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1.  Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content 
Human molecular genetics  2007;16(22):2720-2728.
Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disorder characterized by death of motoneurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the mechanisms whereby they induce disease are not fully understood. Here, we use time-lapse microscopy to monitor for the first time the effect of mutant SOD1 on fast axonal transport (FAT) of bona fide cargoes in living neurons. We analyzed FAT of mitochondria that are a known target for damage by mutant SOD1 and also of membrane-bound organelles (MBOs) using EGFP-tagged amyloid precursor protein as a marker. We studied FAT in motor neurons derived from SOD1G93A transgenic mice that are a model of ALS and also in cortical neurons transfected with SOD1G93A and three further ALS-associated SOD1 mutants. We find that mutant SOD1 damages transport of both mitochondria and MBOs, and that the precise details of this damage are cargo-specific. Thus, mutant SOD1 reduces transport of MBOs in both anterograde and retrograde directions, whereas mitochondrial transport is selectively reduced in the anterograde direction. Analyses of the characteristics of mitochondrial FAT revealed that reduced anterograde movement involved defects in anterograde motor function. The selective inhibition of anterograde mitochondrial FAT enhanced their net retrograde movement to deplete mitochondria in axons. Mitochondria in mutant SOD1 expressing cells also displayed features of damage. Together, such changes to mitochondrial function and distribution are likely to compromise axonal function. These alterations represent some of the earliest pathological features so far reported in neurons of mutant SOD1 transgenic mice.
doi:10.1093/hmg/ddm226
PMCID: PMC4516806  PMID: 17725983
2.  Mutation in the splicing factor Hprp3p linked to retinitis pigmentosa impairs interactions within the U4/U6 snRNP complex 
Human molecular genetics  2007;17(2):225-239.
Mutations in PRPF3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (RP18). Patients with RP18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p C-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked RP families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (CKII). To understand the molecular basis of RP18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of PRPF3 mRNA. We found that Thr494Met does not affect PRPF3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous PRPF3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the C-but not N-terminal region of Hprp3p is indeed phosphorylated by CKII in vitro and in cells. CKII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. Consequently, the Hprp3p C-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the RP18 phenotype.
doi:10.1093/hmg/ddm300
PMCID: PMC4494837  PMID: 17932117 CAMSID: cams268
3.  Stem Cells and Regeneration: Special Review Issue 
Human molecular genetics  2008;17(0):R1-R2.
doi:10.1093/hmg/ddn186
PMCID: PMC4489689  PMID: 18632689
4.  Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis 
Human molecular genetics  2007;16(9):1091-1097.
Coenzyme Q10 (CoQ10) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ10 supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ10 deficiency, a homozygous mutation was identified in the CoQ10 biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ6 concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33–45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ10 supplementation, and growth rate was restored in these cells by either CoQ10 or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ10 deficiency. Moreover, we demonstrate that CoQ10 deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.
doi:10.1093/hmg/ddm058
PMCID: PMC4345105  PMID: 17374725
5.  [No title available] 
PMCID: PMC3888265  PMID: 24057672
6.  Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability 
Human molecular genetics  2014;23(24):6432-6440.
Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r2 = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r2 = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.
doi:10.1093/hmg/ddu360
PMCID: PMC4240195  PMID: 25015100
7.  Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies 
Human molecular genetics  2014;23(23):6139-6146.
Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson’s and Alzheimer’s diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.
doi:10.1093/hmg/ddu334
PMCID: PMC4222357  PMID: 24973356
8.  Genetic comorbidities in Parkinson’s disease 
Human molecular genetics  2013;23(3):831-841.
Parkinson’s disease (PD) has a number of known genetic risk factors. Clinical and epidemiological studies have suggested the existence of intermediate factors that may be associated with additional risk of PD. We construct genetic risk profiles for additional epidemiological and clinical factors using known genome-wide association studies (GWAS) loci related to these specific phenotypes to estimate genetic comorbidity in a systematic review. We identify genetic risk profiles based on GWAS variants associated with schizophrenia and Crohn’s disease as significantly associated with risk of PD. Conditional analyses adjusting for SNPs near loci associated with PD and schizophrenia or PD and Crohn’s disease suggest that spatially overlapping loci associated with schizophrenia and PD account for most of the shared comorbidity, while variation outside of known proximal loci shared by PD and Crohn’s disease accounts for their shared genetic comorbidity. We examine brain methylation and expression signatures proximal to schizophrenia and Crohn’s disease loci to infer functional changes in the brain associated with the variants contributing to genetic comorbidity. We compare our results with a systematic review of epidemiological literature, while the findings are dissimilar to a degree; marginal genetic associations corroborate the directionality of associations across genetic and epidemiological data. We show a strong genetically defined level of comorbidity between PD and Crohn’s disease as well as between PD and schizophrenia, with likely functional consequences of associated variants occurring in brain.
doi:10.1093/hmg/ddt465
PMCID: PMC3888265  PMID: 24057672
9.  Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations suggests distinct pathophysiological mechanisms for craniofacial and limb abnormalities 
Human molecular genetics  2004;13(19):2313-2324.
Gain-of-function missense mutations in FGF receptor 2 (FGFR2) are responsible for a variety of craniosynostosis syndromes including Apert syndrome (AS), Pfeiffer syndrome (PS) and Crouzon syndrome (CS). Unlike the majority of FGFR2 mutations, S252W and P253R AS mutations and a D321A PS mutation retain ligand-dependency and are also associated with severe limb pathology. In addition, a recently identified ligand-dependent S252L/A315S double mutation in FGFR2 was shown to cause syndactyly in the absence of craniosynostosis. Here, we analyze the effect of the canonical AS mutations, the D321A PS mutation and the S252L/A315S double mutation on FGFR2 ligand binding affinity and specificity using surface plasmon resonance. Both AS mutations and the D321A PS mutation, but not the S252L/A315S double mutation, increase the binding affinity of FGFR2c to multiple FGFs expressed in the cranial suture. Additionally, all four pathogenic mutations also violate FGFR2c ligand binding specificity and enable this receptor to bind FGF10. Based on our data, we propose that an increase in mutant FGFR2c binding to multiple FGFs results in craniosynostosis, whereas binding of mutant FGFR2c to FGF10 results in severe limb pathology. Structural and biophysical analysis shows that AS mutations in FGFR2b also enhance and violate FGFR2b ligand binding affinity and specificity, respectively. We suggest that elevated AS mutant FGFR2b signaling may account for the dermatological manifestations of AS.
doi:10.1093/hmg/ddh235
PMCID: PMC4140565  PMID: 15282208
10.  Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability 
Human molecular genetics  2014;23(15):4015-4023.
We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development.
doi:10.1093/hmg/ddu115
PMCID: PMC4082365  PMID: 24626631
11.  Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability 
Human Molecular Genetics  2014;23(24):6432-6440.
Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r2 = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r2 = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.
doi:10.1093/hmg/ddu360
PMCID: PMC4240195  PMID: 25015100
12.  Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies 
Human Molecular Genetics  2014;23(23):6139-6146.
Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson's and Alzheimer's diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.
doi:10.1093/hmg/ddu334
PMCID: PMC4222357  PMID: 24973356
13.  Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability 
Human Molecular Genetics  2014;23(15):4015-4023.
We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development.
doi:10.1093/hmg/ddu115
PMCID: PMC4082365  PMID: 24626631
14.  The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region 
Human molecular genetics  2009;18(22):10.1093/hmg/ddp379.
Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 contains two clusters of imprinted genes within a ~1 Mb domain. This region is conserved on human 11q15.5 where it is implicated in the Beckwith-Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this ~280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del7AI, is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del7AI demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.
doi:10.1093/hmg/ddp379
PMCID: PMC3817080  PMID: 19684026 CAMSID: cams3629
15.  Pathogenic NPHP5 mutations impair protein interaction with Cep290, a prerequisite for ciliogenesis 
Human molecular genetics  2013;22(12):2482-2494.
Mutations in the human NPHP5 gene cause retinal and renal disease but the precise mechanisms by which NPHP5 functions are not understood. We report that NPHP5 is a centriolar protein whose depletion inhibits an early step of ciliogenesis, a phenotype reminiscent of Cep290 loss and contrary to IFT88 loss. Functional dissection of NPHP5 interactions with Cep290 and CaM reveals a requirement of the former for ciliogenesis, while the latter prevents NPHP5 self-aggregation. Disease-causing mutations lead to truncated products unable to bind Cep290 and localize to centrosomes, thereby compromising cilia formation. In contrast, a modifier mutation cripples CaM-binding but has no overt effect on ciliogenesis. Drugs that antagonize negative regulators of the ciliogenic pathway can rescue ciliogenesis in cells depleted of NPHP5, with response profiles similar to those of Cep290- but not IFT88-depleted cells. Our results uncover the underlying molecular basis of disease and provide novel insights into mitigating NPHP5 deficiency.
doi:10.1093/hmg/ddt100
PMCID: PMC3797088  PMID: 23446637 CAMSID: cams3098
16.  A network of dopaminergic gene variations implicated as risk factors for schizophrenia 
Human molecular genetics  2007;17(5):747-758.
We evaluated the hypothesis that dopaminergic polymorphisms are risk factors for schizophrenia (SZ). In stage I, we screened 18 dopamine-related genes in two independent US Caucasian samples: 150 trios and 328 cases/501 controls. The most promising associations were detected with SLC6A3 (alias DAT), DRD3, COMT and SLC18A2 (alias VMAT2). In stage II, we comprehensively evaluated these four genes by genotyping 68 SNPs in all 478 cases and 501 controls from stage I. Fifteen (23.1%) significant associations were found (p ≤ 0.05). We sought epistasis between pairs of SNPs providing evidence of a main effect and observed 17 significant interactions (169 tests); 41.2% of significant interactions involved rs3756450 (5′ near promoter) or rs464049 (intron 4) at SLC6A3. In stage III, we confirmed our findings by genotyping 65 SNPs among 659 Bulgarian trios. Both SLC6A3 variants implicated in the US interactions were overtransmitted in this cohort (rs3756450, p = 0.035; rs464049, p = 0.011). Joint analyses from stages II and III identified associations at all four genes (pjoint < 0.05). We tested 29 putative interactions from stage II and detected replication between seven locus pairs (p ≤ 0.05). Simulations suggested our stage II and stage III interaction results were unlikely to have occurred by chance (p = 0.008 and 0.001, respectively). In stage IV we evaluasted rs464049 and rs3756450 for functional effects and found significant allele-specific differences at rs3756450 using electrophoretic mobility shift assays and dualluciferase promoter assays. Our data suggest that a network of dopaminergic polymorphisms increase risk for SZ.
doi:10.1093/hmg/ddm347
PMCID: PMC3777405  PMID: 18045777
17.  CRISPLD2: a novel NSCLP candidate gene 
Human molecular genetics  2007;16(18):2241-2248.
Non-syndromic cleft lip with or without cleft palate (NSCLP) results from the complex interaction between genes and environmental factors. Candidate gene analysis and genome scans have been employed to identify the genes contributing to NSCLP. In this study, we evaluated the 16q24.1 chromosomal region, which has been identified by multiple genome scans as an NSCLP region of interest. Two candidate genes were found in the region: interferon regulatory factor 8 (IRF8) and cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2). Initially, Caucasian and Hispanic NSCLP multiplex families and simplex parent–child trios were genotyped for single nucleotide polymorphisms (SNPs) in both IRF8 and CRISPLD2. CRISPLD2 was subsequently genotyped in a data set comprised of NSCLP families from Colombia, South America. Linkage disequilibrium analysis identified a significant association between CRISPLD2 and NSCLP in both our Caucasian and Hispanic NSCLP cohorts. SNP rs1546124 and haplotypes between rs1546124 and either rs4783099 or rs16974880 were significant in the Caucasian multiplex population (P = 0.01, P = 0.002 and P = 0.001, respectively). An altered transmission of CRISPLD2 SNPs rs8061351 (P = 0.02) and rs2326398 (P = 0.06) was detected in the Hispanic population. No association was found between CRISPLD2 and our Colombian population or IRF8 and NSCLP. In situ hybridization showed that CRISPLD2 is expressed in the mandible, palate and nasopharynx regions during craniofacial development at E13.5–E17.5, respectively. Altogether, these data suggest that genetic variation in CRISPLD2 has a role in the etiology of NSCLP.
doi:10.1093/hmg/ddm176
PMCID: PMC3755375  PMID: 17616516
18.  Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase 
Human molecular genetics  2006;15(18):2825-2835.
We applied genetic tools available in Drosophila to identify candidate substrates of the UBE3A ubiquitin ligase, the gene responsible for Angelman syndrome (AS). Human UBE3A was expressed in Drosophila heads to identify proteins differentially regulated in UBE3A-expressing versus wild-type extracts. Using two-dimensional gel and MALDI-TOF analysis, we detected 20 proteins that were differentially regulated by over-expression of human UBE3A in Drosophila heads. One protein responsive to UBE3A was the Rho-GEF pebble (pbl). Here, we present three lines of evidence suggesting that UBE3A regulates Pbl. First, we show genetic evidence that UBE3A and the Drosophila de-ubiquitinase fat facets (faf) exert opposing effects on Pbl function. Secondly, we find that both Pbl and ECT2, the mammalian orthologue of Pbl called epithelial cell transforming sequence 2 oncogene, physically interact with their respective ubiquitin E3 ligases. Finally, we show that Ect2 expression is regulated by Ube3a in mouse neurons as the pattern of Ect2 expression is dramatically altered in the hippocampus and cerebellum of Ube3a null mice. These results suggest that an orthologous UBE3A post-translational regulatory pathway regulates neuronal outgrowth in the mammalian brain and that dysregulation of this pathway may result in neurological phenotypes including AS and possibly other autism spectrum disorders.
doi:10.1093/hmg/ddl225
PMCID: PMC3742451  PMID: 16905559
19.  Association of IRF5 in UK SLE families identifies a variant involved in polyadenylation 
Human molecular genetics  2006;16(6):579-591.
Results from two studies have implicated the interferon regulatory gene IRF5 as a susceptibility gene in systemic lupus erythematosus (SLE). In this study, we conducted a family-based association analysis in 380 UK SLE nuclear families. Using a higher density of markers than has hitherto been screened, we show that there is association with two SNPs in the first intron, rs2004640 (P = 3.4 × 10−4) and rs3807306 (P = 4.9 × 10−4), and the association extends into the 3′-untranslated region (UTR). There is a single haplotype block encompassing IRF5 and we show for the first time that the gene comprises two over-transmitted haplotypes and a single under-transmitted haplotype. The strongest association is with a TCTAACT haplotype (T:U = 1.92, P = 5.8 × 10−5), which carries all the over-transmitted alleles from this study. Haplotypes carrying the T alleles of rs2004640 and rs2280714 and the A allele of rs10954213 are over-transmitted in SLE families. The TAT haplotype shows a dose-dependent relationship with mRNA expression. A differential expression pattern was seen between two expression probes located each side of rs10954213 in the 3′-UTR. rs10954213 shows the strongest association with RNA expression levels (P = 1 × 10−14). The A allele of rs10954213 creates a functional polyadenylation site and the A genotype correlates with increased expression of a transcript variant containing a shorter 3′-UTR. Expression levels of transcript variants with the shorter or longer 3′-UTRs are inversely correlated. Our data support a new mechanism by which an IRF5 polymorphism controls the expression of alternate transcript variants which may have different effects on interferon signalling.
doi:10.1093/hmg/ddl469
PMCID: PMC3706933  PMID: 17189288
20.  Polymorphism at the C-reactive protein locus influences gene expression and predisposes to systemic lupus erythematosus 
Human molecular genetics  2003;13(1):137-147.
Relative deficiency of pentraxin proteins is implicated in the pathogenesis of systemic lupus erythematosus. The C-reactive protein (CRP) response is defective in patients with acute flares of disease, and mice with targeted deletions of the serum amyloid P component gene (Sap) develop a lupus-like illness. In humans, the genes for CRP (CRP) and SAP (APCS) map to 1q23.2 within an interval linked with SLE. We have investigated the candidate genes CRP and APCS in two cohorts totalling 586 UK simplex SLE families. The inheritance of an intronic dinucleotide repeat and seven single nucleotide polymorphisms in the CRP and APCS genes was examined by application of family-based tests of association and linkage. Basal levels of CRP were influenced independently by two polymorphisms at the CRP locus, CRP 2 and CRP 4. Furthermore, the latter polymorphism was linked/associated with SLE and antinuclear autoantibody production. Thus, the polymorphism associated with reduced basal CRP was also associated with the development of SLE. These data support the hypothesis that defective disposal of potentially immunogenic material is a contributory factor in lupus pathogenesis. The identification of polymorphisms that determine basal CRP levels has implications in ischaemic heart disease, where CRP level is an important predictor of risk.
doi:10.1093/hmg/ddh021
PMCID: PMC3707088  PMID: 14645206
21.  Dnmt1 Deficiency Promotes CAG Repeat Expansion in the Mouse Germline 
Human molecular genetics  2008;17(9):1306-1317.
Expanded CAG repeat tracts are the cause of at least a dozen neurodegenerative disorders. In humans, long CAG repeats tend to expand during transmissions from parent to offspring, leading to an earlier age of disease onset and more severe symptoms in subsequent generations. Here, we show that the maintenance DNA methyltransferase Dnmt1, which preserves the patterns of CpG methylation, plays a key role in CAG repeat instability in human cells and in the male and female mouse germlines. SiRNA knockdown of Dnmt1 in human cells destabilized CAG triplet repeats, and Dnmt1 deficiency in mice promoted intergenerational expansion of CAG repeats at the murine spinocerebellar ataxia type 1 (Sca1) locus. Importantly, Dnmt1+/− SCA1 mice, unlike their Dnmt1+/+ SCA1 counterparts, closely reproduced the intergenerational instability patterns observed in human SCA1 patients. In addition, we found aberrant DNA and histone methylation at sites within the CpG island that abuts the expanded repeat tract in Dnmt1-deficient mice. These studies suggest that local chromatin structure may play a role in triplet repeat instability. These results are consistent with normal epigenetic changes during germline development contributing to intergenerational instability of CAG repeats in mice and in humans.
doi:10.1093/hmg/ddn019
PMCID: PMC3686304  PMID: 18252747
22.  Nxnl2 splicing results in dual functions in neuronal cell survival and maintenance of cell integrity 
Human Molecular Genetics  2012;21(10):2298-2311.
The Rod-derived Cone Viability Factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration arises due to loss of trophic support from RdCVF2. Contrarily, the progressive loss of rod visual function of the Nxnl2−/− mouse results from a decrease in outer segment length, mediated by a cell-autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2−/− mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.
doi:10.1093/hmg/dds050
PMCID: PMC3664437  PMID: 22343139
Animals; Cell Survival; genetics; Cells, Cultured; Eye Proteins; genetics; metabolism; Mice; RNA Splicing; Retinal Rod Photoreceptor Cells; metabolism; Sensory Receptor Cells; cytology; metabolism; Thioredoxins; genetics; metabolism
23.  [No title available] 
PMCID: PMC3643966  PMID: 16339215
24.  Novel pycnodysostosis mouse model uncovers cathepsin K function as a potential regulator of osteoclast apoptosis and senescence 
Human molecular genetics  2007;16(4):410-423.
Pycnodysostosis is a genetic bone disease featuring the unique bone homeostasis disorders of osteolysis and osteopetrosis in the same organism. The pathomechanism for pycnodysostosis has been largely unknown due to the unavailability of a pycnodysostosis mouse model with all the traits of the disease. We generated cathepsin K−/− mouse strains in the 129/Sv and C57BL/6J backgrounds and found that, only in the 129/Sv background, cathepsin K−/− mice exhibit many characteristics of the human pycnodysostosis-like phenotype. Our data indicated that 129/Sv cathepsin K−/− osteoclasts (OCs) lacked normal apoptosis and senescence and exhibited over-growth both in vitro and in vivo. These abnormalities resulted in an unusually high OC number, which is consistent with a recent case study of human pycnodysostosis. Our results show that cathepsin K function has different effects around the skeleton due to site-specific variations in bone homeostasis, such as phenotypes of osteopetrosis in tibiae and osteolysis in calvariae as a result of cathepsin K mutation. Our data demonstrated that the expression levels of p19, p53 and p21 were significantly reduced in 129/Sv cathepsin K−/− OCs and forced expression of cathepsin K in pre-OCs induced premature senescence and increased expression of p19, p53 and p21. This is the first evidence that cathepsin K plays a key role in OC apoptosis and senescence, revealing the importance of OC senescence in bone homeostasis. The finding of this novel cathepsin K function provides insight into the pathomechanism of pycnodysostosis and may provide new drug targets for diseases involved in OC-related abnormal bone homeostasis.
doi:10.1093/hmg/ddl474
PMCID: PMC3578583  PMID: 17210673
25.  hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction 
Human molecular genetics  2007;17(2):266-280.
Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of our fly model in elucidating the pathophysiology underlying motor neuron degeneration in humans.
doi:10.1093/hmg/ddm303
PMCID: PMC3516386  PMID: 17947296

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