p15、细胞周期蛋白D1和转移生长因子β1在非小细胞肺癌中的表达及其意义

一、材料与方法1.材料 :实验材料取自内蒙古医学院病理教研室在1996～ 1998年间的病理组织存档蜡块 ,共 16 5份。按照 1980年ＷＨＯ肺癌分类标准进行组织学分类 ,并均经病理证实。其中鳞癌 80例 ,腺癌 49例 ,不典型增生 19例 ,鳞状上皮化生17例 ;在 12 9例非小细胞肺癌中 ,同一病例手术切取标本存档蜡块不仅有肺癌组织蜡块也有淋巴结组织蜡块 ;其中淋巴结组织经ＨＥ染色证实有淋巴结转移的 35例 ,无淋巴结转移的 2 8例。其余 6 6例中 ,或同一病例手术切取标本存档蜡块仅有肺癌组织而无淋巴结组织 ,或同一病例标本存档蜡块中虽有淋巴结组织 ,…     

甲状腺多结节性增生的克隆性分析

目的分析甲状腺结节状增生、甲状腺多发性腺瘤样结节和甲状腺结节状增生背景上的腺瘤样增生结节的克隆起源,为甲状腺多结节性增生的临床诊断和治疗提供依据。方法采用激光捕获显微切割技术及基于HUMARA基因多态性克隆性分析检测5例甲状腺结节状增生、5例甲状腺腺瘤、12例甲状腺多发性腺瘤样结节、15例甲状腺结节状增生背景上的腺瘤样增生结节是否为肿瘤性增生。结果HUMARA基因杂合率为86.5%（32/37）。5例单纯结节状增生为多克隆,5例单发包膜完整的腺瘤中1例为HUMARA基因纯合子,其余4例为单克隆。12例多发性腺瘤样结节中2例为HUMARA纯合子,4例多发腺瘤样结节中有多克隆性结节（1/3,1/3,1/2,2/2）,其中4枚包膜包裹不完整,其余6例多枚结节均为单克隆且包膜完整。15例甲状腺结节状增生背景上的腺瘤样增生结节中2例为HUMARA纯合子,另13例结节状增生背景均为多克隆,其中8例结节为多克隆（3例无包膜,5例包膜不完整）,余5例为单克隆（包膜完整）。结论克隆性分析有助于甲状腺结节状增生和腺瘤的鉴别,而多发性腺瘤和结节状增生伴腺瘤形成的诊断还需要结合包膜的完整性做出判断。     

所谓肺硬化性血管瘤组织中两种主要细胞的雄激素受体、磷酸甘油酸酯激酶基因多态性分析

目的探讨所谓肺硬化性血管瘤（PSH）组织内的多角形细胞和表面立方细胞的克隆性组成及意义。方法选PSH患者手术切除标本19例,其中女性17例。石蜡切片HE染色,应用激光捕获显微切割技术获取PSH组织中多角形细胞和表面立方细胞,分别提取基因组DNA,用甲基化敏感的限制性内切酶HhaⅠ或HpaⅡ消化,巢式聚合酶链反应扩增X染色体连锁的雄激素受体（AR）基因及磷酸甘油酸酯激酶（PGK）基因。AR基因产物经变性聚丙烯酰胺凝胶电泳、银染显示其长度多态性;PGK基因产物经Bst XI消化后,琼脂糖电泳显示其酶切位点的多态性。结果17例女性PSH组织,扩增成功AR基因及PGK基因的多态性分别为53％（8／15）和27％（4／15）。对适合于检测的10例标本分别进行AR、PGK位点克隆性分析,所有标本均表现为相同的一条等位基因带完全消失（克隆率=0）或不均衡的甲基化模式（克隆率〈0．25）。结论PSH组织中的多角形细胞和表面立方细胞具有相同的单克隆增生模式,提示二者可能均为肿瘤的实质细胞。     

胃癌及癌前病变的克隆性分析及其与Ki-67表达的相关性

目的 通过人雄激素受体基因位点克隆性分析技术对胃癌及其癌前病变进行克隆性分析,探讨胃癌发生发展过程中单克隆发生率的变化趋势及与Ki-67表达之间的关系及其意义.方法 肠型胃癌根治标本24例,胃镜活检标本150例.采用激光显微切割技术准确获取病变腺上皮细胞,基因组DNA经甲基化敏感的Hpa Ⅱ限制性内切酶消化后,PCR扩增人雄激素受体基因,采用基因扫描技术对PCR产物进行分析.应用免疫组织化学EnVision二步法检测Ki-67在以上病变组织中的表达,并探讨其与克隆性分析结果的相关性.结果 单克隆发生率在胃黏膜肠上皮化生(15.63%,5/32)、低级别上皮内瘤变(22.22%,10/45)、高级别上皮内瘤变(69.44%,25/36)及肠型胃癌(100.0%,20/20)中逐渐增加,除胃黏膜肠上皮化生和低级别上皮内瘤变之间的差异没有统计学意义(P=0.47),其他各组之间差异均有统计学意义(P<0.01).Ki-67的阳性表达率随着病变的发展而不断升高.低级别上皮内瘤变组织中单克隆病例的Ki-67的阳性表达率显著高于多克隆病例(P<0.01),且克隆性与Ki-67的阳性表达率之间存在显著相关性(P<0.01).结论 单克隆的发生率及Ki-67的阳性表达率在胃癌发生发展过程中逐渐增加.且单克隆病变的发生与Ki-67的表达存在一定的相关性,两者的联合可用于胃癌的早期诊断及易感性的预测.     

BIOMED-2聚合酶链反应Ig基因重排对成熟非霍奇金B细胞淋巴瘤诊断的价值

目的 探讨BIOMED-2聚合酶链反应(PCR)在成熟非霍奇金B细胞淋巴瘤(B-NHL)诊断中的价值.方法 收集成熟B-NHL组织标本72例,其中弥漫性大B细胞淋巴瘤37例,黏膜相关淋巴组织结外边缘区淋巴瘤35例为研究对象,并以反应性增生病变25例作为对照.提取以上组织的DNA,并以PCR来检测其完整性和可扩增性,选取质量合格的DNA.85.6%(83/97)的样品DNA长度>300 bp,其中60例成熟B-NHL和23例反应性增生可用于BIOMED-2 PCR检测免疫球蛋白重链(IgH)和kappa轻链(IgK)基因重排的克隆性.结果 利用BIOMED-2 PCR检测的60例成熟B-NHL中,57例存在Ig基因的克隆性重排,其检测敏感性为95%,23例反应性增生病例中未出现Ig基因的克隆性重排,其检测特异性为100%.结论 BIOMED-2 PCR适用于石蜡包埋组织.该方法具有很高的敏感性和特异性,对成熟B-NHL诊断的辅助价值很高.     

Ig/TCR基因重排分析联合EBER原位杂交检测在原发性胃肠道淋巴瘤诊断中的应用

目的 探讨Ig/TCR基因重排分析联合EBER原位杂交检测在原发性胃肠道淋巴瘤(gastrointestinal lymphomas,GIL)中的诊断价值.方法 选取常规石蜡包埋的GIL病理标本35例(包括成熟B淋巴细胞肿瘤29例,成熟T淋巴细胞和NK细胞肿瘤6例),淋巴结反应性增生病变10例,提取DNA,应用BIOMED-2引物系统进行Ig/TCR基因重排的克隆性分析,并采用原位杂交方法检测EB病毒编码的小RNA(EBER).结果 35例GIL中,共检测出34例克隆性重排.其中29例成熟B细胞淋巴瘤均扩增出Ig克隆性重排,敏感性为100%,且联合应用IgH与IgK引物Ig单克隆性重排的检出率最高;6例成熟T淋巴细胞和NK细胞肿瘤中5例扩增出TCR克隆性重排,敏感性为83.3%;10例淋巴结反应性增生病例均未检测到Ig及TCR克隆性重排条带,其检测特异性为100%.29例成熟B细胞淋巴瘤及10例淋巴结反应性增生组织经EBER原位杂交检测均为阴性,6例成熟T淋巴细胞和NK细胞肿瘤中2例EBER原位杂交检测阳性,且均为鼻型NK/T细胞淋巴瘤.结论 BIOMED-2标准化的基因重排分析系统检测石蜡包埋组织中Ig基因和TCR基因克隆性重排的敏感性和特异性均很高,对GIL的诊断和鉴别诊断具有重要的临床应用价值,EBER原位杂交检测对基因克隆性重排阴性的淋巴瘤也具有一定的辅助诊断作用.     

利用女性X染色体失活嵌合性磷酸甘油酸激酶及雄激素受体位点多态性检测骨纤维结构不良的克隆性

目的利用基于女性体细胞构成组织内X染色体失活嵌合性的磷酸甘油酸激酶（PGK）及雄激素受体（AR）基因位点的克隆性检测,探讨骨的纤维结构不良的克隆性,以进一步阐明它的本质。方法21例女性纤维结构不良手术切除标本,均经4％甲醛固定,石蜡包埋,HE染色后应用显微切割技术分离病变及病变周围软组织,提取基因组DNA,经甲基化敏感的限制性内切酶HpaII或HhaI消化,套式聚合酶链反应（nested—PCR）扩增PGK和AR基因。通过Bst XI消化和琼脂糖电泳显示PGK基因单核苷酸多态性;应用变性聚丙烯酰胺凝胶电泳显示AR基因CAG重复序列长度多态性。结果21例纤维结构不良,光镜下均表现其组织学典型特征,即病变主要由梭形纤维样细胞和不成熟骨小梁组成,两者比例各例不一。在梭形细胞问由胶原纤维和鱼钩状或逗点状的不成熟骨小梁,梭形细胞与骨小梁直接移行,小梁周围大多没有骨母细胞围绕。克隆性检测结果表明,2例纤维结构不良在PGK和AR位点均不具有多态性,不能用于分析。其余15例和4例分别在AR和PGK基因位点显示多态性,并均表现为单克隆性,表明其肿瘤性本质。结论纤维结构不良不是一种反应性增生,而是一种真性肿瘤,但还需更多的病例证实。     

人乳头瘤病毒16型 E5基因克隆、原核表达及产物鉴定

目的 克隆HPVl6 E5基因,构建原核重组工程菌并诱导表达,对表达产物HPVl6E5蛋白进行鉴定.方法 提取宫颈癌组织DNA作为模板,用PCR方法扩增HPVl6 E5基因,经BamH I和HindⅢ双酶切后,插入相同酶切的pET21b载体质粒,转化DH5α,筛选阳性克隆.经酶切和测序鉴定后转化大肠埃希菌BL21(DE3),建屯重组工程菌株pET21b-HPVl6E5/BL21(DE3).经IPTG诱导表达,SDS-PAGE和Western印迹检测表达产物.结果 HPVl6 E5基因扩增片段0.27kb.测定序列与HPVl6原型株E5基因比较,出现4处核苷酸变异,分别为3979、4042、4077和4089位,引起144L和165V氨基酸改变.重组质粒经酶切和序列测定证实构建正确.SDS-PAGE分析重组菌在16 kDa处出现蛋白条带.该蛋白条带可被组氨酸标签单克隆抗体特异性识别.结论 成功克隆HPVl6E5基因,并构建原核表达质粒,E5 蛋白在BL21(DE3)中表达.本实验为进一步研究E5生物学活性、转化活性和肿瘤杀伤免疫作用奠定了实验基础.     

克隆性分析在肝细胞腺瘤与局灶性结节性增生鉴别诊断中的应用

目的 探讨肝细胞腺瘤（HA）和局灶性结节性增生（FNH）的克隆构成特征及其在两者鉴别诊断中的应用。方法 2例女性HA标本和发生于3位女性患者的4个FNH标本均经中性福尔马林固定、石蜡包埋、HE染色后应用显微切割技术分离病变及病变周围肝实质,提取基因组DNA,经甲基化敏感的HpaⅡ或Hha Ⅰ消化,巢式PCR扩增磷酸甘油酸激酶（PGK）和雄激素受体（AR）基因。通过Bst Ⅺ消化和琼脂糖电泳显示PGK基因单核苷酸多态性;应用变性聚丙烯酰胺凝胶电泳显示AR基因CAG重复序列长度多态性。选取4例高分化肝细胞癌（HCC）作为对照。结果 2例HA光镜下细胞排列成条索状,厚约2层,局部伴有脂肪变性及糖原贮积。瘤细胞呈圆形或多角形,大小和形态与周围肝细胞相似,未见核分裂象。克隆性分析结果显示,2例HA病变及4例HCC均显示出X染色体失活嵌合性丢失,证明为肿瘤性病变。4个FNH病变均缺乏特征性的中央瘢痕,整个病变显示多克隆性细胞组成。结论 HA属于单克隆性,诊断中需要与FNH及高分化HCC鉴别,除组织学观察外,克隆性分析在肝细胞腺瘤与结构不典型的FNH病变的鉴别诊断中有重要应用价值。     

下一代测序技术在弥漫大B细胞淋巴瘤基因重排检测中的应用

目的探究下一代测序技术(next generation sequencing, NGS)在弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma, DLBCL)辅助诊断中的应用价值。方法收集陕西省人民医院病理科存档的20例组织蜡块,其中15例诊断为DLBCL,5例诊断为淋巴组织反应性增生(reactive lymphoid hyperplasia, RLH)。采用天根公司石蜡包埋组织基因组DNA提取试剂盒进行DNA提取,安捷伦4200评价DNA质量。采用LymphoTrack IGH FR2 Assay和LymphoTrack Dx TCRG Assay进行组织克隆性测定。结果 5例RLH均为多克隆性,11例DLBCL出现NGS IgH FR2单克隆重排(阳性率73.3%,11/15),另有4例未检测出单克隆性重排(2、5、8、15号)。15例中有3例出现TCRG重排性扩增(6、7、14号)。经PCR凝胶电泳法进行验证:4例NGS检测的IgH FR2多克隆性病例均检测到IgH单克隆性基因重排;3例NGS检测到TCRG单克隆性重排病例中,PCR凝胶电泳法只检测到1例呈TCRG克隆性重排。结论高通量测序方法特异性较高,但由于其检测过程复杂,影响因素较多,检测方法的敏感性受到一定影响。虽然该方法可以准确得到基因重排序列,但由于其检测成本较高,对技术平台和人员要求更高,其在临床上的应用尚需时日。     

Clonal analysis of esophageal squamous cell carcinoma with intraepithelial components.

OBJECTIVE: In tumors of the upper aerodigestive tract, field carcinogenesis is a prevailing concept which suggests that such tumors are commonly of multiclonal origin. METHODS: To test this possibility, we applied a PCR-based clonality assay utilizing the polymorphic locus of the human androgen receptor gene (HUMARA) in female patients with esophageal squamous cell carcinomas (SCCs). DNA was extracted from small pieces of tissues microdissected from multiple points of intraepithelial and invasive parts of each tumor and the adjacent epithelia in 12 cases. The HUMARA locus was PCR amplified with or without prior digestion with Hpa II. PCR products were analyzed by a genetic analyzer and polyacrylamide gel electrophoresis followed by silver staining. RESULTS: In each of 8 informative cases, the pattern of X chromosome inactivation of the major cell population in each sample was common among the samples from the invasive part and among those samples, if any, from the intraepithelial part, and was concordant between the intraepithelial and invasive parts in 5 cases and discordant in 1 case. Out of the samples of adjacent epithelia, a monoclonal pattern was demonstrated in 8 basal cell hyperplasias and 3 dysplasias, of which 2 and 1, respectively, showed inactivation patterns discordant with those of the concomitant cancers. CONCLUSION: Esophageal SCCs may often be preceded or accompanied by multiclonal precancerous lesions, and may develop through the outgrowth of single or less commonly multiple dominant clones.     

Clonal analysis of focal nodular hyperplasia of the liver.

Recent evidence suggests that focal nodular hyperplasia of the liver (FNH) may represent a hyperplastic response to a vascular malformation, but the precise etiology remains unclear. We performed a clonal analysis of ten FNHs from nine patients by patterns of X chromosome inactivation. DNA isolated from paraffin-embedded specimens was subjected to polymerase chain reaction amplification for a highly polymorphic region of the human androgen receptor gene (HUMARA). Predigestion of tumor DNA with the methylation-sensitive, restriction enzyme HpaII allowed for selective amplification of the methylated (inactivated) allele. Of the nine patients analyzed, seven were heterozygous for the HUMARA polymorphism and informative for analysis. One informative patient had two lesions, for a total of eight FNHS. Amplification of lesional DNA after HpaII digestion demonstrated clonality in six of the eight informative cases. Paired tissue samples from different lesional areas were available in four of the six FNHs with evidence of clonality. In three of the four cases, DNA extracted from the two tissue samples showed both evidence of clonality and an identical pattern of X chromosome inactivation. In the remaining case, one sample showed evidence of clonality whereas the other was nonclonal. Three hepatic adenomas from two informative patients were also analyzed for comparative purposes, all of which showed evidence of clonality after HpaII digestion. The current study illustrates that most cases of FNH show a uniform pattern of X chromosome inactivation consistent with clonality.     

Molecular evidence that the stromal and epithelial cells in pleomorphic adenomas of salivary gland arise from the same origin: clonal analysis using human androgen receptor gene (HUMARA) assay

Salivary gland pleomorphic adenomas are characterized by a biphasic growth of "epithelial" and "stromal" regions. The "epithelial" region is a compactly organized mixture of both luminal and nonluminal cells, whereas the stromal region is composed predominantly of the nonluminal cells. Using the polymerase chain reaction (PCR)-based HUMARA assay on DNA from formalin-fixed, paraffin-embedded tissues from pleomorphic adnomas of female patients, we intend to clarify the clonal relation between the luminal and nonluminal cells and the clonal nature of the morphologically diverse nonluminal cells in this tumor. HUMARA, the human androgen receptor gene, is located on the X chromosome and contains a segment of polymorphic CAG tandem repeats in exon 1. Several methylation-sensitive HhaI restriction sites are located 5' to these CAG repeats. It is an ideal tool to study clonality of female tissues by examining the methylation pattern. Of the 13 cases analyzed, 3 were homozygous at the HUMARA locus and therefore noninformative. The remaining 10 cases were informative. All 10 cases showed a monoclonal pattern in the stromal area, indicating that the morphologically diverse nonluminal cells are monoclonal. Eight of the 10 cases showed monoclonality in the "epithelial" areas, suggesting a common clonality between luminal and nonluminal cells. Of the remaining 2 samples, 1 was polyclonal for the "epithelial" region, and the other was not amplifiable. Our data provide the first molecular evidence that the luminal and nonluminal cells in pleomorphic adenomas arise from the same clone in most cases, and the morphologically diverse nonluminal cells are monoclonal.     

Gastric neuroendocrine neoplasms: tumour clonality and malignancy-associated large X-chromosomal deletions.

Type I gastric carcinoid tumours associated with corporal (body of stomach) atrophic gastritis (CAG) are benign tumours developing as the final step of a hyperplastic precursor sequence. The neoplastic nature of these tumours has been assumed but never proved. Type III gastric carcinoid tumours and neuroendocrine carcinomas are malignant neoplasms without known precursor lesions. To assess the neoplastic nature of type I carcinoids, the clonal status of 35 tumours from 23 female patients was investigated using the human androgen receptor (HUMARA) gene test, which is based on the pattern of X-chromosome inactivation. For comparison, the same test was also performed on four type III carcinoids and two neuroendocrine carcinomas. DNA extracted from paraffin sections was digested with Hha I restriction enzyme and then amplified by polymerase chain reaction (PCR) using established HUMARA primers. The PCR products were analysed in an automated DNA sequencer. In a complementary analysis of the same tumours, loss of heterozygosity (LOH) on the X chromosome was studied using three polymorphic markers (DXS989, DXS1003, DXS1192) in a PCR-microsatellite-based technique. After exclusion of non-informative cases, 14 of 16 type I carcinoids were found to be monoclonal on the basis of the pattern of X-chromosome inactivation. Monoclonality was also documented in one of three type III carcinoids and in the single neuroendocrine carcinoma, on the basis of LOH at the HUMARA locus, which per se can be regarded as evidence for clonality. Extensive LOH of the X chromosome involving at least two markers, was found in all metastasizing tumours (two type III carcinoids and two neuroendocrine carcinomas), but in none of the 27 benign carcinoids of types I and III. These results indicate that most type I carcinoids are true monoclonal neoplasms and that malignant evolution in gastric neuroendocrine tumours is associated with extensive allelic deletion of one X chromosome.     

Clonality studies in sacral chordoma

Chordomas are rare, slow-growing, primary malignant skeletal neoplasms. Chromosome analysis, telomere reduction and telomere activity, DNA microsatellite, and loss of heterozygosity studies have been performed on chordomas; however, the clonality status (monoclonal versus polyclonal proliferation) is unknown. The primary purpose of this study was to determine whether sacral chordoma is monoclonal or polyclonal in origin with the use of a polymorphic X-linked gene (AR; alias HUMARA) and X-chromosome inactivation studies. DNA was harvested from tumor and corresponding normal tissue from eight women (37-71 years) with chordoma. Clonality was determined using an X chromosome inactivation protocol and a polymorphic human androgen receptor gene (AR) located on the X chromosome. The procedure required a methylation-specific polymerase chain reaction (PCR) and determination of the ratio of active to inactive X chromosomes. Results were informative for seven of the eight women, with two separate X-linked alleles seen for the AR gene in the normal tissue. Expression of AR gene alleles from each of the two X chromosomes was present in the chordoma tumor, indicating a polyclonal proliferation in all seven women. Most solid tumors and skeletal neoplasms are polyclonal in nature. Our study indicates that chordoma is polyclonal in its pattern of proliferation.     

Assessing the inactivation pattern in chromosome X among symptomatic carriers and women with haemophilia

X chromosome inactivation is a stochastic event that occurs early in female embryo development to achieve dosage compensation with males. Certain genetic mechanisms affect the normal process causing a skewed X inactivation pattern which has clinical relevance in female carriers of X-linked recessive disorders, like haemophilia. The most commonly used assay to evaluate the X inactivation pattern is the PCR amplification of the human androgen receptor gene (HUMARA). The use of this technique in bleeding carriers and women with haemophilia allows identifying if their hemorrhagic symptoms are due to an unfavourable lyonization. Furthermore, these studies are important for understanding the X chromosome inactivation process in humans.     

Pathogenetic mechanism for a female patient with hemophilia A北大核心CSCD

Objective: To explore the pathogenetic mechanism for a female patient affected with hemophilia A (hemophilia A, HA). Methods: Potential genetic defect was detected with inverse shifting-polymerase chain reaction (IS-PCR). The pattern of X chromosome inactivation was determined with a human androgen receptor assay (HUMARA assay). G-banded karyotyping was carried out to exclude potential chromosome aberrations. Results: IS-PCR showed that the defect of FVIII gene was the distal type of intron 22 inversion. The HUMARA assay showed that the X chromosome inactivation was non-random, and that the mother's X chromosome activity was lower than that of the father's X chromosome which has carried the inverted FVIII gene. No abnormalities were found with G-banded chromosomes. Conclusion: The prevalence of female HA patient may be caused by non-random inactivation of X chromosomes. © 2016, West China University of Medical Sciences. All rights reserved.     

Non-skewed X-inactivation may cause mental retardation in a female carrier of X-linked alpha-thalassemia/mental retardation syndrome (ATR-X): X-inactivation study of nine female carriers of ATR-X

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X) is a syndromic form of X-linked mental retardation. We investigated the X-inactivation status of nine female ATR-X carriers by methylation-specific PCR of the HUMARA gene. Six carriers demonstrated a skewed X-inactivation pattern (>90:10) and one showed a non-skewed pattern (72:28), while two were uninformative because of homozygosity for the CAG repeat polymorphic alleles in the HUMARA. Only the carrier mother who showed non-skewed X-inactivation had moderate mental retardation. These findings suggest that mutations in ATRX may cause mental retardation in females, if the X chromosome carrying mutated ATRX is not properly inactivated.     

Molecular analysis of clonality in Kaposi's sarcoma.

BACKGROUND: Kaposi''s sarcoma is considered to be an angioproliferative disease associated with a novel herpesvirus (KSHV/HHV8), but the precise pathophysiology of the lesion remains unclear. The study of clonality in Kaposi''s sarcoma using X linked DNA polymorphism has been difficult so far, because of a very strong prevalence of the disease in males. AIMS: To study the clonality of Kaposi''s sarcoma lesions. METHODS: An assay based on a methyl sensitive restriction digest followed by polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor (HUMARA) gene was used. Tissues from Kaposi''s sarcoma lesions and control tissues from the same patients were obtained from seven females, four with classic Kaposi''s sarcoma and three with AIDS associated Kaposi''s sarcoma. A cutaneous angiosarcoma was also analysed, for comparative purposes, and showed evidence of clonality after HpaII digestion. RESULTS: All patients were heterozygous for the HUMARA polymorphism and informative for analysis. In all patients, including four with a nodular form of Kaposi''s sarcoma and more than 70% spindle cells in the lesion, a polyclonal pattern of inactivation could be demonstrated. CONCLUSIONS: The Kaposi''s sarcoma lesion is first of all a polyclonal cell proliferation.     

Skewed X inactivation in manifesting carriers of Duchenne muscular dystrophy

We studied X inactivation patterns in manifesting carriers of familial and sporadic Duchenne muscular dystrophy (DMD) or unaffected carriers of DMD by analysis of the methylation of Hpall sites in the first exon of the human androgen-receptor gene (HUMARA) from peripheral blood samples. Three of the four manifesting carriers, four of the five asymptomatic carriers, and 31 of the 32 female controls were heterozygous for the CAG repeat of HUMARA. All manifesting carriers showed skewed X inactivation, while all unaffected carriers showed almost symmetrical inactivation. One family studied over three generations is noteworthy because it includes two mother/daughter pairs, one an affected pair with skewed X inactivation, and the other a phenotypically normal carrier pair with random X inactivation. On the other hand, the extent of X inactivation for each X chromosome in 31 female controls was widely distributed. These data suggest that in carriers of DMD, both affected and unaffected, it is valuable to analyze the pattern of skewed X inactivation because it provides important prognostic information. Carriers of DMD with skewed X inactivation might show slowly progressive myopathy with advancing age.