A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

doi:10.1038/ng.2754

. 2013 Oct;45(10):1226-1231.

doi: 10.1038/ng.2754. Epub 2013 Sep 8.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Sohela Shah^#^{1

2}, Kasmintan A Schrader^#^{1

2}, Esmé Waanders^#^{3

4}, Andrew E Timms^#⁵, Joseph Vijai^#^{1

2}, Cornelius Miething^#¹, Jeremy Wechsler⁵, Jun Yang⁶, James Hayes¹, Robert J Klein^{1

2}, Jinghui Zhang⁷, Lei Wei^{7

3}, Gang Wu⁷, Michael Rusch⁷, Panduka Nagahawatte⁷, Jing Ma⁸, Shann-Ching Chen³, Guangchun Song³, Jinjun Cheng^{3

8}, Paul Meyers⁹, Deepa Bhojwani¹⁰, Suresh Jhanwar¹¹, Peter Maslak¹², Martin Fleisher¹³, Jason Littman², Lily Offit², Rohini Rau-Murthy², Megan Harlan Fleischut², Marina Corines², Rajmohan Murali¹¹, Xiaoni Gao¹, Christopher Manschreck², Thomas Kitzing¹, Vundavalli V Murty¹⁴, Susana Raimondi³, Roland P Kuiper⁴, Annet Simons⁴, Joshua D Schiffman¹⁵, Kenan Onel¹⁶, Sharon E Plon¹⁷, David Wheeler¹⁷, Deborah Ritter¹⁷, David S Ziegler¹⁸, Kathy Tucker¹⁹, Rosemary Sutton²⁰, Georgia Chenevix-Trench²¹, Jun Li²¹, David G Huntsman²², Samantha Hansford²², Janine Senz²², Thomas Walsh²³, Ming Lee²³, Christopher N Hahn²⁴, Kathryn Roberts³, Mary-Claire King²³, Sarah M Lo²⁵, Ross L Levine²⁶, Agnes Viale²⁷, Nicholas D Socci²⁸, Katherine L Nathanson²⁹, Hamish S Scott²⁴, Mark Daly³⁰, Steven M Lipkin³¹, Scott W Lowe¹, James R Downing³, David Altshuler³⁰, John T Sandlund^#¹⁰, Marshall S Horwitz^#⁵, Charles G Mullighan^#³, Kenneth Offit^#^{1

2}

Affiliations

¹ Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
² Clinical Genetics Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
³ Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁴ Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Radboud Institute for Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
⁵ Department of Pathology, University of Washington, Seattle, Washington, USA.
⁶ Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁷ Department of Computational Biology and Bioinformatics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁸ Pediatric Cancer Genome Project Laboratory, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁹ Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹⁰ Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
¹¹ Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹² Hematology Laboratory Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹³ Clinical Chemistry Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹⁴ Department of Pathology and Cell Biology, Columbia University, New York, New York, USA.
¹⁵ High Risk Pediatric Cancer Clinic, Huntsman Cancer Institute/Primary Children's Medical Center, University of Utah, Salt Lake City, Utah, USA.
¹⁶ Pediatrics, University of Chicago, Chicago, IL , USA.
¹⁷ Texas Children's Cancer Center and Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA.
¹⁸ Kids Cancer Centre, Sydney Children's Hospital, Sydney, New South Wales, Australia, Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Sydney, New South Wales, Australia.
¹⁹ Hereditary Cancer Clinic Prince of Wales Hospital, Randwick, Australia.
²⁰ Children's Cancer Institute Australia for Medical Research, University of New South Wales, Randwick, Australia.
²¹ Cancer Genetics Laboratory, The Queensland Institute of Medical Research, Herston, Australia.
²² Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
²³ Departments of Medicine and Genome Sciences, University of Washington, Seattle, Washington, USA.
²⁴ Department of Molecular Pathology, SA Pathology and Centre for Cancer Biology, Adelaide, Australia.
²⁵ Department of Pediatrics, Weill Cornell College of Medicine, New York, New York, USA.
²⁶ Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁷ Genomics Core Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁸ Bioinformatics Core, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁹ Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
³⁰ Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
³¹ Department of Medicine, Weill Cornell College of Medicine, New York, New York, USA.

^# Contributed equally.

PMID: 24013638
PMCID: PMC3919799
DOI: 10.1038/ng.2754

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Sohela Shah et al. Nat Genet. 2013 Oct.

. 2013 Oct;45(10):1226-1231.

doi: 10.1038/ng.2754. Epub 2013 Sep 8.

Authors

Affiliations

¹ Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
² Clinical Genetics Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
³ Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁴ Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Radboud Institute for Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
⁵ Department of Pathology, University of Washington, Seattle, Washington, USA.
⁶ Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁷ Department of Computational Biology and Bioinformatics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁸ Pediatric Cancer Genome Project Laboratory, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁹ Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹⁰ Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
¹¹ Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹² Hematology Laboratory Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹³ Clinical Chemistry Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
¹⁴ Department of Pathology and Cell Biology, Columbia University, New York, New York, USA.
¹⁵ High Risk Pediatric Cancer Clinic, Huntsman Cancer Institute/Primary Children's Medical Center, University of Utah, Salt Lake City, Utah, USA.
¹⁶ Pediatrics, University of Chicago, Chicago, IL , USA.
¹⁷ Texas Children's Cancer Center and Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA.
¹⁸ Kids Cancer Centre, Sydney Children's Hospital, Sydney, New South Wales, Australia, Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Sydney, New South Wales, Australia.
¹⁹ Hereditary Cancer Clinic Prince of Wales Hospital, Randwick, Australia.
²⁰ Children's Cancer Institute Australia for Medical Research, University of New South Wales, Randwick, Australia.
²¹ Cancer Genetics Laboratory, The Queensland Institute of Medical Research, Herston, Australia.
²² Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
²³ Departments of Medicine and Genome Sciences, University of Washington, Seattle, Washington, USA.
²⁴ Department of Molecular Pathology, SA Pathology and Centre for Cancer Biology, Adelaide, Australia.
²⁵ Department of Pediatrics, Weill Cornell College of Medicine, New York, New York, USA.
²⁶ Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁷ Genomics Core Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁸ Bioinformatics Core, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
²⁹ Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
³⁰ Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
³¹ Department of Medicine, Weill Cornell College of Medicine, New York, New York, USA.

^# Contributed equally.

PMID: 24013638
PMCID: PMC3919799
DOI: 10.1038/ng.2754

Abstract

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

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Figures

**Figure 1. Familial Pre-B cell ALL associated with i(9)(q10) and dic(9;v) in two families harboring a novel, recurrent germline p.Gly183Ser variant**
a. Family 1 of Puerto Rican ancestry. The proband is denoted by an arrow. Exome sequencing was undertaken in germline DNA from all available affected (IV1, IV5, IV6, III5) and unaffected (IV9, III3, III4) individuals as well as the diagnostic leukemic sample from IV6. p.Gly183Ser variant status denoted by (+/−). b. Family 2 of African-American ancestry. The proband is denoted by an arrow. Exome sequencing was undertaken in diagnostic, remission and relapse leukemic samples from individuals III4, IV1, and IV2. p.Gly183Ser variant status denoted by (+/−). c. Chromosome 9 copy number heat map for SNP6.0 microarray data of germline and tumor samples from three members of Family 2. These data demonstrate the common feature of loss of 9p in the tumor specimens. Note the focal dark blue band denoting homozygous loss of CDKN2A/B in all samples. Blue indicates deletions and red indicates gains. G, germline; D, diagnosis; R, relapse sample. d. The haplotype flanking the p.Gly183Ser mutation. A five SNP haplotype rs7850825 to rs7020413 (Chr9:36.997-37.002 Mb) proximal to the mutation was concordant in both family 1 and family 2. However, the distal end flanking the mutation rs6476606 was discordant.

**Figure 2. Recurrent *PAX5* mutations in ALL**
a. Gene schematic of *PAX5* showing the exons (upper grey numbers), amino acid residues (lower grey numbers), protein domains (as denoted by colored legend) and position of the germline p.Gly183Ser variant (in red) in relation to the somatic *PAX5* mutations described in this study (n=13, arrows) and somatic mutations described previously in B-ALL^,,. Primary leukemic samples with confirmed retention of the germline p.Gly183Ser variant denoted by the square shape (Family 1) and diamond shape (Family 2). In one case of i(9)/dic(9) ALL, we found both a heterozygous Val26Gly and a heterozygous Gln350fs mutation, indicating polyclonality of the tumor. b. Conservation of the octapeptide domain in selected PAX family members.

**Figure 3. Attenuated transcriptional activity of *PAX5* p.Gly183Ser**
a. Transcriptional activity of *PAX5* variants compared to wild-type using a Pax5-dependent reporter gene assay in 293T cells. Bars show mean (± s.e.m.) luciferase activity of six individual experiments with triplicate measurements (*PAX5* p.Gly183Val and *PAX5* d2-6 four experiments with triplicate measurements). Asterisks indicate significant difference calculated by Dunnett's test (P<0.0001). MIR, MSCV-IRES-mRFP empty vector. b. Transcriptional activity of *PAX5* variants using CD79A-dependent sIgM expression in the murine J558LμM plastocytoma cell line. Percentages indicate proportion of mRFP positive cells that show sIgM expression. Bars show mean (± s.e.m.) sIgM expression in two individual experiments with triplicate replicates each. Asterisks indicate significant difference calculated by Dunnett's test (P<0.0001). MIR, MSCV-IRES-mRFP empty vector. c. *PAX5*-dependent reporter gene assay of *PAX5* wild-type and *PAX5* p.Gly183Ser run in triplicate as above, with or without co-transfection of 0.05μg of Grg4 as indicated. A *PAX5* p.Tyr179Glu (Y179E) mutant that is deficient in binding to Grg4 and empty vector were used as controls. Asterisks indicate significant differences by two-tailed t-test (p<0.0001). d. Heatmap of *PAX5* activated genes in mature B cells. Four samples from family 2 (diagnosis and relapse samples from individuals IV1 and IV2) show differential expression of *PAX5* activated genes when compared to a group of 139 sporadic B-ALL cases. This indicates an effect of the pGly183Ser mutation on *PAX5* function. Red indicates high expression, blue represents low expression. *PAX5* mutation status is indicated by the colors above the samples. Green indicates wild type *PAX5*, yellow indicates heterozygosity for a *PAX5* mutation, and pink indicates biallelic *PAX5* mutations.

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Comment in

Germline PAX5 mutations and B cell leukemia.
Hyde RK, Liu PP. Hyde RK, et al. Nat Genet. 2013 Oct;45(10):1104-5. doi: 10.1038/ng.2778. Nat Genet. 2013. PMID: 24071841

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References

1. Mullighan CG, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;446:758–64. - PubMed
1. Mullighan CG, et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 2009;360:470–80. - PMC - PubMed
1. Kuiper RP, et al. High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Leukemia. 2007;21:1258–66. - PubMed
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[1] Mullighan CG, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;446:758–64. - PubMed

[2] Mullighan CG, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;446:758–64. - PubMed

[3] Mullighan CG, et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 2009;360:470–80. - PMC - PubMed

[4] Mullighan CG, et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 2009;360:470–80. - PMC - PubMed

[5] Kuiper RP, et al. High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Leukemia. 2007;21:1258–66. - PubMed

[6] Kuiper RP, et al. High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Leukemia. 2007;21:1258–66. - PubMed

[7] Hemminki K, Jiang Y. Risks among siblings and twins for childhood acute lymphoid leukaemia: results from the Swedish Family-Cancer Database. Leukemia. 2002;16:297–8. - PubMed

[8] Hemminki K, Jiang Y. Risks among siblings and twins for childhood acute lymphoid leukaemia: results from the Swedish Family-Cancer Database. Leukemia. 2002;16:297–8. - PubMed

[9] Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371:1030–43. - PubMed

[10] Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371:1030–43. - PubMed

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A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

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A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

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