| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Bieder, Andrea, et al.
(författare)
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Rare variants in dynein heavy chain genes in two individuals with situs inversus and developmental dyslexia : a case report
- 2020
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Ingår i: BMC Medical Genetics. - : Springer. - 1471-2350. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Developmental dyslexia (DD) is a neurodevelopmental learning disorder with high heritability. A number of candidate susceptibility genes have been identified, some of which are linked to the function of the cilium, an organelle regulating left-right asymmetry development in the embryo. Furthermore, it has been suggested that disrupted left-right asymmetry of the brain may play a role in neurodevelopmental disorders such as DD. However, it is unknown whether there is a common genetic cause to DD and laterality defects or ciliopathies. Case presentation Here, we studied two individuals with co-occurring situs inversus (SI) and DD using whole genome sequencing to identify genetic variants of importance for DD and SI. Individual 1 had primary ciliary dyskinesia (PCD), a rare, autosomal recessive disorder with oto-sino-pulmonary phenotype and SI. We identified two rare nonsynonymous variants in the dynein axonemal heavy chain 5 gene (DNAH5): a previously reported variant c.7502G > C; p.(R2501P), and a novel variant c.12043 T > G; p.(Y4015D). Both variants are predicted to be damaging. Ultrastructural analysis of the cilia revealed a lack of outer dynein arms and normal inner dynein arms. MRI of the brain revealed no significant abnormalities. Individual 2 had non-syndromic SI and DD. In individual 2, one rare variant (c.9110A > G;p.(H3037R)) in the dynein axonemal heavy chain 11 gene (DNAH11), coding for another component of the outer dynein arm, was identified. Conclusions We identified the likely genetic cause of SI and PCD in one individual, and a possibly significant heterozygosity in the other, both involving dynein genes. Given the present evidence, it is unclear if the identified variants also predispose to DD and further studies into the association between laterality, ciliopathies and DD are needed.
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| 3. |
- Buckley, Patrick G, et al.
(författare)
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A full-coverage, high-resolution human chromosome 22 genomic microarrayfor clinical and research applications
- 2002
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 11:25, s. 3221-3229
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Tidskriftsartikel (refereegranskat)abstract
- We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.
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| 4. |
- Goumidi, Louisa, et al.
(författare)
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Study of estrogen receptor-α and receptor-β gene polymorphisms on Alzheimer's disease.
- 2011
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Ingår i: Journal of Alzheimer's Disease. - 1387-2877 .- 1875-8908. ; 26:3, s. 431-9
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Tidskriftsartikel (refereegranskat)abstract
- Estrogen treatment can modulate the risk for developing dementia in women. Therefore, single nucleotide polymorphisms (SNPs) in the estrogen receptor genes may constitute genetic susceptibility factors to Alzheimer's disease (AD). Thus, we investigated the impact of the genetic variability of the estrogen receptor α 1 (ESR1) and estrogen receptor α 2 (ESR2) genes on late onset AD risk. We analyzed 39 SNPs in ESR1 and 5 SNPs in ESR2 in a French case-control study of sporadic AD (1007 cases/647 controls). Individuals carrying the minor allele of rs7450824 had a lower risk of AD than homozygous subjects for the major allele (age, gender, and APOE ε4 allele adjusted odds ratio = 0.71 [0.57-0.89], p = 0.003). However, this association did not resist Bonferroni correction for multiple testing (p-threshold < 0.001). Consistently, no significant association could be detected when considering age of onset. We also tested for possible interactions between the ESR SNPs and APOE status (ε4 allele) or gender but no significant interaction could be observed. Even after stratifying the sample on APOE status or gender, no significant association with AD risk could be detected. Finally, we searched for potential gene-gene interactions between ESR1 and ESR2 SNPs but no significant interaction could be detected. Our results reinforce the notion that SNPs in the ESR1 or ESR2 genes do not seem to play a major role in the genetic susceptibility of AD.
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| 5. |
- Grigelioniene, Giedre, et al.
(författare)
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Analysis of short stature homeobox-containing gene ( SHOX) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity
- 2001
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Ingår i: Human Genetics. - : Springer Science and Business Media LLC. - 1432-1203 .- 0340-6717. ; 109:5, s. 551-558
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Tidskriftsartikel (refereegranskat)abstract
- Dyschondrosteosis (DCO; also called Leri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.
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| 6. |
- Hofmeister, Wolfgang, et al.
(författare)
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CTNND2-a candidate gene for reading problems and mild intellectual disability.
- 2015
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Ingår i: Journal of Medical Genetics. - : BMJ. - 0022-2593 .- 1468-6244. ; 52:2, s. 111-22
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cytogenetically visible chromosomal translocations are highly informative as they can pinpoint strong effect genes even in complex genetic disorders.METHODS AND RESULTS: Here, we report a mother and daughter, both with borderline intelligence and learning problems within the dyslexia spectrum, and two apparently balanced reciprocal translocations: t(1;8)(p22;q24) and t(5;18)(p15;q11). By low coverage mate-pair whole-genome sequencing, we were able to pinpoint the genomic breakpoints to 2 kb intervals. By direct sequencing, we then located the chromosome 5p breakpoint to intron 9 of CTNND2. An additional case with a 163 kb microdeletion exclusively involving CTNND2 was identified with genome-wide array comparative genomic hybridisation. This microdeletion at 5p15.2 is also present in mosaic state in the patient's mother but absent from the healthy siblings. We then investigated the effect of CTNND2 polymorphisms on normal variability and identified a polymorphism (rs2561622) with significant effect on phonological ability and white matter volume in the left frontal lobe, close to cortical regions previously associated with phonological processing. Finally, given the potential role of CTNND2 in neuron motility, we used morpholino knockdown in zebrafish embryos to assess its effects on neuronal migration in vivo. Analysis of the zebrafish forebrain revealed a subpopulation of neurons misplaced between the diencephalon and telencephalon.CONCLUSIONS: Taken together, our human genetic and in vivo data suggest that defective migration of subpopulations of neuronal cells due to haploinsufficiency of CTNND2 contribute to the cognitive dysfunction in our patients.
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| 7. |
- Massinen, Satu, et al.
(författare)
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Functional interaction of DYX1C1 with estrogen receptors suggests involvement of hormonal pathways in dyslexia
- 2009
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 18:15, s. 2802-2812
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Tidskriftsartikel (refereegranskat)abstract
- Dyslexia, or specific reading disability, is the unexpected failure in learning to read and write when intelligence and senses are normal. One of the susceptibility genes, DYX1C1, has been implicated in neuronal migration, but little is known about its interactions and functions. As DYX1C1 was suggested to interact with the U-box protein CHIP (carboxy terminus of Hsc70-interacting protein), which also participates in the degradation of estrogen receptors alpha (ERalpha) and beta (ERbeta), we hypothesized that the effects of DYX1C1 might be at least in part mediated through the regulation of ERs. ERs have shown to be important in brain development and cognitive functions. Indeed, we show that DYX1C1 interacts with both ERs in the presence of 17beta-estradiol, as determined by co-localization, co-immunoprecipitation and proximity ligation assays. Protein levels of endogenous ERalpha or exogenous ERbeta were reduced upon over-expression of DYX1C1, resulting in decreased transcriptional responses to 17beta-estradiol. Furthermore, we detected in vivo complexes of DYX1C1 with ERalpha or ERbeta at endogenous levels along neurites of primary rat hippocampal neurons. Taken together, our data suggest that DYX1C1 is involved in the regulation of ERalpha and ERbeta, and may thus affect the brain development and regulate cognitive functions. These findings provide novel insights into the function of DYX1C1 and link neuronal migration and developmental dyslexia to the estrogen-signaling effects in the brain.
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| 8. |
- Massinen, Satu, et al.
(författare)
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Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons.
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.
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| 9. |
- Tammimies, Kristiina, et al.
(författare)
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Molecular networks of DYX1C1 gene show connection to neuronal migration genes and cytoskeletal proteins.
- 2013
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Ingår i: Biological Psychiatry. - : Elsevier BV. - 0006-3223 .- 1873-2402. ; 73:6, s. 583-90
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The dyslexia susceptibility 1 candidate 1 (DYX1C1) gene has recently been associated with dyslexia and reading scores in several population samples. The DYX1C1 has also been shown to affect neuronal migration and modulate estrogen receptor signaling.METHODS: We have analyzed the molecular networks of DYX1C1 by gene expression and protein interaction profiling in a human neuroblastoma cell line.RESULTS: We find that DYX1C1 can modulate the expression of nervous system development and neuronal migration genes such as RELN and associate with a number of cytoskeletal proteins. We also show by live cell imaging that DYX1C1 regulates cell migration of the human neuroblastoma cell line dependent on its tetratricopeptide repeat and DYX1 protein domains. The DYX1 domain is a novel highly conserved domain identified in this study by multiple sequence alignment of DYX1C1 proteins recovered from a wide range of eukaryotic species.CONCLUSIONS: Our results contribute to the hypothesis that dyslexia has a developmental neurobiological basis by linking DYX1C1 with many genes involved in neuronal migration disorders.
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| 10. |
- Tapia Paez, Isabel
(författare)
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Characterization of human chromosome 22 : cloning of breakpoints of the constitutional translocation t(11;22)(q23;q11) and detection of small constitutional delections by microarray CGH
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chromosome 22 is the second smallest human chromosome, composing approximately 1.5% of the genome. The short arm of this acrocentric chromosome harbors ribosomal genes and the long arm contains the protein coding genes. This chromosome is gene-rich in comparison to the majority of other chromosomes, containing approximately 600 so far characterized genes. Many of these are involved in the etiology of a wide spectrum of diseases such as congenital and psychiatric disorders as well as cancers. The constitutional translocation t(11;22) is the most common reciprocal translocation in humans. This translocation is often found in families but can also occur de novo. Translocation carriers are normal and usually become diagnosed in connection with infertility problems or a birth of a genetically unbalanced child. In addition, an increased risk to breast cancer has been reported in some carriers, which suggests that the translocation might have an effect on a gene(s) involved in the etiology of breast cancer. We characterized the breakpoints of this translocation and found that the breakpoint region on chromosome 22 lies within an unclonable gap. The breakpoint on chromosome 11 is also located within an unstable region, as all BACs containing this segment are rearranged. We identified one BAC from chromosome 11 spanning the translocation breakpoint and two BAC clones from chromosome 22, which contain sequences similar/identical to the sequences mediating the translocations breakpoints on chromosome 22. A cosmid library from one translocation carrier was also constructed and chimeric cosmids from both derivative chromosomes were isolated. Their analysis revealed that no gene(s) seems to be disrupted by the translocation breakpoints. We also show that the breakpoints on both chromosomes occur at the tip of hairpins, which are formed due to the presence of long inverted repeats/palindromes. The formation of these structures is the likely reason behind "unclonability" of this region on chromosome 22 and the instability of BACs derived from chromosome 11. Furthermore, based on fiber-FISH experiments we conclude that the breakpoints of the translocations are highly conserved among carriers. The second aspect of the thesis is related to detection of micro-deletions and micro- gains, which cause a large number of genetic disorders. In order to improve the detection of such rearrangements, we applied and further developed the microarray-CGH methodology. We constructed three microarrays: one covering 7 Mb region in the vicinity of the NF2 gene in 22q12; the second is a full coverage chromosome 22 array; and the third is an array covering 6 Mb from the 22q11 region, including the typically deleted region in DiGeorgeVelo-Cardio-Facial syndrome. The latter region is particularly challenging, due to the presence of low copy repeats, high content of common repeats and unclonable sequences. Three types of targets were used in the arrays: i) genomic clones; ii) non-redundant, repeat-free pools of genomic DNA amplified by PCR; and iii) cDNA-based targets, single as well as in pools. We used the arrays to study neurofibromatosis type 2, acral melanoma, dermatofibrosarcoma, and DiGeorge/Velo-Cardio-Facial syndrome. We were able to detect homozygous/heterozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of an imbalanced translocation. Using the novel approach with repeat-free, PCR-generated sequences, we detected heterozygous deletions using as little as 11.5 kb of genomic target sequence. We conclude that the array-CGH is a powerful method for the detection of gene-dosage imbalances. Our results also suggest that most, if not all, medically important segments of our genome will be accessible for analysis using high-resolution microarray-based CGH.
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