Immune cells lacking Y chromosome show dysregulation of autosomal gene expression

Funder: Kjell och Märta Beijers Stiftelse (SE)Funder: Hjärnfonden; doi: http://dx.doi.org/10.13039/501100003792Funder: Cancerfonden; doi: http://dx.doi.org/10.13039/501100002794Funder: Vetenskapsrådet; doi: http://dx.doi.org/10.13039/501100004359Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599Funder: Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse (SE)Funder: Science for Life Laboratory (SE)Funder: Fundacja na rzecz Nauki Polskiej (PL)Funder: Uppsala UniversityAbstract: Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease

Genetic predisposition to mosaic Y chromosome loss in blood.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism1-5, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.This research has been conducted using the UK Biobank Resource under application 9905 and 19808. This work was supported by the Medical Research Council [Unit Programme number MC_UU_12015/2]. Full study-specific and individual acknowledgements can be found in the supplementary information

Sequence based analysis of neurodevelopmental disorders

In this thesis the main focus is the use of methods and applications of next generation sequencing in order to study three of the most common neurodevelopmental disorders: intellectual disability, epilepsy and schizophrenia. A large fraction of the genes in our genome produce several distinct transcript isoforms through the process of splicing and there is an increasing amount of evidence pinpointing mutations affecting splicing as a mechanism of disease.  In Paper I we used exome capture of RNA in combination with sequencing in order to enrich for coding sequences. We show that this approach enables us to detect lowly expressed transcript and splice events that would have been missed in regular RNA sequencing using the same coverage.  In Paper II we selectively depleted the different transcripts of Quaking (QKI), a gene previously associated to schizophrenia. Using RNA sequencing we show that the effects of depletion differ between transcripts and that the QKI gene is a potential regulator of the Glial Fibrillary Acidic Protein (GFAP), a gene implicated in several diseases in the central nervous system. De-novo mutations are frequently reported to be causative in neurodevelopmental disorders with a strong genetic component, such as epilepsy and intellectual disability. In Paper III we used exome sequencing in family trios where the child was diagnosed with both intellectual disability and epilepsy, focusing on finding de-novo mutations. We identified several previously unknown disease causing mutations in genes previously known to cause disease and used previously published interaction and mutation data to prioritize novel candidate genes. The most interesting result from this study are the implication of the HECW2 gene as a candidate gene in intellectual disability and epilepsy. In Paper IV we used RNA sequencing of post mortem brain tissue in a large cohort of schizophrenics and controls.  In this study we could show that the immune system and more specifically the complement system was dysregulated in a large fraction of patients. Further, using co-expression network we also found some evidence suggesting genes involved in axon development and maintenance.

Sequence based analysis of neurodevelopmental disorders

In this thesis the main focus is the use of methods and applications of next generation sequencing in order to study three of the most common neurodevelopmental disorders: intellectual disability, epilepsy and schizophrenia. A large fraction of the genes in our genome produce several distinct transcript isoforms through the process of splicing and there is an increasing amount of evidence pinpointing mutations affecting splicing as a mechanism of disease.  In Paper I we used exome capture of RNA in combination with sequencing in order to enrich for coding sequences. We show that this approach enables us to detect lowly expressed transcript and splice events that would have been missed in regular RNA sequencing using the same coverage.  In Paper II we selectively depleted the different transcripts of Quaking (QKI), a gene previously associated to schizophrenia. Using RNA sequencing we show that the effects of depletion differ between transcripts and that the QKI gene is a potential regulator of the Glial Fibrillary Acidic Protein (GFAP), a gene implicated in several diseases in the central nervous system. De-novo mutations are frequently reported to be causative in neurodevelopmental disorders with a strong genetic component, such as epilepsy and intellectual disability. In Paper III we used exome sequencing in family trios where the child was diagnosed with both intellectual disability and epilepsy, focusing on finding de-novo mutations. We identified several previously unknown disease causing mutations in genes previously known to cause disease and used previously published interaction and mutation data to prioritize novel candidate genes. The most interesting result from this study are the implication of the HECW2 gene as a candidate gene in intellectual disability and epilepsy. In Paper IV we used RNA sequencing of post mortem brain tissue in a large cohort of schizophrenics and controls.  In this study we could show that the immune system and more specifically the complement system was dysregulated in a large fraction of patients. Further, using co-expression network we also found some evidence suggesting genes involved in axon development and maintenance.

Sequence based analysis of neurodevelopmental disorders

In this thesis the main focus is the use of methods and applications of next generation sequencing in order to study three of the most common neurodevelopmental disorders: intellectual disability, epilepsy and schizophrenia. A large fraction of the genes in our genome produce several distinct transcript isoforms through the process of splicing and there is an increasing amount of evidence pinpointing mutations affecting splicing as a mechanism of disease.  In Paper I we used exome capture of RNA in combination with sequencing in order to enrich for coding sequences. We show that this approach enables us to detect lowly expressed transcript and splice events that would have been missed in regular RNA sequencing using the same coverage.  In Paper II we selectively depleted the different transcripts of Quaking (QKI), a gene previously associated to schizophrenia. Using RNA sequencing we show that the effects of depletion differ between transcripts and that the QKI gene is a potential regulator of the Glial Fibrillary Acidic Protein (GFAP), a gene implicated in several diseases in the central nervous system. De-novo mutations are frequently reported to be causative in neurodevelopmental disorders with a strong genetic component, such as epilepsy and intellectual disability. In Paper III we used exome sequencing in family trios where the child was diagnosed with both intellectual disability and epilepsy, focusing on finding de-novo mutations. We identified several previously unknown disease causing mutations in genes previously known to cause disease and used previously published interaction and mutation data to prioritize novel candidate genes. The most interesting result from this study are the implication of the HECW2 gene as a candidate gene in intellectual disability and epilepsy. In Paper IV we used RNA sequencing of post mortem brain tissue in a large cohort of schizophrenics and controls.  In this study we could show that the immune system and more specifically the complement system was dysregulated in a large fraction of patients. Further, using co-expression network we also found some evidence suggesting genes involved in axon development and maintenance.

Reply to Veitia

In 2020, we published a paper in EJHG (DOI: 10.1038/s41431-019-0533-z). A commentary article discussing our results was published in the same journal (DOI: 10.1038/s41431-020-00801-w) and the present paper is our reply.   </p

Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues

Background: The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression. Results: We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics. Conclusion: Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues

Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues

Background: The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression. Results: We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics. Conclusion: Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues

Linkage and exome analysis implicate multiple genes in non-syndromic intellectual disability in a large Swedish family

Background Non-syndromic intellectual disability is genetically heterogeneous with dominant, recessive and complex forms of inheritance. We have performed detailed genetic studies in a large multi-generational Swedish family, including several members diagnosed with non-syndromic intellectual disability. Linkage analysis was performed on 22 family members, nine affected with mild to moderate intellectual disability and 13 unaffected family members. Methods Family members were analyzed with Affymetrix Genome-Wide Human SNP Array 6.0 and the genetic data was used to detect copy number variation and to perform genome wide linkage analysis with the SNP High Throughput Linkage analysis system and the Merlin software. For the exome sequencing, the samples were prepared using the Sure Select Human All Exon Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Ion Proton (TM) System. Validation of identified variants was performed with Sanger sequencing. Results The linkage analysis results indicate that intellectual disability in this family is genetically heterogeneous, with suggestive linkage found on chromosomes 1q31-q41, 4q32-q35, 6p25 and 14q24-q31 (LOD scores of 2.4, simulated p-value of 0.000003 and a simulated genome-wide p-value of 0.06). Exome sequencing was then performed in 14 family members and 7 unrelated individuals from the same region. The analysis of coding variation revealed a pathogenic and candidate variants in different branches of the family. In three patients we find a known homozygous pathogenic mutation in the Homo sapiens solute carrier family 17 member 5 (SLC17A5), causing Salla disease. We also identify a deletion overlapping KDM3B and a duplication overlapping MAP3K4 and AGPAT4, both overlapping variants previously reported in developmental disorders. Conclusions DNA samples from the large family analyzed in this study were initially collected based on a hypothesis that affected members shared a major genetic risk factor. Our results show that a complex phenotype such as mild intellectual disability in large families from genetically isolated populations may show considerable genetic heterogeneity

Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development

Background Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon. Methods We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8–11 weeks post-gestation. Results We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system. Conclusions This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS