Routine application of a novel MLPA-based first-line screening test uncovers clinically relevant copy number aberrations in haematological malignancies undetectable by conventional cytogenetics

Objective

The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis.

Methods

A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis.

Results

More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed.

Discussion

Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected.     

Use of amplified Mycobacterium tuberculosis direct test in respiratory samples from HIV-infected patients in Brazil

OBJECTIVE:

To compare the accuracy of the amplified Mycobacterium tuberculosis direct (AMTD) test with reference methods for the laboratory diagnosis of tuberculosis in HIV-infected patients.

METHODS:

This was a study of diagnostic accuracy comparing AMTD test results with those obtained by culture on Löwenstein-Jensen (LJ) medium and by the BACTEC Mycobacteria Growth Indicator Tube 960 (BACTEC MGIT 960) system in respiratory samples analyzed at the Bioassay and Bacteriology Laboratory of the Oswaldo Cruz Foundation Evandro Chagas Clinical Research Institute in the city of Rio de Janeiro, Brazil.

RESULTS:

We analyzed respiratory samples collected from 118 patients, of whom 88 (74.4%) were male. The mean age was 36.6 ± 10.6 years. Using the AMTD test, the BACTEC MGIT 960 system, and LJ culture, we identified M. tuberculosis complex in 31.0%, 29.7%, and 27.1% of the samples, respectively. In comparison with LJ culture, the AMTD test had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.5%, 89.4%, 75.7%, and 95.0%, respectively, for LJ culture, whereas, in comparison with the BACTEC MGIT 960 system, it showed values of 88.6%, 92.4%, 83.8%, and 94.8%, respectively.

CONCLUSIONS:

The AMTD test showed good sensitivity and specificity in the population studied, enabling the laboratory detection of M. tuberculosis complex in paucibacillary respiratory specimens.     

外周血游离DNA检测非小细胞肺癌患者EGFR基因突变研究进展

外周血游离DNA检测非小细胞肺癌人群表皮生长因子受体基因突变法因受到标本质量、采集运输分离条件、DNA提取方法、基因突变检测方法的多种限制,检测敏感性低,假阴性比例较高,但特异性较好。因此,外周血游离DNA的基因突变检测需针对特殊人群,优化采集运输分离步骤,并尝试使用较好的DNA提取（如改良酚一氯仿法、QIAmpCirculatingNucleicAcidKit）及基因检测方法（如蝎形探针扩增阻滞突变系统、高效液相色谱法、高分辨溶解曲线分析技术、突变一富集PCR等）。本文就外周血游离DNA表皮生长因子受体基因突变检测的现状及发展方向进行综述。     

MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy

Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60–65% of mutations, duplications for 5–10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009–2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45–54 and 3–21, whereas most duplications involved exons 3–18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots – different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers.     

Mechanical elasticity as a physical signature of conformational dynamics in a virus particle

In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications.     

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth.     

Atypical copy number abnormalities in 22q11.2 region: Report of three cases

The 22q11.2 Deletion Syndrome (22q11.2DS) is the most common microdeletion syndrome in humans, with a highly variable phenotype. This chromosomal region contains low copy repeat (LCR) sequences that mediate non-allelic homologous recombination which predispose to copy number abnormalities at this locus. This article describes three patients investigated for suspicion of 22q11.2DS presenting atypical copy number abnormalities overlapping or not with the common ～3 Mb deletion. They were investigated by G-banding karyotype, Multiplex-ligation dependent probe amplification (MLPA) and array Genomic Hibridization (aGH). Clinical and molecular data were compared with literature, in order to contribute to genotype–phenotype correlation. Atypical chromosomal abnormalities were detected: 3.6 Mb deletion at 22q11.21-q11.23 between LCRs B–F in patient 1 and approximately 1.5 Mb deletion at 22q11.21-q11.22 between LCRs D–E in patients 2 and 3. The breakpoints detected in patient 1 have not been previously described. These findings exemplify the complexity and genetic heterogeneity observed in 22q11.2 region and corroborates the idea that genetic modifiers contribute to the phenotypic variability observed in proximal and distal 22q11.2 deletion syndromes.     

MLPA技术在检测胰腺癌细胞系9p21大片段缺失中的应用

目的:评价MLPA技术检测染色体9p21区大片段缺失的有效性及判断缺失边界的准确性,并探讨染色体9p21区大片段缺失的意义.方法:利用MLPA技术检测胰腺癌细胞系PANC-1和BxPC3的9p21区缺失情况,并利用常规PCR实验对MLPA缺失边界进行验证.结果:MLPA检测发现PANC-1和BxPC3均存在累及MTAP、CDKN2A和CDKN2B等基因大片段缺失的情况.PCR实验证实MLPA对PANC-1端粒侧的缺失边界判断准确.PANC-1细胞系染色体9p21区大片段缺失的端粒侧缺失断点位于MTAP基因内.结论:MLPA技术是检测染色体9p21大片段缺失的有效方法,能准确判断大片段缺失边界范围,适合于aCGH检测后大样本筛查.PANC-1细胞系中染色体9p21区大片段缺失可能改变MTAP基因结构.     

The Dopaminergic Neuronal System Regulates the Inflammatory Status of Mouse Lacrimal Glands in Dry Eye Disease

PurposeComparison of the parasympathetic and sympathetic neurons, including the dopaminergic neural system, in dry eye (DE)–induced pathophysiology has not been elucidated well. This study investigated the presence of dopamine receptors (DRs) and their functional roles in the lacrimal glands (LGs) of DE-induced mice.MethodsAfter DE was induced in B6 mice for 2 weeks, the expression of tyrosine hydroxylase (TH), dopamine, and DRs (DR1, DR2, etc.) in the LGs and corneas were measured by quantitative RT-PCR, immunoblot, and ELISA. Using flow cytometry and ELISA, immune cell infiltration and inflammatory cytokine expression were determined in DE-induced LGs with or without DR blockers, SCH-23390 (DR1i), or melperone (DR2i). Corneal erosion scores were also investigated.ResultsThe mRNA and protein levels of TH significantly increased in DE-induced LGs. The dopamine concentration of LGs was 9.51 pmol in DE (versus naive: 1.39 pmol; P < 0.001). Both DR1 and DR2 mRNA expression were significantly enhanced in desiccating stress compared with those in naive (3.7- and 2.1-fold, P < 0.001). Interestingly, DR1 and DR2 immunostaining patterns stained independently in DE-induced LGs. CD3⁺ and CD19⁺ cell infiltration was significantly increased by DR2i (P < 0.001) but not by DR1i. Furthermore, IFN-γ, IL-17, and TNF-α were significantly upregulated by DR2i compared with the blow-only condition. The severity of corneal erosion and inflammation was also aggravated by DR2i.ConclusionsUpregulation of DR1 and DR2 was observed in DE-induced mouse LGs. As the inflammatory conditions are aggravated by the inhibition of DRs, especially DR2, their activity may be an important factor preserving ocular surface homeostasis.     

泪道探通术联合妥布霉素滴眼液治疗先天性泪囊炎

目的:探讨泪道探通术联合妥布霉素滴眼液治疗先天性泪囊炎的临床疗效。方法:选取2017-01/2019-06来我院就诊的先天性泪囊炎患儿228例250眼作为研究对象,年龄为6月龄~3岁。按年龄分为3组:6月龄~<1岁133例149眼,1~<2岁62例64眼,2~3岁33例37眼。三组均实施表面麻醉下泪道探通术联合术后妥布霉素滴眼液治疗。结果:先天性泪囊炎患儿中6月龄~<1岁治愈率为97.3%(145/149),1~<2岁治愈率为92.2%(59/64),2~3岁治愈率为83.8%(31/37),无不良事件发生。三组患儿治愈率比较,6月龄~<1岁患儿治愈率最高(P=0.009)。结论:泪道探通术治疗先天性泪囊炎疗效显著,术后联合妥布霉素滴眼液安全可靠。以6月龄~1岁为最佳治疗时机,其手术操作简单,并发症少,具有较高的临床价值。