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Inherited coding variants at the CDKN2A locus influence susceptibility to acute lymphoblastic leukaemia in children.

Xu H, Zhang H, Yang W, Yadav R, Morrison AC, Qian M, Devidas M, Liu Y, Perez-Andreu V, Zhao X, Gastier-Foster JM, Lupo PJ, Neale G, Raetz E, Larsen E, Bowman WP, Carroll WL, Winick N, Williams R, Hansen T, Holm JC, Mardis E, Fulton R, Pui CH, Zhang J, Mullighan CG, Evans WE, Hunger SP, Gupta R, Schmiegelow K, Loh ML, Relling MV, Yang JJ - Nat Commun (2015)

Bottom Line: There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated.Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23).Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA [2] Department of Laboratory Medicine, National Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.

ABSTRACT
There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16(INK4A), increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A-CDKN2B tumour suppressors in leukaemogenesis.

No MeSH data available.


Related in: MedlinePlus

Functional characterization of ALL risk variant at rs3731249.(a) Mouse haematopoietic progenitor cell Ba/f3 overexpressing wildtype, variant p16INK4A, or transfected with control vector was transduced with leukaemia oncogenic BCR–ABL1 fusion gene. Cell proliferation in the absence of cytokine IL3 was measured daily as an indicator of leukaemic transformation. Ectopic expression of p16INK4A (p.148 T, green) significantly potentiated leukaemic transformation by BCR–ABL1, compared with cells expressing wild-type p16INK4A (p.148A, blue), consistent with the association of this allele with susceptibility to ALL. Data represent the mean of three replicates±s.e.m. (b) Allele-specific expression of p16INK4A in ALL blasts was determined by comparing the number of sequence reads for transcripts containing C or T alleles at rs3731249 (p16INK4Ap.148A versus p16INK4Ap.148 T), in 15 childhood ALL cases with heterozygous genotype in the germline DNA at this SNP. Each dot represents an ALL case (red indicates cases with somatic deletion (loss of heterozygosity) and blue indicates cases without copy number change in tumour) and the line of identity indicates equal expression of both alleles. P-value was estimated by paired t-test based on the number of sequence reads for each allele.
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f2: Functional characterization of ALL risk variant at rs3731249.(a) Mouse haematopoietic progenitor cell Ba/f3 overexpressing wildtype, variant p16INK4A, or transfected with control vector was transduced with leukaemia oncogenic BCR–ABL1 fusion gene. Cell proliferation in the absence of cytokine IL3 was measured daily as an indicator of leukaemic transformation. Ectopic expression of p16INK4A (p.148 T, green) significantly potentiated leukaemic transformation by BCR–ABL1, compared with cells expressing wild-type p16INK4A (p.148A, blue), consistent with the association of this allele with susceptibility to ALL. Data represent the mean of three replicates±s.e.m. (b) Allele-specific expression of p16INK4A in ALL blasts was determined by comparing the number of sequence reads for transcripts containing C or T alleles at rs3731249 (p16INK4Ap.148A versus p16INK4Ap.148 T), in 15 childhood ALL cases with heterozygous genotype in the germline DNA at this SNP. Each dot represents an ALL case (red indicates cases with somatic deletion (loss of heterozygosity) and blue indicates cases without copy number change in tumour) and the line of identity indicates equal expression of both alleles. P-value was estimated by paired t-test based on the number of sequence reads for each allele.

Mentions: To experimentally evaluate the effects of rs3731249 on ALL leukaemogenesis, we directly compared the effect of wildtype versus variant allele p16INK4A (p.148A versus p.148 T) on BCR–ABL1-mediated leukaemic transformation in vitro. We chose mouse haematopoietic progenitor Ba/f3 cell line because it is inherently p16Ink4a-defective due to methylation at the Ink4a-Arf locus22, and ectopic expression of BCR-ABL1 in Ba/f3 cells efficiently induces exogenous cytokine (interleukin 3 (IL3))-independent proliferation. Over-expression of wild-type p16INK4A(p.148A) significantly inhibited leukaemic transformation by BCR–ABL1 (Fig. 2a, Supplementary Fig. 2), consistent with its role as a critical tumour suppressor in ALL. In contrast, Ba/f3 cells overexpressing variant p16INK4A(p.148 T) were significantly more susceptible to BCR–ABL1 transformation measured by IL3-independent growth, suggesting that the p.148 T variant is likely hypomorphic with reduced tumour suppressor function. In Ba/f3 cells transfected with both variant and wild-type p16INK4A, the relative ratio of the p.148 T (variant) to p.148A (wildtype) transcript increased substantially upon BCR–ABL1-mediated transformation (Supplementary Fig. 3), consistent with the increased leukaemia risk conferred by the variant allele at rs3731249. To further examine the potential susceptibility to ALL conferred by the rs3731249 in patients, we compared the genotype distribution in RNA and DNA from primary leukaemic blasts and matched germline samples from children with ALL (Fig. 2b). Of 15 cases with the heterozygous germline genotype at this SNP, six exhibited somatic deletion of one copy of CDKN2A, all of which retained the risk allele in tumour cells. Even in cases not affected by somatic copy number loss at this locus, the variant p16INK4A(c.442 T) was preferentially transcribed relative to wildtype (c.442C), with allele-biased expression ranging from 61 to 100%, Fig. 2b). Altogether, these results pointed to the possibility that cells carrying the hypomorphic risk allele at rs3731249 might have been enriched during leukaemogenesis.


Inherited coding variants at the CDKN2A locus influence susceptibility to acute lymphoblastic leukaemia in children.

Xu H, Zhang H, Yang W, Yadav R, Morrison AC, Qian M, Devidas M, Liu Y, Perez-Andreu V, Zhao X, Gastier-Foster JM, Lupo PJ, Neale G, Raetz E, Larsen E, Bowman WP, Carroll WL, Winick N, Williams R, Hansen T, Holm JC, Mardis E, Fulton R, Pui CH, Zhang J, Mullighan CG, Evans WE, Hunger SP, Gupta R, Schmiegelow K, Loh ML, Relling MV, Yang JJ - Nat Commun (2015)

Functional characterization of ALL risk variant at rs3731249.(a) Mouse haematopoietic progenitor cell Ba/f3 overexpressing wildtype, variant p16INK4A, or transfected with control vector was transduced with leukaemia oncogenic BCR–ABL1 fusion gene. Cell proliferation in the absence of cytokine IL3 was measured daily as an indicator of leukaemic transformation. Ectopic expression of p16INK4A (p.148 T, green) significantly potentiated leukaemic transformation by BCR–ABL1, compared with cells expressing wild-type p16INK4A (p.148A, blue), consistent with the association of this allele with susceptibility to ALL. Data represent the mean of three replicates±s.e.m. (b) Allele-specific expression of p16INK4A in ALL blasts was determined by comparing the number of sequence reads for transcripts containing C or T alleles at rs3731249 (p16INK4Ap.148A versus p16INK4Ap.148 T), in 15 childhood ALL cases with heterozygous genotype in the germline DNA at this SNP. Each dot represents an ALL case (red indicates cases with somatic deletion (loss of heterozygosity) and blue indicates cases without copy number change in tumour) and the line of identity indicates equal expression of both alleles. P-value was estimated by paired t-test based on the number of sequence reads for each allele.
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f2: Functional characterization of ALL risk variant at rs3731249.(a) Mouse haematopoietic progenitor cell Ba/f3 overexpressing wildtype, variant p16INK4A, or transfected with control vector was transduced with leukaemia oncogenic BCR–ABL1 fusion gene. Cell proliferation in the absence of cytokine IL3 was measured daily as an indicator of leukaemic transformation. Ectopic expression of p16INK4A (p.148 T, green) significantly potentiated leukaemic transformation by BCR–ABL1, compared with cells expressing wild-type p16INK4A (p.148A, blue), consistent with the association of this allele with susceptibility to ALL. Data represent the mean of three replicates±s.e.m. (b) Allele-specific expression of p16INK4A in ALL blasts was determined by comparing the number of sequence reads for transcripts containing C or T alleles at rs3731249 (p16INK4Ap.148A versus p16INK4Ap.148 T), in 15 childhood ALL cases with heterozygous genotype in the germline DNA at this SNP. Each dot represents an ALL case (red indicates cases with somatic deletion (loss of heterozygosity) and blue indicates cases without copy number change in tumour) and the line of identity indicates equal expression of both alleles. P-value was estimated by paired t-test based on the number of sequence reads for each allele.
Mentions: To experimentally evaluate the effects of rs3731249 on ALL leukaemogenesis, we directly compared the effect of wildtype versus variant allele p16INK4A (p.148A versus p.148 T) on BCR–ABL1-mediated leukaemic transformation in vitro. We chose mouse haematopoietic progenitor Ba/f3 cell line because it is inherently p16Ink4a-defective due to methylation at the Ink4a-Arf locus22, and ectopic expression of BCR-ABL1 in Ba/f3 cells efficiently induces exogenous cytokine (interleukin 3 (IL3))-independent proliferation. Over-expression of wild-type p16INK4A(p.148A) significantly inhibited leukaemic transformation by BCR–ABL1 (Fig. 2a, Supplementary Fig. 2), consistent with its role as a critical tumour suppressor in ALL. In contrast, Ba/f3 cells overexpressing variant p16INK4A(p.148 T) were significantly more susceptible to BCR–ABL1 transformation measured by IL3-independent growth, suggesting that the p.148 T variant is likely hypomorphic with reduced tumour suppressor function. In Ba/f3 cells transfected with both variant and wild-type p16INK4A, the relative ratio of the p.148 T (variant) to p.148A (wildtype) transcript increased substantially upon BCR–ABL1-mediated transformation (Supplementary Fig. 3), consistent with the increased leukaemia risk conferred by the variant allele at rs3731249. To further examine the potential susceptibility to ALL conferred by the rs3731249 in patients, we compared the genotype distribution in RNA and DNA from primary leukaemic blasts and matched germline samples from children with ALL (Fig. 2b). Of 15 cases with the heterozygous germline genotype at this SNP, six exhibited somatic deletion of one copy of CDKN2A, all of which retained the risk allele in tumour cells. Even in cases not affected by somatic copy number loss at this locus, the variant p16INK4A(c.442 T) was preferentially transcribed relative to wildtype (c.442C), with allele-biased expression ranging from 61 to 100%, Fig. 2b). Altogether, these results pointed to the possibility that cells carrying the hypomorphic risk allele at rs3731249 might have been enriched during leukaemogenesis.

Bottom Line: There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated.Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23).Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA [2] Department of Laboratory Medicine, National Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.

ABSTRACT
There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16(INK4A), increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A-CDKN2B tumour suppressors in leukaemogenesis.

No MeSH data available.


Related in: MedlinePlus