Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.     

Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia

Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity—a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development.     

Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.     

X-linked congenital ptosis and associated intellectual disability,short stature,microcephaly, cleft palate,digital and genital abnormalities define novel Xq25q26 duplication syndrome

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.     

Pure interstitial duplication of chromosome 7q (7q31.2-->q33) in a 4-year-old girl with growth restriction, short stature, speech delay and intellectual disability

We report the cytogenetic and molecular characterization of a 22.3-Mb pure interstitial duplication of chromosome 7q, dup(7)(q31.2-->q33) in a 4-year-old girl with growth restriction, short stature, speech delay, inguinal hernia, strabismus and intellectual disability. We speculate that the gene dosage increase effect of the ING3 and LEP genes may be partially responsible for the phenotype of growth restriction and short stature in this patient.     

The hereditary spastic paraplegia protein strumpellin: Characterisation in neurons and of the effect of disease mutations on WASH complex assembly and function

Mutations in the gene encoding strumpellin cause autosomal dominant hereditary spastic paraplegia (HSP), in which there is degeneration of corticospinal tract axons. Strumpellin is a component of the WASH complex, an actin-regulating complex that is recruited to endosomes by interactions with the retromer complex. The WASH complex and its relationship to retromer have not been fully characterised in neurons, and the molecular pathological mechanism of strumpellin mutation is unclear. Here we demonstrate that the WASH complex assembles in the brain, where it interacts with retromer. Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments. We show that strumpellin is not required for normal transferrin receptor traffic, but is required for the correct subcellular distribution of the β-2-adrenergic receptor. However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation. Models of the WASH complex indicate that it contains a single strumpellin molecule, so in patients with strumpellin mutations, complexes containing wild-type and mutant strumpellin should be present in equal numbers. In most cell types this would provide sufficient functional WASH to allow normal cellular physiology. However, owing to the demands on membrane traffic imposed by their exceptionally long axons, we suggest that corticospinal neurons are especially vulnerable to reductions in functional WASH.     

CC2D2A, encoding a coiled-coil and C2 domain protein, causes autosomal-recessive mental retardation with retinitis pigmentosa

Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca(2+)-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR.     

Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein

The identification of SPG7 as the gene defective in a recessive form of spastic paraplegia has drawn attention to the yeast protein family of ATP-dependent zinc metalloproteases. The protein encoded by SPG7, paraplegin, shows high homology to members of this protein family. Recently, many mammalian ATP-dependent zinc metalloproteases have been identified and considered as possible candidates for defects in other forms of hereditary spastic paraplegia and possibly other neurodegenerative disorders. So far only a partial sequence has been available for one of those genes, ATPase family gene-3, yeast-like-1 (AFG3L1). We have carried out detailed molecular analysis of this gene and identified and characterized its mouse orthologue, Afg3l1. Our data indicate that AFG3L1 is transcribed into four mRNA isoforms that are not translated in humans. Afg3l1 encodes a protein with high homology to paraplegin and the other members of the ATP-dependent zinc metalloprotease family. Like the other ATP-dependent zinc metalloproteases, Afg3l1 localizes to the mitochondria.     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Missense mutation of SPAST protein (I344K) results in loss of ATPase activity and prolonged the half-life,implicated in autosomal dominant hereditary spastic paraplegia

The spastin protein (SPAST) contains an ATPase with diverse cellular activities (AAA) domain and regulates microtubule dynamics. Missense mutations of the SPAST gene are frequently detected in patients with hereditary spastic paraplegias (HSPs) and represent the main reason of loss of SPAST function; however, the pathogenicity of mutant SPAST is heterogeneous. Here, SPAST variant with an I344K mutation (I344K-SPAST) was identified in a Korean family with autosomal dominant-type HSP. We investigated the role of the I344K-SPAST in HSP to provide a therapeutic mechanism. The I344K-SPAST mutation prolonged the half-life of the protein compared to wild-type SPAST (WT-SPAST) in cells by modulating post-translational modifications for proteasomal degradation. I344K-SPAST was localized in microtubule but defective in microtubule severing and ATPase activity compared to WT-SPAST in vitro and in cells. Mutant M87 isoform harboring the same mutation with I344K-M1 SPAST also increased protein stability and loss of MT severing activity, but the pathogenicity was not stronger than I344K-M1 SPAST in neurite outgrowth. Overexpression of I344K-SPAST resulted in microtubule accumulation following inhibited neurite growth in neuroblastoma, neural progenitor cells and mouse primary cortical neurons. Conversely, these pathogenic effects of I344K-SPAST were reduced by overexpression of WT-M1 SPAST in a dose dependent manner since WT-SPAST could interact with I344K-SPAST. Our data therefore provide proof-of-concept that gene transfer of WT-M1 SPAST may serve as a valid therapeutic option for HSPs.     

The AtRbx1 protein is part of plant SCF complexes,and its down-regulation causes severe growth and developmental defects

Recently in yeast and animal cells, one particular class of ubiquitin ligase (E3), called the SCF, was demonstrated to regulate diverse processes including cell cycle and development. In plants SCF-dependent proteolysis is also involved in different developmental and hormonal regulations. To further investigate the function of SCF, we characterized at the molecular level the Arabidopsis RING-H2 finger protein AtRbx1. We demonstrated that the plant gene is able to functionally complement a yeast knockout mutant strain and showed that AtRbx1 protein interacts physically with at least two members of the Arabidopsis cullin family (AtCul1 and AtCul4). AtRbx1 also associates with AtCul1 and the Arabidopsis SKP1-related proteins in planta, indicating that it is part of plant SCF complexes. AtRbx1 mRNAs accumulate in various tissues of the plant, but at higher levels in tissues containing actively dividing cells. Finally to study the function of the gene in planta, we either overexpressed AtRbx1 or reduced its expression by a dsRNA strategy. Down-regulation of AtRbx1 impaired seedling growth and development, indicating that the gene is essential in plants. Furthermore, the AtRbx1-silenced plants showed a reduced level of AtCul1 protein, but accumulated higher level of cyclin D3.     

8.6Mb interstitial deletion of chromosome 4q13.3q21.23 in a boy with cognitive impairment, short stature, hearing loss, skeletal abnormalities and facial dysmorphism

We describe a 16-year-old boy with an 8.6Mb interstitial deletion of chromosome 4q 13.3q21.23 identified by oligo array-CGH. The patient presents psychomotor developmental delay, absent speech, marked progressive growth restriction, hearing loss, skeletal defects and minor facial anomalies. The patient required surgical treatment for cleft lip and palate, bilateral cryptorchidism and a neurofibroma. The analysis of the presented patient against previously published cases allowed us to expand further on the phenotype and to reevaluate previously proposed critical overlapping region at 4q21. As an addition to PRKG2 and RASGEFIB genes, we propose to include BMP3 gene as the principal determinant of the observed common phenotype. BMP3 haploinsufficiency appears to be causative of hearing loss and peculiar skeletal abnormalities including hemivertebrae and brachydactyly.     

Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin

Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.     

Recessive <Emphasis Type="Italic">VARS2</Emphasis> mutation underlies a novel syndrome with epilepsy,mental retardation,short stature,growth hormone deficiency,and hypogonadism

Background

Most mitochondrial and cytoplasmic aminoacyl-tRNA synthetases (aaRSs) are encoded by nuclear genes. Syndromic disorders resulting from mutation of aaRSs genes display significant phenotypic heterogeneity. We expand aaRSs-related phenotypes through characterization of the clinical and molecular basis of a novel autosomal-recessive syndrome manifesting severe mental retardation, ataxia, speech impairment, epilepsy, short stature, microcephaly, hypogonadism, and growth hormone deficiency.

Results

A G>A variant in exon 29 of VARS2 (c.3650G>A) (NM_006295) was identified in the index case. This homozygous variant was confirmed by Sanger sequencing and segregated with disease in the family studied. The c.3650G>A change results in alteration of arginine to histidine at residue 1217 (R1217H) of the mature protein and is predicted to be pathogenic.

Conclusions

These findings contribute to a growing list of aaRSs disorders, broadens the spectrum of phenotypes attributable to VARS2 mutations, and provides new insight into genotype-phenotype correlations among the mitochondrial synthetase genes.