Closing in on the BPES gene on 3q23: mapping of a de Novo reciprocal translocation t(3;4)(q23;p15.2) breakpoint within a 45-kb cosmid and mapping of three candidate genes, RBP1, RBP2, and beta'-COP, distal to the breakpoint

BPES is a genetic disorder presenting with blepharophimosis, ptosis of the eyelids, epicanthus inversus, and telecanthus. BPES type I is associated with female infertility, whereas type II presents without additional symptoms. Hitherto, it remains unknown whether BPES type I results from a defect in a single gene or from a contiguous gene syndrome. Previous cytogenetic and linkage analyses have assigned a BPES locus to 3q23, in a 5-cM interval between D3S1615 and D3S1316. In this report, we describe the molecular and physical characterization of the 3q23 breakpoint in a BPES patient with a t(3;4)(q23;p15.2) translocation. Eight YACs located around and within the D3S1615-D3S1316 interval were mapped relative to the 3q23 breakpoint; 5 YACs spanning the 3q23 breakpoint were identified. Thirteen STSs and ESTs were localized on the YAC map. Subsequent hybridization of 2 YACs spanning the breakpoint to the Human RPCI1 PAC Library and the Human Chromosome 3 LLNL Cosmid Library resulted in the identification of 12 PACs and 50 cosmids respectively, allowing the construction of a detailed PAC and cosmid physical map. A refined position-telomeric to the breakpoint-of 3 candidate genes, cellular retinol-binding proteins 1 and 2 (RBP1, RBP2) and the coatomer beta' subunit (beta'-COP), was obtained on this physical map. Furthermore, a PAC and cosmid contig encompassing the breakpoint was constructed. PAC 169-C 10 and cosmid 11-L 10 crossing the breakpoint have sizes of 110 and 45 kb, respectively. The isolation of coding sequences in these clones and in the rest of the contig will greatly facilitate further efforts toward positional cloning of the gene(s) involved in BPES.     

Refinement of a Translocation Breakpoint Associated with Blepharophimosis–Ptosis–Epicanthus Inversus Syndrome to a 280-kb Interval at Chromosome 3q23

Blepharophimosis syndrome (BPES) is an autosomal dominant disorder of craniofacial development, the features of which include blepharophimosis, ptosis, and epicanthus inversus. Although it has been suggested that BPES is genetically heterogeneous, a major locus for this condition resides at chromosome 3q23. We have previously mapped a translocation breakpoint associated with BPES to the D3S1316–D3S1615 interval. The markers in this region have subsequently been shown to lie in a different order, with the BPES locus mapping to the 1-cM D3S1576 and D3S1316 interval. In the current investigation, a physical map, consisting of 60 yeast artificial chromosome (YAC) clones and 1 bacterial artificial chromosome, that spans this region has been constructed. Ten expressed sequence tags and the cellular retinol-binding protein I locus have been mapped to the contig. YAC end isolation has led to the creation of novel STSs that have been used to reduce the size of the BPES critical region to a 280-kb interval, which has been cloned in two nonchimeric YACs.     

Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES)

Blepharophimosis, ptosis, epicanthus inversus syndrome type I (BPES; OMIM 110100) is an autosomal dominant disorder affecting craniofacial development and ovarian function. We have identified a patient with BPES who carried a de novo reciprocal translocation [46, XX,t(3;21)(q23;q22.1)]. Fluorescence in situ hybridization analysis at band 3q23 using probes derived from BAC 175G20 (Research Genetics), PACs 108L15 and 169C10 (RPCI1), and cosmids AC174D4, AC68D3, AC44F5, and AC125C5 (Lawrence Livermore National Laboratory) was performed. The patient's breakpoint was found to lie within the overlapping region of the BAC and PACs but centromeric to all the cosmids. However, a 10.5-kb BamHI-digested fragment, common to the BAC and PAC clones, was shown to cross the breakpoint. The results have placed our patient's breakpoint proximal to that of the previously reported patient [46,XY,t(3;4)(q23;p15.2)] and within a 10.5-kb interval. This is the second patient in which a breakpoint was refined by molecular cytogenetics. Our findings emphasize the significance of this region for BPES.     

FOXL2、SOX14及BPESC1 mRNA在小睑裂综合征患者眼睑组织中表达水平的研究

目的 研究小睑裂综合征（blephamphimosis—ptosis—epicanthusinversus syndrome,BPES）患者与正常人相比眼睑组织中FOX12、SOX14及BPESC13个基因mRNA的相对表达水平,探讨这3个基因与BPES的相关性,以及可能存在的发病机制。方法TRIzol法抽提眼睑组织总RNA,反转录为cDNA,应用实时荧光定量PCR技术分别检测15例BPES患者（A组）及15例正常人眼睑组织（B组）中FOXL2、SOX14和BPESClmRNA的表达水平,用两配对样本wilcoxon符号秩检验法和2-AAC,法分析两组数据及其差异。结果FOXL2、SOX14和BPESCI3个基因tuRNA的表达水平在A、B两组间的差异均具有统计学意义,P值均〈0．05。A、B两组间,FOXL2基因的△Ct之差（△△ct）,负秩与证秩的平均秩分别为6．00和8．50,SOX14基[天1的△△Ct负秩与正秩的平均秩分别为4．20和9．33,BPESC1基因的△△ct负秩与正秩的平均秩分别为8．23和6．50,可认为A组FOXL2基因和SOXl4基因的△ct值大于B组,BPESCI基因的△ct值小于B组,即A组的FOXL2基因和SOX14基因的表达水平低于B组,BPESCI基因表达水平高于B组。结论FOXL2、BPESC1及SOX14均与BPES具有一定相关性,FOXL2和SOX14的低表达以及BPESC1的高表达可能与BPES的发病相关。     

Cloning, Function, and Expression of sanS: A Gene Essential for Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 2-kb SmaI DNA fragment was cloned from the cosmid library of Streptomyces ansochromogenes. This DNA fragment contains a complete open reading frame which is 1275 bp in length, designated sanS (GenBank accession no. AF322179). The deduced SanS protein consists of 424 amino acids and belongs to a superfamily of enzymes with an unusual ATP-grasp fold. The disruption and complementation of sanS indicated that sanS is essential for nikkomycin biosynthesis in Streptomyces ansochromogenes. The sanS gene was subcloned into expression vector pET23b and overexpressed in E. coli BL21 (DE3). The protein was then purified and showed ATPase activity.     

Fine mapping of the constitutional translocation t(11;22)(q23;q11)

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.     

Co-segregation of trichorhinophalangeal syndrome with a t(8;13)(q23.3;q21.31) familial translocation that appears to increase TRPS1 gene expression

Trichorhinophalangeal syndrome type I (TRPS I) is a rare autosomal dominant syndrome caused by haploinsufficiency of TRPS1 due to point mutations or deletions. Here, we report the first familial TRPS I due to a t(8;13)(q23.3;q21.31) translocation breakpoint <100 kb from the 5′ end of TRPS1. Based on the additional abnormalities observed exclusively in the index patient that are mainly compatible with clinical features of TRPS, her phenotype was defined as expanded TRPS I including brain malformations and intellectual disability. Initial analyses did not reveal any genetic defect affecting TRPS1 or any genomic alteration within the breakpoint regions or elsewhere in the genome. The pathogenic chromosome 8q23.3 breakpoint is at position g.116,768,309_116,768,310 within a transposon type I element, 87 kb from the TRPS1 5′ end. The 13q21.31 breakpoint is within a tandem repeat region at position g.65,101,509_65,101,510 (genome assembly GRCh37/hg19). This breakpoint is flanked by protocadherin 9 (PCDH9) and protocadherin 20 (PCDH20). As an outcome of the translocation, an evolutionarily conserved non-coding VISTA enhancer element from 13q21.31 is placed within the TRPS1 5′ region, 1,294 bp from the breakpoint. The increased expression of TRPS1 found by three independent methods is most probably translocation allele derived and driven by the translocated enhancer element. The index patient’s expanded phenotype presumably involves the epithelial-to-mesenchymal transition pathway that may be due to TRPS1 overexpression. Together, these findings support that the reported translocation-associated phenotypes are “cis-ruption” and TRPS1 overexpression related, the latter most probably caused by the novel enhancer element in the TRPS1 5′ region.     

A gene for blepharophimosis-ptosis-epicanthus inversus syndrome maps to chromosome 3q23

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant malformation of the eyelids that may severely impair visual function. Chromosomal aberrations involving chromosomes 3q23, 3p25 and 7p34 have been reported in BPES but the disease gene has not been hitherto localized by linkage analysis. We have mapped a gene for BPES to chromosome 3q23 in a large French pedigree (Z _max = 4.62 at =0 for probe AFM 182yc5 at locus D3S1549). The best estimate for the location of the disease gene is at locus D3S1549, between the loci D3S1292 and D3S1555 (maximum lod score of 5.10).     

Fc receptor gene translocation in a t(1;19) pre-B ALL cell line

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1;19)(g23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.