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L265P mutation of the MYD88 gene is frequent in Waldenström's macroglobulinemia and its absence in myeloma.

Mori N, Ohwashi M, Yoshinaga K, Mitsuhashi K, Tanaka N, Teramura M, Okada M, Shiseki M, Tanaka J, Motoji T - PLoS ONE (2013)

Bottom Line: We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma.No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32).BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT
L265P mutation in the MYD88 gene has recently been reported in Waldenström's macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström's macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström's macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström's macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström's macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström's macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström's macroglobulinemia than in those with myeloma (p=1.3x10(-10)). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström's macroglobulinemia patients (72%). In the 25 Waldenström's macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10(-4)), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10(-2)). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström's macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

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BSiE1 digestion of the MYD88 gene in Waldenström’s macroglobulinemia and lymphoma.Ten μl of PCR products was digested with BSiE1, separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination. The size of the products is indicated on the left. (A) Sensitivity of BSiE1 digestion. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Aberrant bands were detected in samples containing 0.5% or more of the L265P mutation. Lane 1, 100 bp ladder; lane 2, 0%; lane 3, 0.1%; lane 4, 0.5%; lane 5, 1%; lane 6, 5%; lane 7, WM5. (B) Aberrant bands were detected in 10 of 14 samples from WM patients (lanes 2, 5, 6, 8, 9, 10, 11, 12, 14, and 15). Lane 1, 100 bp ladder; lane 2, WM1; lane 3, WM2; lane 4, WM3; lane 5, WM4; lane 6, WM5; lane 7, WM6; lane 8, WM7; lane 9, WM8; lane 10, WM9; lane 11, WM10; lane 12, WM11; lane 13, WM12, lane 14, WM13; lane 15, WM14.(C) Aberrant bands were detected in 8 of 9 samples from WM patients (lanes 2, 3, 4, 5, 6, 8, 9, and 10) and non-Hodgkin’s lymphoma patient (NHL3, lane 12). Lane 1, 100 bp ladder; lane 2, WM15; lane 3, WM16; lane 4, WM17; lane 5, WM18; lane 6, WM19; lane 7, WM20; lane 8, WM21; lane 9, WM22; lane 10, WM23; lane 11, NHL1; lane 12, NHL3; lane 13, normal lymphocyte 1; lane 14, water.(D) Strength of aberrant bands varied in accordance with percentages of L265P-positive DNA. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Lane 1, 100 bp ladder; lane 2, 50%; lane 3, 80%; lane 4, 90%; lane 5, 100%.
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pone-0080088-g002: BSiE1 digestion of the MYD88 gene in Waldenström’s macroglobulinemia and lymphoma.Ten μl of PCR products was digested with BSiE1, separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination. The size of the products is indicated on the left. (A) Sensitivity of BSiE1 digestion. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Aberrant bands were detected in samples containing 0.5% or more of the L265P mutation. Lane 1, 100 bp ladder; lane 2, 0%; lane 3, 0.1%; lane 4, 0.5%; lane 5, 1%; lane 6, 5%; lane 7, WM5. (B) Aberrant bands were detected in 10 of 14 samples from WM patients (lanes 2, 5, 6, 8, 9, 10, 11, 12, 14, and 15). Lane 1, 100 bp ladder; lane 2, WM1; lane 3, WM2; lane 4, WM3; lane 5, WM4; lane 6, WM5; lane 7, WM6; lane 8, WM7; lane 9, WM8; lane 10, WM9; lane 11, WM10; lane 12, WM11; lane 13, WM12, lane 14, WM13; lane 15, WM14.(C) Aberrant bands were detected in 8 of 9 samples from WM patients (lanes 2, 3, 4, 5, 6, 8, 9, and 10) and non-Hodgkin’s lymphoma patient (NHL3, lane 12). Lane 1, 100 bp ladder; lane 2, WM15; lane 3, WM16; lane 4, WM17; lane 5, WM18; lane 6, WM19; lane 7, WM20; lane 8, WM21; lane 9, WM22; lane 10, WM23; lane 11, NHL1; lane 12, NHL3; lane 13, normal lymphocyte 1; lane 14, water.(D) Strength of aberrant bands varied in accordance with percentages of L265P-positive DNA. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Lane 1, 100 bp ladder; lane 2, 50%; lane 3, 80%; lane 4, 90%; lane 5, 100%.

Mentions: Since the percentage of lymphocytes (median 27.3%, 13.5-96.4) in the bone marrow varied in each WM patient, the mutation may have been undetectable by direct sequencing in some cases. Thus we tested for the mutation with BSiE1 digestion. To determine sensitivity, DNA from the L265P-positive clone (WM5) was serially diluted into DNA from a wild-type clone (WM2 or WM3) to the following percentages: 0%, 0.1%, 0.5%, 1%, and 5%. Sensitivity to the L265P mutation was 0.1-0.5% in our study (Figure 2 A). To compare the sensitivity of direct sequencing with BSiE1 digestion, we used the same tube of PCR products for both analyses. We used the same tube of PCR products to screen for the mutation in other samples. The mutation was repeatedly confirmed with different tubes of PCR products. Aberrant bands corresponding to the mutation were detected in 21 of the 28 patients: 19 of the 25 WM (76%), one of the 2 non-IgM-secreting LPL, and B-cell lymphoma with IgG M-protein (Tables 1-2, Figure 2 B-D). All but one patient (WM21) with aberrant bands had hemizygous mutation (Table 2). Of the 21 patients with aberrant bands, 9 had the mutation detected by direct sequencing. Most patients with relatively strong bands were shown to have the mutation by direct sequencing. All of the 9 patients, in whom the L265P mutation was detected by direct sequencing, also showed the mutation by BSiE1 digestion (Table 2). To confirm these results, several samples without aberrant bands by digestion were also sequenced; however, only the wild-type sequences were obtained. We also examined in the 38 patients with myeloma with BSiE1 digestion; however, no aberrant bands were observed.


L265P mutation of the MYD88 gene is frequent in Waldenström's macroglobulinemia and its absence in myeloma.

Mori N, Ohwashi M, Yoshinaga K, Mitsuhashi K, Tanaka N, Teramura M, Okada M, Shiseki M, Tanaka J, Motoji T - PLoS ONE (2013)

BSiE1 digestion of the MYD88 gene in Waldenström’s macroglobulinemia and lymphoma.Ten μl of PCR products was digested with BSiE1, separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination. The size of the products is indicated on the left. (A) Sensitivity of BSiE1 digestion. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Aberrant bands were detected in samples containing 0.5% or more of the L265P mutation. Lane 1, 100 bp ladder; lane 2, 0%; lane 3, 0.1%; lane 4, 0.5%; lane 5, 1%; lane 6, 5%; lane 7, WM5. (B) Aberrant bands were detected in 10 of 14 samples from WM patients (lanes 2, 5, 6, 8, 9, 10, 11, 12, 14, and 15). Lane 1, 100 bp ladder; lane 2, WM1; lane 3, WM2; lane 4, WM3; lane 5, WM4; lane 6, WM5; lane 7, WM6; lane 8, WM7; lane 9, WM8; lane 10, WM9; lane 11, WM10; lane 12, WM11; lane 13, WM12, lane 14, WM13; lane 15, WM14.(C) Aberrant bands were detected in 8 of 9 samples from WM patients (lanes 2, 3, 4, 5, 6, 8, 9, and 10) and non-Hodgkin’s lymphoma patient (NHL3, lane 12). Lane 1, 100 bp ladder; lane 2, WM15; lane 3, WM16; lane 4, WM17; lane 5, WM18; lane 6, WM19; lane 7, WM20; lane 8, WM21; lane 9, WM22; lane 10, WM23; lane 11, NHL1; lane 12, NHL3; lane 13, normal lymphocyte 1; lane 14, water.(D) Strength of aberrant bands varied in accordance with percentages of L265P-positive DNA. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Lane 1, 100 bp ladder; lane 2, 50%; lane 3, 80%; lane 4, 90%; lane 5, 100%.
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Show All Figures
getmorefigures.php?uid=PMC3818242&req=5

pone-0080088-g002: BSiE1 digestion of the MYD88 gene in Waldenström’s macroglobulinemia and lymphoma.Ten μl of PCR products was digested with BSiE1, separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination. The size of the products is indicated on the left. (A) Sensitivity of BSiE1 digestion. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Aberrant bands were detected in samples containing 0.5% or more of the L265P mutation. Lane 1, 100 bp ladder; lane 2, 0%; lane 3, 0.1%; lane 4, 0.5%; lane 5, 1%; lane 6, 5%; lane 7, WM5. (B) Aberrant bands were detected in 10 of 14 samples from WM patients (lanes 2, 5, 6, 8, 9, 10, 11, 12, 14, and 15). Lane 1, 100 bp ladder; lane 2, WM1; lane 3, WM2; lane 4, WM3; lane 5, WM4; lane 6, WM5; lane 7, WM6; lane 8, WM7; lane 9, WM8; lane 10, WM9; lane 11, WM10; lane 12, WM11; lane 13, WM12, lane 14, WM13; lane 15, WM14.(C) Aberrant bands were detected in 8 of 9 samples from WM patients (lanes 2, 3, 4, 5, 6, 8, 9, and 10) and non-Hodgkin’s lymphoma patient (NHL3, lane 12). Lane 1, 100 bp ladder; lane 2, WM15; lane 3, WM16; lane 4, WM17; lane 5, WM18; lane 6, WM19; lane 7, WM20; lane 8, WM21; lane 9, WM22; lane 10, WM23; lane 11, NHL1; lane 12, NHL3; lane 13, normal lymphocyte 1; lane 14, water.(D) Strength of aberrant bands varied in accordance with percentages of L265P-positive DNA. L265P-positive DNA (WM5) was diluted into wild-type DNA (WM3) before amplification. Lane 1, 100 bp ladder; lane 2, 50%; lane 3, 80%; lane 4, 90%; lane 5, 100%.
Mentions: Since the percentage of lymphocytes (median 27.3%, 13.5-96.4) in the bone marrow varied in each WM patient, the mutation may have been undetectable by direct sequencing in some cases. Thus we tested for the mutation with BSiE1 digestion. To determine sensitivity, DNA from the L265P-positive clone (WM5) was serially diluted into DNA from a wild-type clone (WM2 or WM3) to the following percentages: 0%, 0.1%, 0.5%, 1%, and 5%. Sensitivity to the L265P mutation was 0.1-0.5% in our study (Figure 2 A). To compare the sensitivity of direct sequencing with BSiE1 digestion, we used the same tube of PCR products for both analyses. We used the same tube of PCR products to screen for the mutation in other samples. The mutation was repeatedly confirmed with different tubes of PCR products. Aberrant bands corresponding to the mutation were detected in 21 of the 28 patients: 19 of the 25 WM (76%), one of the 2 non-IgM-secreting LPL, and B-cell lymphoma with IgG M-protein (Tables 1-2, Figure 2 B-D). All but one patient (WM21) with aberrant bands had hemizygous mutation (Table 2). Of the 21 patients with aberrant bands, 9 had the mutation detected by direct sequencing. Most patients with relatively strong bands were shown to have the mutation by direct sequencing. All of the 9 patients, in whom the L265P mutation was detected by direct sequencing, also showed the mutation by BSiE1 digestion (Table 2). To confirm these results, several samples without aberrant bands by digestion were also sequenced; however, only the wild-type sequences were obtained. We also examined in the 38 patients with myeloma with BSiE1 digestion; however, no aberrant bands were observed.

Bottom Line: We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma.No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32).BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT
L265P mutation in the MYD88 gene has recently been reported in Waldenström's macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström's macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström's macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström's macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström's macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström's macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström's macroglobulinemia than in those with myeloma (p=1.3x10(-10)). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström's macroglobulinemia patients (72%). In the 25 Waldenström's macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10(-4)), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10(-2)). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström's macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

Show MeSH
Related in: MedlinePlus