Clinical and genetic analysis of essential hypertension with CYB gene m.15024G>A mutation / 浙江大学学报·医学版

OBJECTIVES@#To explore the role of mitochondrial CYB 15024G>A mutation in the development of essential hypertension.@*METHODS@#Mitochondrial genome sequences of hypertensive patients were obtained from previous studies. Clinical and genetic data of a hypertensive patient with mitochondrial CYB 15024G>A mutation and its pedigree were analyzed. Lymphocytes derived from patient and family members were transformed into immortalized lymphoblastoid cell lines, and the levels of adenosine triphosphate (ATP), mitochondrial membrane potential and intracellular reactive oxygen species (ROS) were detected.@*RESULTS@#The penetrance of this essential hypertension family was 42.9%, and the age of onset was 46-68 years old. Mitochondrial genome sequencing results showed that all maternal members carried a highly conserved mitochondrial CYB 15024G>A mutation. This mutation could affect the free energy of mitochondrial CYB for secondary and tertiary structure and protein folding, thereby changing its structural stability and the structure of the electron transfer function area around the mutation site. Compared with the control, the cell line carrying the mitochondrial CYB 15024G>A mutation showed significantly decreased levels of mitochondrial CYB, ATP and mitochondrial membrane potential, and increased levels of ROS (P<0.01).@*CONCLUSIONS@#Mitochondrial CYB 15024G>A mutation may affect the structure of respiratory chain subunits and mitochondrial function, leading to cell dysfunction, which suggests that the mutation may play a synergistic role in essential hypertension.

A non-invasive method for detecting mitochondrial tRNA / 南方医科大学学报

OBJECTIVE@#To explore the feasibility of detecting maternal hereditary mitochondrial tRNA@*METHODS@#We performed sequence analysis of mitochondrial DNA in blood samples from 2070 cases of maternal hereditary mitochondrial disease in the First Affiliated Hospital of Wenzhou Medical University, and identified 3 patients with m.15927G>A mutation.Buccal swabs and blood samples were obtained from the 3 patients (mutation group) and 3 normal volunteers (control group).After extracting whole genomic DNA from all the samples, the DNA concentration and purity were analyzed.The PCR products were subjected to dot blot hybridization, Southern blot hybridization, and DNA sequencing analysis to verify the feasibility of detecting m.15927G>A mutation using buccal swabs.@*RESULTS@#There was no significant difference in DNA concentration extracted from buccal swabs and blood samples in either the mutation group or the control group (@*CONCLUSIONS@#Buccal swabs collection accurate is an accurate and sensitive method for the detection of m.15927G>A mutation.

Nucleotide modification of mitochondrial tRNA and mitochondrial diseases / 中华医学遗传学杂志

A high proportion of modified nucleotides has been found in mitochondrial tRNA. Such modification can promote accurate folding of tRNA and its stability, while unmodified mitochondrial tRNA may fold into various 2D-structures with impaired functions. Therefore, modification of mitochondrial tRNA is closely related to mitochondrial diseases. Particularly, positions 9, 34, 37, 54 and 55 of the mitochondrial tRNA are critical for such modification. Mutations at these positions are important cause for mitochondrial dysfunction and have been associated with various mitochondrial diseases.

Mutations of mitochondrial tRNAand their connection with hearing loss / 中华医学遗传学杂志

Mitochondrial tRNAgene mutation is closely related to acoustic nerve deafness. Some mutations can affect the structure and transcriptional processing of tRNA, for instance m.7444G>A mutation in tRNAprecursor 3' side, m.7472 insC as well as m.7511T>C mutations in the stem and ring of tRNA, may influence tRNAstability, thus affect the synthesis of mitochondrial peptides, reduce the production of ATP and cause deafness. This article focuses on mitochondrial tRNAgene mutations as well as the mechanism underlying hearing loss.

Progress in research on pathogenic genes and gene therapy for inherited retinal diseases / 中华医学遗传学杂志

Inherited retinal diseases (IRDs), including retinitis pigmentosa, Usher syndrome, Cone-Rod degenerations, inherited macular dystrophy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy are the most common and severe types of hereditary ocular diseases. So far more than 200 pathogenic genes have been identified. With the growing knowledge of the genetics and mechanisms of IRDs, a number of gene therapeutic strategies have been developed in the laboratory or even entered clinical trials. Here the progress of IRD research on the pathogenic genes and therapeutic strategies, particularly gene therapy, are reviewed.

The role of MT-ND1 m.3635G>A mutation in Leber's hereditary optic neuropathy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the role of MT-ND1 m.3635G>A mutation in the pathogenesis of Leber's hereditary optic neuropathy (LHON).</p><p><b>METHODS</b>Biochemical characteristics including the activity of complex Ⅰ, ATP production and oxygen consumption rate among lymphoblastoid cell lines derived from 3 carriers, 3 affected matrilineal relatives of the families and 3 controls were compared.</p><p><b>RESULTS</b>Comparison of mitochondrial functions in lymphoblastoid cell lines of the carriers, patients and controls showed a 51.0% decrease in the activity of complex Ⅰ in patients compared with controls (P<0.05). The m.3635G>A mutation has resulted in decreased efficiency of ATP synthesis (P<0.05). Comparison of oxygen consumption rate showed that the basal OCR (P<0.05), ATP-linked OCR (P<0.05) and the maximum OCR (P<0.05) have all reduced to some extent compared with the controls.</p><p><b>CONCLUSION</b>These results showed that m.3635G>A, as a LHON-associated mutation, can lead to mitochondrial dysfunction.</p>

A novel mutation T8821G in mitochondrial DNA may be associated with Leber's hereditary optic neuropathy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report on clinical, genetic and molecular characterization of two Chinese families with Leber's hereditary optic neuropathy.</p><p><b>METHODS</b>Ophthalmological examinations have revealed variable severity and age at onset of visual loss among the probands and other matrilineal relatives of both families. The entire mitochondrial genome of the two probands was amplified with PCR in 24 overlapping fragments using sets of oligonucleotide primers.</p><p><b>RESULTS</b>The ophthalmological examinations showed that penetrance was 12.5% and 30.0% respectively in the two families. Sequence analysis of the complete mitochondrial genomes in these pedigrees has identified unreported homoplasmic T8821G mutation in the ATPase 6 gene and distinct sets of polymorphisms belonging to haplogroups M10a. The T8821G mutation has occurred at the extremely conserved nucleotide (conventional position 99) of the ATPase6. Thus, this mutation may alter structural formation of ATPase6, thereby leading to failure in the synthesis of ATP involved in visual impairment.</p><p><b>CONCLUSION</b>Above observations have suggested that the ATPase6 T8821G mutation may be involved in the pathogenesis of optic neuropathy in these families.</p>

Identification of mitochondrial DNA ND1 T3866C mutation in three ethnic Han Chinese families affected with Leber's hereditary optic neuropathy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report on the clinical, genetic and molecular characteristics of three ethnic Han Chinese families affected with Leber's hereditary optic neuropathy (LHON).</p><p><b>METHODS</b>The three families were all diagnosed with LHON. Ophthalmologic examinations were conducted on the probands . The ND1, ND4 and ND6 genes of the mitochondrial DNA (mtDNA) were amplified with PCR respectively for the screening of three primary mutations G3460A, G11778A and T14484C. The entire mtDNA of the probands were also amplified by PCR.</p><p><b>RESULTS</b>Analysis of mtDNA in the three pedigrees has failed to find the presence of the three LHON associated mutations but presence of a homoplastic ND1 T3866C mutation in all probands and their matrilineal relatives . The probands had different levels of visual impairment. The penetrance in the three families has been calculated as 12.5%, 11.1% and 33.3%, respectively. The T3866C mutation has resulted in replacement of isoleucine at position 187 with theronine. The isoleucine at position 187 is located at one of the transmembrane domains of ND1 polypeptide.</p><p><b>CONCLUSION</b>Above results have suggested that the ND1 T3866C mutation might have been involved in the pathogenesis of LHON in the three Chinese families studied.</p>

Mitochondrial tRNA(Thr)T15943C mutation may be a new position that affects the phenotypic expression of deafness associated 12s rRNA A1555G mutation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify secondary mutations associated with deafness in a Chinese family affected with deafness.</p><p><b>METHODS</b>The family has been subjected to clinical and molecular analyses, in addition with measurement of reactive oxygen species and doubling time after establishment of immortalized lymphocyte cell lines.</p><p><b>RESULTS</b>The results showed that the hearing loss level and audiometric configuration were discrepant among the family members with maternally transmitted hearing loss. The penetrance of hearing loss in this family was respectively 66.7% and 44.4% when aminoglycoside-induced hearing loss was included or excluded. Analysis of whole mitochondrial genome has found 33 variants as previously reported polymorphisms, except for a 12s rRNA A1555G mutation and a tRNA(Thr)T15943C mutation. Haplotype evolutionary tree has verified that this family belonged to East-Asian haplogroup F. 15943 position was located on the T-stem of the tRNA(Thr), which has destroyed the extremely conserved T-A base pair when T changed to C at this position. However, functional experiments indicated that the population doubling time in special galactose and glucose were longer, whilst the level of reactive oxygen species has increased. Compared with the control cell line groups and a family only carrying the 12s rRNA A1555G mutation, all of the three groups belonged to the same haplogroup.</p><p><b>CONCLUSION</b>Mitochondrial tRNA(Thr)T15943C mutation may act as a potential modifying factor and interact with 12s rRNA A1555G mutation, and thereby enhance the penetrance and expression of deafness.</p>

[Mitochondrial genome analysis in the probands of six Chinese families with MELAS].

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a genetically heterogeneous disorder. The most prevalent mitochondrial DNA (mtDNA) mutation associated with MELAS is the m.3243A>G transition in the mitochondrial tRNA(Leu(UUR)) gene. Here, we report the clinical, genetic and molecular characterization of six probands from Han Chinese families with MELAS. Four of six probands carried the heteroplasmic m.3243A>G mutation. The levels of mutation load ranged from 29% to 59%, which were correlated with the severity of the clinical phenotypes. Two probands with MELAS/Leigh overlap were 3243 A>G negative, whose severity and relapse were greater than the other 4 probands. One proband with MELAS/Leigh harbored the known ND5 m.13094T>C mutation, which is related to MELAS/Leigh overlap and cerebella ataxia. Sequence analysis of entire mtDNA showed the distinct sets of variants including some variants that may be associated with diabetes, hearing loss, seizures, cardiomyopathy, and Leigh syndrome. Our data suggested that the phenotype and severity of MELAS mainly depend on the mutation load, and some variants may partially contribute to the phenotype and diversity. Our finding also highlighted the complexity of the relationship between genotype and phenotype in MELAS.

Mitochondrial DNA mutation associated with hypertension in tRNA(Ile) and tRNA(Gln) genes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the relationship between mitochondrial DNA (mtDNA) mutations and hypertension.</p><p><b>METHODS</b>Clinical data of two pedigrees with maternally transmitted hypertension was collected. Whole mtDNA sequence was analyzed.</p><p><b>RESULTS</b>The family members on the maternal side presented with various levels of hypertension, with the onset age ranging from 44 to 55 years old. Analysis of the mtDNA sequence of the two families members showed all patients have carried a matrilineal 4329C> G mutation of the tRNA(Ile) and tRNA(Gln) genes. The same mutation was not found in 366 healthy controls. The 4329C site of mtDNA is highly conserved across species, and has been associated with the fidelity of amino acid accept arm of the tRNAs, as well as functionality and stability in the formation of tRNAs.</p><p><b>CONCLUSION</b>The 4329C> G point mutation in tRNA(Ile) and tRNA(Gln) probably has contributed to the pathogenesis of hypertension, possibly in association with other modifying factors.</p>

Function study of non-syndromic deafness associated mitochondrial 12S rRNA A839G mutation / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the correlation between nonsyndromic deafness and mitochondrial 12s rRNA A839G mutation.</p><p><b>METHODS</b>According to the clinical manifestations of mitochondrial DNA sequencing and analysis to find and determine family containing mitochondrial 12s rRNA A839G mutation. Harvested its family members blood and transferred their lymphocytes into lymphoblastoid cell lines, followed by cells cultured, cell doubling experiment, susceptibility testing, cellular oxygen consumption rate experiment, ROS and mitochondrial membrane potential experimental tests were progressed to explore the correlation between the A839G mutation and nonsyndromic deafness.</p><p><b>RESULTS</b>The mitochondrial 12s rRNA A839G mutation pedigrees were determined through the full sequence detections of the Mitochondrial DNA, further phylogenetic analysis showed that 839 point conservative index (CI) up to 78.6%; in RPMI-galactose medium containing A839G gene mutant cell line, the doubling time was significantly longer than the control group, and the difference was significant (P = 0.033). The effect to cell lines containing the A839G mutation of aminoglycoside drugs was not obvious. When compared with the control group, cell lines containing the A839G mutation significantly reduced cellular oxygen consumption rate(P = 0.033); compared with the control group, the ROS levels of cell lines containing the A839G mutation appeared more substantial elevated with significan difference (P < 0.01). The mitochondrial membrane potential of cells of experimental group was significantly reduced than the control group.</p><p><b>CONCLUSION</b>The present study proved that the mitochondria 12s rRNA A839G mutations affect the function of the mitochondrial respiratory chain at the cell level, which might reduce the growth rate of the mutant cell lines, result in hearing.</p>

Multiplex allele-specific PCR assays for the identification of mitochondrial 12S rRNA mutations / 中华检验医学杂志

Objective To investigate the clinical application of multiplex allele-specific PCR assays for simultaneous detection of the mitochondrial 12S rRNA A1555G and C1494T mutations associated with aminoglycoside-induced hearing impairment.Methods Three standard plasmids of different genotypes (wild-type, A1555G mutant and C1494T mutant) were constructed for templates and allele-specific primers aiming directly at wild-type and mutant of mitochondrial DNA nt1555 and nt1494 were designed for developing a multiplex allele-specific PCR technique to detect the A1555G and C1494T mutations.Then the method was applied to clinical screening of 138 non-syndromic hearing loss subjects and confirmed by DNA sequencing.Results Multiplex allele-specific PCR was successfully applied to the detection of A1555G and C1494T mutations in a cohort of 138 Han Chinese genetically unrelated hearing-loss subjects.Finally, 11(7.97%) unrelated affected subjects harbored the A1555G and C1494T mutations in the 12S rRNA gene(10 cases for A1555G and 1 cases for C1494T), which was well consistent with results of DNA sequencing [7.97%(11/138), Kappa=1.000, P＜0.01].Conclusion This study indicates that the multiplex allele-specific PCR assay is useful, convenient and reliable in the detection of the A1555G and C1494T mutations, which could identify the subjects at risk and effectively prevent of aminoglycoside-induced hearing loss.

Voltage-dependent anion channel (VDAC) is involved in apoptosis of cell lines carrying the mitochondrial DNA mutation.

BACKGROUND: The mitochondrial voltage-dependent anion channel (VDAC) is increasingly implicated in the control of apoptosis. We have studied the effects the mitochondrial DNA (mtDNA) tRNAIle mutation on VDAC expression, localization, and apoptosis. METHODS: Lymphoblastoid cell lines were derived from 3 symptomatic and 1 asymptomatic members of a family with hypertension associated with the A4263G tRNAIle mutation as well as from control subjects. Mitochondrial potential (DeltaPsim) and apoptosis were measured by flow cytometry; co-localization of VDAC and Bax was evaluated by confocal microscopy. RESULTS: Expression of VDAC and Bax in mtDNA cell lines was found to be increased compared to controls, while expression of the small conductance calcium-dependant potassium channel (sKCa) was unchanged. Confocal imaging revealed co-localization of VDAC/Bax on the outer mitochondrial membrane of A4263G cell lines but not from controls. Flow cytometry indicated that the mitochondrial potential was decreased by 32% in mutated cells versus controls while rates of apoptosis were increased (P < 0.05). The difference was attenuated by Cyclosporin A (CsA, 2 muM), a blocker of VDAC. CONCLUSION: We conclude that increased expression of mitochondrial VDAC and subcellular co-localization of VDAC/Bax increases mitochondrial permeability and apoptosis in cell lines carrying the mtDNA tRNAIle A4263G mutation.

Variations of mitochondrial gene ATP6 in type 2 diabetes mellitus / 中华检验医学杂志

Objective To investigate the correlation between the variations of mitochondrial gene ATP6 and type 2 diabetes mellitus ( T2DM ) and chronic complications. Methods Genomic DNA were extracted from 254 T2DM patients and 165 age-matched controls. After amplification of ATP6 by PCR and direct sequencing, all sequences were compared with the reference sequence (rCRS) to find out the variations. Bioinformatics and statistic method were used to analyze these variations. Results Many variations were detected respectively in T2DM patients and controls, a part of them only appeared in T2DM patients in low frequency, which has not been reported previously. Most of these variations are located in thethird and forth transmembrane helix of ATP synthase subunit 6 (ATPase6). Interestingly, these variationsalmost were detected in the non-obese T2DM patients with hypotension, including G8557A, A8563G,T8594C, C8609T, A8689G, G8998A and G9139A. Conclusions There were many variations in geneATP6 and must of them are mitochondrial SNP, while variations A8689G, T8825C, G8920A, G8998A andG9139A may be mild mutations which my increase the susceptibility of T2DM. G8557A, A8563G,T8594C, C8609T, A8689G, G8998A and G9139A may be associated with the biogenetics diseases suchdiabetes and hypertension.