To prepare the ESA (European Space Agency) spaceflight project "Wound healing and Sutures in Unloading Conditions", we studied mechanisms of apoptosis in wound healing models based on ex vivo skin ...tissue cultures, kept for 10 days alive in serum-free DMEM/F12 medium supplemented with bovine serum albumin, hydrocortisone, insulin, ascorbic acid and antibiotics at 32 °C. The overall goal is to test: (i) the viability of tissue specimens; (ii) the gene expression of activators and inhibitors of apoptosis and extracellular matrix components in wound and suture models; and (iii) to design analytical protocols for future tissue specimens after post-spaceflight download. Hematoxylin-Eosin and Elastica-van-Gieson staining showed a normal skin histology with no signs of necrosis in controls and showed a normal wound suture. TdT-mediated dUTP-biotin nick end labeling for detecting DNA fragmentation revealed no significant apoptosis. No activation of caspase-3 protein was detectable.
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mRNAs were not altered in epidermis and dermis samples with and without a wound compared to 0 day samples (specimens investigated directly post-surgery).
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mRNAs were downregulated in epidermis/dermis samples with and/or without a wound compared to 0 day samples.
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were upregulated in 10 day wound samples compared to 0 day samples in epidermis/dermis.
mRNAs were elevated in 10 day wound and no wound samples compared to 0 day samples in dermis. In conclusion, we demonstrate that it is possible to maintain live skin tissue cultures for 10 days. The viability analysis showed no significant signs of cell death in wound and suture models. The gene expression analysis demonstrated the interplay of activators and inhibitors of apoptosis and extracellular matrix components, thereby describing important features in ex vivo sutured wound healing models. Collectively, the performed methods defining analytical protocols proved to be applicable for post-flight analyzes of tissue specimens after sample return.
•Keratinocytes from Sca-1 knockout mice have reduced clonogenic potential.•Sca-1 knockout transcriptome differs from the wildtype transcriptome.•Sca-1 knockout mice have normal epidermal morphology.
...Stem Cell Antigen-1 (SCA-1) is a central positive marker for isolating stem cells in several tissues in the mouse. However, for the epidermis, this appears to be the opposite since lack of SCA-1 has been shown to identify keratinocyte populations with progenitor characteristics.
This study investigates the effect of SCA-1 knockout in murine keratinocytes. We compared Sca-1EGFP/EGFP knockout and wildtype mice with respect to the three-dimensional morphology of the epidermis, performed functional assays, and generated gene expression profiles on FACS sorted cells.
There were no morphological abnormalities on skin, fur, or hair follicles in transgenic knockout mice compared to wild type mice. SCA-1 knockout keratinocytes showed significantly reduced colony-forming efficiency, colony size and proliferation rate in vitro, however, SCA-1 knockout did not alter wound healing efficiency or keratinocyte proliferation rate in vivo. Moreover, gene expression profiling shows that the effect from knockout of SCA-1 in keratinocytes is dissimilar from what has been observed in other tissues. Additionally, tumor assay indicated that SCA-1 knockout decreases the number of formed papillomas.
The results indicate a more complex role for SCA-1, which might differ between epidermal keratinocytes during homeostasis and activated conditions.
The YTH domain family member 3 gene (YTHDF3) encodes a reader of the abundant N6‐methyladenosine (m6A) modification of eukaryotic mRNA, which plays an essential role in regulating mRNA stability and ...is necessary to achieve normal development of the central nervous system in animal models. YTHDF3 has not previously been implicated in Mendelian disease despite a high probability of loss of function intolerance and statistical evidence of enrichment for gene‐disruptive de novo variants in large‐scale studies of individuals with intellectual disability and/or developmental delay. We report four individuals with deletion of 8q12.3, deletion size 1.38–2.60 Mb, encompassing YTHDF3, three of them were de novo, and in one case, the inheritance was unknown. Common features of the individuals (age range, 4–22 years) were developmental delay and/or intellectual disability. Two individuals underwent squint surgery. We suggest that haploinsufficiency of YTHDF3 causes a neurodevelopmental disorder with developmental delay and intellectual disability of variable degree.
Mono‐allelic deletions in 8q12.3 including YTHDF3 cause intellectual disability or developmental delay with no major malformations.
Current differentiation protocols for generating mesencephalic dopaminergic (mesDA) neurons from human pluripotent stem cells result in grafts containing only a small proportion of mesDA neurons when ...transplanted in vivo. In this study, we develop lineage-restricted undifferentiated stem cells (LR-USCs) from pluripotent stem cells, which enhances their potential for differentiating into caudal midbrain floor plate progenitors and mesDA neurons. Using a ventral midbrain protocol, 69% of LR-USCs become bona fide caudal midbrain floor plate progenitors, compared to only 25% of human embryonic stem cells (hESCs). Importantly, LR-USCs generate significantly more mesDA neurons under midbrain and hindbrain conditions in vitro and in vivo. We demonstrate that midbrain-patterned LR-USC progenitors transplanted into 6-hydroxydopamine-lesioned rats restore function in a clinically relevant non-pharmacological behavioral test, whereas midbrain-patterned hESC-derived progenitors do not. This strategy demonstrates how lineage restriction can prevent the development of undesirable lineages and enhance the conditions necessary for mesDA neuron generation.
Mosaics and moles SUNDE, Lone; NIEMANN, Isa; STAEHR HANSEN, Estrid ...
European journal of human genetics,
10/2011, Letnik:
19, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Hydatidiform mole (HM) is an abnormal human pregnancy, where the placenta presents with vesicular swelling of the chorionic villi. A fetus is either not present, or malformed and not viable. Most ...moles are diploid androgenetic as if one spermatozoon fertilized an empty oocyte, or triploid with one maternal and two paternal chromosome sets as if two spermatozoa fertilized a normal oocyte. However, diploid moles with both paternal and maternal markers of the nuclear genome have been reported. Among 162 consecutively collected diploid moles, we have earlier found indications of both maternal and paternal genomes in 11. In the present study, we have performed detailed analysis of DNA-markers in tissue and single cells from these 11 HMs. In 3/11, we identified one biparental cell population only, whereas in 8/11, we demonstrated mosaicism: one biparental cell population and one androgenetic cell population. One mosaic mole was followed by persistent trophoblastic disease (PTD). In seven of the mosaics, one spermatozoon appeared to have contributed to the genomes of both cell types. Our observations make it likely that mosaic conceptuses, encompassing an androgenetic cell population, result from various postzygotic abnormalities, including paternal pronuclear duplication, asymmetric cytokinesis, and postzygotic diploidization. This corroborates the suggestion that fertilization of an empty egg is not mandatory for the creation of an androgenetic cell population. Future studies of mosaic conceptuses may disclose details about fertilization, early cell divisions and differentiation. Apparently, only a minority of diploid moles with both paternal and maternal markers are 'genuine' diploid biparental moles (DiBiparHMs).
The bulge region of adult murine hair follicles harbors epidermal stem cells with multipotent capacity; however, the restricted contributions of these cells under homeostatic conditions indicates ...that additional stem or progenitor cell populations may be required to maintain squamous and sebaceous lineages. We have identified a distinct population of murine hair follicle keratinocytes residing in the upper isthmus (UI) between the infundibulum and bulge regions that are distinguished by low α6 integrin levels and are negative for CD34 and Sca-1. Purified UI cells give rise to long-term, stable epidermal, follicular and sebaceous lineages and can self-renew in vivo. These cells are non-quiescent and possess a unique transcript profile compared with bulge stem cells and may represent a distinct reservoir of epidermal stem or progenitor cells.
ObjectivesCharcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system, yet no studies have compared the mortality in patients with CMT with that of the ...general population, and prevalence estimates vary considerably. We performed a nationwide register-based study to investigate the prevalence, incidence and mortality of CMT in Denmark.DesignWe used the Danish National Patient Registry to select all records with primary diagnostic codes for CMT between 1977 and 2012 given at a neurological, neurophysiological, paediatric or clinical genetic clinic. The prevalence was estimated by 31 December 2012, and the incidence rate was calculated based on data from 1988 to 2012. We calculated a standardised mortality ratio (SMR) and an absolute excess mortality rate (AER) stratified according to age categories and disease duration.ResultsA total of 1534 patients (652 women) were identified. The prevalence proportion was 22.5 per 100 000 (95% CI 21.2 to 23.7) and the incidence rate was 0.98 (95% CI 0.93 to 1.04) per 100 000 person-years. The SMR was 1.36 (95% CI 1.21 to 1.53), and the AER was 4.87 per 1000 person-years (95% CI 2.77 to 6.96). We found a significantly higher SMR in cases below 50 years of age, and in cases with disease duration of more than 10 years.ConclusionsWe found a reduced life expectancy among patients diagnosed with CMT. To our knowledge, this is the first study of CMT to use nationwide register-based data, and the first to report an SMR and an AER.
Every year more than 800 patients in Denmark are diagnosed with renal cell carcinoma (RCC) of which 3-5% are expected to be part of a hereditary renal cancer syndrome. We performed genetic screening ...of causative and putative RCC-genes (VHL, FH, FLCN, MET, SDHB, BAP1, MITF, CDKN2B) in RCC-patients suspected of a genetic predisposition.
The cohort consisted of forty-eight Danish families or individuals with early onset RCC, a family history of RCC, a family history of RCC and melanoma or both RCC- and melanoma diagnosis in the same individual. DNA was extracted from peripheral blood samples or cancer-free formalin-fixed paraffin-embedded tissue.
One start codon variant of unknown clinical significance (VUS) (c.3G>A, p.Met1Ile) and one missense VUS (c.631A>C, p.Met211Leu) was found in VHL in a patient with RCC-onset at twenty-eight years of age but without other manifestations or family history of von Hippel-Lindau (VHL). Furthermore, in three families we found three different variants in BAP1, one of which was a novel non-segregating missense variant (c.1502G>A, p.Ser501Asn) in a family with two brothers affected with RCC. Finally, we found the known E318K-substitution in MITF in a RCC-affected member of a family with multiple melanomas. No variants were detected in CDKN2B.
Although we did find three VUS's in BAP1 in three families and a pathogenic variant in MITF in one family, pathogenic germline variants in BAP1, MITF or CDKN2B are not frequent causes of hereditary renal cancer in Denmark. It is possible that the high prevalence of risk factors such as male gender, smoking and obesity has influenced the development of cancer in the patients of the current study. Further investigations into putative predisposing genes and risk factors of RCC are necessary to enable better prediction of renal cancer risk or presymptomatic testing of relatives in hereditary renal cancer families.
Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype.
We identified two families ...with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells.
Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability.
This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.
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