Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.     

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).     

Mutations in LMF1 cause combined lipase deficiency and severe hypertriglyceridemia

Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.     

The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides

Bitter taste generally causes aversion, which protects humans from ingesting toxic substances. But bitter flavors also contribute to the palatability of food and beverages, thereby influencing nutritional habits in humans. Although many studies have examined bitter taste, the underlying receptor mechanisms remain poorly understood. Anatomical, functional and genetic data from rodents suggest the existence of a family of receptors that are responsive to bitter compounds. Here we report that a human member of this family, TAS2R16, is present in taste receptor cells on the tongue and is activated by bitter beta-glucopyranosides. Responses to these phytonutrients show a similar concentration dependence and desensitization in transfected cells and in experiments assessing taste perception in humans. Bitter compounds consisting of a hydrophobic residue attached to glucose by a beta-glycosidic bond activate TAS2R16. Thus, TAS2R16 links the recognition of a specific chemical structure to the perception of bitter taste. If the ability of TAS2R16 to detect substances with common molecular properties is typical of the bitter receptor family, it may explain how a few receptors permit the perception of numerous bitter substances.     

Fras1 deficiency results in cryptophthalmos,renal agenesis and blebbed phenotype in mice

Loss of tight association between epidermis and dermis underlies several blistering disorders and is frequently caused by impaired function of extracellular matrix (ECM) proteins. Here we describe a new protein in mouse, Fras1, that is specifically detected in a linear fashion underlying the epidermis and the basal surface of other epithelia in embryos. Loss of Fras1 function results in the formation of subepidermal hemorrhagic blisters as well as unilateral or bilateral renal agenesis during mouse embryogenesis. Postnatally, homozygous Fras1 mutants have fusion of the eyelids and digits and unilateral renal agenesis or dysplasia. The defects observed in Fras1-/- mice phenocopy those of the existing bl (blebbed) mouse mutants, which have been considered a model for the human genetic disorder Fraser syndrome. We show that bl/bl homozygous embryos are devoid of Fras1 protein, consistent with the finding that Fras1 is mutated in these mice. In sum, our data suggest that perturbations in the composition of the extracellular space underlying epithelia could account for the onset of the blebbed phenotype in mouse and Fraser syndrome manifestation in human.     

Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.     

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.     

Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1

Genomic imprinting is an epigenetic modification that results in expression from only one of the two parental copies of a gene. Differences in methylation between the two parental chromosomes are often observed at or near imprinted genes. Beckwith-Wiedemann syndrome (BWS), which predisposes to cancer and excessive growth, results from a disruption of imprinted gene expression in chromosome band 11p15.5. One third of individuals with BWS lose maternal-specific methylation at KvDMR1, a putative imprinting control region within intron 10 of the KCNQ1 gene, and it has been proposed that this epimutation results in aberrant imprinting and, consequently, BWS1, 2. Here we show that paternal inheritance of a deletion of KvDMR1 results in the de-repression in cis of six genes, including Cdkn1c, which encodes cyclin-dependent kinase inhibitor 1C. Furthermore, fetuses and adult mice that inherited the deletion from their fathers were 20-25% smaller than their wildtype littermates. By contrast, maternal inheritance of this deletion had no effect on imprinted gene expression or growth. Thus, the unmethylated paternal KvDMR1 allele regulates imprinted expression by silencing genes on the paternal chromosome. These findings support the hypothesis that loss of methylation in BWS patients activates the repressive function of KvDMR1 on the maternal chromosome, resulting in abnormal silencing of CDKN1C and the development of BWS.     

Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot-Marie-Tooth disease type 4A/8q21.

We previously localized and fine-mapped Charcot Marie Tooth 4A (CMT4A), the autosomal recessive, demyelinating peripheral neuropathy, to chromosome 8. Through additional positional cloning, we have identified a good candidate gene, encoding ganglioside-induced differentiation-associated protein-1 (GDAP1). We found three different mutations in four different Tunisian families-two nonsense and one missense mutation. How mutations in GDAP1 lead to CMT4A remains to be understood.     

The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a DNA duplication at chromosome 17p11.2. In view of the point mutation in the gene for peripheral myelin protein pmp-22/gas-3 in Trembler mice, a murine model for CMT1A, we have analysed whether this gene is altered in CMT1A. Here we show that the human homologue of the murine pmp-22 gene is located within the CMT1A DNA duplication, which is a direct repeat and does not interrupt the coding region of PMP-22. Expression of PMP-22 in CMT1A fibroblasts is similar to expression in control fibroblasts. Increased gene dosage or altered PMP-22 expression in the peripheral nervous system are therefore possible mechanisms by which PMP-22 is involved in CMT1A.     

Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.     

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.