Engineered telomere degradation models dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4-5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.     

Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.     

POT of gold: modeling dyskeratosis congenita in the mouse

Dyskeratosis congenita (DC) is a rare syndrome, characterized by cutaneous abnormalities and premature death caused by bone marrow failure. In this issue of Genes & Development, Hockemeyer and colleagues (pp. 1773-1785) report a new mouse model that reconstitutes key features of DC. Disease phenotypes are generated by a POT1b deletion in a telomerase-deficient background that accelerates the shortening of telomeres by degradation.     

Three novel truncating TINF2 mutations causing severe dyskeratosis congenita in early childhood

Dyskeratosis congenita (DC) is a telomere biology disorder characterized by a mucocutaneous triad, aplastic anemia, and predisposition to cancer. Mutations in a narrow segment of TINF2 exon 6 have been recognized to cause often-severe DC that is either sporadic or autosomal dominant. We describe three children with very early presentations of DC, including one with the severe variant known as Revesz syndrome. Although most TINF2 mutations reported to date are missense changes, each of our patients carried a novel heterozygous nonsense or frameshift mutation, revealing a new 5' boundary to the affected gene segment in patients with DC. Examination of patient-derived lymphoblastoid cell lines revealed stable expression of the predicted truncated TIN2 proteins. In co-immunoprecipitation assays, the ability of a truncation mutant to interact with TRF1 was severely impaired, whereas the ability of the most common DC-associated mutant was much less affected. This suggests that the disruption of TIN2-TRF1 interaction may contribute to the severe clinical phenotype observed in the context of the TIN2 truncation mutation, but is unlikely to be the primary cause of telomere shortening associated with the more prevalent TIN2 missense mutations. Telomere flow-fluorescent in situ hybridization (FISH) analysis of one pedigree showed the dramatic effect a de novo nonsense TINF2 mutation had on telomere length in early development. These cases underscore the severe manifestations of truncating TINF2 mutations.     

Telomere length measurement can distinguish pathogenic from non-pathogenic variants in the shelterin component, TIN2

Dyskeratosis congenita (DC) is a heterogeneous bone marrow failure syndrome with seven disease-causing genes identified to date, six of which are linked to telomere maintenance. Mutations in one of these genes (TINF2), which encodes a component of the shelterin complex, are associated with particularly short telomeres. Among the 224 consecutive patients with different forms of bone marrow failure (46 with DC, 122 with aplastic anaemia and 57 with some features of DC), we have identified 16 new families with variants in exon 6 of the TINF2 gene, eight of which are novel. We observe that the phenotype associated with these mutations extends to a severe early presentation, not always classified as DC. In addition, we see that some of the variants identified are not associated with short telomeres and are also found in asymptomatic individuals. In the absence of any direct functional assay, the data indicates that the telomere length measurement can inform us as to which variants in TINF2 are pathogenic and which may be non-pathogenic.     

端粒相关蛋白TIN2

TIN2(TRF1相互作用核蛋白2)是一重要的端粒相关蛋白。人TIN2蛋白包括N端,TRF1交互作用区(TRF1-Int)和C端3个结构域。它在TRF1复合物、TRF2复合物和Shelterin的功能中扮演关键角色,协同其它端粒蛋白维持端粒长度、结构和功能。TIN2与个体发育、细胞分化和肿瘤发生密切相关。     

Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1

Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.     

Minishelterins separate telomere length regulation and end protection in fission yeast

The conserved shelterin complex is critical for chromosome capping and maintaining telomere length homeostasis. In fission yeast, shelterin is comprised of five proteins. Taz1, Rap1, and Poz1 function as negative regulators of telomere elongation, whereas Pot1 and Tpz1 are critical for end capping and telomerase recruitment. How the five proteins work together to safeguard chromosome ends and promote telomere length homeostasis is a matter of great interest. Using a combination of deletions, fusions, and tethers, we define key elements of shelterin important for telomere length regulation. Surprisingly, deletion of the entire Rap1 and Poz1 proteins does not impair telomere length regulation as long as a static bridge is provided between Taz1 and Tpz1. Cells harboring minishelterin display wild-type telomere length and intact subtelomeric silencing. However, protection against end fusions in G1 is compromised in the absence of Rap1. Our data reveal a remarkable plasticity in shelterin architecture and separate functions in length regulation and end protection.     

Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.     

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

6A8α-甘露糖苷酶表达对人鼻咽癌细胞CNE-2L2端粒酶活性和端粒长度的影响

探讨端粒长度与端粒酶活性在人鼻咽癌细胞CNE-2L2恶性行为改变前后的变化，建立研究恶性行为改变与端粒长度与端粒酶活性间关系的细胞模型。与6A8α-甘露糖苷酶表达正常的CNE-2L2细胞(野生型细胞W，转导空载体的细胞M及转导无关DNA片段的细胞S)相比，6A8α-甘露糖苷酶表达低下的细胞(AS)接种裸鼠皮下后的肿瘤性生长受抑。用Telo TAGGG Telomere Length Assay Kit及Telomerase PCR ELISA Kit分别测定端粒长度及端粒酶活性，用RT-PCR方法分析端粒重复序列结合因子(TRF)的转录水平。见AS细胞的端粒明显缩短(6．78Kb，W细胞为8．40Kb，M细胞为8．34kb，S细胞为9．56kb)，但端粒酶活性及端粒重复序列结合因子l和2(TRFl和2)的转录水平未见改变。实验表明，恶性行为降低的CNE-2L2细胞的端粒变短，但与端粒酶活性及TRF1／2无关，提示在CNE-2L2细胞中可能存在着端粒酶及TRF1／2以外的调节端粒长度的机制。这为研究肿瘤细胞恶性行为改变与端粒长度与端粒酶活性间关系提供了一个模型。     

先天性厚甲症Ⅱ型一家系KRT17基因突变研究

目的 研究1个先天性厚甲症Ⅱ型家系的基因突变情况.方法 收集该家系的详细临床资料,外周血提取基因组DNA,PCR扩增KRT17热点突变区,通过PCR扩增产物直接测序方法对该家系患者、正常成员和100名无亲缘关系的正常人进行KRT17基因突变检测.结果 在该家系患者KRT17基因的第1外显子上发现了1个错义突变(296 T→C),导致KRT17的1A区亮氨酸由脯氨酸替代(L99P),而家系中正常成员和家系外100名正常对照中均未能发现该突变.结论 该家系患者的临床表现为KRT17发生突变(L99P)所致,结合文献复习证实部分PC-Ⅱ家系基因型与表现型之间存在一定相关性.

Abstract：

Objective To investigate the keratin 17 gene (KRT17)mutation in a pedigree with pachyonychia congenita type 2 (PC-Ⅱ ). Methods DNA was extracted from the blood samples of the patients, unaffected members of the pedigree, and 100 unrelated healthy controls. PCR was performed to amplify the hot spots in KRT17 gene. PCR products were directly sequenced to detect mutation. Results A heterozygous 296T--C mutation was found in all the affected members of this family, which resulted in the substitution of leucine by proline in codon 99 (L99P) in the 1A domain of the KRT17, but not in the healthy individuals from the family and the 100 unrelated controls. Conclusion The mutation of KRT17 may play a major role in the pathogenesis of this pedigree with pachyonychia congenita type 2.     

Measurement of telomere length in cells from pleural effusion: Asbestos exposure causes telomere shortening in pleural mesothelial cells

We estimated the telomere lengths of neoplastic and non‐neoplastic mesothelial cells and examined their correlation with asbestos exposure and the expression of markers of mesothelial malignancy. Cell blocks of pleural effusion obtained from 35 cases of non‐neoplastic disease (NN), 12 cases of malignant mesothelioma (MM) and 12 cases of carcinomatous effusion due to lung adenocarcinoma (LA) were examined. Fifteen of the 35 NN cases had pleural plaques (NNpp+) suggestive of asbestos exposure, and the other 20 cases had no pleural plaques (NNpp‐). Telomere length was measured using the tissue quantitative fluorescence in situ hybridization method, and expressed as normalized telomere‐to‐centromere ratio. NN cases had significantly longer telomeres than MM (P < 0.001) and LA (P < 0.001) cases. Telomeres in NNpp+ cases were slightly shorter than those of NNpp‐ cases (P = 0.047). MM and LA showed almost the same telomere length. NN cases with shorter telomeres tended to show aberrant expression of epithelial membrane antigen (EMA), CD146, glucose transporter 1 (GLUT1) and IGF‐II messenger RNA‐binding protein 3 (IMP3). These results suggest that telomere shortening and subsequent genetic instability play an important role in the development of MM. Measurement of telomere length of cells in pleural effusion might be helpful for earlier detection of MM.     

COL 2A1基因突变致SEDC的产前分子诊断

目的本研究对COL2A1基因（typeⅡcollagen gene）G504S突变导致的先天性脊柱骨骺发育不良（SEDC）家系的1例中期妊娠者进行产前分子诊断,从而预防SEDC的发生。方法患者妻于20孕周在B超下进行羊膜囊穿刺,抽取羊水10ml,提取羊水细胞基因组DNA。选择3个多态性STR位点,即D3S1358、D16S539和D21S11,排除母体细胞污染。在此基础上,对COL2A1基因外显子23进行扩增,PCR产物进行正、反向测序。结果该例胎儿COL2A1的外显子23测序结果显示,胎儿未带有与父亲同样的COL2A1基因突变。胎儿产前基因诊断结果与B超监测结果一致。结论对于有SEDC风险的胎儿进行产前分子诊断非常重要,可以在B超诊断前了解胎儿基因型并明确诊断。     

Deficiency in DNA mismatch repair increases the rate of telomere shortening in normal human cells

DNA mismatch repair (MMR) is essential for genome stability and inheritance of a mutated MMR gene, most frequently MSH2 or MLH1, results in cancer predisposition known as Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC). Tumors that arise through MMR deficiency show instability at simple tandem repeat loci (STRs) throughout the genome, known as microsatellite instability (MSI). The STR instability is dominated by errors that accumulate during replication in the absence of effective MMR. In this study we show that there is a high level of instability within telomeric DNA with a tendency toward deletions in tumor-derived MMR defective cell lines. We downregulated MSH2 expression in a normal fibroblast cell line and isolated four clones, with differing levels of MSH2 depletion. The telomere-shortening rate was measured at the Xp/Yp, 12q, and 17p telomeres in the MSH2 depleted and three control clones. Interestingly the mean telomere-shortening rate in the clones with MSH2 depletion was significantly greater than in the control clones. This is the first demonstration that MSH2 deficiency alone can lead to accelerated telomere shortening in normal human cells.     

A CLCN1 mutation in dominant myotonia congenita impairs the increment of chloride conductance during repetitive depolarization

Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.     

端锚酶和端粒酶反义寡核苷酸联合作用对人肺腺癌细胞 A549 端粒的影响

目的探讨端锚酶及端粒酶反义寡核苷酸联合作用对A549细胞端粒相关蛋白表达和翻译及对端粒缩短和细胞周期的作用。方法将A549细胞随机分组为空白对照、sTANKS、shTERT、asTANKS、ashTERT和asTANKS ashTERT组,经不同处理,检测hTERTmRNA、端粒酶及端锚酶活性、端粒平均长度及A549细胞寿命。结果ashTERT能抑制hTERTmRNA表达及蛋白质活性;asTANKS不影响hTERTmRNA表达,却能抑制端锚酶活性。asTANKS或ashTERT均可致A549端粒长度缩短,asTANKS ashTERT则缩短更为明显,其减少A549细胞平均传代数的作用也明显大于asTANKS或ashTERT。结论asTANKS对A549端粒长度的抑制有别于端粒酶途径,不仅能通过影响端锚酶活性缩短肿瘤细胞A549端粒的平均长度,而且可与端粒酶抑制剂协同作用明显缩短肿瘤细胞的生存周期,这可能成为抗癌作用的新靶点。     

Myotonia congenita and myotonic dystrophy in the same family: coexistence of a CLCN1 mutation and expansion in the CNBP (ZNF9) gene

Myotonia is characterized by hyperexcitability of the muscle cell membrane. Myotonic disorders are divided into two main categories: non-dystrophic and dystrophic myotonias. The non-dystrophic myotonias involve solely the muscle system, whereas the dystrophic myotonias are characterized by multisystem involvement and additional muscle weakness. Each category is further subdivided into different groups according to additional clinical features or/and underlying genetic defects. However, the phenotypes and the pathological mechanisms of these myotonic disorders are still not entirely understood. Currently, four genes are identified to be involved in myotonia: the muscle voltage-gated sodium and chloride channel genes SCN4A and CLCN1, the myotonic dystrophy protein kinase (DMPK) gene, and the CCHC-type zinc finger, nucleic acid binding protein gene CNBP. Additional gene(s) and/or modifying factor(s) remain to be identified. In this study, we investigated a large Norwegian family with clinically different presentations of myotonic disorders. Molecular analysis revealed CCTG repeat expansions in the CNBP gene in all affected members, confirming that they have myotonic dystrophy type 2. However, a CLCN1 mutation c.1238C>G, causing p.Phe413Cys, was also identified in several affected family members. Heterozygosity for p.Phe413Cys seems to exaggerate the severity of myotonia and thereby, to some degree, contributing to the pronounced variability in the myotonic phenotype in this family.     

X连锁性铁粒幼细胞贫血家系新发现ALAS2基因G514A突变

目的明确2例患有铁粒幼细胞贫血同胞兄弟系X连锁性铁粒幼细胞贫血,并鉴定其家系的ALAS2基因。方法利用PCR-SSCP技术分析该家系先证者,外祖父母,父母及其患病兄弟6人的ALAS2基因,确定存在基因突变,之后通过测序分析明确具体突变的位点及碱基。结果该两例患儿在ALAS2基因第5外显子都存在突变,该突变来自于母系家族,其母亲和外祖母为携带者;通过测序分析进一步明确具体的突变为ALAS2基因G514A突变,导致甲硫氨酸被异亮氨酸所替代。结论该家系患者为ALAS2基因突变所致的X连锁性铁粒幼细胞贫血,存在一种新的突变基因-ALAS2基因第5外显子G514A突变。