BCR-ABL kinase domain mutations in tyrosine kinase inhibitors-naïve and -exposed Southeast Asian chronic myeloid leukemia patients

BCR-ABL kinase domain (KD) mutation is the main mechanism associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients. This study targeted a large cohort of CML (n = 171) comprising 80 naïve CML cases without prior TKI exposure as well as 91 cases undergoing 1st generation (imatinib) and/or 2nd generation (nilotinib/dasatinib) TKI therapy. KD mutations were analyzed by denaturing high performance liquid chromatography followed by direct sequencing. Twenty-one types of mutations were found in 37 patients including 13 known mutations and 8 previously unidentified mutations. Thirty cases had a single mutation while 7 cases had multiple mutations. Twenty-three percent of patients receiving first-line imatinib, 69% of imatinib-resistant patients receiving 2nd generation TKI, and 75% of advanced phase patients treated with front-line 2nd generation TKI had KD mutations. Interestingly, 9% of TKI-naïve CML cases were also discovered to carry the KD mutations including 5 novel variants. Patients who received hydroxyurea had a 2-fold increase in KD mutations as compared to newly diagnosed patients but they still had a lower mutation frequency than TKI-exposed cases. Mutations in the naïve cases were mainly localized in the C-helix domain and SH3 contact site whereas in exposed cases predominantly in the drug contact site, P-loop, and catalytic domain. T315I resistant mutation was identified only in TKI-exposed cases. In conclusion, several known and novel BCR-ABL KD mutations were discovered in the TKI-naïve and -exposed Southeast Asian CML patients, supporting the concept that naturally occurring KD mutations were present in leukemic cells prior to drug exposure. T315I resistant mutation was completely undetectable in this naïve Southeast Asian cohort; its incidence, however, increases with drug exposure.     

Treatment with dasatinib or nilotinib in chronic myeloid leukemia patients who failed to respond to two previously administered tyrosine kinase inhibitors – a single center experience

OBJECTIVE:

To evaluate hematological, cytogenetic and molecular responses as well as the overall, progression-free and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitor after failing to respond to imatinib and nilotinib/dasatinib.

METHODS:

Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed at baseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor. Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNet recommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing.

RESULTS:

We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and a second tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinib as a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13% achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1 mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351T mutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free and event-free survivals were 86, 54 and 22% (p<0.0001), respectively, for chronic phase patients and 66%, 66% and 0% (p<0.0001), respectively, for accelerated phase patients. All blast crisis patients died within 6 months of treatment. Fifty-six percent of the chronic phase patients lost their hematologic response within a median of 23 months.

CONCLUSIONS:

Although the responses achieved by the third tyrosine kinase inhibitor were not sustainable, a third tyrosine kinase inhibitor may be an option for improving patient status until a donor becomes available for transplant. Because the long-term outcome for these patients is poor, the development of new therapies for resistant chronic myeloid leukemia patients is necessary.     

Gaucher disease: expression and characterization of mild and severe acid beta-glucosidase mutations in Portuguese type 1 patients

Type 1 Gaucher disease (GD), the most prevalent lysosomal storage disease, results from the deficient activity of acid beta-glucosidase. Molecular analysis of 12 unrelated Portuguese patients with type 1 GD identified three novel acid beta-glucosidase mutations (F109V, W184R and R395P), as well as three previously reported, but uncharacterized, lesions (R359Q, G377S and N396T). The type 1 probands were either heteroallelic for the well-characterized common lesion, N370S, and the F109V, W184R, R359Q or N396T lesions or homoallelic for the G377S or N396T mutations. Expression of the W184R, R359Q and R395P mutations revealed very low specific activities based on cross-reacting immunologic material (CRIM SAs of 0.0004, 0.016 and 0.045, respectively), consistent with their being found only in type 1 patients who had a neuroprotective N370S allele. In contrast, the F109V, G377S and N396T alleles had significant acid beta-glucosidase activity (CRIM specific activities of 0.15, 0.17, 0.14, respectively), in agreement with their being mild type 1 alleles. Thus, these studies identified additional acid beta-glucosidase mutations in the Portuguese population and demonstrated that the G377S and N396T mutations were neuroprotective, consistent with the mild clinical phenotypes of the type 1 patients who were homoallelic for the G377S and N396T lesions.     

Bosutinib: A Review of Its Use in Patients with Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia

Bosutinib (Bosulif^®) is an orally administered small molecule tyrosine kinase inhibitor (TKI) of BCR–ABL and SRC family kinases. It is indicated for the treatment of adult patients with chronic-, accelerated-, or blast-phase Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapy (imatinib, dasatinib, or nilotinib) [USA] or for a small subpopulation of these patients for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options (EU). In a multinational pivotal trial (n = 547), bosutinib treatment resulted in a major cytogenetic response (MCyR) at 24 weeks in one-third of all treated patients with imatinib-resistant chronic-phase CML who had no previous exposure to any TKIs other than imatinib (primary endpoint), with similar results observed in chronic-phase CML patients who were intolerant of imatinib and naïve to all other TKIs. MCyRs were also seen in more than one–quarter of evaluable patients with chronic-phase CML previously treated with multiple TKIs. Most of the patients with chronic-phase CML achieved a complete hematologic response with bosutinib and some patients with advanced phases of CML achieved an overall hematologic response. Responses were seen irrespective of the type of BCR-ABL mutation at baseline, except T315I. Bosutinib had a manageable tolerability profile in the pivotal trial, with ≤21 % of patients with chronic-phase CML discontinuing the treatment because of adverse events. Diarrhea was the most common adverse event but was generally manageable, with only few patients discontinuing the treatment because of diarrhea. Therefore, bosutinib is a useful TKI option for patients with Ph+ CML in second-line or greater settings.     

GATA4 mutations in 486 Chinese patients with congenital heart disease

Recent studies have reported germline mutations in GATA4 gene in some types of congenital heart disease (CHD). However, the prevalence of GATA4 mutations in CHD and the correlation between the GATA4 genotype and CHD phenotype have not been extensively studied. We screened germline mutations in the coding exons and the flanking intron sequences of the GATA4 gene in 486 CHD patients by denaturing high-performance liquid chromatography (DHPLC), and confirmed the mutations by sequencing. Nine distinct mutations including one small deletion mutation (46delS), two small insertion mutations (118–119insA and 125–126insAA), and six non-synonymous mutations (A6V, P163S, E359K, P407Q, S429T and A442V) were identified in 12 of the 486 patients (nine with ventricular septal defect, two with Tetralogy of Fallot, and one with endocardial cushion defect). Of them, two patients carrying E359K mutation were from two generations in one family with ventricular septal defect (VSD). Interestingly, a nucleotide insertion of c.1146 + 25insA in exon 6 was detected in five VSD patients, but not in 486 normal healthy controls. Our findings are useful in understanding the prevalence of GATA4 mutations and the correlation between the GATA4 genotype and the CHD phenotype in Chinese patients.     

Spotlight on Dasatinib in Chronic Myeloid Leukemia and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemiay

Dasatinib (Sprycel?) is an orally administered small molecule inhibitor of multiple tyrosine kinases, including BCR-ABL and SRC family kinases, which is indicated for the treatment of adults with newly diagnosed chronic-phase chronic myeloid leukemia (CML), CML (chronic-, accelerated- or blast-phase) with resistance or intolerance to prior therapy, including imatinib, or Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) with resistance or intolerance to prior therapy. Dasatinib is ≈325-fold more active than imatinib in inhibiting wild-type BCR-ABL kinase in vitro and is active against a wide variety of imatinib-resistant BCR-ABL mutants, except for T315I. This article reviews the efficacy and tolerability of dasatinib in the treatment of patients with newly diagnosed chronic-phase CML or imatinib-resistant or -intolerant CML or Ph+ ALL, as well as summarizing its pharmacologic properties. In clinical trials, oral dasatinib was effective in achieving major or complete cytogenetic responses in both newly diagnosed and imatinib-resistant or -intolerant chronic-phase CML. Dasatinib was likewise effective in achieving major or overall hematologic responses in imatinib-resistant or -intolerant, accelerated- or blast-phase CML, or Ph+ ALL. Responses were rapidly achieved within 1-3 months and were durable over 1-5 years of follow-up. The majority of adverse events with dasatinib were of mild or moderate severity. Fluid retention (including pleural effusion) was the most common adverse event. Hematologic abnormalities were common and cytopenias were the most common grade 3/4 adverse events. Dasatinib 100?mg administered once daily was as effective as dasatinib 70?mg administered twice daily, and was better tolerated, being associated with lower incidences of pleural effusion and grade 3/4 thrombocytopenia, in particular. Dasatinib was more effective than high-dose imatinib in the treatment of patients with imatinib-resistant chronic-phase CML and was more effective than standard dosages of imatinib, as well as being associated with less frequent fluid retention, in patients with newly diagnosed chronic-phase CML. Dasatinib was generally equally effective in patients with or without BCR-ABL mutations at baseline. Therefore, oral dasatinib is a highly effective, once-daily therapy for the first-line treatment of newly diagnosed patients with chronic-phase CML, as well as for the treatment of patients with imatinib-resistant or -intolerant chronic- and advanced-phase CML or Ph+ ALL.     

Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors

Point mutations in the kinase domain of BCR-ABL were described in 40?C90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We herein describe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinase inhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detected in 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experiments proved that the method is specific and reproducible, with maximum sensitivity of 1?×?10^?3. The developed assay is a convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together with sequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.     

Identification of recurrent and novel mutations in the LDL receptor gene in Japanese familial hypercholesterolemia

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 T→C), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population. Hum Mutat 14:87, 1999. © 1999 Wiley‐Liss, Inc.     

A new rapid and sensitive assay for detecting the T315I BCR-ABL kinase domain mutation in chronic myeloid leukaemia

A significant minority of chronic myeloid leukaemia patients eventually develop resistance to imatinib, often as a result of point mutations within the BCR-ABL kinase domain. Second-line tyrosine kinase inhibitors (TKIs) are effective against mutations that confer imatinib resistance; however, the T315I BCR-ABL mutant has proved resistant to all available TKIs. An assay facilitating early identification of BCR-ABL(T315I) would therefore aid in identifying high-risk patients who may benefit from alternative therapy. This report describes the development of a sensitive T315I mutation detection methodology based on real-time PCR with self-probing fluorescent primers. The technique demonstrated complete concordance with direct sequencing, correctly identifying 34 T315I-positive samples from a total of 61 samples screened. In a limiting dilution assay, the mutated clone was detectable to a level of 1% of total cells. The data show that Scorpions PCR enables rapid screening for BCR-ABL(T315I) in chronic myeloid leukaemia patients and is appropriate for use in a clinical setting.     

三氧化二砷对伊马替尼耐药细胞株32Dp210T315I的体外抑制作用研究

目的 探讨三氧化二砷( ATO)对伊马替尼(IM)产生耐药性的慢性粒细胞性白血病(CML)细胞是否具有抑制细胞生长的作用.方法 小鼠髓系造血细胞株32D细胞经处理使其变为表达p210Bcr-Abl的32Dp210细胞,以此作为野生型细胞.32Dp210和伊马替尼耐药细胞株32Dp210T315I分别在含有10％ FBS,2％ L-glutamine和1％ Penicillin-Streptomycin的RPMI1640和IMDM培养体系中培养.细胞株分别在不同浓度的三氧化二砷、伊马替尼作为对照的没有药物作用的培养体系中培养.在24h和48 h后,MTT实验检测细胞增殖,Annexin-V staining实验检测细胞凋亡.结果 在体外试验中,我们发现三氧化二砷对32Dp210和32Dp210T315I具有剂量依赖的抑制作用,对32Dp210T315I细胞的抑制比对32Dp210细胞更为有效.三氧化二砷对32Dp210T315I细胞的50％抑制浓度(IC50)是1μmol/L,远低于32Dp210的IC50值(4μmol/L).2μmol/L三氧化二砷可以明显诱导约90％的32Dp210T315I细胞凋亡,比32Dp210的凋亡率(30％)高很多.结论 ATO具有作为一种生长抑制剂和诱导伊马替尼耐药细胞凋亡的特性,也使其成为一种新的治疗伊马替尼耐药CML患者的治疗途径.     

Tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 x g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.     

Terapia przewlekłej białaczki szpikowej – teraźniejszość i wyzwania na przyszłość

Introduction of imatinib to the treatment of chronic myelogenous leukemia (CML) more than a decade ago may be considered as one of the milestones in the history of cancer treatment and oncology. Small molecule inhibitor, which specifically inhibits BCR/ABL oncogenic tyrosine kinase, responsible for malignant transformation of hematopoietic stem cell proved to be unexpectedly effective in the majority of patients and has revolutionized CML therapy. Unfortunately, a significant group of patients develops resistance or is intolerant to the drug which necessitate search for new better drugs. In the last 5 years 2^nd generation inhibitors have been approved (dasatinib, nilotinib and bosutinib), clinical trials are ongoing with ^3rd generation inhibitors (among them ponatinib, active against BCR/ABL with T315I mutation) and allosteric inhibitors. None of the available drugs eliminates leukemia stem cells, which are the roots of the disease, therefore therapy must be continued indefinitely. Since ultimate goal is to cure the disease there are number of trials to eradicate the disease with combination therapies. We may expect that such like imatinib opened new therapeutic horizons in oncology, complete eradication of CML will help to find cure other cancers.     

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1_V3Lib) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1_V3Lib at passage 17 could not be suppressed even at 10 μM (> 1400-fold resistance), while HIV-1_JR-FL at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1_V3Lib-P17 contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.     

Hereditary thrombophilia, anti-beta2 glycoprotein 1 IgM, and anti-annexin V antibodies in recurrent pregnancy loss

Citation Karata S, Aydin Y, Ocer F, Buyru A, Balci H. Hereditary Thrombophilia, anti‐beta2 glycoprotein 1 IgM, and anti‐annexin V antibodies in recurrent pregnancy loss. Am J Reprod Immunol 2012; 67: 251–255 Problem We investigated the beta2‐glycoprotein I and anti‐annexin V antibodies as anti‐phospholipid–cofactor antibodies; and factor V G1691A Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T mutations as hereditary thrombophilia in recurrent pregnancy losses (RPL). Method of study Study group consisted of 84 women with recurrent pregnancy loss and control group consisted of 84 women having at least one live birth. Results Methylenetetrahydrofolate reductase C677T homozygous mutation was detected in 28.5% of the study group and in 14.2% of the controls, and the difference was highly significant (P < 0.001). Heterozygous mutation of this gene was found in 64.3% of the study population and in 38.1% of the controls, and difference in heterozygous mutation frequency was also significant (P < 0.001). Both homozygous and heterozygous mutations of PT G20210A and factor V G1691A were not different between the groups. There was no significant difference in anti‐annexin V levels and anti‐beta2‐gp 1 levels of the groups. Conclusion We concluded that both homozygous and heterozygous mutations of MTHFR C677T were related with RPL in Caucasian women.     

Imatinib resistance in a novel translocation der(17)t(1;17)(q25;p13) with loss of TP53 but without BCR/ABL kinase domain mutation in chronic myelogenous leukemia

We describe here two novel translocations, t(7;14)(p22;q13) and der(17)t(1;17)(q25;p13), in a 41-year-old man with an accelerated phase (AP) of chronic myelogenous leukemia (CML). Chromosome analysis initially showed 46,XY,t(7;14)(p13;q22),t(9;22)(q34;q11.2)[20]. In 3 years, the karyotype evolved to 45,X,−Y,der(7)t(7;14)(p13;q22),t(9;22)(q34;q11.2),−14,der(17)t(1;17)(q25;p13),+der(22)t(9;22)[20], accompanied with a resistance to imatinib mesylate. The TP53 was deleted from the der(17)t(1;17)(q25;p13), but there was no mutation of TP53 in the remaining allele. Mutations in the BCR/ABL kinase domain could not be detected as well. Morphologically, dysplastic changes including pseudo-Pelger–Huët anomaly appeared in the bone marrow cells. These findings suggest that the t(7;14)(p22;q13) translocation had a crucial role in the progression to CML-AP, and that the resistance to imatinib may be due to the additional cytogenetic abnormalities, including der(17)t(1;17)(q25;p13), but not to BCR/ABL mutations.     

BCR/ABL amplification in chronic myelocytic leukemia blast crisis following imatinib mesylate administration

The onset of accelerated phase or blast crisis of chronic myelocytic leukemia (CML) is usually associated with the acquisition of new chromosome abnormalities in addition to the t(9;22)(q34;q11) that is characteristic of the chronic phase CML. We describe the cytogenetic and molecular genetic findings in two cases of myelocytic blast crisis of CML, one occurring 6 months after commencing treatment with the ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI571, Glivec, or Gleevec) and the second treated with imatinib mesylate for established blast crisis. In both cases, multiple secondary cytogenetic abnormalities were observed at transformation, with homogeneously staining regions that were shown to contain BCR/ABL amplification by fluorescence in situ hybridization appearing after imatinib mesylate administration. BCR/ABL amplification is emerging as an important mechanism of acquired resistance to imatinib mesylate.     

Large‐scale study of clinical and biochemical characteristics of Chinese patients diagnosed with Krabbe disease

Krabbe disease (KD) is a rare disease caused by the deficiency of β‐galactocerebrosidase. This study investigated 22 unrelated Chinese patients, including their clinical presentations, plasma psychosine levels and β‐galactocerebrosidase gene mutations. We found the late‐onset form of KD present in 82% of the patients in our study, which was more prevalent than in patients from other populations. Plasma psychosine levels were elevated in KD, which were correlated with the severity of clinical presentations. Sanger sequencing identified 8 novel mutations, including 7 missense mutations, p.H253Y, p.S259L, p.P318L, p.F350V, p.T428A, p.L530P, p.G586D, and 1 splicing mutation, c.1251+1G>A. Quantitative real‐time polymerase chain reaction (PCR) and multiplex ligation‐dependent probe amplification identified a novel exon 12 and 14 deletion, separately. Next generation sequencing, applied at the final step, revealed 2 missense mutant alleles missed using Sanger sequencing. The most common mutation in Chinese population is p.P154H, which accounts for 20.5% of alleles. Consistent with the higher prevalence of the late‐onset form of KD, missense mutations predominated in our study, different with the common mutation types in Europe and Japan. This work was the first large‐scale study of Chinese KD patients describing their clinical, biochemical and genetic characteristics, which furthered our understanding of this classical neurological lysosomal storage disease.     

Functional impairment of two novel mutations detected in lipoprotein-associated phospholipase A2 (Lp-PLA2) deficiency patients

Plasma lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor (PAF) acetylhydrolase (PAF-AH), is a member of the serine-dependent class of A2 phospholipases that hydrolyze sn2-ester bonds of fragmented or oxidized phospholipids at sites where atherosclerotic plaques are forming. Most circulating Lp-PLA2 is bound to low-density lipoprotein (LDL) particles in plasma and the rest to high-density lipoprotein (HDL). Deficiency of Lp-PLA2 is a predisposing factor for cardiovascular diseases in the Japanese population. We describe here two novel mutations of the gene encoding Lp-PLA2, InsA191 and I317N in Japanese subjects. The first patient, with partial Lp-PLA2 deficiency, was heterozygous for the InsA191 mutation; macrophages from this patient secreted only half the normal amount of Lp-PLA2 in vitro. The other patient, who showed complete Lp-PLA2 deficiency, was a compound heterozygote for the novel I317N mutation and a common V279F mutation; macrophages from that patient failed to secrete any Lp-PLA2. Measurement of Lp-PLA2 mass, activity and Western blotting verified impaired production and secretion of the enzyme after transfection of mutant construct into COS-7 cells. These results indicated that both novel mutants, InsA191 and I317N, impair function of the Lp-PLA2 gene.