从母系遗传研究肾阴虚型慢性再生障碍性贫血的发病机制**☆

背景：多项研究表明恶性血液病可以出现线粒体的突变,但尚未有关于慢性再生障碍性贫血中线粒体变化的研究。目的：研究肾阴虚和肾阳虚型慢性再生障碍性贫血患者线粒体突变情况,探讨母系遗传的本质——线粒体与肾阴虚型慢性再障发生、发展的关系,以期进一步研究慢性再障的发病机制。方法：收集10例诊断明确的肾阴虚型5例肾阳虚型慢性再生障碍性贫血患者骨髓和口腔黏膜上皮,提取DNA,进行线粒体DNA的全测序,比较线粒体基因。结果与结论：肾阴虚型慢性再生障碍性贫血患者线粒体全测序表明许多患者的突变位点发生在与线粒体氧化呼吸链密切相关的区域,涵盖了还原态烟酰胺腺嘌呤二核苷酸脱氢酶1~2、4~6,细胞色素B等多个线粒体DNA的编码基因。而肾阳虚型慢性再生障碍性贫血患者线粒体突变不明显。提示线粒体基因突变引起的呼吸链酶复合体表达水平的改变,造成细胞能量代谢障碍,可能在造血干细胞衰竭的发生发展中起到关键作用。而这一变化是与肾阴虚——母系遗传息息相关的。     

再生障碍性贫血患者骨髓单个核细胞端粒长度及P53、P21的表达

目的:研究再生障碍性贫血(AA)患者骨髓单个核细胞(BMMNC)的端粒长度以及P53和P21表达水平,探讨它们与AA发病的关系。方法:采用实时荧光定量聚合酶链反应(RT-qPCR)检测60例AA患者[其中非重型再障(NSAA)38例,重型再障(SAA)22例]和25例对照者BMMNC中端粒长度以及P53和P21的mRNA表达情况;采用Western blot法检测P53和P21蛋白表达情况;并进行相关性比较。骨髓活检术检测骨髓造血细胞成分分布情况;流式细胞术检测CD34~+细胞占有核细胞百分比情况。结果:AA患者端粒长度较对照组明显缩短骨髓造血细胞成分及CD34~+细胞占有核细胞百分比较对照组明显减少(P0.05);NSAA、SAA患者与对照组相比端粒长度均明显缩短,骨髓造血细胞成分及CD34~+细胞占有核细胞百分比均明显减少(P0.05);SAA与NSAA比较,端粒长度明显缩短,骨髓造血细胞成分及CD34~+细胞占有核细胞百分比明显减少(P0.05)。AA患者P53和P21的mRNA及蛋白表达水平均较对照组明显升高(P0.05);NSAA和SAA患者P53和P21的mRNA及蛋白表达水平与对照组相比均明显升高(P0.05);SAA患者P53和P21的mRNA及蛋白表达水平较NSAA明显升高(P0.05)。端粒长度与P53表达水平或P21表达水平之间没有相关性(P0.05);P53表达水平与P21表达水平之间呈正相关(P0.05)。结论:端粒长度的改变以及P53和P21可能参与了再障的发病经过,推测可能是通过抑制造血干细胞的增殖和分化,从而引发造血细胞凋亡。     

营养组合物对再生障碍性贫血大鼠基因损伤的修复作用

目的研究营养组合物对再生障碍性贫血(aplastic anemia,AA)大鼠基因损伤修复的影响,探讨其治疗再生障碍性贫血的作用机制。方法选取SD大鼠,随机分为正常对照组,再障模型组,营养组合物高剂量组,中剂量组和低剂量组。留取各组大鼠肝脏、骨髓、脑组织,采用RT-PCR法分析各组织修复基因表达情况。结果(1)营养组合物对再障大鼠肝脏组织中核苷酸切除修复基因Xpc、骨髓和脑组织中核苷酸修复基因Ercc2及碱基切除修复基因Ogg1、MTH1具有损伤修复作用;(2)营养组合物对再障大鼠肝脏、骨髓及脑组织中错配修复基因MSH2、MSH3、MLH1、PMS2具有损伤修复作用;(3)营养组合物对再障大鼠肝脏、骨髓及脑组织中重组修复基因Rad51、Rad52、Xrcc1具有损伤修复作用;(4)营养组合物对再障大鼠肝脏及骨髓SOS修复基因Rec A具有损伤修复作用。结论营养组合物对再障大鼠肝脏、骨髓、脑组织基因损伤具有修复作用,为治疗再生障碍性贫血提供理论依据。     

骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫抑制

背景：骨髓间充质干细胞对再生障碍性贫血患者T细胞增殖的影响国内报道较少,而骨髓间充质干细胞是否通过抑制T细胞增殖实现对再生障碍性贫血患者的免疫调节目前尚无定论。 目的：观察人骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫调节作用。 方法：体外分离培养、扩增人骨髓间充质干细胞并通过形态学特征以及流式细胞术进行表面标志鉴定,将骨髓间充质干细胞分别与正常人和再生障碍性贫血患者外周血提取的T淋巴细胞共培养7 d。应用ELISA法检测各培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素、白细胞介素4及白细胞介素10水平。 结果与结论：再生障碍性贫血组患者培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素水平明显高于正常人(P < 0.05),白细胞介素4、白细胞介素10低于正常人(P < 0.05)。骨髓间充质干细胞可下调白细胞介素2、γ-干扰素表达,同时上调白细胞介素4、白细胞介素10表达,从而调节再生障碍性贫血患者的免疫紊乱。     

重组人γ-干扰素联合白消安诱导建立小鼠重型再生障碍性贫血模型

背景:建立理想的重型再生障碍性贫血动物模型对探究再生障碍性贫血发病机制及筛选有效防治药物尤为重要。目的:应用注射用重组人γ-干扰素联合白消安建立小鼠重型再生障碍性贫血模型。方法:选择健康雌性昆明小鼠60只,随机分为再生障碍性贫血造模组(n=50)和对照组(n=10)。再生障碍性贫血造模组予重组人γ-干扰素1×104 U/d腹腔注射,以白消安18 mg/(kg·d)灌胃,均连续用药7 d;对照组给予同等容量生理盐水灌胃及腹腔注射。比较两组间一般情况、体质量及血细胞计数,并观察再生障碍性贫血造模组小鼠骨髓细胞形态学及骨髓活组织病理学的变化。结果与结论:给药第7天时,再生障碍性贫血造模组小鼠体质量、白细胞、血红蛋白、血小板及网织红细胞计数均较对照组明显下降,差异有显著性意义(P0.05);骨髓细胞形态学及骨髓活组织病理学检查显示再生障碍性贫血造模组小鼠骨髓增生极度减低,非造血细胞团相对易见,油滴明显增多,脂肪空泡明显。提示联合应用重组人γ-干扰素和白消安能成功建立小鼠再生障碍性贫血模型,该造模方法操作简便,费用低,稳定性好。     

端粒缩短与骨髓衰竭性疾病的研究进展

端粒缩短是骨髓衰竭性疾病的重要发病机制,以端粒为诊疗靶点可能成为骨髓衰竭性疾病的研究新方向.端粒缩短与临床常见的获得性骨髓衰竭性疾病,包括再生障碍性贫血、骨髓增生异常综合征和免疫相关性全血细胞减少症,以及部分先天性骨髓衰竭性疾病,包括先天性角化不良和范科尼贫血有关.     

乙酰苯肼、X射线和环磷酰胺联合复制小鼠再生障碍性贫血模型

目的以乙酰苯肼、X射线、环磷酰胺联合应用的方法建立小鼠再生障碍性贫血动物模型。方法BALB/c小鼠,采用皮下注射乙酰苯肼100mg/kg,次日X射线2.0Gy照射后,于试验第5天环磷酰胺80 mg/kg腹腔注射,第15天重复以上步骤但不给予射线处理。观察试验小鼠的血象、骨髓象、造血细胞内的线粒体、造血干细胞集落的形成及肝脏、脾脏的病理变化。结果与正常对照组相比,再障模型组小鼠外周血象三系细胞均有显著降低:促红细胞生成素(EPO)含量升高;骨髓有核细胞数显著降低,造血干细胞集落的形成减低,肝脾出现再障的病理改变。结论本方法建立的再生障碍性贫血小鼠模型,简便,成功率高,外周血和骨髓的病理变化持续、稳定,符合再生障碍性贫血的临床表现。     

营养组合物对再生障碍性贫血小鼠骨髓造血细胞线粒体形态及跨膜电位的影响

目的研究营养组合物对再生障碍性贫血(aplasbic anemia,AA)小鼠骨髓造血细胞线粒体形态及跨膜电位的影响,探讨其治疗再生障碍性贫血的作用机制。方法 BALB/c小鼠100只,适应性喂养1w后,随机分为:正常对照组、再障模型组及不同剂量的营养组合物组。采用皮下注射乙酰苯肼100mg/kg,次日X射线2.0Gy照射后,于试验第5天环磷酰胺80mg/kg腹腔注射,第15天重复以上步骤但不给予射线处理,建立再生障碍性贫血小鼠模型,以铅砖屏蔽施以假照射及单纯等量生理盐水相应部位注射为正常对照组。试验第7天开始,营养组合物高、中、低剂量组小鼠每天灌胃,分别给予1445.55,963.7,674.59mg/(kg·d)营养组合物,直至第45天,颈椎脱臼法处死小鼠,观察试验小鼠的骨髓象、透射电镜观察骨髓造血细胞线粒体超微结构,流式细胞术检测线粒体跨膜电位。结果①骨髓透射电镜显示,再障模型组小鼠造血细胞内线粒体数目较正常对照组明显减少(P0.01),线粒体肿胀变性,呈空泡样变或髓样变,嵴不清楚或消失,内质网及核模有不同程度扩张。而各营养组合物组与再障模型组比较,此类细胞内线粒体数目明显增多,线粒体肿胀变性显著减轻。对照组骨髓造血细胞线粒体结构无明显异常。②各营养组合物组骨髓造血细胞随着营养组合物剂量的升高,线粒体膜电位明显升高,呈量效关系,抑制细胞的凋亡,而再障模型组细胞膜电位明显降低。结论营养组合物能够明显改善AA小鼠骨髓造血细胞线粒体数量和结构,抑制跨膜电位下降及细胞凋亡,此可能为其治疗AA的作用机制之一。     

一个同时携带线粒体DNA A1555G突变和GJB2235delC单杂合突变家系的基因型与听力表型

目的 调查一个同时携带线粒体DNA A1555G突变和GJB2 235delC突变的非综合征型耳聋家系,分析其基因型和听力表型的关系.方法 对家系成员进行临床听力测试,收集家系中8名成员的外周静脉血样本,从白细胞中提取DNA,聚合酶链反应扩增GJB2基因和线粒体DNA(mitochondric DNA,mtDNA)目的 片段,对扩增片段直接测序进行GJB2基因、mtDNA 12S rRNA及tRNASer(UCN)基因突变分析.结果 此家系先证者存在mtDNA A1555G突变和GJB2 235delC杂合突变,听力表型为极重度感音神经性耳聋.其他母系成员携带mtDNA A1555G突变,未发现tRNASer(UCN)基因突变,家系中其他母系成员听力表型为双侧对称高频下降或听力正常.结论 GJB2 235delC单杂合突变可能参与了mtDNA A1555G的听力损害.     

一个同时携带线粒体DNA A1555G突变和GJB2235delC单杂合突变家系的基因型与听力表型

目的 调查一个同时携带线粒体DNA A1555G突变和GJB2 235delC突变的非综合征型耳聋家系,分析其基因型和听力表型的关系.方法 对家系成员进行临床听力测试,收集家系中8名成员的外周静脉血样本,从白细胞中提取DNA,聚合酶链反应扩增GJB2基因和线粒体DNA(mitochondric DNA,mtDNA)目的 片段,对扩增片段直接测序进行GJB2基因、mtDNA 12S rRNA及tRNASer(UCN)基因突变分析.结果 此家系先证者存在mtDNA A1555G突变和GJB2 235delC杂合突变,听力表型为极重度感音神经性耳聋.其他母系成员携带mtDNA A1555G突变,未发现tRNASer(UCN)基因突变,家系中其他母系成员听力表型为双侧对称高频下降或听力正常.结论 GJB2 235delC单杂合突变可能参与了mtDNA A1555G的听力损害.     

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non- coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.      

Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment

Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing loss cases. Approximately half result from mutations in the connexin 26 (Cx26) gene, GJB2, in Caucasian populations. Heterozygous mutations in GJB2 occasionally co-occur with a deletion of part of GJB6 (connexin 30; Cx30). It is estimated that approximately 1% of deafness is maternally inherited, due to mutations in mitochondrial DNA (mtDNA). Few studies have focused on the frequency of mutations in connexins or mtDNA in African American (AA) and Caribbean Hispanic (CH) admixture populations. In this study, we performed bidirectional sequencing of the GJB2 gene and polymerase chain reaction (PCR) screening for the common GJB6 deletion, as well as PCR/RFLP analysis for three mutations in mtDNA (A1555G, A3243G, A7445G), in 109 predominantly simplex AA and CH individuals. Variations found were a 101T > C (M34T; 1/101 cases), 109G > A (V37I; 1/101), 35delG (mutation; 4/101, (3/4) of non-AA/CH ethnicity), 167delT (mutation; 1/101), 139G > T (mutation; E47X; 1/101 homozygote, consanguineous), -15C > T (1/101), 79G > A (V27I; 9/101), 380G > A (R127H; 4/101; Guyana, India, Pakistan ethnicity), 670A > C (Indeterminate; K224Q; 1/101), 503A > G (novel; K168R; 3/101) and 684C > A (novel; 1/101). All but one of the AA and CH patients had monoallelic variations. There were no hemizygous GJB6 deletions in those with monoallelic GJB2 variations. We also did not identify any patients with the three mutations in mtDNA. Bidirectional sequencing of the GJB2 gene was performed in 187 AA and Hispanic healthy individuals. Our results reveal that GJB2 mutations, GJB6 deletions, and mtDNA mutations may not be significant in these minority admixture populations.     

Maternally inherited deafness associated with a T1095C mutation in the mDNA

Hearing loss is a relatively frequent defect in children with a genetic or predisposition basis in about 50% of cases. Mitochondrial DNA (mtDNA)-associated disorder often present with sensorineural hearing loss (SNHL) either in isolation or as a part of a multisystem disorder in adults but the frequency in pediatric cases is unknown. We analysed deafness-related mtDNA mutations in 80 deaf children to assess the relative frequency of alterations in childhood-onset SNHL. In 16 patients in whom maternal inheritance was possible, we screened for new mutations likely to affect mitochondrial protein synthesis. In one child we detected a novel mutation (T1095C) in the 12S rRNA gene. This mutation fulfils the suggested criteria for definition of a disease-related nucleotide variant. No mutations were found in other patients. Although we cannot exclude the presence of still undefined new mtDNA mutations, our data suggest that mtDNA defect are not common in childhood-onset SNHL.     

Mutations in mitochondrially encoded complex I enzyme as the second common cause in a cohort of Chinese patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes

The mutation pattern of mitochondrial DNA (mtDNA) in mainland Chinese patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) has been rarely reported, though previous data suggested that the mutation pattern of MELAS could be different among geographically localized populations. We presented the results of comprehensive mtDNA mutation analysis in 92 unrelated Chinese patients with MELAS (85 with classic MELAS and 7 with MELAS/Leigh syndrome (LS) overlap syndrome). The mtDNA A3243G mutation was the most common causal genotype in this patient group (79/92 and 85.9%). The second common gene mutation was G13513A (7/92 and 7.6%). Additionally, we identified T10191C (p.S45P) in ND3, A11470C (p. K237N) in ND4, T13046C (p.M237T) in ND5 and a large-scale deletion (13025-13033:14417-14425) involving partial ND5 and ND6 subunits of complex I in one patient each. Among them, A11470C, T13046C and the single deletion were novel mutations. In summary, patients with mutations affecting mitochondrially encoded complex I (MTND) reached 12.0% (11/92) in this group. It is noteworthy that all seven patients with MELAS/LS overlap syndrome were associated with MTND mutations. Our data emphasize the important role of MTND mutations in the pathogenicity of MELAS, especially MELAS/LS overlap syndrome.     

Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma

Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive the pathogenesis and progression of human malignancies. Previous investigations have revealed a high frequency of somatic mutations in the D‐loop control region of mtDNA in osteosarcoma. However, little is known with regard to whether or not somatic mutations also occur in the coding regions of mtDNA in osteosarcoma. To test this possibility, in the present study we screened somatic mutations over the full‐length mitochondrial genome of 31 osteosarcoma tumour tissue samples, and corresponding peripheral blood samples from the same cohort of patients. We detected a sum of 11 somatic mutations in the mtDNA coding regions in our series. Nine of them were missense or frameshift mutations that have the potential to hamper mitochondrial respiratory function. In combination with our earlier observations on the D‐loop fragment, 71.0% (22/31) of patients with osteosarcoma carried at least one somatic mtDNA mutation, and a total of 40 somatic mutations were identified. Amongst them, 29 (72.5%) were located in the D‐loop region, two (5%) were in the sequences of the tRNA genes, two (5%) were in the mitochondrial ATP synthase subunit 6 gene and seven (17.5%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, somatic mtDNA mutation was not closely associated with the clinicopathological characteristics of osteosarcoma. Together, these findings suggest that somatic mutations are highly prevalent events in both coding and non‐coding regions of mtDNA in osteosarcoma. Some missense and frameshift mutations are putatively harmful to proper mitochondrial activity and might play vital roles in osteosarcoma carcinogenesis.     

Development of a new multiplex quantitative real‐time PCR assay for the detection of the mtDNA4977 deletion in coronary artery disease patients: A link with telomere shortening

Mitochondrial DNA (mtDNA) and telomere shortening have been proposed as important contributors to vascular disease and atherogenesis. The role of mitochondrial and telomere alterations has been examined frequently, but usually separately. Recently, an integrated model in which DNA damage and metabolic pathways intersect in age‐associated cardiovascular disease has been proposed. In this study we developed a fast and reliable real‐time PCR‐based procedure to investigate relative quantification of the 4,977 bp mitochondrial DNA deletion (also indicated as “mtDNA⁴⁹⁷⁷ deletion”), employing TaqMan probes with a multiplex approach. As a validation of the assay, a nested PCR coamplification was performed. Telomere shortening was evaluated by a real‐time monochrome multiplex PCR technique employing a SybrGreen‐based analysis. The study of mtDNA⁴⁹⁷⁷ deletion and telomere shortening was carried out in atrial biopsies from 11 patients undergoing coronary artery (n = 5) and valve surgery (n = 6). The relative quantifications showed that the amount of mtDNA⁴⁹⁷⁷ deletion was greater in tissue of patients with coronary artery disease (CAD) (P = 0.01) and that telomere length (expressed as telomere length relative to a single copy reference gene) was significantly shorter in tissue of CAD patients, compared to patients without CAD (P = 0.03). Moreover, most conventional risk factors were significantly more frequent in CAD patients, smoking and dyslipidemia having the strongest association with the degree of mtDNA⁴⁹⁷⁷deletion and a significant correlation with telomere attrition (P = 0.02 and P = 0.006, respectively). In conclusion, the present study suggests that mtDNA⁴⁹⁷⁷ deletion and telomere shortening may represent additional and synergic major risk factors for the pathogenesis of CAD and its complications. Environ. Mol. Mutagen. 54:299–307, 2013. © 2013 Wiley Periodicals, Inc.     

Variants in mitochondrial tRNAGlu, tRNAArg, and tRNAThr may influence the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese families with hearing loss

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing loss including severity, age-at-onset, audiometric configuration in these subjects. Penetrances of hearing loss in BJ107, BJ108, and BJ109 pedigrees are 35%, 63%, and 67%, respectively. Mutational analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mitochondrial DNA (mtDNA) variants belonging to haplogroups N, F, and M, respectively. Of these variants, the A14693G mutation in the tRNA(Glu), the T15908C mutation in the tRNA(Thr), and the T10454C mutation in the tRNA(Arg) are of special interest as these mutations occur at positions which are highly evolutionarily conserved nucleotides of corresponding tRNAs. These homoplasmic mtDNA mutations were absent among 156 unrelated Chinese controls. The A14693G and T10454C mutations occur at the highly conserved bases of the TpsiC-loop of tRNA(Glu) and tRNA(Arg), respectively. Furthermore, the T15908C mutation in the tRNA(Thr) disrupts a highly conserved A-U base-pairing at the D-stem of this tRNA. The alteration of structure of these tRNAs by these mtDNA mutations may lead to a failure in tRNA metabolism, thereby causing impairment of mitochondrial translation. Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA mutations. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.     

Leber's hereditary optic neuropathy with 14484 mutation in Central Java,Indonesia

Leber's hereditary optic neuropathy (LHON) is a maternally inherited late-onset form of blindness characterized by acute or subacute bilateral retinal degradation resulting in a permanent loss of central vision. G11778A, C3460A, and T14484C mutations on mitochondrial DNA (mtDNA) are specific for LHON and account for most, but not all, worldwide LHON cases. A six-generation Indonesian LHON family with the T14484C mutation was analyzed. Polymerase chain reaction/restriction fragment length polymorphism analysis showed that all of the maternal lineages had the T14484C mutation in a homoplasmic form. Penetrance of the disease (33.3%) and male predominance (3:1) was similar to other worldwide LHON with the T14484C mutation. The incidence of offspring born to affected mothers was no different from that of unaffected mothers, and the age distribution of cases was no higher than that of asymptomatic carriers. Eight secondary mutations were sought but not detected. The patients of this family belonged to haplogroup M. These findings support the idea that the mtDNA backgrounds involved in the expression of LHON mutations in southeast Asians are different from those of Europeans.T. Nishioka and M. Tasaki contributed equally to this work     

Detection of deafness-causing mutations in the Greek mitochondrial genome

Mitochondrion harbors its own DNA, known as mtDNA, encoding certain essential components of the mitochondrial respiratory chain and protein synthesis apparatus. mtDNA mutations have an impact on cellular ATP production and many of them are undoubtedly a factor that contributes to sensorineural deafness, including both syndromic and non-syndromic forms. Hot spot regions for deafness mutations are the MTRNR1 gene, encoding the 12S rRNA, the MTTS1 gene, encoding the tRNA for Ser^{(UCN)}, and the MTTL1 gene, encoding the tRNA for Leu^{(UUR)}. We investigated the impact of mtDNA mutations in the Greek hearing impaired population, by testing a cohort of 513 patients suffering from childhood onset prelingual or postlingual, bilateral, sensorineural, syndromic or non-syndromic hearing loss of any degree for six mitochondrial variants previously associated with deafness. Screening involved the MTRNR1 961delT/insC and A1555G mutations, the MTTL1 A3243G mutation, and the MTTS1 A7445G, 7472insC and T7510C mutations. Although two patients were tested positive for the A1555G mutation, we failed to identify any subject carrying the 961delT/insC, A3243G, A7445G, 7472insC, or T7510C mutations. Our findings strongly support our previously raised conclusion that mtDNA mutations are not a major risk factor for sensorineural deafness in the Greek population.     

Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement

The activity of deoxyguanosine kinase (DGUOK), a mitochondrial enzyme involved in the anabolism of mitochondrial (mt) deoxyribonucleotides, governs the maintenance of the mtDNA. Deleterious mutations of the DGUOK gene are thus associated with mtDNA depletion and result in combined deficiencies of mtDNA-encoded respiratory chain enzymes. With the aim to estimate the prevalence of DGUOK mutations in a cohort of 30 patients with hepatocerebral disease and combined respiratory chain deficiencies, we studied the DGUOK gene and identified previously unreported mutations in five families. Two patients and their affected sibs, born to non-consanguineous parents, were homozygous for a missense mutation (M1T, and L250S, respectively). One patient presented a homozygous 4 pb insertion (796 insTGAT) and two other patients, and their affected sibs, were compound heterozygous (E165V/L266R and E211G/L266R, respectively). These findings allowed us to propose prenatal diagnosis in two families. In conclusion, we observed a high prevalence of DGUOK mutations (17%) in patients with hepatic involvement and combined respiratory chain deficiencies with hepatic involvement.