In order to investigate whether DNA methylation marks could contribute to the incomplete penetran... more In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p,3 10 28 ) between carriers and noncarriers. The three sites replicated (p,2 10 27 ) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10 211 ,p,3.02 10 24 ) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p.0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.
To determine whether distinct single nucleotide polymorphisms (SNPs) within the glutamate recepto... more To determine whether distinct single nucleotide polymorphisms (SNPs) within the glutamate receptor ionotropic NMDA 1 gene (GRIN1) are associated with NMDA receptor (NMDAR) encephalitis and whether these same variants are associated with variability in the clinical presentation and course of affected patients. We performed clinical follow-up on 48 patients with NMDAR encephalitis and NMDAR autoantibodies detected in serum or CSF. All RefSeq GRIN1 coding exons were sequenced in 39 Caucasian-European patients, and the frequencies of SNPs were compared with those of an ethnically similar population using a case-control study design. Predetermined clinical variables were compared between patients with and without identified SNPs. Two SNPs were identified in GRIN1: 24 (62%) Caucasian-European patients with NMDAR encephalitis had alternate alleles at both rs6293 (exon 6) and rs1126442 (exon 7; exon numbering according to NM_001185090). The SNPs were in complete linkage disequilibrium. The ...
Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder resulting from loss of normal... more Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder resulting from loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus and infertility. Clinical features may be subtle and highly variable making the diagnosis of PCD challenging. The diagnosis can be confirmed with ciliary ultrastructure analysis and/or molecular genetic testing of 32 PCD associated genes. However, due to this genetic heterogeneity, comprehensive molecular genetic testing is not considered standard of care, and the most efficient molecular approach has yet to be elucidated. Here, we propose a cost-effective and time-efficient molecular genetic algorithm in solving PCD cases. We conducted targeted copy number variation (CNV) analysis and/or whole exome sequencing (WES) on 20 families (22 patients) from a subset of 45 families (52 patients) with a clinical diagnosis of PCD who did not have a molecular genetic diagnosis after ...
Allelic discrimination using TaqMan 5'-nuclease assay chemistry has been in routine use for m... more Allelic discrimination using TaqMan 5'-nuclease assay chemistry has been in routine use for many years, and the catalog of Life Technologies' predesigned SNP genotyping assays now exceeds 4 million entries. However, predesigned assays are often not available for genomic regions with a high GC content, nor can an assay necessarily be designed in this type of region using the manufacturer's design pipelines. Additionally, when an assay is available, the performance can be poor when using standard protocols. Here we report a modified allelic discrimination protocol for variants that reside in extremely GC-rich (GC > 75%) regions. The approach resolves fluorescent signal from reference and variant alleles, allowing all samples to be successfully assigned a genotype call. This protocol modification adds an extra step to the standard workflow, but the increased time is a productive compromise to generate high-quality data.
To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment... more To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment epithelium (RPE) in two families. Two families with autosomal-dominant PD were identified. Eight members of family 1 (five affected) were subjected to whole-genome SNP genotyping; multipoint genome-wide linkage analysis identified 7 regions of potential linkage, and genotyping four additional individuals from family 1 resulted in a maximum logarithm of odds score of 2.09 observed across four chromosomal regions. Exome sequencing of two affected family 1 members identified 15 shared non-synonymous rare coding sequence variants within the linked regions; candidate genes were prioritised and further analysed. Sanger sequencing confirmed a novel heterozygous missense variant (E79K) in orthodenticle homeobox 2 (OTX2) that segregated with the disease phenotype. Family 2 with PD (two affected) harboured the same missense variant in OTX2. A shared haplotype of 19.68 cM encompassing OTX2 was ide...
PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRP... more PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRPS1-related disorders associated with reduced activity includes Arts syndrome, Charcot-Marie-Tooth disease-5 (CMTX5) and X-linked non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype associated with decreased PRS-1 function in two affected male siblings. Using whole exome and Sanger sequencing techniques, we identified a novel missense mutation in PRPS1. The clinical phenotype in our patients is characterized by high prenatal maternal α-fetoprotein, intrauterine growth restriction, dysmorphic facial features, severe intellectual disability and spastic quadraparesis. Additional phenotypic features include macular coloboma-like lesions with retinal dystrophy, severe short stature and diabetes insipidus. Exome sequencing of the two affected male siblings identified a shared putative pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally inherited. Follow-up testing showed normal levels of hypoxanthine in urine samples and uric acid levels in blood serum. The PRS activity was significantly reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes revealed abnormally low guanosine triphosphate and guanosine diphosphate. This presentation is the most severe form of PRPS1-deficiency syndrome described to date and expands the spectrum of PRPS1-related disorders.
Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict pat... more Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict patients of all ages, but more commonly adolescents, and comprise the main differential diagnosis of common juvenile myoclonic epilepsy. Genetic or minimally invasive pathologic diagnoses are available for many but not all teenage-onset progressive myoclonus epilepsies. We describe a multiplex family with autosomal recessive teenage-onset progressive myoclonus epilepsy that had remained undiagnosed despite extensive genetic and pathologic testing. We describe whole exome sequencing combined with homozygosity mapping to identify the disease gene directly and diagnose the family. The affected gene is CLN6, previously known to underlie variant late-infantile and adult-onset neuronal ceroid lipofuscinoses. Combined with other recent work, our results add CLN6 to the genetic mutations causing teenage-onset progressive myoclonus epilepsy, expand the group of teenage-onset progressive myoclonus epilepsy patients who can be diagnosed by genetic testing, and extend the clinical spectrum of CLN6 mutations to include teenage-onset progressive myoclonus epilepsy. This work also exemplifies the potentiality of nextgeneration sequencing in the genetic identification and diagnosis of patients with neurologic diseases of unknown cause.
The Charadriiformes is a large and diverse order of shorebirds currently classified into 19 famil... more The Charadriiformes is a large and diverse order of shorebirds currently classified into 19 families, including morphologically aberrant forms that are of uncertain phylogenetic placement within non-passerine birds in general. Recent attempts using morphological characters have failed to recover a well-supported phylogeny depicting higher level relationships within Charadriiformes and the limits to the order, primarily because of inconsistency and homoplasy in these data. Moreover, these trees are incongruent with the relationships presented in the DNA hybridization tapestry of, including the location of the root and the branching order of major clades within the shorebirds. To help clarify this systematic confusion we therefore sequenced the large RAG-1 nuclear exon (2850 bp) from 36 species representing 17 families of shorebirds for which DNA was available. Trees built with maximum parsimony, maximum likelihood or Bayesian methods are topologically identical and fully resolved, with high support at basal nodes. This further attests to the phylogenetic utility of the RAG-1 sequences at higher taxonomic levels within birds. The RAG-1 tree is topologically similar to the DNA hybridization tree in depicting three major subordinal clades of shorebirds, the Charadrii (thick-knees, sheathbills, plovers, oystercatchers, and allies), Scolopaci (sandpipers and jacanas) and the Lari (coursers, pratincoles, gulls, terns, skimmers, and skuas). However, the basal split in the RAG-1 tree is between Charadrii and (Scolopaci+Lari), whereas in the DNA hybridization tree Scolopaci is the sister group to the (Charadrii+Lari). Thus in both of these DNA-based trees the Alcidae (auks, murres, and allies) are not basal among shorebirds as hypothesized in morphological trees, but instead are placed as a tip clade within Lari. The enigmatic buttonquails (Turnicidae), variously hypothesized as being allied to either the Galliformes, Gruiformes, or Charadriiformes, are shown to be a basal lineage in the more conventional Lari clade. Divergence times estimated with rate-smoothing methods and minimum time constraints imposed at nodes with key fossils suggest that Charadriiformes originated in Gondwanaland.
Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morp... more Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morphological characters, recent studies have turned to DNA sequences to resolve the systematic uncertainties of family-level relationships in this group. However, trees constructed using nuclear genes or the mitochondrial Cytochrome b gene suggest deep-level relationships of shorebirds that differ from previous studies based on morphology or DNA-DNA hybridization distances. To test phylogenetic hypotheses based on nuclear genes (RAG-1, myoglobin intron-2) and single mitochondrial genes (Cytochrome b), approximately 13,000 bp of mitochondrial sequence was collected for one exemplar species of 17 families of Charadriiformes plus potential outgroups. Maximum likelihood and Bayesian analyses show that trees constructed from long mitochondrial sequences are congruent with the nuclear gene topologies [Chardrii (Lari, Scolopaci)]. Unlike short mitochondrial sequences (such as Cytochrome b alone), longer sequences yield a well-supported phylogeny for shorebirds across various taxonomic levels. Examination of substitution patterns among mitochondrial genes reveals specific genes (especially ND5, ND4, ND2, and COI) that are better suited for phylogenetic analyses among shorebird families because of their relatively homogeneous nucleotide composition among lineages, slower accumulation of substitutions at third codon positions, and phylogenetic utility in both closely and distantly related lineages. For systematic studies of birds in which family and generic levels are examined simultaneously, we recommend the use of both nuclear and mitochondrial sequences as the best strategy to recover relationships that most likely reflect the phylogenetic history of these lineages.
In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present... more In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one-third of the level of that in DNA from normal white matter. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimer's, Huntington's, and Parkinson's was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2-fold higher than the DNA demethylase from normal, Alzheimer's, Huntington's and Parkinson's disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain. V V C 2007 Wiley-Liss, Inc.
The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in re... more The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare…
Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known gen... more Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known genetic risk has been traced to rare variants, principally copy number variants (CNVs). To identify common risk variation, the Autism Genome Project (AGP) Consortium genotyped 1558 rigorously defined ASD families for 1 million single-nucleotide polymorphisms (SNPs) and analyzed these SNP genotypes for association with ASD. In one of four primary association analyses, the association signal for marker rs4141463, located within MACROD2, crossed the genome-wide association significance threshold of P…
The reticulon-4 receptor, encoded by RTN4R, limits axonal sprouting and neural plasticity by inhi... more The reticulon-4 receptor, encoded by RTN4R, limits axonal sprouting and neural plasticity by inhibiting the outgrowth of neurites. Human association studies have implicated mutations in RTN4R in the development of schizophrenia, including the identification of several rare nonconservative missense mutations of RTN4R in schizophrenia patients. To investigate the effects of missense mutation of the reticulon-4 receptor on phenotypes relevant to schizophrenia, we behaviourally characterized a novel Rtn4r mutant mouse line with an amino acid substitution (R189H) in the Nogo-66 binding site. Behavioural assays included prepulse inhibition of acoustic startle, locomotor activity, social interaction and spatial cognition. When compared with wildtype littermates, Rtn4r mutant mice exhibited greater social preference, which may reflect a social-anxyolitic effect, and a mild impairment in spatial cognition. Given the mild effect of the R189H mutation of Rtn4r on behavioural phenotypes relevant to schizophrenia, our results do not support missense mutation of RTN4R as a strong risk factor in the pathogenesis of schizophrenia.
We report a consanguineous couple that has experienced three consecutive pregnancy losses followi... more We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a rec...
Haplotypes are often critical for the interpretation of genetic laboratory observations into medi... more Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalon...
In order to investigate whether DNA methylation marks could contribute to the incomplete penetran... more In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p,3 10 28 ) between carriers and noncarriers. The three sites replicated (p,2 10 27 ) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10 211 ,p,3.02 10 24 ) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p.0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.
To determine whether distinct single nucleotide polymorphisms (SNPs) within the glutamate recepto... more To determine whether distinct single nucleotide polymorphisms (SNPs) within the glutamate receptor ionotropic NMDA 1 gene (GRIN1) are associated with NMDA receptor (NMDAR) encephalitis and whether these same variants are associated with variability in the clinical presentation and course of affected patients. We performed clinical follow-up on 48 patients with NMDAR encephalitis and NMDAR autoantibodies detected in serum or CSF. All RefSeq GRIN1 coding exons were sequenced in 39 Caucasian-European patients, and the frequencies of SNPs were compared with those of an ethnically similar population using a case-control study design. Predetermined clinical variables were compared between patients with and without identified SNPs. Two SNPs were identified in GRIN1: 24 (62%) Caucasian-European patients with NMDAR encephalitis had alternate alleles at both rs6293 (exon 6) and rs1126442 (exon 7; exon numbering according to NM_001185090). The SNPs were in complete linkage disequilibrium. The ...
Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder resulting from loss of normal... more Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder resulting from loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus and infertility. Clinical features may be subtle and highly variable making the diagnosis of PCD challenging. The diagnosis can be confirmed with ciliary ultrastructure analysis and/or molecular genetic testing of 32 PCD associated genes. However, due to this genetic heterogeneity, comprehensive molecular genetic testing is not considered standard of care, and the most efficient molecular approach has yet to be elucidated. Here, we propose a cost-effective and time-efficient molecular genetic algorithm in solving PCD cases. We conducted targeted copy number variation (CNV) analysis and/or whole exome sequencing (WES) on 20 families (22 patients) from a subset of 45 families (52 patients) with a clinical diagnosis of PCD who did not have a molecular genetic diagnosis after ...
Allelic discrimination using TaqMan 5'-nuclease assay chemistry has been in routine use for m... more Allelic discrimination using TaqMan 5'-nuclease assay chemistry has been in routine use for many years, and the catalog of Life Technologies' predesigned SNP genotyping assays now exceeds 4 million entries. However, predesigned assays are often not available for genomic regions with a high GC content, nor can an assay necessarily be designed in this type of region using the manufacturer's design pipelines. Additionally, when an assay is available, the performance can be poor when using standard protocols. Here we report a modified allelic discrimination protocol for variants that reside in extremely GC-rich (GC > 75%) regions. The approach resolves fluorescent signal from reference and variant alleles, allowing all samples to be successfully assigned a genotype call. This protocol modification adds an extra step to the standard workflow, but the increased time is a productive compromise to generate high-quality data.
To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment... more To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment epithelium (RPE) in two families. Two families with autosomal-dominant PD were identified. Eight members of family 1 (five affected) were subjected to whole-genome SNP genotyping; multipoint genome-wide linkage analysis identified 7 regions of potential linkage, and genotyping four additional individuals from family 1 resulted in a maximum logarithm of odds score of 2.09 observed across four chromosomal regions. Exome sequencing of two affected family 1 members identified 15 shared non-synonymous rare coding sequence variants within the linked regions; candidate genes were prioritised and further analysed. Sanger sequencing confirmed a novel heterozygous missense variant (E79K) in orthodenticle homeobox 2 (OTX2) that segregated with the disease phenotype. Family 2 with PD (two affected) harboured the same missense variant in OTX2. A shared haplotype of 19.68 cM encompassing OTX2 was ide...
PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRP... more PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRPS1-related disorders associated with reduced activity includes Arts syndrome, Charcot-Marie-Tooth disease-5 (CMTX5) and X-linked non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype associated with decreased PRS-1 function in two affected male siblings. Using whole exome and Sanger sequencing techniques, we identified a novel missense mutation in PRPS1. The clinical phenotype in our patients is characterized by high prenatal maternal α-fetoprotein, intrauterine growth restriction, dysmorphic facial features, severe intellectual disability and spastic quadraparesis. Additional phenotypic features include macular coloboma-like lesions with retinal dystrophy, severe short stature and diabetes insipidus. Exome sequencing of the two affected male siblings identified a shared putative pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally inherited. Follow-up testing showed normal levels of hypoxanthine in urine samples and uric acid levels in blood serum. The PRS activity was significantly reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes revealed abnormally low guanosine triphosphate and guanosine diphosphate. This presentation is the most severe form of PRPS1-deficiency syndrome described to date and expands the spectrum of PRPS1-related disorders.
Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict pat... more Progressive myoclonus epilepsies are severe, intractable, and neurodegenerative. They afflict patients of all ages, but more commonly adolescents, and comprise the main differential diagnosis of common juvenile myoclonic epilepsy. Genetic or minimally invasive pathologic diagnoses are available for many but not all teenage-onset progressive myoclonus epilepsies. We describe a multiplex family with autosomal recessive teenage-onset progressive myoclonus epilepsy that had remained undiagnosed despite extensive genetic and pathologic testing. We describe whole exome sequencing combined with homozygosity mapping to identify the disease gene directly and diagnose the family. The affected gene is CLN6, previously known to underlie variant late-infantile and adult-onset neuronal ceroid lipofuscinoses. Combined with other recent work, our results add CLN6 to the genetic mutations causing teenage-onset progressive myoclonus epilepsy, expand the group of teenage-onset progressive myoclonus epilepsy patients who can be diagnosed by genetic testing, and extend the clinical spectrum of CLN6 mutations to include teenage-onset progressive myoclonus epilepsy. This work also exemplifies the potentiality of nextgeneration sequencing in the genetic identification and diagnosis of patients with neurologic diseases of unknown cause.
The Charadriiformes is a large and diverse order of shorebirds currently classified into 19 famil... more The Charadriiformes is a large and diverse order of shorebirds currently classified into 19 families, including morphologically aberrant forms that are of uncertain phylogenetic placement within non-passerine birds in general. Recent attempts using morphological characters have failed to recover a well-supported phylogeny depicting higher level relationships within Charadriiformes and the limits to the order, primarily because of inconsistency and homoplasy in these data. Moreover, these trees are incongruent with the relationships presented in the DNA hybridization tapestry of, including the location of the root and the branching order of major clades within the shorebirds. To help clarify this systematic confusion we therefore sequenced the large RAG-1 nuclear exon (2850 bp) from 36 species representing 17 families of shorebirds for which DNA was available. Trees built with maximum parsimony, maximum likelihood or Bayesian methods are topologically identical and fully resolved, with high support at basal nodes. This further attests to the phylogenetic utility of the RAG-1 sequences at higher taxonomic levels within birds. The RAG-1 tree is topologically similar to the DNA hybridization tree in depicting three major subordinal clades of shorebirds, the Charadrii (thick-knees, sheathbills, plovers, oystercatchers, and allies), Scolopaci (sandpipers and jacanas) and the Lari (coursers, pratincoles, gulls, terns, skimmers, and skuas). However, the basal split in the RAG-1 tree is between Charadrii and (Scolopaci+Lari), whereas in the DNA hybridization tree Scolopaci is the sister group to the (Charadrii+Lari). Thus in both of these DNA-based trees the Alcidae (auks, murres, and allies) are not basal among shorebirds as hypothesized in morphological trees, but instead are placed as a tip clade within Lari. The enigmatic buttonquails (Turnicidae), variously hypothesized as being allied to either the Galliformes, Gruiformes, or Charadriiformes, are shown to be a basal lineage in the more conventional Lari clade. Divergence times estimated with rate-smoothing methods and minimum time constraints imposed at nodes with key fossils suggest that Charadriiformes originated in Gondwanaland.
Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morp... more Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morphological characters, recent studies have turned to DNA sequences to resolve the systematic uncertainties of family-level relationships in this group. However, trees constructed using nuclear genes or the mitochondrial Cytochrome b gene suggest deep-level relationships of shorebirds that differ from previous studies based on morphology or DNA-DNA hybridization distances. To test phylogenetic hypotheses based on nuclear genes (RAG-1, myoglobin intron-2) and single mitochondrial genes (Cytochrome b), approximately 13,000 bp of mitochondrial sequence was collected for one exemplar species of 17 families of Charadriiformes plus potential outgroups. Maximum likelihood and Bayesian analyses show that trees constructed from long mitochondrial sequences are congruent with the nuclear gene topologies [Chardrii (Lari, Scolopaci)]. Unlike short mitochondrial sequences (such as Cytochrome b alone), longer sequences yield a well-supported phylogeny for shorebirds across various taxonomic levels. Examination of substitution patterns among mitochondrial genes reveals specific genes (especially ND5, ND4, ND2, and COI) that are better suited for phylogenetic analyses among shorebird families because of their relatively homogeneous nucleotide composition among lineages, slower accumulation of substitutions at third codon positions, and phylogenetic utility in both closely and distantly related lineages. For systematic studies of birds in which family and generic levels are examined simultaneously, we recommend the use of both nuclear and mitochondrial sequences as the best strategy to recover relationships that most likely reflect the phylogenetic history of these lineages.
In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present... more In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one-third of the level of that in DNA from normal white matter. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimer's, Huntington's, and Parkinson's was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2-fold higher than the DNA demethylase from normal, Alzheimer's, Huntington's and Parkinson's disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain. V V C 2007 Wiley-Liss, Inc.
The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in re... more The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare…
Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known gen... more Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known genetic risk has been traced to rare variants, principally copy number variants (CNVs). To identify common risk variation, the Autism Genome Project (AGP) Consortium genotyped 1558 rigorously defined ASD families for 1 million single-nucleotide polymorphisms (SNPs) and analyzed these SNP genotypes for association with ASD. In one of four primary association analyses, the association signal for marker rs4141463, located within MACROD2, crossed the genome-wide association significance threshold of P…
The reticulon-4 receptor, encoded by RTN4R, limits axonal sprouting and neural plasticity by inhi... more The reticulon-4 receptor, encoded by RTN4R, limits axonal sprouting and neural plasticity by inhibiting the outgrowth of neurites. Human association studies have implicated mutations in RTN4R in the development of schizophrenia, including the identification of several rare nonconservative missense mutations of RTN4R in schizophrenia patients. To investigate the effects of missense mutation of the reticulon-4 receptor on phenotypes relevant to schizophrenia, we behaviourally characterized a novel Rtn4r mutant mouse line with an amino acid substitution (R189H) in the Nogo-66 binding site. Behavioural assays included prepulse inhibition of acoustic startle, locomotor activity, social interaction and spatial cognition. When compared with wildtype littermates, Rtn4r mutant mice exhibited greater social preference, which may reflect a social-anxyolitic effect, and a mild impairment in spatial cognition. Given the mild effect of the R189H mutation of Rtn4r on behavioural phenotypes relevant to schizophrenia, our results do not support missense mutation of RTN4R as a strong risk factor in the pathogenesis of schizophrenia.
We report a consanguineous couple that has experienced three consecutive pregnancy losses followi... more We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a rec...
Haplotypes are often critical for the interpretation of genetic laboratory observations into medi... more Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalon...
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Papers by Tara Paton