Fanconi Anemia and Ubiquitination

Fanconi anemia （FA） is a rare recessive hereditary disease characterized clinically by congenital defects, progressive bone-marrow failure, and cancer predisposition. Cells from FA patients exhibit hypersensitivity to DNA cross-linking agents, such as mitomycin C （MMC）. To date, at least 12 FA genes have been found deleted or mutated in FA cells, and 10 FA gene products form a core complex involved in FA/BRCA2 DNA repair pathway-FA pathway. The ubiquitin E3 ligase FANCL, an important factor of FA core complex, co-functions with a new ubiquitin conjugating enzyme UBE2T to catalyze the monoubiquitination of FANCD2. FANCD2-Ub binds BRCA2 to form a new complex located in chromatin foci and then take part in DNA repair process. The deubiquitylating enzyme USP1 removes the mono-ubiquitin from FANCD2-Ub following completion of the repair process, then restores the blocked cell cycle to normal order by shutting off the FA pathway. In a word, the FANCD2 activity adjusted exquisitely by ubiquitination and/or deubiquitination in vivo may co-regulate the FA pathway involving in variant DNA repair pathway.

DTL facilitates the Fanconi anemia pathway for ultraviolet-induced DNA repair in retinal pigment epithelial cells

The excessive energy of light,especially the invisible rays with lower wavelength,is basically absorbed by retinal pigment epithelium(RPE)and usually causes DNA damage.The molecular mechanism behind DNA damage repair response to this frequent stress in RPE is not clearly understood.In this study,we determined that the Fanconi anemia(FA)pathway was activated in human RPE ARPE-19 cells after ultraviolet(UV)B and C treatment.Moreover,immunoprecipitation(IP)of FANCD2 indicated that denticleless E3 ubiquitin protein ligase homolog(DTL)closely interacted with FANCD2.Knockdown of DTL weakened the activity of the FA pathway in ARPE-19 cells responding to UV treatment.Finally,the DTL promoter was incubated with a biotin-labeled probe and pulled down by streptavidin beads followed by the genomic DNA sonication.p53 was indicated by mass spectrum and further determined by chromatin IP assay.Taken together,our results demonstrated that DTL regulated by p53 could activate the FA pathway for UV-induced DNA damage repair in retinal pigment epithelial cells.

Effect of a Nutrient Mixture on Fanconi Anemia Fibroblast and Normal Human Dermal Fibroblast: A Comparison

Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a specific nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF. We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A: PD20, FA-A: PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 μg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 μg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 μg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose-dependent manner with total block at 1000 μg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 μg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.

Fanconi anemia manifesting as a squamous cell carcinoma of the mandible: a case report

Progressive bone marrow failure and development of malignancies, particularly acute myeloid leukemia and solid tumors the most important features of Fan- coni’s Anemia (FA). This paper reports the case of a 16-year-old patient with FA who developped squa- mous cell carcinoma of the mandible, ten years after the bone marrow transplantation (BMT).

Disulfiram enhances the antitumor activity of cisplatin by inhibiting the Fanconi anemia repair pathway

A series of chemotherapeutic drugs that induce DNA damage,such as cisplatin(DDP),are standard clinical treatments for ovarian cancer,testicular cancer,and other diseases that lack effective targeted drug therapy.Drug resistance is one of the main factors limiting their application.Sensitizers can overcome the drug resistance of tumor cells,thereby enhancing the antitumor activity of chemotherapeutic drugs.In this study,we aimed to identify marketable drugs that could be potential chemotherapy sensitizers and explore the underlying mechanisms.We found that the alcohol withdrawal drug disulfiram(DSF)could significantly enhance the antitumor activity of DDP.JC-1 staining,propidium iodide(PI)staining,and western blotting confirmed that the combination of DSF and DDP could enhance the apoptosis of tumor cells.Subsequent RNA sequencing combined with Gene Set Enrichment Analysis(GSEA)pathway enrichment analysis and cell biology studies such as immunofluorescence suggested an underlying mechanism:DSF makes cells more vulnerable to DNA damage by inhibiting the Fanconi anemia(FA)repair pathway,exerting a sensitizing effect to DNA damaging agents including platinum chemotherapy drugs.Thus,our study illustrated the potential mechanism of action of DSF in enhancing the antitumor effect of DDP.This might provide an effective and safe solution for combating DDP resistance in clinical treatment.

The Fanconi anemia pathway and DNA interstrand cross-link repair

Fanconi anemia(FA)is an autosomal or X-linked recessive disorder characterized by chromosomal instability,bone marrow failure,cancer susceptibility,and a profound sensitivity to agents that produce DNA interstrand cross-link(ICL).To date,15 genes have been identified that,when mutated,result in FA or an FA-like syndrome.It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FAlike proteins.Here,we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.

Diagnosis of Fanconi anemia in children with atypical clinical features： a primary study

Background Fanconi anemia is a severe congenital disorder associated with mutations in a cluster of genes responsible for DNA repair.Arriving at an accurate and timely diagnosis can be difficult in cases of Fanconi anemia with atypical clinical features.It is very important to increase the rate of accurate diagnosis for such cases in a clinical setting.The purpose of this study is to explore the clinical diagnosis of Fanconi anemia in children with atypical clinical features.Methods Six cases of Fanconi anemia with atypical clinical features were enrolled in the study,and their clinical features were recorded,their FANCA gene transcription was assessed by RT-PCR,and FANCA mutations and the ubiquitination of FANCD2 protein were analyzed using DNA sequencing and western blotting respectively.Results All six cases showed atypical clinical features including no apparent deformities,lack of response to immune therapy,and progressively increasing bone marrow failure.They also have significantly increased fetal hemoglobin,negative mitomycin-induced fracture test results,and carry a FANCA gene missense mutation.Single protein ubiquitination of FANCD2 was not observed in those patients.Conclusion The combination of clinical features,FANCA pathogenic gene mutation genotype and the absence of FANCD2 protein ubiquitination are helpful in the accurate and timely diagnosis of Fanconi anemia in children.

Fanconi anemia gene-associated germline predisposition in aplastic anemia and hematologic malignancies

Whether Fanconi anemia(FA)heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is a crucial but unsettled issue in cancer prevention and family consulting.We retrospectively analyzed rare possibly significant variations(PSVs)in the five most obligated FA genes,BRCA2,FANCA,FANCC,FANCD2,and FANCG,in 788 patients with aplastic anemia(AA)and hematologic malignancy.Sixty-eight variants were identified in 66 patients(8.38%).FANCA was the most frequently mutated gene(n=29),followed by BRCA2(n=20).Compared with that of the ExAC East Asian dataset,the overall frequency of rare PSVs was higher in our cohort(P=0.016).BRCA2 PSVs showed higher frequency in acute lymphocytic leukemia(P=0.038),and FANCA PSVs were significantly enriched in AA and AML subgroups(P=0.020;P=0.008).FA-PSV-positive MDS/AML patients had a higher tumor mutation burden,higher rate of cytogenetic abnormalities,less epigenetic regulation,and fewer spliceosome gene mutations than those of FA-PSV-negative MDS/AML patients(P=0.024,P=0.029,P=0.024,and P=0.013).The overall PSV enrichment in our cohort suggests that heterozygous mutations of FA genes contribute to hematopoietic failure and leukemogenesis.

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay

Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we introduced two novel diagnostic methods,including single-cell sequencing and capillary nano-immunoassay.One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied.The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing.There were two homozygous mutations(FANCM/FANCD1).Furthermore,the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins.Abnormal FANCM and FANCD1 expressions simultaneously existed.This case was thus diagnosed as FA-D1/FA-M dual subtype.Compared with mixed cell sequencing,single-cell sequencing data shows more accuracy for the FA subtype evaluation,while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.

DNAH2 facilitates the homologous recombination repair of Fanconi anemia pathway through modulating FANCD2 ubiquitination

Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA.The assays of homologous recombination repair,mitomycin C(MMC)sensitivity,immunofluorescence,and ubiquitination modification were performed in U2OS and DR-U2OS cell lines.In MMC-treated U2OS cells,the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks.We also observed the reduced enrichment of FANCD2 protein to DNA damage sites.Furthermore,the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2.Thus,our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2.DNAH2 may act as a novel co-pathogenic gene of FA patients.

Fanconi’s Anemia—Rare Aplastic Anemia at Ten Year-Old Boy in Mogadishu-Somalia: Case Report

Fanconi’s anemia (FA) alson called Fanconi Pancytopenia is a rare, potentially life-threatening failure of haemopoiesis characterized by aplastic anemia that is associated with a variety of congenital abnormalities (Cafe-au-lait spots, abnormalities of fingers, hyperpigmentation of the skin, short stature, microcephaly, deformities of the ear, hypogenitalism, renal anomalies, etc.) and a high risk of developing of malignancy and chromosomal instability. FA is the first described in 1927 by Guido Funconi reported 3 brothers with pancytopenia and physical anomalies. The diagnosis is based on morphological abnormalities, hematologic abnormalities and genetic tests. The present case report describes a 10 years old Somali boy was diagnosed with a Fanconi anemia after recurrent blood transfusion. Though aplastic anaemia in children is an important haematological disorder, there is no study having been undertaken in Somalia and this is the first reported by the patient with Fanconi’s anemia in Somalia. We report this case to create awareness among clinicians the presence of this disease and have a consideration when it comes differentiatal diagnosis of recurrent blood transfusion patients with pancytopenia because it’s a rare genetic disease in Mogadishu and around the world.

异基因造血干细胞移植联合地西他滨维持治疗成功救治范可尼贫血进展为急性髓系白血病1例报告

目的探讨异基因造血干细胞移植(allo-HSCT)后序贯地西他滨用于治疗范可尼贫血(FA)进展为急性髓系白血病(AML)患儿的可行性及有效性。方法回顾分析1例FA进展为AML患儿接受同胞弟弟作为供体的单倍体移植,移植后予地西他滨维持治疗。结果患儿生后6岁出现贫血,8岁确诊范可尼贫血,按照再生障碍性贫血治疗(环孢素、安雄)6年无效,且进行性三系下降,血小板无效输注,骨髓检查诊断为AML。骨髓二代基因测序检测,仍然表现为FANCA基因c.3348+1G>A纯合变异。遂予FLAG方案(氟达拉滨+阿糖胞苷+粒细胞集落刺激因子)化疗后桥接以TBI为基础的清髓性预处理方案,+15天粒细胞及血小板植入,移植后给予患者减量环孢素,保持Ⅱ度皮排排异直至移植后1年,移植后6个月开始予每2个月予地西他滨,共计6次,同时监测WT1的表达水平。现已经移植后近6年,患儿无事件生存中。结论allo-HSCT后联合地西他滨序贯治疗能够有效治疗FA进展为AML。

范可尼贫血患儿异基因造血干细胞移植治疗21例长期随访分析

目的 探讨儿童范可尼贫血(FA)移植预后及相关危险因素。方法 回顾性分析2016年1月—2019年12月在上海交通大学医学院附属上海儿童医学中心经基因检测确诊FA并进行异基因造血干细胞移植(allo-HSCT)的21例患者。结果 患者起病中位年龄3.8(0.1-10.2)岁,移植中位年龄6.7(0.7-11.2)岁。所有患儿均以全血细胞减少为移植指征,移植前均未发生肿瘤。均应用BU 2天/TBI 3Gy+FC为基础的减低剂量预处理方案。15例患儿接受无关供者移植,5例亲缘供体移植,1例脐血移植。19例采用环孢素A联合甲氨蝶呤,2例采用环孢素A联合吗替麦考酚酯预防移植物抗宿主病(GVHD)。急性GVHD和慢性GVHD累积发生率分别为33%和29%。其中,Ⅲ-Ⅳ度aGVHD累积发生率24%(5例),中重度cGVHD发生率为24%(5例)。中位随访时间为49(2-106.2)个月,总体生存率为62%。死亡原因包括4例死于肠道aGVHD,1例死于肺部cGVHD,1例死于植入失败,1例死于植入物功能不良和1例死于CMV脑炎,未发现继发肿瘤发生。多因素分析提示移植前年龄越大及移植前高血清铁蛋白含量是预后的不良因素(P=0.006&P=0.02)。结论 早期明确诊断,及时开展allo-HSCT治疗有助于改善FA长期治疗效果。

范可尼贫血:从遗传疾病到癌症关联的DNA修复通路探索与治疗展望

范可尼贫血(FA)是一种遗传性疾病,其特征包括骨髓衰竭、发育异常和易患癌症。这种疾病是由基因突变引起的,导致修复DNA链间交联(ICLs)异常。DNA损伤反应失调会导致基因组不稳定,增加突变率和致癌风险。FA通路是DNA损伤应答的重要组成部分,在DNA链间交联修复和基因组稳定性方面发挥着关键作用。任何一个编码FA蛋白的基因胚系突变都会导致FA。随着体细胞癌中FA基因表达异常的普遍发生和不断开展的FA通路激活与化疗耐药相关性的研究,FA通路与癌症之间的联系得到了进一步确认,并且基于FA通路基因缺陷的靶向治疗也在逐步开发和应用。本文综述了FA蛋白在ICLs修复、FA信号网络调节以及其在癌症发病和预后中的重要作用,并探讨了靶向FA途径的小分子抑制剂的潜在应用。

Fanconis贫血病人造血干细胞DNA修复基因hHR21^(sp)表达研究

目的 :研究正常人外周血单核细胞在UV或γ辐射后hHR2 1sp基因的转录表达 ,进一步分析hHR2 1sp基因在fanconisanemia(FA)单核细胞和造血干细胞的转录表达水平及发病机制中的作用。方法 :对正常人外周血单核细胞在UV或γ辐射后不同时间提取细胞总RNA ,通过RT -PCR、southern杂交、以 β -actin为内参照放射影像对hHR2 1sp基因的转录表达水平进行检测 ,进一步检测FA病人外周血单核细胞和骨髓造血细胞hHR2 1sp基因的表达水平。结果 :正常外周血单核细胞在UV或γ -辐射后不同时间hHR2 1sp基因转录表达都有所变化。正常人外周血单核细胞与对照组比较在 80J/m2 UV辐射hHR2 1sp表达于 3h开始增加 ;在 6h表达水平最高 ,与正常对照有 2倍多差异 ,而在 9h表达有所降低 ;而γ -辐射 5Gy辐射 6h增加明显 ,在 9h后有所降低与正常对照有 2倍差异。检测发现FA病人单核细胞与正常人外周血单核细胞hHR2 1sp基因表达无明显差异 ;但FA病人造血干细胞与正常组骨髓细胞相比hHR2 1sp基因表达降低显著 ,有 1倍多差异。结论 :实验结果表明hHR2 1sp在FA病人造血干细胞表达缺陷 ,提示hHR2 1sp基因表达降低可影响FA病人造血干细胞和DNA双链断裂修复功能是FA发病机制之一。

血细胞减少伴多发畸形2例

患儿1,女,10岁,因全血细胞减少伴反复鼻衄就诊,有反复上呼吸道感染史,有皮肤咖啡斑、小头畸形,基因检测发现DNA连接酶IV(ligase IV,LIG4)基因存在复合杂合变异,诊断为LIG4综合征。患儿2,女,6岁,因血小板减少2年余就诊,有身材矮小、皮肤黝黑、手部畸形等,染色体断裂试验检查结果为阳性,诊断为范可尼贫血互补组A型。该2例患儿临床表现相似,最终诊断为两类疾病,提示血细胞减少伴畸形的患儿并非仅是血液病,需警惕免疫系统等其他疾病。

Fanconi贫血家族与白血病

本文报道了2例Fanconi贫血（FA）兄弟，发病于10岁前。都患有全血细胞减少伴皮肤色素沉着，拇指缺如，生殖器发育不良等先天性畸形。经中西医治疗5年后，其中1例转变为急性白血病死亡，另1例一般健康状态良好。FA转变成白血病至今国内未见报道。本例家族的情形说明FA与原发性再障有质的不同。

Fanconi贫血的细胞遗传学研究

作者对５例Ｆａｎｃｏｎｉ贫血患者的外周血淋巴细胞染色体的自发畸变及ＭＭＣ诱变后的染色体畸变进行了观察，发现其自发染色体畸变可见裂隙、断裂、内复制、ＳＣＥ频率增高等，经ＭＭＣ诱变后染色体畸变率明显升高，出现三射体、四射体等异常结构，ＳＣＥ频率骤增。

Fanconi贫血7例染色体畸变及手纹改变

作者对7例Fanconi贫血患者染色体及手纹改变进行了观察.6例作了染色体检查,发现5例有染色体数目改变,呈二倍体2例,超二倍体2例,二倍体及超二倍体同时存在1例.7例都作了手纹改变观察,有通贯手及双手掌褶网状改变者5例,有掌轴t″变化者2例.作者就Fanconi贫血患者临床表现及染色体和手纹改变意义作了简要讨论.

范可尼贫血相关基因和早发性卵巢功能不全的研究进展

早发性卵巢功能不全(premature ovarian insufficiency,POI)是引起女性不孕的重要原因,发病率>1%。范可尼贫血(Fanconi anemia,FA)是一种以骨髓衰竭、出生缺陷、癌症倾向以及细胞对DNA链间交联剂敏感性增加为特征的疾病。FA相关基因以其在DNA链间交联修复的重要作用而闻名,已被证明参与生殖细胞发育,约半数的FA女性生育力受损,而FA男性基本上都不能生育。目前为止已经发现至少22个FA相关基因,FA相关基因缺陷小鼠模型的生育力受到不同程度的影响,轻者生育力下降但仍能孕育后代,重者性腺先天异常,完全丧失生育能力。FA相关基因缺陷引起的不孕病例报道也越来越多。原始生殖细胞增殖和减数分裂过程的异常被认为是引起FA患者不孕的主要原因。有研究表明参与减数分裂、DNA损伤修复及有丝分裂的基因是引起POI的一大基因家族。因此,FA相关基因在卵泡发育及生育力维持方面的作用需要得到重视。