Lars Bolund

Aarhus University, Aarhus, Central Jutland, Denmark

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Publications (313)1596.89 Total impact

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    ABSTRACT: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.
    03/2015; 42(1):14-21. DOI:10.5653/cerm.2015.42.1.14
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    ABSTRACT: Building a population-specific catalogue of single nucleotide variants (SNVs), indels and structural variants (SVs) with frequencies, termed a national pan-genome, is critical for further advancing clinical and public health genetics in large cohorts. Here we report a Danish pan-genome obtained from sequencing 10 trios to high depth (50 × ). We report 536k novel SNVs and 283k novel short indels from mapping approaches and develop a population-wide de novo assembly approach to identify 132k novel indels larger than 10 nucleotides with low false discovery rates. We identify a higher proportion of indels and SVs than previous efforts showing the merits of high coverage and de novo assembly approaches. In addition, we use trio information to identify de novo mutations and use a probabilistic method to provide direct estimates of 1.27e-8 and 1.5e-9 per nucleotide per generation for SNVs and indels, respectively.
    Nature Communications 01/2015; 6:5969. DOI:10.1038/ncomms6969 · 10.74 Impact Factor
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    ABSTRACT: Logistic regression analysis based on data from 822 Han Chinese oldest old aged 92+ demonstrated that interactions between carrying FOXO1A-266 or FOXO3-310 or FOXO3-292 and tea drinking at around age 60 or at present time were significantly associated with lower risk of cognitive disability at advanced ages. Associations between tea drinking and reduced cognitive disability were much stronger among carriers of the genotypes of FOXO1A-266 or FOXO3-310 or FOXO3-292 compared with noncarriers, and it was reconfirmed by analysis of three-way interactions across FOXO genotypes, tea drinking at around age 60, and at present time. Based on prior findings from animal and human cell models, we postulate that intake of tea compounds may activate FOXO gene expression, which in turn may positively affect cognitive function in the oldest old population. Our empirical findings imply that the health benefits of particular nutritional interventions, including tea drinking, may, in part, depend upon individual genetic profiles.
    The Journals of Gerontology Series A Biological Sciences and Medical Sciences 06/2014; 70(4). DOI:10.1093/gerona/glu060 · 4.98 Impact Factor
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    ABSTRACT: The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTauP301L-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.
    BioTechniques 05/2014; 56(5):263-8. DOI:10.2144/000114169 · 2.75 Impact Factor
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    ABSTRACT: To analyze the correlation between the genetic constitution and the phenotype in triploid pregnancies. 158 triploid pregnancies identified in hospitals in Western Denmark from April 1986 to April 2010. Clinical data and karyotypes were collected retrospectively and archived samples were retrieved. The parental origin of the genome, either double paternal contribution (PPM) or double maternal contribution (MMP) was determined by analyzing methylation levels at imprinted sites. There were significantly more PPM than MMP cases (p<0.01). In MMP cases the possible karyotypes had similar frequencies, whereas in PPM cases 43% had the karyotype 69,XXX, 51% the karyotype 69,XXY, and 6% the karyotype 69,XYY. Molar phenotype was only seen in PPM cases. However, PPM cases with a non-molar phenotype were also seen. For both parental genotypes, various fetal phenotypes were seen at autopsy. Levels of human chorionic gonadotropin in maternal serum (MS-hCG) were low in MMP cases and varying in PPM cases, some being as low as in the MMP cases. In a triploid pregnancy, suspicion of hydatidiform mole at US, by macroscopic inspection of the evacuated tissue, at histology, or due to a high MS-hCG level each predict the parental type PPM with a very high specificity. In contrast the sensitivity of these observations is less than 100%.
    American journal of obstetrics and gynecology 03/2014; 211(4). DOI:10.1016/j.ajog.2014.03.039 · 3.97 Impact Factor
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    ABSTRACT: Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.
    Reproduction Fertility and Development 03/2014; 26(3):469-84. DOI:10.1071/RD13037 · 2.58 Impact Factor
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    ABSTRACT: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. Time-lapse analysis. Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p < 0.001), displayed longer duration of the 3-cell stage (p < 0.001), and arrested development from the compaction stage and onwards (p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells (p < 0.001). This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos.
    Journal of Assisted Reproduction and Genetics 01/2014; 31(4). DOI:10.1007/s10815-014-0178-3 · 1.77 Impact Factor
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    ABSTRACT: In this paper, we mine full mtDNA sequences from an exome capture data set of 2000 Danes, showing that it is possible to get high-quality full-genome sequences of the mitochondrion from this resource. The sample includes 1000 individuals with type 2 diabetes and 1000 controls. We characterise the variation found in the mtDNA sequence in Danes and relate the variation to diabetes risk as well as to several blood phenotypes of the controls but find no significant associations. We report 2025 polymorphisms, of which 393 have not been reported previously. These 393 mutations are both very rare and estimated to be caused by very recent mutations but individuals with type 2 diabetes do not possess more of these variants. Population genetics analysis using Bayesian skyline plot shows a recent history of rapid population growth in the Danish population in accordance with the fact that >40% of variable sites are observed as singletons.European Journal of Human Genetics advance online publication, 22 January 2014; doi:10.1038/ejhg.2013.282.
    European journal of human genetics: EJHG 01/2014; 22(8). DOI:10.1038/ejhg.2013.282 · 4.23 Impact Factor
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    ABSTRACT: Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control. A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems. This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
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    ABSTRACT: Biomimetic nanofibrous scaffolds combined with stem cells are promising for bone tissue engineering. In the present study, we have employed nano-hydroxyapatite (nHAp) contained polycaprolactone (PCL) nanofibers as a biomimetic nanofibrous scaffold, and mesenchymal stem cells derived from human induced pluripotent stem cells (hiPS-MSCs) as the novel stem cells sources. The response of hiPS-MSCs on the nanofibrous scaffolds in terms of cell proliferation and differentiation into the osteoblastic phenotype was investigated by XTT assay, scanning electron microscopy (SEM), osteogenic genes expression (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen I (COL1A1), and osteocalcin (OC)), ALP activity, and calcium deposition. It is clearly shown that the hiPS-MSCs attached, and proliferated on the nanofibrous scaffolds. Compared with PCL nanofibers without nHAp, the cells on the nHAp contained nanofibers demonstrated superior capabilites to differentiate to form calcified extrac
    RSC Advances 01/2014; 4(11-11):5734-5739. DOI:10.1039/C3RA44181D · 3.71 Impact Factor
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    ABSTRACT: Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Tamoxifen resistance may be the result of acquisition of epigenetic regulation within breast cancer cells, such as DNA methylation, resulting in changed mRNA expression of genes pivotal for estrogen-dependent growth. Alternatively, tamoxifen resistance may be due to selection of pre-existing resistant cells, or a combination of the two mechanisms. To evaluate the contribution of these possible tamoxifen resistance mechanisms, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massive parallel sequencing to analyze a well-established tamoxifen-resistant cell line model (TAMR), consisting of 4 resistant and one parental cell line. Another tamoxifen-resistant cell line model system (LCC1/LCC2) was used to validate the DNA methylation and gene expression results. Significant differences were observed in global gene expression and DNA methylation profiles between the parental tamoxifen-sensitive cell line and the 4 tamoxifen-resistant TAMR sublines. The 4 TAMR cell lines exhibited higher methylation levels as well as an inverse relationship between gene expression and DNA methylation in the promoter regions. A panel of genes, including NRIP1, HECA and FIS1, exhibited lower gene expression in resistant vs. parental cells and concurrent increased promoter CGI methylation in resistant vs. parental cell lines. A major part of the methylation, gene expression, and pathway alterations observed in the TAMR model were also present in the LCC1/LCC2 cell line model. More importantly, high expression of SOX2 and alterations of other SOX and E2F gene family members, as well as RB-related pocket protein genes in TAMR highlighted stem cell-associated pathways as being central in the resistant cells and imply that cancer-initiating cells/cancer stem-like cells may be involved in tamoxifen resistance in this model. Our data highlight the likelihood that resistant cells emerge from cancer-initiating cells/cancer stem-like cells and imply that these cells may gain further advantage in growth via epigenetic mechanisms. Illuminating the expression and DNA methylation features of putative cancer-initiating cells/cancer stem cells may suggest novel strategies to overcome tamoxifen resistance.
    Breast cancer research: BCR 12/2013; 15(6):R119. DOI:10.1186/bcr3588 · 5.88 Impact Factor
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    ABSTRACT: To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.
    Aging cell 12/2013; DOI:10.1111/acel.12186 · 5.94 Impact Factor
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    ABSTRACT: When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set or two paternal and one maternal set but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples. Using Methylation specific Multiplex Ligation-dependent Probe Amplification we measured methylation levels at different imprinted sites. We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed. We present Methylation specific Multiplex Ligation-dependent Probe Amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 hours; it is inexpensive, simple, and easy to use and parental samples are not needed. This article is protected by copyright. All rights reserved.
    Prenatal Diagnosis 12/2013; 33(12). DOI:10.1002/pd.4206 · 2.51 Impact Factor
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    ABSTRACT: Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.
    PLoS ONE 10/2013; 8(10):e76098. DOI:10.1371/journal.pone.0076098 · 3.53 Impact Factor
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    ABSTRACT: Women with mutation in both alleles of the NLRP7 or C6orf221/KHDC3L genes are predisposed to diploid biparental moles, but it has also been suggested that mutation in these genes can predispose to diploid androgenetic or triploid moles and to other kinds of reproductive wastage. We have investigated the association between molar pregnancy and recurrent miscarriages regarding changes in the NLRP7 and C6orf221/KHDC3L genes. Our study group can be divided into three sub-cohorts: 1) women having had at least one molar pregnancy and at least two non-mole miscarriages, 2) women having had recurrent androgenetic hydatidiform moles and 3) women having had one diploid androgenetic hydatidiform mole and a relative having had a hydatidiform mole (familial hydatidiform moles).We observed a statistically non-significant tendency of non-synonymous variants in NLRP7 to be more frequent in women with familial hydatidiform mole and in women with female family members with hydatidiform mole or non-mole miscarriage compared to women with no family history of mole or miscarriage. However, we did not find any unequivocal pathogenic mutations (the term 'unequivocal pathogenic mutations' refers to mutations which indubitably have a pathogenic effect on the affected woman) in NLRP7 or C6orf221/KHDC3L in any of the women in the study group. This indicates that recurrent miscarriages plus hydatidiform mole, recurrent androgenetic hydatidiform moles and familial androgenetic hydatidiform moles in general do not have the same monogenetic etiology as familiar diploid biparental moles.
    Molecular Human Reproduction 08/2013; DOI:10.1093/molehr/gat056 · 3.48 Impact Factor
  • Science translational medicine 07/2013; 5(166):1-10. · 14.41 Impact Factor
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    ABSTRACT: We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.
    Scientific Reports 07/2013; 3:2243. DOI:10.1038/srep02243 · 5.08 Impact Factor
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    Dataset: det132supp
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    ABSTRACT: How does tetraploidy develop in hydatidiform moles (HMs), and what is the frequency of the different origins? Most molar pregnancies with tetraploid cells appear to be produced by somatic endoreduplications, while a minority originate from a tetraploid zygote. The frequency of zygotic tetraploidy was estimated to be 0.7. The parental origin of the genome in tetraploid HMs has only been evaluated in a few cases, most showing three genome sets from the father (PPPM). Estimates of the proportion of HMs that are tetraploid vary between 2 and 28. From 1986 to 2010, unfixed samples of clinically suspected molar pregnancies were forwarded to the Danish Mole Project. For this cohort study 442 samples fulfilled the following criteria for inclusion: macroscopic appearance of HM and 10 vesicular chorionic villi with a diameter of 1 mm. Of 403 karyotyped samples, 21 cases disclosed 2 tetraploid metaphases. The 21 cases were scrutinized by karyotyping, flow cytometry (FC) and DNA-marker analysis. Among 20 HMs, 3 showed the genotype PPPM: one with the sex chromosomes XXYY and two with XXXY, indicating that they originated in tetraploid zygotes. In 14 androgenetic, one likely androgenetic and two mosaics, the tetraploid cells likely developed by endoreduplications of diploid cells. One case did not fulfil the histopathological criteria for HM. As an inclusion criterion was the macroscopic observation of vesicular chorionic villi, some non-molar hydropic placentas may have been included and some early moles may have been excluded. In future, studies to determine that an HM is tetraploid and discriminate cases of mosaicism and to deduce the origin of the tetraploidy must use the techniques of karyotyping, DNA-marker analysis and FC in combination. No external funding was sought to support this study. None of the authors has any conflict of interest to declare.
    Human Reproduction 04/2013; 28(7). DOI:10.1093/humrep/det132 · 4.59 Impact Factor

Publication Stats

10k Citations
1,596.89 Total Impact Points

Institutions

  • 1979–2015
    • Aarhus University
      • • Institute of Human Genetics
      • • Department of Genetics and Biotechnology
      Aarhus, Central Jutland, Denmark
  • 2011
    • BGI Human Genome Center
      Peping, Beijing, China
  • 2006–2009
    • Beijing Genomics Institute
      Bao'an, Guangdong, China
  • 2007
    • Chinese Academy of Sciences
      Peping, Beijing, China
  • 1998
    • Medical College of Wisconsin
      • Department of Biochemistry
      Milwaukee, Wisconsin, United States
  • 1997
    • IT University of Copenhagen
      København, Capital Region, Denmark
  • 1996
    • Swedish University of Agricultural Sciences
      • Institutionen för husdjursgenetik
      Uppsala, Uppsala, Sweden
  • 1992–1995
    • Aarhus University Hospital
      Aarhus, Central Jutland, Denmark
  • 1994
    • Baylor College of Medicine
      Houston, Texas, United States
  • 1990
    • Duke University
      Durham, North Carolina, United States