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Stephen W Scherer

Publications and source records attributed to Stephen W Scherer.

At least 73 records · Page 4Linked to original sources
PubMed2003

Mutations in NHLRC1 cause progressive myoclonus epilepsy.

Lafora progressive myoclonus epilepsy is characterized by pathognomonic endoplasmic reticulum (ER)-associated polyglucosan accumulations. We previously discovered that mutations in EPM2A cause Lafora disease. Here, we identify a second gene associated with this disease, NHLRC1 (also called EPM2B), which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Laforin and malin colocalize to the ER, suggesting they operate in a related pathway protecting against polyglucosan accumulation and epilepsy.

Base Sequence↗
PubMed2003

Recent segmental and gene duplications in the mouse genome.

BACKGROUND: The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (>/= 5 kb) and recent (>/= 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies. RESULTS: We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice. CONCLUSION: Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the identified duplication content presented in this work. This data will also be relevant to the growing number of investigators who use the draft genome sequence for experimental design and analysis.

Animals↗
PubMed2003

Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements.

The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.

Animals↗
PubMed2003

Genescript: DNA sequence annotation pipeline.

UNLABELLED: Genescript uses a number of publicly available analysis programs to annotate a DNA sequence. It provides an integrated display of results from each program, and includes an evidence-based scoring system that gives informative summaries of predicted gene models. AVAILABILITY: Genescript is available for download from http:://tcag.bioinfo.sickkids.on.ca/genescript/

Computer Graphics↗
PubMed2003

Human chromosome 7: DNA sequence and biology.

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

Animals↗
PubMed2003

The mouse genome sequence--the end of the tail, or just the beginning?

The recent flurry of papers on the mouse genome includes the description of the full genome assembly, analysis of the mouse transcriptome, the origin of interstrain variation, initial analysis of conserved non-coding regions and high-throughput expression analysis of a subset of genes. Each illustrates how the availability of the genome sequence will change the way mouse biologists do business in future.

Alternative Splicing↗
PubMed2003

Genome-wide detection of segmental duplications and potential assembly errors in the human genome sequence.

BACKGROUND: Previous studies have suggested that recent segmental duplications, which are often involved in chromosome rearrangements underlying genomic disease, account for some 5% of the human genome. We have developed rapid computational heuristics based on BLAST analysis to detect segmental duplications, as well as regions containing potential sequence misassignments in the human genome assemblies. RESULTS: Our analysis of the June 2002 public human genome assembly revealed that 107.4 of 3,043.1 megabases (Mb) (3.53%) of sequence contained segmental duplications, each with size equal or more than 5 kb and 90% identity. We have also detected that 38.9 Mb (1.28%) of sequence within this assembly is likely to be involved in sequence misassignment errors. Furthermore, we have identified a significant subset (199,965 of 2,327,473 or 8.6%) of single-nucleotide polymorphisms (SNPs) in the public databases that are not true SNPs but are potential paralogous sequence variants. CONCLUSION: Using two distinct computational approaches, we have identified most of the sequences in the human genome that have undergone recent segmental duplications. Near-identical segmental duplications present a major challenge to the completion of the human genome sequence. Potential sequence misassignments detected in this study would require additional efforts to resolve.

Artifacts↗
PubMed2003

Molecular characterization of a 12q22-q24 deletion associated with congenital deafness: confirmation and refinement of the DFNA25 locus.

The DFNA25 locus for autosomal dominant nonsyndromic hereditary hearing loss has been mapped to 12q21-q24 by linkage analysis. A de novo deletion in a six-year-old boy with congenital hearing loss as well as mental and motor retardation now provides independent confirmation of this genetic localization and narrows the critical interval to 13 cM in the 12q22-q24.1 region. Mapping of the deletion was performed using fluorescence in situ hybridization (FISH) analysis with region-specific yeast artificial chromosome (YAC) clones. Ten YACs 929_e_4, 959_c_3, 746_h_7, 817_h_10, 886_a_6, 916_h_9, 969_c_12, 747_e_2, 812_h_12, and 959_f_8 were absent from one chromosome 12 from the patient. Molecular analyses of eight polymorphic markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 12 is of paternal origin. Several known genes including ATP2A2, UBE3B, and VR-OAC that map in the 12q22-q24.1 region are included in the deletion. These results provide evidence that haploinsufficiency for a gene or genes in 12q22-q24.1 is associated with autosomal dominant deafness.

Child↗
PubMed2003

GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome.

Children with constitutional trisomy 21 (Down syndrome) have an approximately 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia. Unique to newborn infants with Down syndrome is a transient leukemia (TL), also referred to as transient myeloproliferative syndrome, that undergoes spontaneous remission in the majority of cases but in approximately 20% is followed by AMKL later in life. Recently mutations of the gene encoding the hematopoietic transcription factor GATA1 were shown to be specific for AMKL of Down syndrome. Here, we demonstrate that GATA1 mutations are present in blasts of TL and show the identical GATA1 mutation in sequential samples collected from a patient during TL and subsequent AMKL. These findings suggest a model of malignant transformation in Down syndrome AMKL in which GATA1 mutations are an early event and AMKL arises from latent TL clones following initial apparent remission.

Cell Transformation, Neoplastic↗
PubMed2003

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5).

Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No. NW_000039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31, which has been shown to confer susceptibility to Crohn's disease. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohn's disease.

Animals↗
PubMed2003

Heterogeneity of the 7q36 breakpoints in the t(7;12) involving ETV6 in infant leukemia.

The t(7;12)(q36;p13) is a recurrent chromosome abnormality in infant leukemia. In these cases, the involvement of ETV6, with disruption of the gene consistently at its 5' end, has been reported by several groups. A fusion transcript between ETV6 and HLXB9 has been detected in some, but not all, reported cases of t(7;12). We report here a study based on fluorescence in situ hybridization (FISH) mapping of the translocation breakpoints in seven patients and detailed molecular studies using Southern blotting on two of these patients. The FISH studies have shown a cluster of breakpoints within a cosmid contig proximal to the HLXB9 gene. Southern blotting analysis enabled us to define two distinct breakpoints within the area covered by the cosmid contig in two patients. The analysis of an unusual case of t(7;12)(q22;p13) [full karyotype: 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36)] also revealed a break in 7q36, although in a region proximal to the overlapping cosmids. 5' RACE PCR in one patient has shown a rearrangement involving the ETV6 allele not involved in the t(7;12), suggesting that no functional ETV6 allele might be present in this case. These data show some heterogeneity in the distribution of breakpoints in 7q36, indicating that the generation of a fusion gene might not be the mechanism responsible for leukemogenesis in the t(7;12), at least in some cases.

Base Sequence↗
PubMed2003

Cloning and characterization of human CADPS and CADPS2, new members of the Ca2+-dependent activator for secretion protein family.

The recent identification of some of the components involved in regulated and constitutive exocytotic pathways has yielded important insights into the mechanisms of membrane trafficking and vesicle secretion. To understand precisely the molecular events taking place during vesicle exocytosis, we must identify all of the proteins implicated in these pathways. In this paper we describe the full-length cloning and characterization of human CADPS and CADPS2, two new homologs of the mouse Cadps protein involved in large dense-core vesicle (LDCV)-regulated exocytosis. We show that these two genes have disparate RNA expression patterns, with CADPS restricted to neural and endocrine tissues and CADPS2 expressed ubiquitously. We also identify a C2 domain, a known protein motif involved in calcium and phospholipid interactions, in both CADPS and CADPS2. We propose that CADPS functions as a calcium sensor in regulated exocytosis, whereas CADPS2 acts as a calcium sensor in constitutive vesicle trafficking and secretion. CADPS and CADPS2 were determined to span 475 kb and 561 kb on human chromosomes 3p21.1 and 7q31.3, respectively. The q31-q34 of human chromosome 7 has recently been identified to contain a putative susceptibility locus for autism (AUTS1). The function, expression profile, and location of CADPS2 make it a candidate gene for autism, and thus we conducted mutation screening for all 28 exons in 90 unrelated autistic individuals. We identified several nucleotide substitutions, including only one that would affect the amino acid sequence. No disease-specific variants were identified.

Amino Acid Sequence↗
PubMed2003

Identification of a novel protein interacting with laforin, the EPM2a progressive myoclonus epilepsy gene product.

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.

Amino Acid Sequence↗
PubMed2003

Molecular basis for expression of common and rare fragile sites.

Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.

Alleles↗
PubMed2003

Remarkable sequence signatures in archaeal genomes.

Complete archaeal genomes were probed for the presence of long (> or = 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non-archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species-specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non-coding regions. Thus, archaea appear to possess a non-coding genomic signature that is absent in bacterial species. The identification of a species-specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity.

Base Sequence↗
PubMed2002

Chromosomal regions containing high-density and ambiguously mapped putative single nucleotide polymorphisms (SNPs) correlate with segmental duplications in the human genome.

We have explored the National Center for Biotechnology Information (NCBI) single nucleotide polymorphisms (SNPs) database for a correlation between the density of putative SNPs, as well as SNPs that map to different chromosomal locations (ambiguously mapped SNPs), and segmental duplications of DNA in chromosome regions involved in genomic disorders. A high density of SNPs (14.4 and 12.4 SNPs per kb) was detected in the low copy repeats (LCRs) responsible for the chromosome 17p12 duplication and deletion that cause peripheral neuropathies. None of the SNPs at the PMP22 gene were ambiguously mapped, but 93% of the SNPs at LCRs mapped on both LCR copies, indicating that they are in fact variants in paralogous sequences. Similarly, a high SNP density was found in the LCR regions flanking the neurofibromatosis type 1 (NF1) gene, with 80% of SNPs mapping on both LCR copies. A high density of SNPs was found within LCR sequences involved in the deletions that mediate contiguous gene syndromes on chromosomes 7q11, 15q11-q13 and 22q11. We have analyzed the whole sequence of chromosome 22, which contains 14% of ambiguously mapped SNPs, and have found a good correlation between these SNPs and segmental duplications detected by BLAST analysis. We have identified several segments of ambiguously mapped SNPs, four corresponding to LCRs involved in the chromosome 22q11 microdeletion syndromes. Our data indicate that most SNPs in LCR segments are in fact paralogous sequence variants (PSVs), and suggest that a significant proportion of the SNPs in the NCBI database correspond to PSVs within segmental duplications of the human genome sequence.

Centromere↗
PubMed2002

Identification and characterization of an imprinted antisense RNA (MESTIT1) in the human MEST locus on chromosome 7q32.

Imprinted gene(s) on human chromosome 7 are thought to be involved in Russell-Silver syndrome (RSS), based on the fact that approximately 10% of patients have maternal uniparental disomy of chromosome 7. However, involvement of the known imprinted genes (GRB10 at 7p12, PEG10 at 7q21.3 and MEST at 7q32) in RSS has yet to be established. To screen for new imprinted genes, we are initially using somatic cell hybrids containing a paternal or maternal human chromosome 7. Transcripts located between D7S530 and D7S649 (a 1.5 Mb interval encompassing MEST ) were subjected to RT-PCR analysis using somatic cell hybrids. One transcript named MESTIT1 (for MEST intronic transcript 1) reproducibly showed paternal-specific expression. Upon further analysis, we found MESTIT1 to be (1) paternally (and not maternally) expressed in all fetal tissues and fibroblasts examined, (2) to be located in an intron of one of the two isoforms of MEST but transcribed in the opposite direction, (3) to be composed of at least two exons without any significant open reading frame, and (4) to exist as a 4.2 kb transcript in many fetal and adult tissues. We could also identify two isoforms of the mouse Mest gene as observed in humans, but it is still unknown if a murine ortholog of MESTIT1 exists. We also examined the imprinting status of MEST isoforms as a first step in assessing whether MESTIT1 might influence the allelic expression pattern of the sense transcript. MEST isoform 1 was determined to be exclusively expressed from the paternal allele in all fetal tissues and cell lines examined, whereas MEST isoform 2 was only preferentially expressed from the paternal allele in a tissue/cell-type-specific manner. Our results suggest that MESTIT1 is a paternally expressed non-coding RNA that may be involved in the regulation of MEST expression during development. MESTIT1 (also known as PEG1-AS) is now the third independent transcript (with MEST and COPG2IT1) identified at human chromosome 7q32 demonstrating paternal chromosome-specific expression.

Blotting, Northern↗