Diffuse idiopathic skeletal hyperostosis in elderly Icelanders and its association with the metabolic syndrome: the AGES-Reykjavik Study.

Objective: To describe the prevalence of diffuse idiopathic skeletal hyperostosis (DISH) in a large population-based study of elderly Icelanders, with particular reference to weight-related factors and the metabolic syndrome.Method: The study population comprised 5321 participants aged 68-96 years (2276 males, mean ± sd age 76 ± 5 , and 3045 females, age 77 ± 6) from the AGES-Reykjavik Study. DISH diagnosis was based on computed tomography (CT) scans, and interpreted strictly by the Resnick criteria and additional suggestions for CT interpretation by Oudkerk et al. Radiology readings were taken by a radiology resident and sample readings by two experienced radiologists.Results: A diagnosis of DISH was made in 13.7% of males and 2.8% of females. There was no association with age, but a strong association was seen with the metabolic syndrome [odds ratio (OR) 2.12, 95% confidence interval (CI) 1.69-2.64, p = 3.9 × 10-11]. Among the components of the metabolic syndrome, the association with DISH was significant for the insulin resistance criterion (OR 1.66, 95% CI 1.32-2.01, p < 0.001) and the body mass index (BMI) criterion (OR 2.16, 95% CI 1.70-2.74, p < 0.001). Other weight-related variables (midlife BMI, weight, and abdominal circumference) showed similar associations.Conclusions: This study, which to our knowledge is the largest published study on the prevalence of DISH, shows an association with the metabolic syndrome, particularly with the insulin resistance and BMI criteria. This is analogous with previous reports linking DISH with metabolic causes. In this age category, we did not observe any increase in prevalence with age.

Publisher Correction: A short isoform of ATG7 fails to lipidate LC3/GABARAP.

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A short isoform of ATG7 fails to lipidate LC3/GABARAP.

Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.

MODY in Iceland is associated with mutations in HNF-1alpha and a novel mutation in NeuroD1.

AIMS/HYPOTHESIS: Five different types of maturity-onset diabetes of the young (MODY) have been identified until now but mutation screening suggests that more MODY genes exist. Mutations in genes encoding transcription factors essential for normal development and function of pancreatic beta cells has recently become important in studying the genetics of Type II (non-insulin-dependent) diabetes mellitus. Patients with MODY and their families in Iceland were screened for mutations in the transcription factor genes. METHODS: Clinical and biochemical information on individuals with MODY was collected and their family trees constructed. Linkage analysis was carried out on chromosomal regions known to harbour genes previously shown to be associated with MODY. Mutations were identified by direct sequencing. RESULTS: Three families were identified. Two of these showed linkage to chromosome 12 and carried mutations in exon 4 of the HNF-1alpha gene (290fsdelC and R272C). However, the third family showed no linkage to the previously described MODY genes but shared a novel mutation in the NeuroD1 gene on chromosome 2q32. This mutation, a glutamate to lysine substitution at codon 110, resides in the basic domain of the protein. CONCLUSION/INTERPRETATION: Mutations in MODY subjects have been identified in the Icelandic population. In addition this study identified the NeuroD1 gene as the gene responsible for the sixth type of MODY.

Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus.

Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. Here we report the complete exon/intron structure of the mouse Mitf gene and show it to be similar to the human gene. We also found that the mouse gene is transcriptionally complex and is capable of generating at least 13 different Mitf isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia mutations. Two of the mutations are reported for the first time here (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws), Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data presented here, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal.

Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.

The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes.

The murine Bin1 gene functions early in myogenesis and defines a new region of synteny between mouse chromosome 18 and human chromosome 2.

We cloned and functionally characterized the murine Bin1 gene as a first step to investigate its physiological roles in differentiation, apoptosis, and tumorigenesis. The exon-intron organization of the >/=55-kb gene is similar to that of the human gene. Consistent with a role for Bin1 in apoptosis, the promoter included a functional consensus motif for activation by NF-kappaB, an important regulator of cell death. A muscle regulatory module defined in the human promoter that includes a consensus recognition site for myoD family proteins was not conserved in the mouse promoter. However, Bin1 is upregulated in embryonic development by E10.5 in myotomes, the progenitors of skeletal muscle, supporting a role in myogenesis and suggesting that the mouse and human genes may be controlled somewhat differently during development. In C2C12 myoblasts antisense Bin1 prevents induction of the cell cycle kinase inhibitor p21WAF1, suggesting that it acts at an early time during the muscle differentiation program. Interspecific mouse backcross mapping located the Bin1 locus between Mep1b and Apc on chromosome 18. Since the human gene was mapped previously to chromosome 2q14, the location of Bin1 defines a previously unrecognized region of synteny between human chromosome 2 and mouse chromosome 18.

The bHLH-Zip transcription factor Tfeb is essential for placental vascularization.

Tfeb is a member of the basic Helix-Loop-Helix-Zipper family of transcription factors. In vitro studies have shown that TFEB can bind DNA as a homodimer or as a heterodimer with three closely related family members: MITF, TFE3 and TFEC. While mutations of Mitf have been shown to affect the development of a number of cell types including melanocytes, osteoclasts, and masts cells, little is known about the phenotypic consequences of mutations at Tfe3, Tfeb and Tfec. Here we show that mice with a targeted disruption of Tfeb die between 9.5 and 10.5 days in embryonic development and have severe defects in placental vascularization. Tfeb is expressed at low levels in the embryo but at high levels in the labyrinthine trophoblast cells of the placenta. While labyrinthine cells are present in the mutant Tfeb placenta, they fail to express VEGF, a potent mitogen required for normal vasculogenesis of the embryo and extraembryonic tissues. In Tfeb mutant embryos the embryonic vasculature forms normally but few vessels are seen entering the placenta and those that do enter fail to thrive and branch normally. Our results indicate that Tfeb plays a critical role in the signal transduction processes required for normal vascularization of the placenta.

NEUROD2 and NEUROD3 genes map to human chromosomes 17q12 and 5q23-q31 and mouse chromosomes 11 and 13, respectively.

NEUROD2 and NEUROD3 are transcription factors involved in neurogenesis that are related to the basic helix-loop-helix protein NEUROD. NEUROD2 maps to human chromosome 17q12 and mouse chromosome 11. NEUROD3 maps to human chromosome 5q23-q31 and mouse chromosome 13.

Cloning and characterization of two vertebrate homologs of the Drosophila eyes absent gene.

The Drosophila eyes absent (eya) gene plays an essential role in the events that lead to proper development of the fly eye and embryo. Here we report the analysis of two human and two mouse homologs of the fly eya gene. Sequence comparison reveals a large domain of approximately 270 amino acids in the carboxyl terminus of the predicted mammalian proteins that shows 53% identity between the fly sequence and all of the vertebrate homologs. This Eya-homology domain is of novel sequence, with no previously identified motifs. RNA hybridization studies indicate that the mouse genes are expressed during embryogenesis and in select tissues of the adult. Both mouse Eya genes are expressed in the eye, suggesting that these genes may function in eye development in vertebrates as eya does in the fly. The mouse Eya2 gene maps to chromosome 2 in the region syntenic with human chromosome 20q13, and the mouse Eya2 gene maps to chromosome 4 in the region syntenic with human chromosome 1p36. Our findings support the notion that several families of genes (Pax-6/eyeless, Six-3/sine oculis, and Eya) play related and critical roles in the eye for both files and vertebrates.

The semidominant Mi(b) mutation identifies a role for the HLH domain in DNA binding in addition to its role in protein dimerization.

The mouse microphthalmia (mi) locus encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor called MITF (microphthalmia transcription factor). Mutations at mi affect the development of several different cell types, including melanocytes, mast cells, osteoclasts and pigmented epithelial cells of the eye. Here we describe the phenotypic and molecular characterization of the semidominant Microphthalmia(brwnish) (Mi(b)) mutation. We show that this mutation primarily affects melanocytes and produces retinal degeneration. The mutation is a G to A transition leading to a Gly244Glu substitution in helix 2 of the HLH dimerization domain. This location is surprising since other semidominant mi mutations characterized to date have been shown to affect DNA binding or transcriptional activation domains of MITF and act as dominant negatives, while mutations that affect MITF dimerization are inherited recessively. Gel retardation assays showed that while the mutant MITF(Mi-b) protein retains its dimerization potential, it is defective in its ability to bind DNA. Computer modeling suggested that the Gly244Glu mutation might disrupt DNA binding by interfering with productive docking of the protein dimer onto DNA. The Mi(b) mutation therefore appears to dissociate a DNA recognition function of the HLH domain from its role in protein dimerization.

The NEUROD gene maps to human chromosome 2q32 and mouse chromosome 2.

The Neurod gene is a basic-helix-loop-helix gene that regulates neurogenesis and is identical to the hamster beta2 gene that was cloned as a regulator of insulin transcription. Here we report the cloning of human NEUROD and mapping of the gene to human chromosome 2q32 and to mouse chromosome 2.

Mild osteopetrosis in the microphthalmia-oak ridge mouse. A model for intermediate autosomal recessive osteopetrosis in humans.

Mutations at the mouse microphthalmia (mi) locus affect coat color, eye development, and mast cells. The original allele, mi, also shows severe osteopetrosis. Mice homozygous for the microphthalmia-Oak Ridge (Mior) mutation are white, microphthalmic animals with retarded incisor development. To investigate whether this mutation causes osteopetrosis, we examined skeletal tissues of the Mior mouse. A typical osteopetrotic lesion, accumulation of unresorbed primary spongiosa, was found at the metaphyses of long bones and at the costochondral junctions in Mior/Mior mice from 10 days to 37 days of age, whereas no accumulation was seen at the mid-diaphyses in these bones. The osteopetrotic conditions of Mior/Mior mice increased progressively during the first 5 weeks after birth. However, adult Mior/Mior mice 3 months or older showed improvement of the osteopetrotic condition, although the disease was not completely resolved. Ultrastructurally, osteoclasts of Mior/Mior mice had well developed ruffled borders. These results show that the Mior mutation has milder osteopetrotic changes than the original mi mutation, a surprising observation given that both mutations affect the same functional domain of the mi protein, a basic-Helix-Loop-Helix-Zipper transcription factor. The Mior phenotype resembles the intermediate autosomal recessive osteopetrosis in humans.

Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.

The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element.

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.

Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells.

Murine chromosomal location of five bHLH-Zip transcription factor genes.

The genes for the bHLH-Zip transcription factors Tfap4, Mxi1, Tcfeb, Usf1, and Usf2 have been mapped in mouse by interspecific backcross analysis. Mxi1, Usf1, and Usf2 have been mapped previously by in situ hybridization, but their positions on the meiotic linkage map had not been determined. The other two genes have not previously been mapped in mouse. These transcription factors belong to a growing family of transcriptional regulators, some of which are known to form a complex network of interacting proteins that control cell proliferation and apoptosis. As expected, based on mapping studies of other bHLH-Zip genes, these loci were well distributed among mouse chromosomes. In addition, some of the probes used in this study detected multiple, independently segregating loci, suggesting the possible existence of additional family members or species-specific pseudogenes.

Meso1, a basic-helix-loop-helix protein involved in mammalian presomitic mesoderm development.

To identify genes involved in the regulation of early mammalian development, we have developed a dominant-negative mutant basic-helix-loop-helix (bHLH) protein probe for interaction cloning and have isolated a member of the bHLH family of transcription factors, Meso1. Meso1-E2A heterodimers are capable of binding to oligonucleotide probes that contain a bHLH DNA recognition motif. In mouse embryos, Meso1 is expressed prior to MyoD1 family members. Meso1 expression is first detected at the neural plate stage of development in the paraxial mesoderm of the head and in presomitic mesodermal cells prior to their condensation into somites. Our findings suggest that Meso1 may be a key regulatory gene involved in the early events of vertebrate mesoderm differentiation.