIST1 gene analysis revealed a Pro136His mutation in all patients. Hearing evaluation showed peripherial and mixed hearing loss in two of the patients, one of them with severe unilateral microtia. Computed tomography scan showed structural middle ear anomalies, and MRI showed distortion of the skull contour as well as some of the brain structures. Conclusions: We report a previously undescribed TWIST1 gene mutation in patients with SCS. There is evidence that indicates hearing loss (conductive and mixed) can be related both with middle ear (microtia, high jugular bulb, and enlarged vestibules) as well as with brain stem anomalies. Here we discuss the relationship between the gene mutation and the clinical, imaging, speech, and hearing findings.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

A comparison of family functioning, temperament, and childhood conditions in monozygotic twin pairs discordant for lifetime bulimia nervosa

Objective: The authors investigated differences between twins in nine pairs of female monozygotic twins in the Australian Twin Registry who were discordant for lifetime bulimia nervosa. Method: The twins affected and unaffected by lifetime bulimia nervosa were compared on self-report measures, including a measure of parental bonding, four measures of temperament, and six early-childhood medical conditions. Results: No twins had current bulimia nervosa, and there was no difference in weight or eating status between the affected and unaffected twins. The affected twins reported significantly lower self-esteem and less warmth but more overprotection by their mothers during childhood. Conclusions: Although limited by the small number of discordant twin pairs and the inability to detect causal relationships, these results suggest that environmental influences that promote low self-esteem may also increase the risk for bulimia nervosa. These temperamental differences may explain the discrepancies in parenting or perceived parenting.

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the overexpression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1

GCR1 has been tentatively identified in Arabidopsis thaliana as the first plant G-protein coupled receptor (GPCR) (Josefsson and Rask 1997) implicated in the cytokinin sensory pathway (Plakidou-Dymock et al. 1998). A protein fusion of GCR1 and green fluorescent protein has been expressed in Arabidopsis and shown GCR1 to be located on the plasma membrane. Studies of plants with altered GCR1 expression have led us to question GCR1's involvement in cytokinin signaling. Transgenic Arabidopsis plants containing sense and antisense constructs for GCR1 have been produced and over- and under-expression confirmed. The analysis of 12 antisense and 17 sense lines has failed to reveal the previously reported Dainty phenotype or altered cytokinin sensitivity. We have used the Gauntlet approach to test the plants' response to various plant hormones although this has not yet identified a mutant phenotype. The yeast-two hybrid system has been used and so far there is no evidence to suggest GCR1 interacts with heterotrimeric G proteins. Before GCR1 can be identified as genuine G-protein coupled receptor, the identification of a ligand and a proof of association with heterotrimeric G-proteins should be obtained.

Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation

The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (TRAP) in osteoclasts. Several lines of evidence were consistent with this model. The combination of MITF and PU.1 synergistically activated the TRAP promoter in transient assays. This activation was dependent on intact binding sites for both factors in the TRAP promoter. MITF and PU.1 physically interacted when coexpressed in COS cells or in vitro when purified recombinant proteins were studied. The minimal regions of MITF and PU.1 required for the interaction were the basic-helix-loop-helix zipper domain and the Ets DNA binding domain, respectively. Significantly, mice heterozygous for both the mutant mi allele and a PU.1 null allele developed osteopetrosis early in life which resolved with age. The size and number of osteoclasts were not altered in the double heterozygous mutant mice, indicating that the defect lies in mature osteoclast function. Taken in total, the results afford an example of how lineage-specific gene regulation can be achieved by the combinatorial action of two broadly expressed transcription factors.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Estimating Two-Stage Models for Genetic Influences on Alcohol, Tobacco or Drug Use Initiation and Dependence Vulnerability in Twin and Family Data

Genetic research on risk of alcohol, tobacco or drug dependence must make allowance for the partial overlap of risk-factors for initiation of use, and risk-factors for dependence or other outcomes in users. Except in the extreme cases where genetic and environmental risk-factors for initiation and dependence overlap completely or are uncorrelated, there is no consensus about how best to estimate the magnitude of genetic or environmental correlations between Initiation and Dependence in twin and family data. We explore by computer simulation the biases to estimates of genetic and environmental parameters caused by model misspecification when Initiation can only be defined as a binary variable. For plausible simulated parameter values, the two-stage genetic models that we consider yield estimates of genetic and environmental variances for Dependence that, although biased, are not very discrepant from the true values. However, estimates of genetic (or environmental) correlations between Initiation and Dependence may be seriously biased, and may differ markedly under different two-stage models. Such estimates may have little credibility unless external data favor selection of one particular model. These problems can be avoided if Initiation can be assessed as a multiple-category variable (e.g. never versus early-onset versus later onset user), with at least two categories measurable in users at risk for dependence. Under these conditions, under certain distributional assumptions., recovery of simulated genetic and environmental correlations becomes possible, Illustrative application of the model to Australian twin data on smoking confirmed substantial heritability of smoking persistence (42%) with minimal overlap with genetic influences on initiation.

Developmental expression of a class IV POU gene in the gastropod Haliotis asinina supports a conserved role in sensory cell development in bilaterians

POU-IV genes regulate neuronal development in a number of deuterostomes (chordates) and ecdysozoans (arthropods and nematodes). Currently their function and expression in the third bilaterian clade, the Lophotrochozoa, comprising molluscs, annelids and. their affiliates, is unclear. Herein we characterise the developmental expression of HasPOU-IV in the gastropod mollusc, Haliotis asinina. The POU-IV gene is transiently expressed in I I distinct larval territories during the first 3 days of development. HasPOU-IV is first expressed in sets of ventral epidermal cells in the newly hatched trochophore larvae. As larval morphogenesis proceeds, we observe HasPOU-IV transcripts in cells that putatively form a range of sensory systems including chemo- and mechanosensory cells in the foot, cephalic tentacles, the ctenidia. the geosensory statocyst and the eyes. By comparing HasPOU-IV expression with POU-IV genes in other bilaterians we infer that this class of POU-domain genes had an ancestral role in regulating sensory cell development.

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum . Dimethyl sulphide dehydrogenase was shown to contain bis (molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop-terin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB , ddhD and ddhC . DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c (2) mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Concordance of iron indices in homozygote and heterozygote sibling pairs in hemochromatosis families: implications for family screening

Background/Aims: Concordance of iron indices between same sex siblings homozygous for the cysteine-to-tyrosine substitution at amino acid 282 (C282Y) mutation suggests that the variable phenotype in hereditary hemochromatosis is caused by genetic factors. Concordance of iron indices between same-sex heterozygous sibling pairs would provide further evidence of genetic modifiers of disease expression, and guidance for family screening strategies of subjects heterozygous for the C282Y mutation. Methods: We compared the iron indices of 35 C282Y homozygous and 35 C282Y heterozygous same-sex sibling pairs. To clarify whether concordance between siblings was due to environmental or genetic factors we compared the iron indices of 164 C282Y homozygous-normal, same-sex dizygotic twins. Results: Serum ferritin (r = 0.50, P = 0.003), hepatic iron concentration (r = 0.61, P = 0.025) and hepatic iron index (r = 0.67, P = 0.01) were highly concordant in C282Y homozygotes. Heterozygote siblings were concordant for serum ferritin (r = 0.76, P = 0.0001) and transferrin saturation (r = 0.79, P = 0.0001). Homozygote-normal same-sex dizygotic twins were concordant for serum ferritin (r = 0.62, P = 0.0001) but not for transferrin saturation. Conclusions: Concordance of iron indices exists in C282Y homozygote and heterozygote sibling pairs. Siblings of expressing C282Y heterozygotes require phenotypic assessment. These data provide evidence for modifying genes influencing disease expression in hemochromatosis. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.