DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation

This work was funded by The Medical Research Council UK (MR/K000292/1), Children with Cancer UK (2013/144), Barts and The London Charity (845/1796), and Leukaemia Lymphoma Research/Bloodwise (14032)

Health and population effects of rare gene knockouts in adult humans with related parents.

Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.The study was funded by the Wellcome Trust (WT102627 and WT098051), Barts Charity (845/1796), Medical Research Council (MR/M009017/1). This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Collaboration for Applied Health Research and Care (CLAHRC) for Yorkshire and Humber. Core support for Born in Bradford is also provided by the Wellcome Trust (WT101597). V.N. was supported by the Wellcome Trust PhD Studentship (WT099769). D.G.M. and K.K. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM104371. E.R.M. is funded by NIHR Cambridge Biomedical Research Centre. H.H. is supported by awards to establish the Farr Institute of Health Informatics Research, London, from the Medical Research Council, Arthritis Research UK, British Heart Foundation, Cancer Research UK, Chief Scientist Office, Economic and Social Research Council, Engineering and Physical Sciences Research Council, NIHR, National Institute for Social Care and Health Research, and Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via https://doi.org/10.1126/science.aac862

Number of coding, rare and LoF variants across 24 candidate genes selected from exome sequencing of 75 individuals from multiply affected families.

<p>* MAF as defined in controls.</p><p>Number of coding, rare and LoF variants across 24 candidate genes selected from exome sequencing of 75 individuals from multiply affected families.</p

Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing

Many disease-associated variants identified by genome-wide association (GWA) studies are expected to regulate gene expression. Allele-specific expression (ASE) quantifies transcription from both haplotypes using individuals heterozygous at tested SNPs. We performed deep human transcriptome-wide resequencing (RNA-seq) for ASE analysis and expression quantitative trait locus discovery. We resequenced double poly(A)-selected RNA from primary CD4(+) T cells (n = 4 individuals, both activated and untreated conditions) and developed tools for paired-end RNA-seq alignment and ASE analysis. We generated an average of 20 million uniquely mapping 45 base reads per sample. We obtained sufficient read depth to test 1371 unique transcripts for ASE. Multiple biases inflate the false discovery rate which we estimate to be ∼50% for random SNPs. However, after controlling for these biases and considering the subset of SNPs that pass HapMap QC, 4.6% of heterozygous SNP-sample pairs show evidence of imbalance (P < 0.001). We validated four findings by both bacterial cloning and Sanger sequencing assays. We also found convincing evidence for allelic imbalance at multiple reporter exonic SNPs in CD6 for two samples heterozygous at the multiple sclerosis-associated variant rs17824933, linking GWA findings with variation in gene expression. Finally, we show in CD4(+) T cells from a further individual that high-throughput sequencing of genomic DNA and RNA-seq following enrichment for targeted gene sequences by sequence capture methods offers an unbiased means to increase the read depth for transcripts of interest, and therefore a method to investigate the regulatory role of many disease-associated genetic variants

Summary of non-parametric linkage results in twelve multiply affected disease pedigrees.

<p>Summary of linkage data for the twelve families included in the NPL analysis. The linkage <i>p</i>-values were computed using Merlin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116845#pone.0116845.ref033" target="_blank">33</a>]. The power was assessed using simulations (see Methods).</p><p>Summary of non-parametric linkage results in twelve multiply affected disease pedigrees.</p

Top three most significant genes for the aggregate test for rare LoF variants only between cases (one per multiply affected family) and controls (with MAF <0.5% in 1000G).

<p>Top three most significant genes for the aggregate test for rare LoF variants only between cases (one per multiply affected family) and controls (with MAF <0.5% in 1000G).</p

Segregation result for a novel c.70G>A (p.V24I) SNV in <i>IL21R</i> in the entire Neu4801 family.

<p>All other individuals carry homozygous GG wild type alleles (genotypes not shown on figure).</p

Analytical design of our study of rare variation in CeD.

<p>Only post quality filtered SNVs and indels were included in each analytical test. A, Not in dbSNP132, <5% MAF in 1000G, <10% MAF in coeliac exomes, not in 101 control exomes (54 ultra rare diseases from Kings College London and 47 Environmental Genome Project samples from University of Washington). B, Rare allele defined as MAF <0.5% in 1000G (n = 1092) for 220 controls and 41 unrelated coeliac exomes. C, MAF <0.5%, only variants predicted to be damaging and regions without duplications.</p

Nonsynonymous missense SNVs located in linkage regions (<i>p</i> < 0.01).

<p>* Candidate genes selected for deep amplicon resequencing</p><p>Nonsynonymous missense SNVs located in linkage regions (<i>p</i> < 0.01).</p

Top 15 most significant genes for the aggregate test for rare LoF variants in immune genes between cases (one per multiply affected family) and controls (with MAF <0.5% in 1000G).

<p>* Candidate genes selected for deep amplicon resequencing.</p><p>Top 15 most significant genes for the aggregate test for rare LoF variants in immune genes between cases (one per multiply affected family) and controls (with MAF <0.5% in 1000G).</p