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In JoVE (1)
Other Publications (17)
- Nucleic Acids Research
- American Journal of Human Genetics
- American Journal of Human Genetics
- Human Molecular Genetics
- Human Molecular Genetics
- Annual Review of Genomics and Human Genetics
- Mammalian Genome : Official Journal of the International Mammalian Genome Society
- Human Genetics
- Human Molecular Genetics
- The American Journal of Psychiatry
- PLoS Genetics
- American Journal of Human Genetics
- Biological Psychiatry
- PloS One
- Biological Psychiatry
- Journal of Neurodevelopmental Disorders
- Human Molecular Genetics
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Articles by Silvia Paracchini in JoVE
JoVE General
Genetik Dernekleri Fonksiyonel Dayanak Test bir Allel özel Gen İfadesi Testi
Wellcome Trust Centre for Human Genetics, University of Oxford
Genetik dernekler genellikle işlevsel bir düzeyde açıklanamayan kalır. Bu yöntem, analiz ederek transkripsiyonu SNP için heterozigot hücrelere gen ekspresyonu fenotipi ile ilişkili genetik belirteçlerin etkisini değerlendirmek amaçlamaktadır. Teknoloji allel spesifik astar uzatma ürünleri ölçmek için MALDI-TOF kütle spektrometresi doğru ölçüm sağlar.
Other articles by Silvia Paracchini on PubMed
Hierarchical High-throughput SNP Genotyping of the Human Y Chromosome Using MALDI-TOF Mass Spectrometry
We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.
A Predominantly Neolithic Origin for Y-chromosomal DNA Variation in North Africa
We have typed 275 men from five populations in Algeria, Tunisia, and Egypt with a set of 119 binary markers and 15 microsatellites from the Y chromosome, and we have analyzed the results together with published data from Moroccan populations. North African Y-chromosomal diversity is geographically structured and fits the pattern expected under an isolation-by-distance model. Autocorrelation analyses reveal an east-west cline of genetic variation that extends into the Middle East and is compatible with a hypothesis of demic expansion. This expansion must have involved relatively small numbers of Y chromosomes to account for the reduction in gene diversity towards the West that accompanied the frequency increase of Y haplogroup E3b2, but gene flow must have been maintained to explain the observed pattern of isolation-by-distance. Since the estimates of the times to the most recent common ancestor (TMRCAs) of the most common haplogroups are quite recent, we suggest that the North African pattern of Y-chromosomal variation is largely of Neolithic origin. Thus, we propose that the Neolithic transition in this part of the world was accompanied by demic diffusion of Afro-Asiatic-speaking pastoralists from the Middle East.
A 77-kilobase Region of Chromosome 6p22.2 is Associated with Dyslexia in Families from the United Kingdom and from the United States
Several quantitative trait loci (QTLs) that influence developmental dyslexia (reading disability [RD]) have been mapped to chromosome regions by linkage analysis. The most consistently replicated area of linkage is on chromosome 6p23-21.3. We used association analysis in 223 siblings from the United Kingdom to identify an underlying QTL on 6p22.2. Our association study implicates a 77-kb region spanning the gene TTRAP and the first four exons of the neighboring uncharacterized gene KIAA0319. The region of association is also directly upstream of a third gene, THEM2. We found evidence of these associations in a second sample of siblings from the United Kingdom, as well as in an independent sample of twin-based sibships from Colorado. One main RD risk haplotype that has a frequency of approximately 12% was found in both the U.K. and U.S. samples. The haplotype is not distinguished by any protein-coding polymorphisms, and, therefore, the functional variation may relate to gene expression. The QTL influences a broad range of reading-related cognitive abilities but has no significant impact on general cognitive performance in these samples. In addition, the QTL effect may be largely limited to the severe range of reading disability.
The Chromosome 6p22 Haplotype Associated with Dyslexia Reduces the Expression of KIAA0319, a Novel Gene Involved in Neuronal Migration
Dyslexia is one of the most prevalent childhood cognitive disorders, affecting approximately 5% of school-age children. We have recently identified a risk haplotype associated with dyslexia on chromosome 6p22.2 which spans the TTRAP gene and portions of THEM2 and KIAA0319. Here we show that in the presence of the risk haplotype, the expression of the KIAA0319 gene is reduced but the expression of the other two genes remains unaffected. Using in situ hybridization, we detect a very distinct expression pattern of the KIAA0319 gene in the developing cerebral neocortex of mouse and human fetuses. Moreover, interference with rat Kiaa0319 expression in utero leads to impaired neuronal migration in the developing cerebral neocortex. These data suggest a direct link between a specific genetic background and a biological mechanism leading to the development of dyslexia: the risk haplotype on chromosome 6p22.2 down-regulates the KIAA0319 gene which is required for neuronal migration during the formation of the cerebral neocortex.
Haplotype-specific Expression of Exon 10 at the Human MAPT Locus
Neurofibrillary tangles composed of exon 10+ microtubule associated protein tau (MAPT) deposits are the characteristic feature of the neurodegenerative diseases progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). PSP, CBD and more recently Alzheimer's disease and Parkinson's disease, are associated with the MAPT H1 haplotype, but the relationship between genotype and disease remains unclear. Here, we investigate the hypothesis that H1 expresses more exon 10+ MAPT mRNA compared to the other haplotype, H2, leading to a greater susceptibility to neurodegeneration in H1 carriers. We performed allele-specific gene expression on two H1/H2 heterozygous human neuronal cell lines, and 14 H1/H2 heterozygous control individual post-mortem brain tissue from two brain regions. In both tissue culture and post-mortem brain tissue, we show that the MAPT H1 haplotype expresses significantly more exon 10+ MAPT mRNA than H2. In post-mortem brain tissue, we show that the total level of MAPT expression from H1 and H2 is not significantly different, but that the H1 chromosome expresses up to 1.43-fold more exon 10+ MAPT mRNA than H2 in the globus pallidus, a brain region highly affected by tauopathy (maximum exon 10+ MAPT H1:H2 transcript ratio=1.425, SD=0.205, P<0.0001), and up to 1.29-fold more exon 10+ MAPT mRNA than H2 in the frontal cortex (maximum exon 10+ MAPT H1:H2 transcript ratio=1.291, SD=0.315, P=0.006). These data may explain the increased susceptibility of H1 carriers to neurodegeneration and suggest a potential mechanism between MAPT genetic variability and the pathogenesis of neurodegenerative disease.
The Genetic Lexicon of Dyslexia
Reading abilities are acquired only through specific teaching and training. A significant proportion of children fail to achieve these skills despite normal intellectual abilities and an appropriate opportunity to learn. Difficulty in learning to read is attributable to specific dysfunctions of the brain, which so far remain poorly understood. However, it is recognized that the neurological basis for dyslexia, or reading disability, is caused in large part by genetic factors. Linkage studies have successfully identified several regions of the human genome that are likely to harbor susceptibility genes for dyslexia. In the past few years there have been exciting advances with the identification of four candidate genes located within three of these linked chromosome regions: DYX1C1 on chromosome 15, ROBO1 on chromosome 3, and KIAA0319 and DCDC2 on chromosome 6. Functional studies of these genes are offering new insights about the biological mechanisms underlying the development of dyslexia and, in general, of cognition.
Alternative Splicing in the Dyslexia-associated Gene KIAA0319
The KIAA0319 gene in chromosome 6p22 has been strongly associated with developmental dyslexia. In this article we show a wide expression pattern of this gene in human adult brain by Northern blot analysis. We also performed RT-PCR analysis to detect alternative splicing variants in human brain. Most of the detected variants involve alternative splicing of the exons at the 5' and the 3' ends. Two main forms differing in the length of the 5' UTR are detected at approximately the same rate. Two variants (B and C) lacking exon 19, which encodes the transmembrane domain, are the main alternative forms detected among those predicted to encode protein. These two variants could be secreted and might be involved in signaling functions. A similar RT-PCR analysis performed in mouse and rat adult brains showed that only some of the alternative splicing variants are equivalent to those found in the human gene.
Y-chromosomal Insights into the Genetic Impact of the Caste System in India
The caste system has persisted in Indian Hindu society for around 3,500 years. Like the Y chromosome, caste is defined at birth, and males cannot change their caste. In order to investigate the genetic consequences of this system, we have analysed male-lineage variation in a sample of 227 Indian men of known caste, 141 from the Jaunpur district of Uttar Pradesh and 86 from the rest of India. We typed 131 Y-chromosomal binary markers and 16 microsatellites. We find striking evidence for male substructure: in particular, Brahmins and Kshatriyas (but not other castes) from Jaunpur each show low diversity and the predominance of a single distinct cluster of haplotypes. These findings confirm the genetic isolation and drift within the Jaunpur upper castes, which are likely to result from founder effects and social factors. In the other castes, there may be either larger effective population sizes, or less strict isolation, or both.
The Dyslexia-associated Gene KIAA0319 Encodes Highly N- and O-glycosylated Plasma Membrane and Secreted Isoforms
The KIAA0319 gene has been recently associated with developmental dyslexia and shown to be involved in neuronal migration. The deduced KIAA0319 protein contains several polycystic kidney disease (PKD) domains which may mediate the interaction between neurons and glial fibres during neuronal migration. We have previously reported the presence of several alternative splicing variants, some of which are predicted to affect the deduced protein. In this study, we over-expressed constructs containing the main form (A) and two alternative variants (B and C) of KIAA0319. We show that the full-length KIAA0319 (A) is a type I plasma membrane protein, a topology consistent with its proposed function in neuronal migration. The oligomeric status of KIAA0319 is mainly dimeric, and this condition depends on the cysteine-rich regions of the protein, especially the transmembrane (TM) domain and surrounding sequence. KIAA0319 is highly glycosylated in different mammalian cell lines. The central region including the PKD domains is N-glycosylated. Furthermore, a short fragment N-terminal to the PKD domains contains mucin-type O-glycosylation. The two alternative isoforms are soluble proteins lacking the TM domain and, interestingly, only isoform B is secreted. KIAA0319-deletion proteins lacking the TM domain were also secreted. These results suggest that KIAA0319 could be involved not only in cell-cell interactions, but also in signalling.
Association of the KIAA0319 Dyslexia Susceptibility Gene with Reading Skills in the General Population
The authors previously identified a haplotype on chromosome 6p22 defined by three single-nucleotide polymorphisms (SNPs) that was associated with dyslexia (reading disability) in two independent samples of families that included at least one sibling with severe reading impairment. The authors also showed that this haplotype is associated with a reduction in expression of the KIAA0319 gene. In addition, a completely independent study detected an association between KIAA0319 markers and reading disability. In the current study, the authors tested whether the KIAA0319 gene influences reading skills in the general population, rather than having an effect restricted to reading disability.
A Common Variant Associated with Dyslexia Reduces Expression of the KIAA0319 Gene
Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.
CMIP and ATP2C2 Modulate Phonological Short-term Memory in Language Impairment
Specific language impairment (SLI) is a common developmental disorder characterized by difficulties in language acquisition despite otherwise normal development and in the absence of any obvious explanatory factors. We performed a high-density screen of SLI1, a region of chromosome 16q that shows highly significant and consistent linkage to nonword repetition, a measure of phonological short-term memory that is commonly impaired in SLI. Using two independent language-impaired samples, one family-based (211 families) and another selected from a population cohort on the basis of extreme language measures (490 cases), we detected association to two genes in the SLI1 region: that encoding c-maf-inducing protein (CMIP, minP = 5.5 x 10(-7) at rs6564903) and that encoding calcium-transporting ATPase, type2C, member2 (ATP2C2, minP = 2.0 x 10(-5) at rs11860694). Regression modeling indicated that each of these loci exerts an independent effect upon nonword repetition ability. Despite the consistent findings in language-impaired samples, investigation in a large unselected cohort (n = 3612) did not detect association. We therefore propose that variants in CMIP and ATP2C2 act to modulate phonological short-term memory primarily in the context of language impairment. As such, this investigation supports the hypothesis that some causes of language impairment are distinct from factors that influence normal language variation. This work therefore implicates CMIP and ATP2C2 in the etiology of SLI and provides molecular evidence for the importance of phonological short-term memory in language acquisition.
Characterization of a Family with Rare Deletions in CNTNAP5 and DOCK4 Suggests Novel Risk Loci for Autism and Dyslexia
Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral deficits and complex genetic etiology. A recent study of 517 ASD families implicated DOCK4 by single nucleotide polymorphism (SNP) association and a microdeletion in an affected sibling pair.
Identification of Candidate Genes for Dyslexia Susceptibility on Chromosome 18
Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis.
DCDC2, KIAA0319 and CMIP Are Associated with Reading-related Traits
Several susceptibility genes have been proposed for dyslexia (reading disability; RD) and specific language impairment (SLI). RD and SLI show comorbidity, but it is unclear whether a common genetic component is shared.
Dissection of Genetic Associations with Language-related Traits in Population-based Cohorts
Recent advances in the field of language-related disorders have led to the identification of candidate genes for specific language impairment (SLI) and dyslexia. Replication studies have been conducted in independent samples including population-based cohorts, which can be characterised for a large number of relevant cognitive measures. The availability of a wide range of phenotypes allows us to not only identify the most suitable traits for replication of genetic association but also to refine the associated cognitive trait. In addition, it is possible to test for pleiotropic effects across multiple phenotypes which could explain the extensive comorbidity observed across SLI, dyslexia and other neurodevelopmental disorders. The availability of genome-wide genotype data for such cohorts will facilitate this kind of analysis but important issues, such as multiple test corrections, have to be taken into account considering that small effect sizes are expected to underlie such associations.
PCSK6 is Associated with Handedness in Individuals with Dyslexia
Approximately 90% of humans are right-handed. Handedness is a heritable trait, yet the genetic basis is not well understood. Here we report a genome-wide association study for a quantitative measure of relative hand skill in individuals with dyslexia [reading disability (RD)]. The most highly associated marker, rs11855415 (P = 4.7 × 10(-7)), is located within PCSK6. Two independent cohorts with RD show the same trend, with the minor allele conferring greater relative right-hand skill. Meta-analysis of all three RD samples is genome-wide significant (n = 744, P = 2.0 × 10(-8)). Conversely, in the general population (n = 2666), we observe a trend towards reduced laterality of hand skill for the minor allele (P = 0.0020). These results provide molecular evidence that cerebral asymmetry and dyslexia are linked. Furthermore, PCSK6 is a protease that cleaves the left-right axis determining protein NODAL. Functional studies of PCSK6 promise insights into mechanisms underlying cerebral lateralization and dyslexia.
