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The AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by sensing the metabolic status of the cell. AMPK is regulated by phosphorylation and dephosphorylation as a result of changing AMP/ATP levels and by removal of inhibitory ubiquitin residues by USP10. In this context, we identified the GID-complex, an evolutionarily conserved ubiquitin-ligase-complex (E3), as a negative regulator of AMPK activity. Our data show that the GID-complex targets AMPK for ubiquitination thereby altering its activity. Cells depleted of GID-subunits mimic a state of starvation as shown by increased AMPK activity and macroautophagic/autophagic flux as well as reduced MTOR activation. Consistently, gid-genes knockdown in C. elegans results in increased organismal lifespan. This study may contribute to understand metabolic disorders such as type 2 diabetes mellitus and morbid obesity and implements alternative therapeutic approaches to alter AMPK activity. Abbreviations ACTB: actin, beta; ADP: adenosine diphosphate; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ARMC8: armadillo repeat containing 8; ATP: adenosine triphosphate; BafA1: bafilomycin A1; BCAA: branched chain amino acid; BICC1: BicC family RNA binding protein 1; BSA: bovine serum albumin; CAMKK2 kinase: calcium/calmodulin dependent protein kinase kinase 2, beta; CHX: cycloheximide; DMEM: Dulbecco’s modified Eagle’s medium; E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3: ubiquitin ligase; ECAR: extracellular acidification rate; FACS: fluorescent associated cell sorter; FBP1: fructose-bisphosphatase 1; FCCP: carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; G6P: glucose-6-phosphate; GDP: guanosine diphosphate; GFP: green fluorescent protein; GID: glucose induced degradation deficient; GMP: guanosine monophosphate; GTP: guanosine triphosphate; HBP1: high mobility group box transcription factor 1; HPRT: hypoxanthine guanine phosphoribosyl transferase; KO: knock out; LE: long exposure; MAEA: macrophage erythroblast attacher; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MKLN1: muskelin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin; NES: normalized enrichment score; OCR: oxygen consumption rate; PBS: phosphate buffered saline; PCK1: phosphoenolpyruvate carboxykinase 1, cytosolic; PCR: polymerase chain reaction; PFA: paraformaldehyde; RANBP9: RAN binding protein 9; RING: really interesting new gene; RMND5: required for meiotic nuclear division5 homolog; RPS6: ribosomal protein S6; RPTOR: regulatory associated protein of MTOR, complex 1; SE: short exposure; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TSC2: tuberous sclerosis complex 2; TUBA4A: tubulin; TUBE: tandem ubiquitin binding entities; Ub: ubiquitin; UPS: ubiquitin proteasome system; WDR26: WD repeat domain 26; WT: wild type.

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.

Whole-exome sequencing allows for an unbiased and comprehensive mutation screening. Although successfully used to facilitate the diagnosis of single-gene disorders, the genetic cause(s) of a substantial proportion of presumed monogenic diseases remain to be identified. We used whole-exome sequencing to examine offspring from a consanguineous marriage featuring a novel combination of congenital hypothyroidism, hypomagnesemia and hypercholesterolemia. Rather than identifying one causative variant, we report the first instance in which three independent autosomal-recessive single-gene disorders were identified in one patient. Together, the causal variants give rise to a blended and seemingly novel phenotype: we experimentally characterized a novel splice variant in the thyroglobulin gene (c.638+5G>A), resulting in skipping of exon 5, and detected a pathogenic splice variant in the magnesium transporter gene TRPM6 (c.2667+1G>A), causing familial hypomagnesemia. Based on the third variant, a stop variant in ABCG5 (p.(Arg446*)), we established a diagnosis of sitosterolemia, confirmed by elevated blood plant sterol levels and successfully initiated targeted lipid-lowering treatment. We propose that blended phenotypes resulting from several concomitant single-gene disorders in the same patient likely account for a proportion of presumed monogenic disorders of currently unknown cause and contribute to variable genotype-phenotype correlations.

Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC-with presumably disturbed cornea growth and differentiation-contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injuries.

Deletions of the chromosomal region 2q37 cause brachydactyly-mental retardation syndrome (BDMR), also known as Albright hereditary osteodystrophy-like syndrome. Recently, histone deacetylase 4 (HDAC4) haploinsufficiency has been postulated to be the critical genetic mechanism responsible for the main clinical characteristics of the BDMR syndrome like developmental delay and behavioural abnormalities in combination with brachydactyly type E (BDE). We report here on the first three generation familial case of BDMR syndrome with inheritance of an interstitial microdeletion of chromosome 2q37.3. The deletion was detected by array comparative genomic hybridization and comprises the HDAC4 gene and two other genes. The patients of this pedigree show a variable severity of psychomotor and behavioural abnormalities in combination with a specific facial dysmorphism but without BDE. Given that only about half of the patients with 2q37 deletions have BDE; we compared our patients with other patients carrying 2q37.3 deletions or HDAC4 mutations known from the literature to discuss the diagnostic relevance of the facial dysmorphism pattern in 2q37.3 deletion cases involving the HDAC4 gene. We conclude that HDAC4 haploinsufficiency is responsible for psychomotor and behavioural abnormalities in combination with the BDMR syndrome-specific facial dysmorphism pattern and that these clinical features have a central diagnostic relevance.European Journal of Human Genetics advance online publication, 28 November 2012; doi:10.1038/ejhg.2012.240.

BACKGROUND: Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder characterised by hypoplastic or absent clavicles, increased head circumference, large fontanels, dental anomalies and short stature. Although CCD is usually caused by mutations leading to haploinsufficiency of RUNX2, the underlying genetic cause remains unresolved in about 25% of cases. METHODS: Array comparative genomic hybridisation was performed to detect copy number variations (CNVs). Identified CNVs were characterised by quantitative PCR and sequencing analyses. The effect of candidate genes on mineralisation was evaluated using viral overexpression in chicken cells. RESULTS: In 2 out of 16 cases, the authors identified microduplications upstream of MSX2 on chromosome 5q35.2. One of the unrelated affected individuals presented with a phenocopy of CCD. In addition to a classical CCD phenotype, the other subject had a complex synpolydactyly of the hands and postaxial polydactyly of the feet which have so far never been reported in association with CCD or CNVs on 5q35.2. The duplications overlap in an approximately 219 kb region that contains several highly conserved non-coding elements which are likely to be involved in MSX2 gene regulation. Functional analyses demonstrated that the inhibitory effect of Msx2 overexpression on mineralisation cannot be ameliorated by forced Runx2 expression. CONCLUSIONS: These results indicate that CNVs in non-coding regions can cause developmental defects, and that the resulting phenotype can be distinct from those caused by point mutations within the corresponding gene. Taken together, these findings reveal an additional mechanism for the pathogenesis of CCD, particularly with regard to the regulation of MSX2.

The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11–/–Hoxd12–/–Hoxd13–/– triple-knockout mice and Hoxd13–/–Hoxa13+/– mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl- D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl- D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates beta(1)-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.

Mental retardation affects 2-3% of the population and shows a high heritability. Neurodevelopmental disorders that include pronounced impairment in language and speech skills occur less frequently. For most cases, the molecular basis of mental retardation with or without speech and language disorder is unknown due to the heterogeneity of underlying genetic factors. We have used molecular karyotyping on 1523 patients with mental retardation to detect copy number variations (CNVs) including deletions or duplications. These studies revealed three heterozygous overlapping deletions solely affecting the forkhead box P1 (FOXP1) gene. All three patients had moderate mental retardation and significant language and speech deficits. Since our results are consistent with a de novo occurrence of these deletions, we considered them as causal although we detected a single large deletion including FOXP1 and additional genes in 4104 ancestrally matched controls. These findings are of interest with regard to the structural and functional relationship between FOXP1 and FOXP2. Mutations in FOXP2 have been previously related to monogenic cases of developmental verbal dyspraxia. Both FOXP1 and FOXP2 are expressed in songbird and human brain regions that are important for the developmental processes that culminate in speech and language. (C) 2010 Wiley-Liss, Inc

Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels.

In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.