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Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: an immediate source for thousands of new mouse models.

Andrews TD, Whittle B, Field MA, Balakishnan B, Zhang Y, Shao Y, Cho V, Kirk M, Singh M, Xia Y, Hager J, Winslade S, Sjollema G, Beutler B, Enders A, Goodnow CC - Open Biol (2012)

Bottom Line: These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation.The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping.We show that exome sequencing data alone are sufficient to identify induced mutations.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics Laboratory, Australian National University, GPO Box 334, Canberra City, Australian Capital Territory, 2601 , Australia. dan.andrews@anu.edu.au

ABSTRACT
Accurate identification of sparse heterozygous single-nucleotide variants (SNVs) is a critical challenge for identifying the causative mutations in mouse genetic screens, human genetic diseases and cancer. When seeking to identify causal DNA variants that occur at such low rates, they are overwhelmed by false-positive calls that arise from a range of technical and biological sources. We describe a strategy using whole-exome capture, massively parallel DNA sequencing and computational analysis, which identifies with a low false-positive rate the majority of heterozygous and homozygous SNVs arising de novo with a frequency of one nucleotide substitution per megabase in progeny of N-ethyl-N-nitrosourea (ENU)-mutated C57BL/6j mice. We found that by applying a strategy of filtering raw SNV calls against known and platform-specific variants we could call true SNVs with a false-positive rate of 19.4 per cent and an estimated false-negative rate of 21.3 per cent. These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation. The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping. Exome sequencing of first-generation mutant mice revealed hundreds of unphenotyped protein-changing mutations, 52 per cent of which are predicted to be deleterious, which now become available for breeding and experimental analysis. We show that exome sequencing data alone are sufficient to identify induced mutations. This approach transforms genetic screens in mice, establishes a general strategy for analysing rare DNA variants and opens up a large new source for experimental models of human disease.

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Nimbus results from a loss of function mutation in the Ptprc gene. (a) Schematic diagram showing the location of single nucleotide mutation at Chr1:139986183 at the +1 intronic position of the exon 17 splice donor sequence and the location of the corresponding region in the encoded CD45 protein (TM, transmembrane domain; FNIII, fibronectin III-like domain; PTP, protein tyrosine phosphatase). (b) Loss of CD45 protein expression. Bold black lines show flow cytometric staining with antibody to the B-cell-specific CD45R isoform on IgM+, IgD+ B lymphocytes in blood from (i) Ptprc+/+ wild-type (wt), (ii) Ptprcnimbus/+ heterozygous or (iii) Ptprcnimbus/nimbus homozygous mouse, compared with negative control staining on CD3+ T cells in the same mouse (thin black line) and compared with positive control staining with the same antibody on B cells in a wt mouse (grey shaded area).
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RSOB120061F5: Nimbus results from a loss of function mutation in the Ptprc gene. (a) Schematic diagram showing the location of single nucleotide mutation at Chr1:139986183 at the +1 intronic position of the exon 17 splice donor sequence and the location of the corresponding region in the encoded CD45 protein (TM, transmembrane domain; FNIII, fibronectin III-like domain; PTP, protein tyrosine phosphatase). (b) Loss of CD45 protein expression. Bold black lines show flow cytometric staining with antibody to the B-cell-specific CD45R isoform on IgM+, IgD+ B lymphocytes in blood from (i) Ptprc+/+ wild-type (wt), (ii) Ptprcnimbus/+ heterozygous or (iii) Ptprcnimbus/nimbus homozygous mouse, compared with negative control staining on CD3+ T cells in the same mouse (thin black line) and compared with positive control staining with the same antibody on B cells in a wt mouse (grey shaded area).

Mentions: To identify the mutation causing the recessive lymphopaenia phenotype in the nimbus strain, we performed Amplifluor assays on each of the three homozygous mutations identified in the proband exome sequence to trace their inheritance in the pedigree. Homozygosity for a C-to-T mutation identified at Chr1 : 139 986 182 bp was found to co-segregate with the lymphopaenia phenotype (table 2). This change lies 1 bp upstream of exon 18 of the Ptprc gene and disrupts the intronic-1 G nucleotide of the consensus splice acceptor sequence [34], which is otherwise absolutely conserved across vertebrates. PCR amplification of the mutant Ptprcnim mRNA showed the first 14 bp of exon 18 were deleted compared with the spliced wild-type mRNA and putatively the AG nucleotides at +13 to 14 of exon 18 from an alternative splice acceptor site. This altered splicing leads to a frameshift in the mutant transcript from the truncated start of exon 18 onwards. Ptprc encodes the CD45 protein, which is a tyrosine phosphatase receptor type C. CD45 is an abundant protein in the plasma membrane of leukocytes and plays critical roles in lymphocyte development in mice and humans (reviewed in [35]). Mice homozygous for the Ptprcnim mutation indeed had almost no CD45 protein on the surface of their B-lymphocytes (2% of wild-type controls) as measured by flow cytometric staining with antibodies to CD45 (figure 5b), while heterozygous mice showed an approximately 50 per cent reduction in the expression of CD45. The lymphopaenia in nimbus homozygotes matches that in mice and humans with other or severe loss-of-function mutations in Ptprc [29,36,37].TableĀ 2.


Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: an immediate source for thousands of new mouse models.

Andrews TD, Whittle B, Field MA, Balakishnan B, Zhang Y, Shao Y, Cho V, Kirk M, Singh M, Xia Y, Hager J, Winslade S, Sjollema G, Beutler B, Enders A, Goodnow CC - Open Biol (2012)

Nimbus results from a loss of function mutation in the Ptprc gene. (a) Schematic diagram showing the location of single nucleotide mutation at Chr1:139986183 at the +1 intronic position of the exon 17 splice donor sequence and the location of the corresponding region in the encoded CD45 protein (TM, transmembrane domain; FNIII, fibronectin III-like domain; PTP, protein tyrosine phosphatase). (b) Loss of CD45 protein expression. Bold black lines show flow cytometric staining with antibody to the B-cell-specific CD45R isoform on IgM+, IgD+ B lymphocytes in blood from (i) Ptprc+/+ wild-type (wt), (ii) Ptprcnimbus/+ heterozygous or (iii) Ptprcnimbus/nimbus homozygous mouse, compared with negative control staining on CD3+ T cells in the same mouse (thin black line) and compared with positive control staining with the same antibody on B cells in a wt mouse (grey shaded area).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376740&req=5

RSOB120061F5: Nimbus results from a loss of function mutation in the Ptprc gene. (a) Schematic diagram showing the location of single nucleotide mutation at Chr1:139986183 at the +1 intronic position of the exon 17 splice donor sequence and the location of the corresponding region in the encoded CD45 protein (TM, transmembrane domain; FNIII, fibronectin III-like domain; PTP, protein tyrosine phosphatase). (b) Loss of CD45 protein expression. Bold black lines show flow cytometric staining with antibody to the B-cell-specific CD45R isoform on IgM+, IgD+ B lymphocytes in blood from (i) Ptprc+/+ wild-type (wt), (ii) Ptprcnimbus/+ heterozygous or (iii) Ptprcnimbus/nimbus homozygous mouse, compared with negative control staining on CD3+ T cells in the same mouse (thin black line) and compared with positive control staining with the same antibody on B cells in a wt mouse (grey shaded area).
Mentions: To identify the mutation causing the recessive lymphopaenia phenotype in the nimbus strain, we performed Amplifluor assays on each of the three homozygous mutations identified in the proband exome sequence to trace their inheritance in the pedigree. Homozygosity for a C-to-T mutation identified at Chr1 : 139 986 182 bp was found to co-segregate with the lymphopaenia phenotype (table 2). This change lies 1 bp upstream of exon 18 of the Ptprc gene and disrupts the intronic-1 G nucleotide of the consensus splice acceptor sequence [34], which is otherwise absolutely conserved across vertebrates. PCR amplification of the mutant Ptprcnim mRNA showed the first 14 bp of exon 18 were deleted compared with the spliced wild-type mRNA and putatively the AG nucleotides at +13 to 14 of exon 18 from an alternative splice acceptor site. This altered splicing leads to a frameshift in the mutant transcript from the truncated start of exon 18 onwards. Ptprc encodes the CD45 protein, which is a tyrosine phosphatase receptor type C. CD45 is an abundant protein in the plasma membrane of leukocytes and plays critical roles in lymphocyte development in mice and humans (reviewed in [35]). Mice homozygous for the Ptprcnim mutation indeed had almost no CD45 protein on the surface of their B-lymphocytes (2% of wild-type controls) as measured by flow cytometric staining with antibodies to CD45 (figure 5b), while heterozygous mice showed an approximately 50 per cent reduction in the expression of CD45. The lymphopaenia in nimbus homozygotes matches that in mice and humans with other or severe loss-of-function mutations in Ptprc [29,36,37].TableĀ 2.

Bottom Line: These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation.The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping.We show that exome sequencing data alone are sufficient to identify induced mutations.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics Laboratory, Australian National University, GPO Box 334, Canberra City, Australian Capital Territory, 2601 , Australia. dan.andrews@anu.edu.au

ABSTRACT
Accurate identification of sparse heterozygous single-nucleotide variants (SNVs) is a critical challenge for identifying the causative mutations in mouse genetic screens, human genetic diseases and cancer. When seeking to identify causal DNA variants that occur at such low rates, they are overwhelmed by false-positive calls that arise from a range of technical and biological sources. We describe a strategy using whole-exome capture, massively parallel DNA sequencing and computational analysis, which identifies with a low false-positive rate the majority of heterozygous and homozygous SNVs arising de novo with a frequency of one nucleotide substitution per megabase in progeny of N-ethyl-N-nitrosourea (ENU)-mutated C57BL/6j mice. We found that by applying a strategy of filtering raw SNV calls against known and platform-specific variants we could call true SNVs with a false-positive rate of 19.4 per cent and an estimated false-negative rate of 21.3 per cent. These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation. The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping. Exome sequencing of first-generation mutant mice revealed hundreds of unphenotyped protein-changing mutations, 52 per cent of which are predicted to be deleterious, which now become available for breeding and experimental analysis. We show that exome sequencing data alone are sufficient to identify induced mutations. This approach transforms genetic screens in mice, establishes a general strategy for analysing rare DNA variants and opens up a large new source for experimental models of human disease.

Show MeSH
Related in: MedlinePlus