亲子鉴定中STR基因座D2S1338突变分析与解决对策

目的： 研究短串联重复序列(short tandem repeats,STR)基因座分型方法在亲子鉴定中的应用。方法：提取基因组DNA,应用IdentifilerTM 15+1和AGCU 21+1二个系统的STR基因座荧光标记试剂盒,对36个常染色体STR基因座进行检测。结果：在IdentifilerTM 15+1系统检验过程中,15个被检基因座除D2S1338基因座的分型结果不符合遗传规律,其余均符合遗传规律。应用AGCU 21+1 STR基因座荧光标记试剂盒补充检测,所检测的21个常染色体STR基因座全部符合遗传规律。结论：当D2S1338基因座发生了突变,应用AGCU 21+1 STR基因座荧光标记试剂盒进行补充检测,还可作为STR基因座发生突变的补救手段。     

聚合酶链式反应-变性高效液相色谱技术在唐氏综合征筛查中的应用

目的:探讨聚合酶链式反应-变性高效液相色谱技术(polymerase chain reaction-denaturing high performance liquidchromatography,PCR-DHPLC)在唐氏综合征筛查中的应用。方法:针对21号染色体上的基因座D21S1409、D21S1422、D21S1435和GATA163G03设计引物,PCR扩增唐氏综合征患儿样本和正常样本,DHPLC仪检测;同步进行外周血染色体核型分析,并以核型分析为金标准,评价PCR-DHPLC技术的准确性。结果:柱温50℃时,唐氏综合征患儿样本DHPLC检测结果呈现特异唐氏综合征峰。结论:PCR-DHPLC方法具有较高的敏感性和特异性,可用于唐氏综合征的高通量快速筛查。     

Application of PCR-DHPLC technology in Down's syndrome screening

目的:探讨聚合酶链式反应-变性高效液相色谱技术(polymerase chain reaction-denaturing high performance liquid chromatography,PCR-DHPLC)在唐氏综合征筛查中的应用.方法:针对21号染色体上的基因座D21S1409、D21S1422、D21S1435和GATA163G03设计引物,PCR扩增唐氏综合征患儿样本和正常样本,DHPLC仪检测；同步进行外周血染色体核型分析,并以核型分析为金标准,评价PCR-DHPLC技术的准确性.结果:柱温50℃时,唐氏综合征患儿样本DHPLC检测结果呈现特异唐氏综合征峰.结论:PCR-DHPLC方法具有较高的敏感性和特异性,可用于唐氏综合征的高通量快速筛查.     

亲子鉴定中Identifiler~（TM）系统15个STR基因座突变分析

目的：观察和分析Identifiler~（TM）系统15个短串联重复序列（short tandem repeat,STR）基因座在亲子鉴定中的突变现象。方法：应用Identifiler~（TM）荧光标记复合扩增试剂盒检测710例亲子鉴定案,对其中发现突变基因座的案件加用STRtyper荧光标记复合扩增试剂盒进行等位基因检测,或6个mini Y-STR基因座检测。结果：在认定亲子关系的615例中,Identifiler~（TM）荧光标记复合扩增试剂盒中的15个基因座确定7例突变,其中vWA基因座2例,D13S17、FGA、D18S51、D21S11、D19S433基因座各1例;一步突变的6例,二步突变的1例。其突变均来自父亲,且年龄均在35岁以上。结论：在亲子鉴定中用Identifiler~（TM）荧光标记复合扩增试剂盒检测到1～2个基因座不符合遗传规律时,有必要增加突变率低、稳定性好的STR基因座进行复核并排除近亲关系。     

肝细胞癌8号染色体微卫星变异的研究(英文)

目的探讨肝细胞癌(HCC)8号染色体微卫星变异的特点及其与临床病理的相关性。方法采用 MegaBACE 500型自动化 DNA 分析系统,对56例 HCC 中8号染色体上10个微卫星的杂合性缺失(LOH)、微卫旱不稳定性(MSI)和等位基因失衡(AI)3种变异特征进行检测。结果 56例 HCC 在8号染色体上10个基因座发生 LOH 的总频率为66.1%(37/56),MSI 的频率为12.5%(7/56),AI 的频率为19.6%(11/56)。LOH 以 D8S261最高为53.5%(23/43),其次为 D8S1721(52.5%)和 D8S1771(52.5%)。D8S277基因座,血清HBsAg 阳性患者的 LOH 频率显著高于 HBsAg 阴性者(P<0.01),D8S261、D8S298和 D8S1733基因座,血清 HBsAg 阴性患者的 LOH频率显著高于 HBsAg 阳性者(P<0.01);D8S298和 D8S1771基因座.肿瘤直径>3cm 的 LOH 率明显高于≤3cm 组(P<0.05和 P<0.01);在 D8S1721基因座,无包膜或包膜不完整的肿瘤的 LOH 显著高于包膜完整的肿瘤(P<0.01);D8S298和 D8S1771基因座,肝内转移者的 LOH 明显高于无肝内转移者(P<0.05)。MSI 和 AI 与 HCC 临床病理学特点无明显相关性。结论 HCC 的8号染色体上存在广泛的微卫星变异,其中以代表肿瘤抑制基因路径的 LOH 方式在 HCC 的发生和发展过程中起重要作用,代表错配修复基因路径的 MSI 的作用次之。特定基因座的 LOH 与临床和病理学参数有一定的相关性。     

肝细胞癌8号染色体微卫星变异的研究

目的　　探讨肝细胞癌(HCC)8号染色体微卫星变异的特点及其与临床病理的相关性。方法采用MegaBACE　500型自动化DNA分析系统,对56例HCC中8号染色体上10个微卫星的杂合性缺失(LOH)、微卫星不稳定性(MSI)和等位基因失衡(AI)3种变异特征进行检测。结果　　56 例HCC 在8 号染色体上10 个基因座发生LOH 的总频率为66.1%(37 /56),MSI 的频率为12.5%(7 /56),AI的频率为19.6%(11/56)。LOH以D8S261最高为53.5%(23/43),其次为D8S1721(52.5%)和D8S1771(52.5%)。D8S277基因座,血清HBsAg阳性患者的LOH频率显著高于HBsAg阴性者(P<0.01),D8S261、D8S298和D8S1733基因座,血清HBsAg阴性患者的LOH频率显著高于HBsAg阳性者(P<0.01);D8S298和D8S1771基因座,肿瘤直径>3cm的LOH率明显高于￣<3cm组(P<0.05和P<0.01);在D8S1721基因座,无包膜或包膜不完整的肿瘤的LOH显著高于包膜完整的肿瘤(P<0.01);D8S298和D8S1771基因座,肝内转移者的LOH明显高于无肝内转移者(P<0.05)。MSI和AI与HCC临床病理学特点无明显相关性。结论　　HCC的8号染色体上存在广泛的微卫星变异,其中以代表肿瘤抑制基因路径的LOH方式在HCC的发生和发展过程中起重要作用,代表错配修复基因路径的MSI的作用次之。特定基因座的LOH与临床和病理学参数有一定的相关性。     

短串联重复序列分型鉴定肝癌肝移植术后超晚期复发肿瘤起源的可行性研究

目的 分析短串联重复序列（short tandem repeat,STR）分型鉴定肝癌肝移植术后超晚期复发肿瘤起源的可行性。方法 回顾性收集本院1例肝癌肝移植术后发生超晚期肿瘤复发患者的原发灶和复发灶样本,采用PCR技术体外扩增15个STR基因座片段,通过分析病灶STR位点一致性进行鉴定。结果 STR基因座的检测和对比结果显示,复发灶中的15个STR位点（D3S1358、vWA、D8S1179、D21S11、D18S51、D2S441、D19S433、TH01、FGA、D22S1045、D5S818、D13S317、D10S1248、D1S1656、D12S391）与原发灶相同,似然率为2.28×1018,因此排除了复发灶为供肝新生肿瘤的可能性,证实其为原发灶起源的复发肿瘤。结论 STR分型可以有效地对肝癌肝移植术后超晚期复发肿瘤的起源进行鉴定。     

肺癌组织中STR基因座变异规律的研究

背景与目的:在DNA水平进行个体同一认定及亲缘鉴定,是目前获得准确结论的最直接的方法,而短串联重复序列(short tandem repeat,STR)复合扩增荧光检测技术是目前应用最广泛的DNA技术.但已有研究表明STR基因座在肿瘤组织中会发生明显高于正常组织或血液的变异.该研究的目的为探索20个常染色体STR基因座及Amel基因座在人肺癌组织中的变异规律,为肺癌组织的个人识别及亲缘鉴定提供依据.方法:收集75例人肺癌组织和相对应的癌旁正常组织,采用组织DNA提取试剂盒提取DNA,MicroreaderTM 21 Direct ID System试剂盒进行PCR扩增,采用3130序列分析仪进行毛细管电泳,用Gene Mapper ID V3.2基因分析软件进行基因分型.结果:75例癌组织中有24例发生了STR位点的变异(32%),在21个常用STR基因座上共检出55次变异,其中等位基因增加10次,杂合性等位基因完全丢失10次,部分性丢失35次.D2S441、Penta E基因座未检出变异.结合目前实验结果与肺癌患者的临床资料分析,发现STR位点的变异与肺癌的分型、患者性别无联系(P>0.05),与肺癌的分期及患者年龄正相关,各组间比较差异有统计学意义(P<0.05).结论:肺癌组织中STR基因座较常发生变异,并且变异更可能在年龄大、恶性程度高的肺癌中发生;该实验检出D2S441、Penta E基因座无变异,在今后的研究中可加大样本量,进一步验证可否将其纳入稳定的STR基因座,为肿瘤组织等特殊检材的鉴定提供依据.     

胃癌及癌前病变组织染色体7q31.1杂合性缺失的意义

目的 检测胃癌及不典型增生细胞染色体7q31.1区域的杂合性缺失（LOH）,绘制胃癌及癌前病变7q31.1区域等位基因缺失图谱,确定其常见最小缺失区域,探索胃黏膜上皮癌变过程不同阶段的分子遗传学改变。方法 在胃癌及胃黏膜组织石蜡切片上行显微切割,获得胃癌及胃黏膜上皮不典型增生细胞。用高密度微卫星标志结合PCR技术检测胃癌及癌前病变细胞染色体7q31.1杂合性缺失,绘制胃癌及癌前病变染色体7q31.1等位基因的缺失图谱。结果 发现胃癌染色体7q31.1至少有一个位点存在杂合性缺失的21例,占70.0%（21/30）;D7S2459、D7S523、D7S2502、D7S486、D7S480、D7S650、D7S2486各位点杂合性缺失频率分别为10.0%、6.7%、23.3%、43.3%、26.7%、26.7%、20.0%;缺失图谱分析显示胃癌常见最小缺失区域位于D7S2502~D7S480之间。在胃黏膜不典型增生组织中,至少一个位点等位基因缺失的11例,占36.7%（11/30）,其中缺失频率最高的微卫星位点是D7S480为23.3%(7/30);不同程度胃黏膜不典型增生患者中染色体7q31.1 LOH阳性率比较差别有统计学意义（P<0.01）。结论 胃癌染色体7q31.1常见最小缺失区域在D7S2502~D7S480之间,在D7S486附近可能存在与胃癌相关的抑癌基因。在胃黏膜癌前病变阶段（不典型增生）可检测出染色体7q31.1区域的杂合性缺失,7q31.1 LOH可能是胃癌发生极早期的分子事件之一。     

甲状腺乳头状癌组织常染色体和性染色体STR基因座变异分析

目的：探讨甲状腺乳头状癌组织中短串联重复序列（STR）基因座变异规律以及显微切割技术在肿瘤组织鉴定中的应用,为甲状腺乳头状癌组织的个体识别及亲缘关系鉴定提供依据。方法：收集43例人甲状腺乳头状癌和癌旁正常组织经激光显微切割获取肿瘤细胞和间质细胞,采用Chelex-100法提取DNA,分别采用Goldeneye^® DNA 22NC、Goldeneye^® DNA 27YB和Goldeneye^® DNA 17X试剂盒进行常染色体和性染色体STR基因座PCR扩增,ABI 3500遗传分析仪检测进行STR分型。结果：甲状腺乳头状癌组织肿瘤间质细胞与对应癌旁组织的STR分型一致。43例癌组织的肿瘤细胞有9例STR基因座发生了变异,在常染色体和X染色体联合检测的37个STR基因座共检出11次变异（常染色体变异7次,X染色体变异4次）,其中等位基因增加3次,部分杂合性丢失6次,出现新等位基因2次,并且同一甲状腺乳头状癌肿瘤细胞可同时发生多种变异。Y染色体STR基因座没有检出变异。结合实验结果与患者临床资料分析,STR基因座的变异与甲状腺乳头状癌患者的临床分期、性别无明显相关关系,与患者的年龄正相关,差异具有统计学意义（P < 0.05）。结论：甲状腺恶性肿瘤组织常染色体和X染色体STR基因座存在变异,而且变异更可能发生在年龄大的甲状腺乳头状癌组织中,在司法鉴定中进行STR分型时应谨慎判型。显微切割技术可准确分离间质细胞,是解决此类恶性肿瘤组织检材涉及的案件进行身源鉴定的有效手段。     

儿童急性淋巴细胞白血病遗传学特征

目的：分析儿童急性淋巴细胞白血病（ALL）遗传学变化的特点.方法：以94例初发ALL患儿为研究对象,采用染色体核型分析及多重PCR技术检测患者骨髓白血病细胞的细胞遗传学和分子生物学变化.结果：63例患儿做了核型分析,有分裂象者55例,其中异常者12例（19.0%）;无分裂象者8例（12.7%）.对93例患儿进行了多重PCR检测,其中包括53例染色体有分裂象患儿和8例无分裂象的患儿,41例正常染色体核型患儿中异常者为17例（占41.5%）,12例染色体核型异常者中融合基因异常者4例（33.3%）,无分裂象患者异常2例,无分裂象检出率与有分裂象者核型分析异常检出率差异无统计学意义;93例患儿中融合基因检出率为34.4%（32/93）,其中TEL/AML1融合基因阳性20例（21.51%）,MLL重排3例（3.23%）,BCR/ABL（p190）融合基因阳性3例（3.23%）,E2A/PBX阳性5例（5.38%）,HOX11阳性1例.94例患儿通过两种检测方法共检出细胞遗传学异常46例（48.94%）.结论：儿童ALL可出现细胞遗传学改变.对于有足够分裂象的患者,常规染色体核型分析仍是可靠的方法.对分裂象质量低或无分裂象的儿童ALL患者,多重PCR检测异常克隆能提高细胞遗传学异常检出率.将染色体核型分析及多重PCR结合起来可提高细胞遗传学异常检出率.     

Frequent allelic loss/imbalance on the long arm of chromosome 21 in oral cancer: evidence for three discrete tumor suppressor gene loci.

Frequent allelic imbalances including loss of heterozygosity (LOH) and microsatellite instability (MI) on the long arm of chromosome 21 (21q) have been found in several types of human cancer. This study was designed to identify tumor suppressor locus (or loci) associated with oral squamous cell carcinoma (SCC) on 21q. Among 38 patients with oral SCC tested, 15 (44%) of 34 informative cases showed LOH at one or more loci. Deletion mapping of these 15 tumors revealed three discrete commonly deleted regions on the chromosome arm. A minimal region with frequent LOH was found at the marker D21S236 mapped on 21q11.1. Another region of frequent deletion was identified between markers D21S11 and D21S1436 on 21q21, and a further commonly deleted region was found at D21S1254 on 21q22.1. In addition, we have detected MI on the chromosome arm in our oral SCC samples with significant correlation with tumor stage. Thus, our results strongly suggest that allelic imbalances on 21q may be involved in the development of oral SCC; and that at least three different putative tumor suppressor genes contributing to the pathogenesis of this disease are present on 21q.     

Chromosome 5 allele loss in familial and sporadic colorectal adenomas

DNA extracted from familial and sporadic colorectal neoplasms was compared with constitutional DNA using a range of hypervariable locus specific probes to assess the extent of allele loss during conversion to malignancy. Chromosome 5 allele loss was observed in 23% of carcinoma samples, as previously found by others. However, we have been able to show for the first time loss of the D5S43 locus on chromosome 5 in adenomas from three patients, two of whom had the precancerous condition adenomatous polyposis coli (APC). These results suggest significant genetic changes involving chromosome 5 are occurring in benign adenomas. Probes for chromosome 1 (loci D1S7 and D1S8) and for chromosome 7 (loci D7S21 and D7S22) revealed no notable alterations in the adenoma samples. Complete loss of alleles for loci on chromosome 7 was not observed in carcinomas but reduced intensity of one parental allele was found in three specimens one of which was known to have multiple copies of this chromosome. Results using probes for chromosome 1 suggest that deletion of the D1S7 or D1S8 loci is not a common event in colorectal carcinogenesis. Loss of chromosome 5 alleles in adenomas from APC patients provides evidence in support of Knudson's hypothesis.     

Deletion of chromosome 11p15, p12, q22, q23-24 loci in human prostate cancer

Loss of heterozygosity (LOH) on chromosome 11 is frequently altered in various epithelial cancers. The present study was designed to investigate LOH on chromosome 11 in microdissected samples of normal prostatic epithelium and invasive carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 38 prostate cancers, amplified by polymerase chain reaction PCR and analyzed for LOH on chromosome 11 using 9 different polymorphic DNA markers (D11S1307, D11S989, D11S1313, D11S898, D11S940, D11S1818, D11S924, D11S1336 and D11S912). LOH on chromosome 11 was identified in 30 of 38 cases (78%) with at least one marker. Four distinct regions of loss detected were: 1) at 11p15, at loci between D11S1307 and D11S989; 2) at 11p12, on locus D11S131 (11p12); 3) at 11q22, on loci D11S898, D11S940 and D11S1818; and 4) at 11q23-24, on loci between D11S1336 and D11S912. We found 25% of the tumors with LOH at 11p15; 39% had LOH at 11p12; 66% had LOH at 11q22; and 47% had LOH at 11q23-24. These deletions at 11p15, 11p12, 11q22 and 11q23-24 loci were not related to the stage or grade of the tumor. Int. J. Cancer 72:283–288, 1997. © 1997 Wiley-Liss, Inc.     

Molecular abnormalities of chromosome-19 in malignant gliomas - preferential involvement of the 19q13.2-q13.4 region

A deletion mapping analysis of chromosome 19 was performed on a series of 101 samples derived from malignant gliomas. A total of 35 tumors displayed different deletions for the loci studied (D19S21, D19S11, D19S74, D19S7, D19S8, CKM, and D19S22). In most instances, losses involving the long arm markers of chromosome 19 were observed, and only four samples were characterized by losses on the short arm. No tumor was found displaying loss of both short and long arm markers. The higher frequency of deletions was detected in tumors with a major oligodendroglial component: 76% of samples included in this group displayed losses at 19q. Among the astrocytic tumors, the frequency of 19q alterations varied as follows: 11% in pilocytic astrocytomas, 17% in astrocytomas grade II, 10% in anaplastic astrocytomas and 21% in glioblastoma multiforme. No ependymoma was found displaying allele loss on chromosome 19. The common region of overlap for the 19q deletions observed involves primarily the distal portion of the long arm, 19q13.2-q13.4. In agreement with previous reports, these data suggest the non-random involvement of a tumor suppressor gene located at 19q13 in the genesis or progression of malignant gliomas.     

Prognostic significance of karyotype analysis in children with acute lymphoblastic leukemia.

Chromosome studies, using bone marrow samples of 26 pretreated children (below 15 years of age) with Acute Lymphoblastic Leukemia were carried out to explore the potentialities of applying chromosomal findings as a prognostic indicator in these patients. Abnormal karyotype was identified in 15 patients (57.6 per cent). The chromosomes frequently involved in non-random numerical abnormalities were Nos. 8, 18 and 21. Structural chromosome changes observed consisted of deletion 6q- and translocation t (4;11). After karyotype analysis, patients were grouped into subsets on the basis of the karyotype pattern observed. They were followed up to evaluate their prognosis and survival period. Patients showing hyperdiploid clone with greater than 51 chromosomes had the best prognosis. Patients with normal karyotype and patients with deletion of the long arm of chromosome 6 showed intermediate prognosis whereas patients showing t (4;11), trisomy 8, trisomy 18, trisomy 21, and hypodiploid karyotype were associated with worst prognosis. Thus, karyotype analysis before treatment helps to classify ALL patients as poor, intermediate and good prognosis groups and on this basis therapy can be designed accordingly.     

10号染色体可能含有多个与胶质母细胞瘤相关的肿瘤抑制基因

目的 寻找胶质母细胞瘤（GBM）10号染色体上可能存在肿瘤抑制基因的杂合性丢失（LOH）区域,为发现和定位肿瘤抑制基因（TSG）提供线索和依据。方法 应用聚合酶链反应（PCR）方法,采用荧光标记的引物和先进的377型DNA序列自动分析仪,分析了21例GBM10号染色体上20个微卫星多态性标记的LOH。结果 在85．7％（18／21例）GBM的10号染色体上观察到LOH,在57．7％（162／281）可提供信息位点上存在LOH。10q的LOH率高于10p,分别是81．0％（17／21）、66．7％（14／21）。在下列位点或区域检测到较高的LOH率（＞60％）：10q22.3-23.3上的D10s185-D10s192间区域,10p14-15.1上的D10s591-D10s249间区域,10q24.2-26.3上的D10s1693-D10s212间区域,10p12.2-14上的D10s547位点,10q21.3上的D10537位点。结论 10号染色体可能在GBM的分子水平发病机制中发挥着重要作用,它上面的多个染色体区域可能存在与GBM相关的多个TSG。