Lanyi A, Li B, Li S, Talmadge C B, Brichacek B, Davis J R, Kozel B A, Trask B, van den Engh G, Uzvolgyi E, Stanbridge E J, Nelson D L, Chinault C, Heslop H, Gross T G, Seemayer T A, Klein G, Purtilo D T, Sumegi J
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198, USA.
Genomics. 1997 Jan 1;39(1):55-65. doi: 10.1006/geno.1996.4466.
X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.
X连锁淋巴增生性疾病（XLP）的特征是对爱泼斯坦-巴尔病毒（EBV）感染具有显著的易感性。XLP患者感染EBV总是会导致致命的单核细胞增多症、无丙种球蛋白血症或恶性淋巴瘤。最初，XLP基因被定位到Xq25中DXS42和DXS37之间的一个10厘摩区域。随后，在一个XLP家族（43）中发现了Xq25的一个细胞遗传学可见的中间缺失。在本研究中，我们通过双激光流式核型分析估计XLP患者43 - 004中的缺失涉及X染色体的2%，即大约3兆碱基的DNA序列。从一个人类染色体Xq25特异性酵母人工染色体（YAC）亚文库中，分离出了5个包含患者43 - 004中缺失的DNA序列的YAC。来自这些YAC的序列标签位点（STS）已被用于识别无关XLP患者中的中间缺失。又发现了3个有中间缺失的家族。其中两名患者（63 - 003和73 - 032）携带一个3.0兆碱基的中间缺失，与43 - 004的缺失重叠。在一名表现出XLP典型的感染后单核细胞增多症表型伴低丙种球蛋白血症和恶性淋巴瘤的XLP患者（30 - 011）中，检测到一个大约250千碱基的缺失，与在患者43 - 004、63 - 003和73 - 032中检测到的缺失重叠。构建了一个跨越XLP关键区域的2.2兆碱基的YAC重叠群，其在X染色体上的方向通过双色荧光原位杂交确定，由15个重叠的YAC克隆组成。构建了该区域的详细限制性酶切图谱。通过反向钳制均匀电场凝胶电泳确定YAC插入片段的大小。通过荧光原位杂交和限制性酶切图谱分析确定YAC的嵌合情况。基于λ亚克隆、YAC末端衍生质粒和平均间距为100千碱基的STS，使用5种稀有切割限制性酶构建了一个长程物理图谱。这些STS和λ亚克隆用于Southern杂交和PCR分析。此处展示的工作极大地细化了XLP的关键区域。具有重叠中间缺失的YAC重叠群构成了构建关键区域转录图谱的基础，并有助于鉴定XLP基因。
