Homozygous Loss Of Function Research Articles

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.100BVES loss-of-function mutations in limb-girdle muscular dystrophy 2X with cardiac conduction disorders

BVES encodes for a 360 amino acid protein also known as POPDC1, which is part of the Popeye domain containing (POPDC) family of proteins. POPDC1, POPDC2 and POPDC3 are cAMP-binding transmembrane proteins that are abundantly expressed in striated muscle. A homozygous missense mutation (p.Ser201Phe) in BVES has been identified in three individuals from one single family, the eldest presenting with an overt limb-girdle muscular dystrophy phenotype and all three of them showing an elevated creatine kinase (CK) level and a second-degree atrioventricular (AV) block. The disease was classified as LGMD2X. We performed whole exome sequencing (WES) in a large cohort of patients with unexplained limb-girdle muscular weakness (LGMW) and/or an elevated CK level. Immunohistochemical, Western blotting and mRNA experiments of patients' skeletal muscle tissue were performed to study the pathogenicity of identified loss-of-function (LOF) variants in BVES. We identified 4 individuals from 3 families harboring homozygous LOF variants in BVES. Patients showed skeletal muscle and cardiac conduction abnormalities of varying nature and severity, but exhibited at least subclinical signs of both skeletal muscle and cardiac involvement. Identification of LOF mutations in BVES, causing an adult-onset skeletal muscle disorder with concomitant cardiac conduction disorders, underlines the role of POPDC family, and POPDC1 in particular, in striated muscle disease. This recessive disorder linked to mutations in BVES appears of low prevalence, but is probably underdiagnosed due to its striking phenotypic variability and often subtle yet clinically relevant manifestations, particularly concerning the cardiac involvement.

Natural Knockouts: Natural Selection Knocked Out.

In functional genomics studies, research is dedicated to unveiling the function of genes using gene-knockouts, model organisms in which a gene is artificially inactivated. The idea is that, by knocking out the gene, the provoked phenotype would inform us about the function of the gene. Still, the function of many genes cannot be elucidated, because disruption of conserved sequences, including protein-coding genes, often does not directly affect the phenotype. Since the phenomenon was first observed in the early nineties of the last century, these so-called ‘no-phenotype knockouts’ have met with great skepticism and resistance by died-in-the-wool selectionists. Still, functional genomics of the late 20th and early 21st centuries has taught us two important lessons. First, two or more unrelated genes can often substitute for each other; and second, some genes are only present in the genome in a silent state. In the laboratory, the disruption of such genes does not negatively influence reproductive success, and does not show measurable fitness effects of the species. The genes are redundant. Genetic redundancy, one of the big surprises of modern biology, can thus be defined as the condition in which the inactivation of a gene is selectively neutral. The no-phenotype knockout is not just a freak of the laboratory. Genetic variants known as homozygous loss-of-function (HLOF) variants are of considerable scientific and clinical interest, as they represent experiments of nature qualifying as “natural knockouts”. Such natural knockouts challenge the conventional NeoDarwinian appraisal that genetic information is the result of natural selection acting on random genetic variation.

Abstract 4671: Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients

Abstract Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4671. doi:10.1158/1538-7445.AM2017-4671

Knockout! Knockout! Who’s Not There?

Knockout! Knockout! Who’s Not There?

Importance of Genetic Studies in Consanguineous Populations for the Characterization of Novel Human Gene Functions.

SummaryConsanguineous offspring have elevated levels of homozygosity. Autozygous stretches within their genome are likely to harbour loss of function (LoF) mutations which will lead to complete inactivation or dysfunction of genes. Studying consanguineous offspring with clinical phenotypes has been very useful for identifying disease causal mutations. However, at present, most of the genes in the human genome have no disorder associated with them or have unknown function. This is presumably mostly due to the fact that homozygous LoF variants are not observed in outbred populations which are the main focus of large sequencing projects. However, another reason may be that many genes in the genome—even when completely “knocked out,” do not cause a distinct or defined phenotype. Here, we discuss the benefits and implications of studying consanguineous populations, as opposed to the traditional approach of analysing a subset of consanguineous families or individuals with disease. We suggest that studying consanguineous populations “as a whole” can speed up the characterisation of novel gene functions as well as indicating nonessential genes and/or regions in the human genome. We also suggest designing a single nucleotide variant (SNV) array to make the process more efficient.

Homozygous loss-of-function variants in European cosmopolitan and isolate populations.

Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation.

Revisiting disease genes based on whole-exome sequencing in consanguineous populations.

Assigning a causal role for genes in disease states is one of the most significant medical applications of human genetics research. The requirement for at least two different pathogenic alleles in the same gene in individuals with a similar phenotype to assign a causal link has not always been fully adhered to, and we now know that even two alleles may not necessarily constitute sufficient evidence. Autozygosity is a rich source of natural "knockout" events by virtue of rendering ancestral loss-of-function (LOF) variants homozygous. In this study, we exploit this phenomenon by examining 523 exomes enriched for autozygosity to call into question previously published disease links for several genes based on the identification of confirmed homozygous LOF variants in the absence of the purported diseases. This study highlights an additional advantage of consanguineous populations in the quest to improve the medical annotation of the human genome.

Abstract 3087: Whole genome sequencing of rhabdoid tumors of the kidney

Abstract Rhabdoid tumors (RT) of the kidney (RTK) are aggressive pediatric solid tumors that predominantly affect infants. There is no effective chemotherapy and the overall 4-year survival rate is 23%. RT has a characteristic loss of SMARCB1 function, found in >90% of the patients. SMARCB1 is a conserved core subunit of the SWI/SNF chromatin-remodeling complex, which in turn is responsible for proper chromatin assembly and dynamic regulation of gene expression. Previous studies showed a remarkable paucity of mutations in coding regions of genomes and a highly penetrant cancer susceptibility in a conditional knockout mouse model. These findings support the interpretation that SMARCB1 is a tumor suppressor whose inactivation is the primary driver in RT and that RT follows a tumorigenesis model in which cancer is driven by aberrant epigenetic regulation and gene expression instead of accumulation of somatic mutations. Characterizing interplays of mutations, gene expression and epigenetic regulation will be important in understanding RT development and biology. To achieve this goal, we will comprehensively characterize genetic and epigenetic aberrations in RT using HiSeq sequencing technology. Our research efforts include profiling whole genome, whole transcriptome, promoter methylation and histone modification in 40 primary RTK samples. Here, we report preliminary results from whole genome analyses. Using an amplification-free library construction method, we sequenced whole genomes of 40 RTK and matched normal cases to an average haploid coverage of 39.4X. The RTK genomes were mostly diploid, but we found 35 loci that are either recurrently focally amplified or deleted using GISTIC 2.0 at FDR ≤0.05. Using the Trans-ABySS de novo short-read assembler, we assembled the RT cases’ whole genomes and identified a total of 19 genes that were recurrently rearranged in 8 out of 40 cases. Eleven of the genes were either known tumor suppressors (e.g. CABIN1, BCR) or associated with developmental or neurodegenerative diseases (e.g. UPB1, SPECC1L). The genome-wide single nucleotide variant and indel analysis showed an average somatic mutation rate at 0.37 per Mb in RTK, comparable to the previous finding of 0.19 per Mb in AT/RT. Approximately 99% of the somatic mutations occurred in non-genic regions. SMARCB1 had homozygous loss of function in 83% of cases by somatic homozygous deletion, or heterozygous deletion or truncating point mutations followed by loss of heterozygosity. The remaining cases appeared to have at least 1 copy of the gene unaffected and analyses are ongoing to investigate the inactivation mechanism in these cases. Citation Format: Hye-Jung E. Chun, Kelsey Zhu, Jenny Q. Qian, Karen L. Mungall, Yussanne Ma, Yong-Jun Zhao, Andrew J. Mungall, Richard A. Moore, Jacquie E. Schein, Daniela S. Gerhard, Elizabeth J. Perlman, Marco A. Marra. Whole genome sequencing of rhabdoid tumors of the kidney. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2014-3087

When Half Is Not Enough: Gene Expression and Dosage in the 22q11 Deletion Syndrome

The 22q11 Deletion Syndrome (22q11DS, also known as DiGeorge or Velo-Cardio-Facial Syndrome) has a variable constellation of phenotypes including life-threatening cardiac malformations, craniofacial, limb, and digit anomalies, a high incidence of learning, language, and behavioral disorders, and increased vulnerability for psychiatric diseases, including schizophrenia. There is still little clear understanding of how heterozygous microdeletion of approximately 30-50 genes on chromosome 22 leads to this diverse spectrum of phenotypes, especially in the brain. Three possibilities exist: 1) 22q11DS may reflect haploinsufficiency, homozygous loss of function, or heterozygous gain of function of a single gene within the deleted region; 2) 22q11DS may result from haploinsufficiency, homozygous loss of function, or heterozygous gain of function of a few genes in the deleted region acting at distinct phenotypically compromised sites; 3) 22q11DS may reflect combinatorial effects of reduced dosage of multiple genes acting in concert at all phenotypically compromised sites. Here, we consider evidence for each of these possibilities. Our review of the literature, as well as interpretation of work from our laboratory, favors the third possibility: 22q11DS reflects diminished expression of multiple 22q11 genes acting on common cellular processes during brain as well as heart, face, and limb development, and subsequently in the adolescent and adult brain.

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells.

Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.