EXOME SEQUENCING REVEALS RECURRENT GERMLINE VARIANTS IN PATIENTS WITH FAMILIAL LYMPHOPLASMACYTIC LYMPHOMA.
(Abstract release date: 05/19/16)
EHA Library. Roccaro A. 06/09/16; 132920; E1371
Dr. Aldo Roccaro
Contributions
Contributions
Abstract
Abstract: E1371
Type: Eposter Presentation
Background
Waldenström’s Macroglobulinemia (WM) represents a B-cell lymphoproliferative disorder, classified as a lymphoplasmacytic lymphoma, according to the WHO classification. Previous studies have identified familial aggregation of WM cases, and the clustering of B-cell lymphoproliferative disorders among first degree relatives of patients with WM, including chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, IgM-MGUS and IgG/IgA-MGUS, thus suggesting a possible contribution of inherited susceptibility to familial WM. Nevertheless, whether relatively common germline variants may contribute specifically to familial WM cases, remains unexplored.
Aims
- To identify the potential contribution for inherited susceptibility to familial WM.- To define the functional relevance of the most recurrent observed variant.
Methods
Whole exome sequencing was performed on germline DNA obtained from four members of a single family with documented coinheritance of WM (three affected; 1 unaffected) and applied bioinformatic tools to identify candidate germline variants likely to have a biological role in WM. Additional 246 independent, unrelated WM cases (50 probands from familial cases; 196 from non-familial cases) were screened for the identified variants. The DNA was size-selected to exonic hybrid capture using SureSelect v2 Exome bait (Agilent). Samples were multiplexed and sequenced on Illumina HiSeq flow cells with the goal of an average depth of coverage of 100x. Allele frequencies (AF) in 1000G, as well as PolyPhen-2 prediction, were used to quantify the deleteriousness of SNVs. Variants with AF >0.05 in 1000G were filtered out, and the remaining SNVs were defined as potential familial WM-associated variants. Gene prioritization was obtained by using GRAIL and global coexpression network COXPRESdb. Functional validation was performed by engineering WM cells for the most recurrent variant, by using site-directed mutagenesis and LentiORF.
Results
LAPTM5(c403t) and HCLS1(g496a) represented the most recurrent variants, present in 3/3 affected members of the index family. Each of these variants was present in 8% of the unrelated familial cases, in 0.5% of the non-familial cases and in <0.05 of a control population.To interrogate a possible involvement of LAPTM5 and HCLS1 in WM biology, we used the HEFalMp database to construct a combined LAPTM5/HCLS1 module and interrogated whether this module is disrupted in WM patients compared to healthy donors (HDs), using an independent gene expression profile dataset (GSE6691). We demonstrated a statistically significant high gene-to-gene connectivity in HDs, while the sub-network was disrupted in WM. Importantly, the change in mean connectivity was significantly different in the WM- versus the HD-modules (P<0.0001). Together, these findings suggest a conserved high interactivity between LAPTM5 and HCLS1 in normal B cells, while WM cells present a disrupted pattern of connectivity, which likely impacts disease biology. Previous studies have reported on LAPTM5 over-expression in patients with B-cell lymphomas. LAPTM5c403t was the most significantly predicted variant to be functionally related to the WM mRNA signature, and therefore dissected its functional relevance, by using site-directed mutagenesis in WM cells. LAPTM5c403t mutated cells presented with enhanced cell growth, NF-kBp65 activation and phospho- NF-kBp65 nuclear translocation, compared to the non-mutated cells, thus suggesting the role of the LAPTM5(c403t) variant in enhancing WM cell proliferation through modulation of NF-kB activation.
Conclusion
LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM. Future studies will be needed to clarify the penetrance of specific alleles as well as possible combinatorial effects.
Session topic: E-poster
Type: Eposter Presentation
Background
Waldenström’s Macroglobulinemia (WM) represents a B-cell lymphoproliferative disorder, classified as a lymphoplasmacytic lymphoma, according to the WHO classification. Previous studies have identified familial aggregation of WM cases, and the clustering of B-cell lymphoproliferative disorders among first degree relatives of patients with WM, including chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, IgM-MGUS and IgG/IgA-MGUS, thus suggesting a possible contribution of inherited susceptibility to familial WM. Nevertheless, whether relatively common germline variants may contribute specifically to familial WM cases, remains unexplored.
Aims
- To identify the potential contribution for inherited susceptibility to familial WM.- To define the functional relevance of the most recurrent observed variant.
Methods
Whole exome sequencing was performed on germline DNA obtained from four members of a single family with documented coinheritance of WM (three affected; 1 unaffected) and applied bioinformatic tools to identify candidate germline variants likely to have a biological role in WM. Additional 246 independent, unrelated WM cases (50 probands from familial cases; 196 from non-familial cases) were screened for the identified variants. The DNA was size-selected to exonic hybrid capture using SureSelect v2 Exome bait (Agilent). Samples were multiplexed and sequenced on Illumina HiSeq flow cells with the goal of an average depth of coverage of 100x. Allele frequencies (AF) in 1000G, as well as PolyPhen-2 prediction, were used to quantify the deleteriousness of SNVs. Variants with AF >0.05 in 1000G were filtered out, and the remaining SNVs were defined as potential familial WM-associated variants. Gene prioritization was obtained by using GRAIL and global coexpression network COXPRESdb. Functional validation was performed by engineering WM cells for the most recurrent variant, by using site-directed mutagenesis and LentiORF.
Results
LAPTM5(c403t) and HCLS1(g496a) represented the most recurrent variants, present in 3/3 affected members of the index family. Each of these variants was present in 8% of the unrelated familial cases, in 0.5% of the non-familial cases and in <0.05 of a control population.To interrogate a possible involvement of LAPTM5 and HCLS1 in WM biology, we used the HEFalMp database to construct a combined LAPTM5/HCLS1 module and interrogated whether this module is disrupted in WM patients compared to healthy donors (HDs), using an independent gene expression profile dataset (GSE6691). We demonstrated a statistically significant high gene-to-gene connectivity in HDs, while the sub-network was disrupted in WM. Importantly, the change in mean connectivity was significantly different in the WM- versus the HD-modules (P<0.0001). Together, these findings suggest a conserved high interactivity between LAPTM5 and HCLS1 in normal B cells, while WM cells present a disrupted pattern of connectivity, which likely impacts disease biology. Previous studies have reported on LAPTM5 over-expression in patients with B-cell lymphomas. LAPTM5c403t was the most significantly predicted variant to be functionally related to the WM mRNA signature, and therefore dissected its functional relevance, by using site-directed mutagenesis in WM cells. LAPTM5c403t mutated cells presented with enhanced cell growth, NF-kBp65 activation and phospho- NF-kBp65 nuclear translocation, compared to the non-mutated cells, thus suggesting the role of the LAPTM5(c403t) variant in enhancing WM cell proliferation through modulation of NF-kB activation.
Conclusion
LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM. Future studies will be needed to clarify the penetrance of specific alleles as well as possible combinatorial effects.
Session topic: E-poster
Abstract: E1371
Type: Eposter Presentation
Background
Waldenström’s Macroglobulinemia (WM) represents a B-cell lymphoproliferative disorder, classified as a lymphoplasmacytic lymphoma, according to the WHO classification. Previous studies have identified familial aggregation of WM cases, and the clustering of B-cell lymphoproliferative disorders among first degree relatives of patients with WM, including chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, IgM-MGUS and IgG/IgA-MGUS, thus suggesting a possible contribution of inherited susceptibility to familial WM. Nevertheless, whether relatively common germline variants may contribute specifically to familial WM cases, remains unexplored.
Aims
- To identify the potential contribution for inherited susceptibility to familial WM.- To define the functional relevance of the most recurrent observed variant.
Methods
Whole exome sequencing was performed on germline DNA obtained from four members of a single family with documented coinheritance of WM (three affected; 1 unaffected) and applied bioinformatic tools to identify candidate germline variants likely to have a biological role in WM. Additional 246 independent, unrelated WM cases (50 probands from familial cases; 196 from non-familial cases) were screened for the identified variants. The DNA was size-selected to exonic hybrid capture using SureSelect v2 Exome bait (Agilent). Samples were multiplexed and sequenced on Illumina HiSeq flow cells with the goal of an average depth of coverage of 100x. Allele frequencies (AF) in 1000G, as well as PolyPhen-2 prediction, were used to quantify the deleteriousness of SNVs. Variants with AF >0.05 in 1000G were filtered out, and the remaining SNVs were defined as potential familial WM-associated variants. Gene prioritization was obtained by using GRAIL and global coexpression network COXPRESdb. Functional validation was performed by engineering WM cells for the most recurrent variant, by using site-directed mutagenesis and LentiORF.
Results
LAPTM5(c403t) and HCLS1(g496a) represented the most recurrent variants, present in 3/3 affected members of the index family. Each of these variants was present in 8% of the unrelated familial cases, in 0.5% of the non-familial cases and in <0.05 of a control population.To interrogate a possible involvement of LAPTM5 and HCLS1 in WM biology, we used the HEFalMp database to construct a combined LAPTM5/HCLS1 module and interrogated whether this module is disrupted in WM patients compared to healthy donors (HDs), using an independent gene expression profile dataset (GSE6691). We demonstrated a statistically significant high gene-to-gene connectivity in HDs, while the sub-network was disrupted in WM. Importantly, the change in mean connectivity was significantly different in the WM- versus the HD-modules (P<0.0001). Together, these findings suggest a conserved high interactivity between LAPTM5 and HCLS1 in normal B cells, while WM cells present a disrupted pattern of connectivity, which likely impacts disease biology. Previous studies have reported on LAPTM5 over-expression in patients with B-cell lymphomas. LAPTM5c403t was the most significantly predicted variant to be functionally related to the WM mRNA signature, and therefore dissected its functional relevance, by using site-directed mutagenesis in WM cells. LAPTM5c403t mutated cells presented with enhanced cell growth, NF-kBp65 activation and phospho- NF-kBp65 nuclear translocation, compared to the non-mutated cells, thus suggesting the role of the LAPTM5(c403t) variant in enhancing WM cell proliferation through modulation of NF-kB activation.
Conclusion
LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM. Future studies will be needed to clarify the penetrance of specific alleles as well as possible combinatorial effects.
Session topic: E-poster
Type: Eposter Presentation
Background
Waldenström’s Macroglobulinemia (WM) represents a B-cell lymphoproliferative disorder, classified as a lymphoplasmacytic lymphoma, according to the WHO classification. Previous studies have identified familial aggregation of WM cases, and the clustering of B-cell lymphoproliferative disorders among first degree relatives of patients with WM, including chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, IgM-MGUS and IgG/IgA-MGUS, thus suggesting a possible contribution of inherited susceptibility to familial WM. Nevertheless, whether relatively common germline variants may contribute specifically to familial WM cases, remains unexplored.
Aims
- To identify the potential contribution for inherited susceptibility to familial WM.- To define the functional relevance of the most recurrent observed variant.
Methods
Whole exome sequencing was performed on germline DNA obtained from four members of a single family with documented coinheritance of WM (three affected; 1 unaffected) and applied bioinformatic tools to identify candidate germline variants likely to have a biological role in WM. Additional 246 independent, unrelated WM cases (50 probands from familial cases; 196 from non-familial cases) were screened for the identified variants. The DNA was size-selected to exonic hybrid capture using SureSelect v2 Exome bait (Agilent). Samples were multiplexed and sequenced on Illumina HiSeq flow cells with the goal of an average depth of coverage of 100x. Allele frequencies (AF) in 1000G, as well as PolyPhen-2 prediction, were used to quantify the deleteriousness of SNVs. Variants with AF >0.05 in 1000G were filtered out, and the remaining SNVs were defined as potential familial WM-associated variants. Gene prioritization was obtained by using GRAIL and global coexpression network COXPRESdb. Functional validation was performed by engineering WM cells for the most recurrent variant, by using site-directed mutagenesis and LentiORF.
Results
LAPTM5(c403t) and HCLS1(g496a) represented the most recurrent variants, present in 3/3 affected members of the index family. Each of these variants was present in 8% of the unrelated familial cases, in 0.5% of the non-familial cases and in <0.05 of a control population.To interrogate a possible involvement of LAPTM5 and HCLS1 in WM biology, we used the HEFalMp database to construct a combined LAPTM5/HCLS1 module and interrogated whether this module is disrupted in WM patients compared to healthy donors (HDs), using an independent gene expression profile dataset (GSE6691). We demonstrated a statistically significant high gene-to-gene connectivity in HDs, while the sub-network was disrupted in WM. Importantly, the change in mean connectivity was significantly different in the WM- versus the HD-modules (P<0.0001). Together, these findings suggest a conserved high interactivity between LAPTM5 and HCLS1 in normal B cells, while WM cells present a disrupted pattern of connectivity, which likely impacts disease biology. Previous studies have reported on LAPTM5 over-expression in patients with B-cell lymphomas. LAPTM5c403t was the most significantly predicted variant to be functionally related to the WM mRNA signature, and therefore dissected its functional relevance, by using site-directed mutagenesis in WM cells. LAPTM5c403t mutated cells presented with enhanced cell growth, NF-kBp65 activation and phospho- NF-kBp65 nuclear translocation, compared to the non-mutated cells, thus suggesting the role of the LAPTM5(c403t) variant in enhancing WM cell proliferation through modulation of NF-kB activation.
Conclusion
LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM. Future studies will be needed to clarify the penetrance of specific alleles as well as possible combinatorial effects.
Session topic: E-poster
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