Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice

The role of the Notch signaling pathway in tumor development is complex, with Notch1 functioning either as an oncogene or as a tumor suppressor in a context-dependent manner. To further define the role of Notch1 in tumor development, we systematically surveyed for tumor suppressor activity of Notch1 in vivo. We combined the previously described Notch1 intramembrane proteolysis-Cre (Nip1::Cre) allele with a floxed Notch1 allele to create a mouse model for sporadic, low-frequency loss of Notch1 heterozygosity. Through this approach, we determined the cell types most affected by Notch1 loss. We report that the loss of Notch1 caused widespread vascular tumors and organism lethality secondary to massive hemorrhage. These findings reflected a cell-autonomous role for Notch1 in suppressing neoplasia in the vascular system and provide a model by which to explore the mechanism of neoplastic transformation of endothelial cells. Importantly, these results raise concerns regarding the safety of chronic application of drugs targeting the Notch pathway, specifically those targeting Notch1, because of mechanism-based toxicity in the endothelium. Our strategy also can be broadly applied to induce sporadic in vivo loss of heterozygosity of any conditional alleles in progenitors that experience Notch1 activation.     

胃癌组织脆性组氨酸三联体基因杂合性丢失的临床意义

目的检测脆性组氨酸三联体(FHIT)基因在胃癌(GC)、不典型增生(DP)和肠上皮化生(IM)组织中的杂合性丢失(loss of heterozygosity,LOH),研究LOH在GC发生中的作用。方法采用PCR方法检测GC、DP和IM组织和正常对照胃黏膜组织中FHIT基因多态性位点D3S1234和D3S1300中的LOH。结果 GC组、DP组和IM组在D3S1234位点的LOH发生率分别为33.0%,29.0%和11.0%;在D3S1300位点的LOH发生率分别为34.0%,33.0%和8.0%。GC组和DP组在D3S1234和D3S1300位点的LOH发生率显著高于IM组,GC组与DP组间差异无统计学意义。结论 FHIT基因的LOH可能是GC形成过程中的早期事件,能用于GC高危人群筛查和早期诊断的研究。     

Multiplex loss of heterozygosity analysis by using single or very few cells

Loss of heterozygosity (LOH) is an indication of tumor suppressor gene inactivation. However, loss of heterozygosity analysis has been limited to either a small scale or to very few genetic markers. To significantly increase the scale of study and to include a large number of markers in the analysis, experimental conditions were established for using single cells or single cell equivalent with 10 markers typed simultaneously. Under these conditions, the allele amplification failure rate was 3.7% when single tissue cultured human cells were used. When 30 cells from a 5-μm paraffin-archived breast tumor tissue section were used, the failure rates were 0% for four of the five heterozygous loci and 10% for the fifth. Small amplification failure rates (6.1% and 6.7% on average) were observed when 5 or 10 cells from paraffin-archived breast tissue were used. These results indicate that with polymerase chain reaction (PCR) primers of high quality, it is possible to obtain reliable results by using single cells from fresh tissue or very few cells from paraffin-archived specimens. The results also show the importance of including replicates, using primers of high quality, and optimizing PCR conditions when a limited amount of material is used for the assay. The feasibility of LOH analysis with very few paraffin-embedded breast cancer tissues was demonstrated.     

Identification of loss of heterozygosity on circulating free DNA in peripheral blood of prostate cancer patients: potential and technical improvements

BACKGROUND: Accurate identification of loss of heterozygosity (LOH) on circulating free DNA is often restricted by technical limitations such as poor quality and quantity of tumor-specific DNA and contamination by normal DNA. However, plasma DNA may harbor tumor-specific genetic alterations and could therefore be an interesting target for noninvasive examinations of tumor DNA. METHODS: By PCR-based fluorescence microsatellite analysis using 12 polymorphic markers, we investigated LOH on cell-free DNA in blood plasma from 59 patients with localized prostate cancer (PCa) and 12 with metastatic disease (MPCa). In addition, plasma DNA from 21 PCa patients was fractionated into high- and low-molecular-weight DNA by 2 different column systems. To avoid appearance of artificial allelic loss and stabilize the amplification, TMAC (tetramethylammonium chloride) was added to each PCR. RESULTS: Overall incidences of LOH at all markers analyzed were 10% in PCa and 12% in MPCa samples. Highest frequencies were found at markers D11S898 (28%) in PCa and D6S1631 (27%) in MPCa. Statistical evaluation showed significant associations between LOH and increasing Gleason scores for the marker combinations D6S1631*D8S286*D9S171 (P = 0.03) and D8S286*D9S171 (P = 0.05). Fractionation of plasma DNA resulted in a higher overall LOH frequency in the low-molecular-weight DNA fraction (23%) compared with the high-molecular-weight DNA (7%). CONCLUSIONS: LOH analysis of circulating DNA can provide tumor-specific genetic information on PCa patients. Fractionation of plasma DNA and addition of TMAC improved LOH detection and general assay performance.     

Pleomorphic carcinoma of the lung associated with loss of heterozygosity of p53 gene

We report a case with pleomorphic carcinoma of the lung in a 70-year-old man. Pleomorphic carcinoma is characterized by a heterogenous composition that includes epithelial and mesechymal malignancies. In the present case, the tumor was composed of a mixture of unequivocal squamous cell carcinoma and spindle cell components resembling sarcomatous overgrowth. The spindle component did not include a heterologous mesenchymal element characterized by overt differentiation for bone, cartilage, neuron or muscle tissue. To evaluate a state of differentiation of the spindle cell component, we immunohistochemically examined expression of the antigens including vimentin, cytokeratin, sarcomeric actin, alpha-smooth muscle actin, S-100 protein, CD34, Factor VIII, and CD68. The results showed sole expression of vimentin in the spindle cell component, suggesting an immature state of the mesenchymal lineage. Furthermore, the spindle cell component of this case was genetically characterized by loss of heterozygosity (LOH) at a codon 234 of exon 7 of the p53 gene. This mutation causes an amino-acid replacement (Tyr to Cys), which was previously proven to attenuate p53 function. The present case may suggest a relation between somatic alteration of the p53 gene and histogenesis of pleomorphic carcinoma.     

Rapid generation of detailed loss of heterozygosity profiles for routine diagnosis of gliomas.

Aberrations in the genomes of gliomas seem to correlate well with clinical parameters. Pertinent studies, however, rely on highly sophisticated methods, they require large amounts of high-quality sample material and/or they demand profound analytical expertise. Consequently, molecular tumour analysis has not yet been widely implemented in routine laboratory applications. We have developed an easy-to-perform approach for the rapid derivation of a detailed loss-of-heterozygosity profile from individual gliomas. DNA of PCR quality is extracted in a one-step procedure from routinely obtained material. A microsatellite-based marker set is used to detect the deletion status of genomic regions (i) with established diagnostic relevance, (ii) recurrently found deleted in gliomas, or (iii) generally associated with tumour suppressor activity. The complete profile comprises 25 regions and is generated from 64 markers multiplexed into 18 reactions. Illustratively, we present findings from an anaplastic oligodendroglioma; the molecular data for this tumour allow refined diagnostic and prognostic statements that could not be derived from histology alone. Our approach should prove useful in the routine diagnosis of gliomas. Simultaneously, research data for many highly relevant regions are generated in a comparatively simple and inexpensive way.     

髓母细胞瘤的分子遗传学研究

髓母细胞瘤是儿童最常见的脑肿瘤.占中枢神经系统肿瘤的10%～20%.在过去的几年中,人们对该肿瘤进行了大量的研究工作并且发现了一些常见的分子遗传学改变,包括17p、8p、6q、10q、11、16号染色体的丢失和17q、7q、14q的获得以及某些与患者预后相关的基因,如c-myc、erbB-2等.随着人们对髓母细胞瘤相关基因功能的不断探索,该肿瘤的发病机制将会更加明确.其临床治疗也会得到进一步的改善.本文对髓母细胞瘤近年来分子遗传学研究结果和不同亚型髓母细胞瘤的遗传学特点及与该亚型肿瘤患者预后的关系作一介绍.     

Mass spectrometry-based loss of heterozygosity analysis of single-nucleotide polymorphism loci in paraffin embedded tumors using the MassEXTEND assay: single-nucleotide polymorphism loss of heterozygosity analysis of the protein tyrosine phosphatase receptor type J in familial colorectal cancer

As the number of identified single-nucleotide polymorphisms (SNPs) increases, high-throughput methods are required to characterize the informative loci in large patient series. We investigated the feasibility of MassEXTEND LOH analysis using Sequenom's MassArray RT software, a mass spectrometry method, as an alternative to determine loss of heterozygosity (LOH). For this purpose, we studied the c.827A>C SNP (1176A>C p.Gln276Pro) in protein tyrosine phosphatase receptor type-J (PTPRJ), which is frequently deleted in human cancers. In sporadic colorectal cancer (CRC), c.827A>C showed allele-specific LOH of the c.827A allele, which is important because LOH of PTPRJ may be an early event during sporadic CRC. To elucidate the impact of this low-penetrance gene on familial CRC, we studied c.827A>C in 222 familial CRC cases and 156 controls. In 6.2% of the A/C genotyped CRC samples, LOH of c.827A was observed with MassEXTEND LOH analysis and confirmed by conventional sequencing. Furthermore, a case with LOH of c.827A showed no LOH in 22 synchronously detected adenomas, including one with malignant transformation. The importance of the PTPRJ- c.827A>C SNP appears to be limited in familial CRC. We conclude that MassEXTEND LOH analysis (using Sequenom's MassARRAY RT software) is a sensitive, high-throughput, and cost-effective method to screen SNP loci for LOH in formalin-fixed paraffin-embedded tissue.     

Molecular diagnostics for hereditary hearing loss in children

Introduction: Hearing loss (HL) is the most common birth defect in industrialized countries with far-reaching social, psychological and cognitive implications. It is an extremely heterogeneous disease, complicating molecular testing. The introduction of next-generation sequencing (NGS) has resulted in great progress in diagnostics allowing to study all known HL genes in a single assay. The diagnostic yield is currently still limited, but has the potential to increase substantially.

Areas covered: In this review the utility of NGS and the problems for comprehensive molecular testing for HL are evaluated and discussed.

Expert commentary: Different publications have proven the appropriateness of NGS for molecular testing of heterogeneous diseases such as HL. However, several problems still exist, such as pseudogenic background of some genes and problematic copy number variant analysis on targeted NGS data. Another main challenge for the future will be the establishment of population specific mutation-spectra to achieve accurate personalized comprehensive molecular testing for HL.     

1种用于分析疾病相关ABO抗原丢失的染色体杂合性缺失方法

目的 建立1种9号染色体杂合性缺失(LOH)的分析方法,以解释因ABO基因附近染色体的LOH导致的疾病相关ABO抗原丢失现象.方法 在9号染色体长臂和短臂上共选取13个微卫星位点,其中10个位点(D9S1677、D9S289、D9S1776、D9S1682、D9S290、D9S164、D9S1818、D9S1826、D9S158、D9S1838)位于ABO血型基因近端,平均间距5.52 cM;另3个位点(D9S1858、D9S171、D9S1843)位于ABO基因远端,平均间距39.3 cM.分别设计合成FAM和HEX荧光标记引物,优化PCR扩增后,以聚丙烯酰胺凝胶毛细管电泳,通过GeneMapper软件分析微卫星不稳定性,观察染色体LOH发生情况.结果 13个微卫星位点均得到了较好的PCR扩增效果,PCR产物长度在75～273 bp之间,各位点均有较高的杂合度,适合于微卫星片段长度分析.13个位点可在2个泳道中进行双荧光同步分析,各位点不同等位基因间互不干扰.分别对1例正常血型标本和2例血液病相关的ABO抗原变异标本(包括口腔黏膜DNA和外周血DNA)进行微卫星分析,正常血型标本的口腔DNA和外周血DNA未发现染色体LOH现象.而2例ABO抗原变异标本,与口腔黏膜DNA相比,外周血DNA均存在不同程度的LOH现象,表明ABO抗原丢失可能源于血液系统疾病相关的9号染色体LOH现象.结论 建立了1种基于微卫星不稳定分析的9号染色体LOH方法,可用于血液系统疾病相关的ABO抗原丢失标本分子机制的研究.     

Molecular and biochemical studies of acute intermittent porphyria in 196 patients and their families

BACKGROUND: Acute intermittent porphyria (AIP) is a metabolic disease with clinical manifestations that mimic other abdominal, neurologic, or mental crises. We studied the diagnostic accuracy of current laboratory tests during an acute attack and in remission. METHODS: Since 1966, we have studied all known Finnish AIP patients (n = 196) and their families (n = 45) and identified the porphobilinogen deaminase (PBGD) mutation in each family. Diagnoses or exclusions of AIP were based on clinical data (including family history), biochemical tests, and in 239 cases, mutation testing. We retrospectively evaluated the diagnostic accuracy of erythrocyte PBGD activity, urinary excretion of porphobilinogen (PBG) and delta-aminolevulinic acid, and urinary and fecal excretion of porphyrins in these patients. RESULTS: Measurement of urinary PBG identified all 35 AIP patients studied during an acute attack. The mean excretion of PBG was 50-fold above the reference interval, although the intraindividual increases were modest (1.6- to 4.0-fold). In the mutation-screened population, urinary PBG analysis identified only 85% of 81 AIP patients studied during remission, but by ROC curve analysis it was nonetheless the best of the biochemical tests. It was increased 相似文献   

散发性特发甲状旁腺机能减退患者体内存在自身抗体

目的 探讨散发性特发甲状旁腺机能减退症患者体内是否存在自身免疫性抗体.方法 对散发性特发甲状旁腺机能减退症患者26例、其直系亲属112例以及健康对照者60例,采用间接免疫荧光技术检测抗甲状旁腺自身免疫性抗体,同时检测血清钙、磷、镁、甲状旁腺激素含量.结果 散发性特发甲状旁腺机能减退症患者血清中抗甲状旁腺自身抗体阳性10例,阳性率为38%,明显高于直系亲属的10%(χ2=13.42,P＜0.01)和健康对照者的7%(χ2=13.46,P＜0.01).患者以女性为主(19/26),抗体阴性患者平均病程0.5～7年(中位数为2.2年),较抗体阳性患者平均病程0.5～4.5年(中位数为1.75)有延长的趋势;血清甲状旁腺激素水平在抗体阴性患者中为(7.06±2.06)ng/L,较抗体阳性患者的(8.86±2.86)ng/L有降低的趋势,差异无统计学意义(P=0.74).结论 散发性特发甲状旁腺机能减退症患者体内有抗甲状旁腺的自身免疫性血清学证据,抗甲状旁腺抗体的出现提示该病与自身免疫有关.     

Somatic mutation of MutYH in Tunisian patients with sporadic colorectal cancer

The MutYH gene is an adenine-specific DNA glycosylase that prevents G/T transversions. Germline mutation in this gene causes MYH-associated polyposis (MAP) that predispose to hereditary colorectal cancer (CRC). This study describes for the first time the association of the MutYH mutation with sporadic CRC. From the 48 Tunisian sporadic CRC cases analyzed, two patients showed somatic mutation of the MutYH gene. In addition, the two hotspot germline mutations MutYH Y165C and G382D seem to be infrequent in sporadic CRC.     

散发性乳腺癌中BRCAl基因甲基化状况及其与蛋白表达的关系

目的探讨散发性乳腺癌中BRCA l基因异常甲基化状况及其与蛋白表达的关系,进而研究散发性乳腺癌中BRCA l基因异常的表遗传学作用。方法用甲基特异性聚合酶链反应(PCR)和免疫组织化学(SP)法研究乳腺癌组织、癌旁正常组织中BRCA l基因启动子甲基化状况及蛋白表达情况。结果在52例散发性乳腺癌中,BRCA l甲基化率为29%,BRCA l蛋白表达下降率为33%;15例BRCA l发生甲基化的癌组织标本中BRCA l的表达下降率为87%,而37例BRCA l未发生甲基化的癌组织中BRCA l蛋白表达下降率11%。在肿瘤分级中,T3分级患者的肿瘤组织中BRCA l甲基化率(45%)及表达下降率(60%)均分别高于T1 T2分级患者肿瘤组织的BRCA l甲基化率(19%)和表达下降率(16%);有淋巴结转移患者的肿瘤组织中BRCA l甲基化率(46%)及蛋白表达下降率(50%)均分别高于无淋巴结转移的甲基化率(14%)和表达下降率(18%)。结论BRCA l基因表遗传学改变是其蛋白表达下降乃至失活的重要原因,并且与散发性乳腺癌的恶性进程和淋巴结转移密切相关。