Figure 1.
Identification of a patient with biallelic PAX5 mutations. (A) Family pedigree of the 19-yr-old patient (II.B) with his asymptomatic mother (I.B). (B) Chromatograms of Sanger sequencing showing segregation of the PAX5-c.G92A (left) and PAX5-c.G724T (right) mutations in the four family members. PCR-amplified DNA from granulocytes was analyzed. In addition, PCR analysis was performed with DNA from epithelial cells of the patient (II.B). R (G + A); K (G + T). (C) PAX5 domain organization. The two mutations on the maternal and paternal PAX5 alleles are indicated together with the paired domain (PD), octapeptide motif (OP), partial homeodomain (HD), transactivation domain (TAD), and inhibitory domain (ID; Dörfler and Busslinger, 1996). (D) Abundance of PAX5-c.G92A and PAX5-c.G724T mRNAs in naive mature B cells of the patient. The frequency of the mutant mRNAs was determined by RT-qPCR amplification and subsequent sequencing of the cloned PCR fragments. (E) Immunoblot analysis of nuclear extracts from EBV-immortalized B cells of the patient and three controls with anti-PAX5 and anti-H3 antibodies. The positions of the full-length PAX5-R31Q and truncated PAX5-E242* proteins are indicated. (F) Expression of wild-type PAX5, PAX5-R31Q, and PAX5-E242* proteins in nuclear extracts of transfected HEK-293T cells, analyzed by immunoblotting with anti-PAX5 and anti-H3 antibodies. (G) Analysis of the transactivation potential of the wild-type and mutant PAX5 proteins in HEK-293T cells, transfected with the indicated PAX5 expression vectors, the plasmid lucCD19 containing three high-affinity PAX5-binding sites upstream of the β-globin TATA box and initiator region linked to the firefly luciferase gene (Czerny and Busslinger, 1995), and a control renilla luciferase plasmid. The firefly and renilla luciferase activities were measured 2 d after transfection. Normalized firefly activity is shown relative to the pcDNA3.1 vector control (set to 1). The pooled data of three independent experiments (each dot corresponding to one transfection assay; n = 9 for each expression vector) are shown as mean values with SEM analyzed by ANOVA with Dunnett’s T3 multiple comparison test; **, P < 0.01; ****, P < 0.0001. For detailed statistical information, see Table S6. (H) Evolutionary conservation of arginine (R) at position 31 in all human PAX proteins; remaining amino acids are abbreviated as follows: asparagine (N), glycine (G), glutamine (Q), proline (P), leucine (L).

Identification of a patient with biallelic PAX5 mutations. (A) Family pedigree of the 19-yr-old patient (II.B) with his asymptomatic mother (I.B). (B) Chromatograms of Sanger sequencing showing segregation of the PAX5-c.G92A (left) and PAX5-c.G724T (right) mutations in the four family members. PCR-amplified DNA from granulocytes was analyzed. In addition, PCR analysis was performed with DNA from epithelial cells of the patient (II.B). R (G + A); K (G + T). (C) PAX5 domain organization. The two mutations on the maternal and paternal PAX5 alleles are indicated together with the paired domain (PD), octapeptide motif (OP), partial homeodomain (HD), transactivation domain (TAD), and inhibitory domain (ID; Dörfler and Busslinger, 1996). (D) Abundance of PAX5-c.G92A and PAX5-c.G724T mRNAs in naive mature B cells of the patient. The frequency of the mutant mRNAs was determined by RT-qPCR amplification and subsequent sequencing of the cloned PCR fragments. (E) Immunoblot analysis of nuclear extracts from EBV-immortalized B cells of the patient and three controls with anti-PAX5 and anti-H3 antibodies. The positions of the full-length PAX5-R31Q and truncated PAX5-E242* proteins are indicated. (F) Expression of wild-type PAX5, PAX5-R31Q, and PAX5-E242* proteins in nuclear extracts of transfected HEK-293T cells, analyzed by immunoblotting with anti-PAX5 and anti-H3 antibodies. (G) Analysis of the transactivation potential of the wild-type and mutant PAX5 proteins in HEK-293T cells, transfected with the indicated PAX5 expression vectors, the plasmid lucCD19 containing three high-affinity PAX5-binding sites upstream of the β-globin TATA box and initiator region linked to the firefly luciferase gene (Czerny and Busslinger, 1995), and a control renilla luciferase plasmid. The firefly and renilla luciferase activities were measured 2 d after transfection. Normalized firefly activity is shown relative to the pcDNA3.1 vector control (set to 1). The pooled data of three independent experiments (each dot corresponding to one transfection assay; n = 9 for each expression vector) are shown as mean values with SEM analyzed by ANOVA with Dunnett’s T3 multiple comparison test; **, P < 0.01; ****, P < 0.0001. For detailed statistical information, see Table S6. (H) Evolutionary conservation of arginine (R) at position 31 in all human PAX proteins; remaining amino acids are abbreviated as follows: asparagine (N), glycine (G), glutamine (Q), proline (P), leucine (L).

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