Research progress on standardized clinical outcome of peritoneal dialysis / 中华肾脏病杂志

As a home treatment and economical and practical treatment mode, peritoneal dialysis (PD) is an effective renal replacement therapy for end-stage kidney disease. The number of PD patients in the world is increasing, and prognosis has been significantly improved. However, compared with the general population, the quality of life of PD patients is not satisfactory, and the disease burden is still very high. There is significant heterogeneity in the reports of clinical outcomes of PD in different countries and regions. The heterogeneity seriously affects the validity of clinical research evidence and the continuous improvement of the quality of PD centers. New progress has been made in the study of standardized clinical outcome of PD in recent years. The article reviews the heterogeneity of PD clinical outcome report, standardized clinical outcome classification and core outcome to standardize the report of PD clinical outcome, improve the clinical research quality and management level of PD, and finally improve the prognosis of patients.

Detection of 3243 A/G and 3316 G/A mitochondrial DNA mutations in Nagpur population

The present study aims to detect 3243 A/G and 3316 G/A mitochondrial DNA (mtDNA) mutations in Nagpurpopulation. Total of 142 patients of type 2 diabetes mellitus and 142 healthy control individuals were selected forthe study from Nagpur city. Selected mutations studied using restriction fragment length polymorphism methodand confirmed by DNA sequencing. Results showed that 3316 G/A mt DNA mutation found in seven patientsof type 2 diabetes mellitus with a 4.92% prevalence, however, found absent in healthy control individuals. ChiSquare and Fisher's exact test showed a significant association between healthy control individuals and type 2diabetes mellitus patients detected with 3316 G/A mutation (p ≤ 0.01). ODDS ratio found significant (for 95%CI; p = 0.05) for 3316 G/A mutation. Furthermore, we did not find 3243 A/G mtDNA mutation in the studiedpopulation. Among studied mutations, 3316 G/A mutation in the ND1 gene is a pathogenic mutation that mayresponsible for type 2 diabetes mellitus in the Nagpur population.

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis

BACKGROUND: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. METHODS: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. RESULTS: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. CONCLUSION: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

Entire Mitochondrial DNA Sequencing on Massively Parallel Sequencing for the Korean Population

Mitochondrial DNA (mtDNA) genome analysis has been a potent tool in forensic practice as well as in the understanding of human phylogeny in the maternal lineage. The traditional mtDNA analysis is focused on the control region, but the introduction of massive parallel sequencing (MPS) has made the typing of the entire mtDNA genome (mtGenome) more accessible for routine analysis. The complete mtDNA information can provide large amounts of novel genetic data for diverse populations as well as improved discrimination power for identification. The genetic diversity of the mtDNA sequence in different ethnic populations has been revealed through MPS analysis, but the Korean population not only has limited MPS data for the entire mtGenome, the existing data is mainly focused on the control region. In this study, the complete mtGenome data for 186 Koreans, obtained using Ion Torrent Personal Genome Machine (PGM) technology and retrieved from rather common mtDNA haplogroups based on the control region sequence, are described. The results showed that 24 haplogroups, determined with hypervariable regions only, branched into 47 subhaplogroups, and point heteroplasmy was more frequent in the coding regions. In addition, sequence variations in the coding regions observed in this study were compared with those presented in other reports on different populations, and there were similar features observed in the sequence variants for the predominant haplogroups among East Asian populations, such as Haplogroup D and macrohaplogroups M9, G, and D. This study is expected to be the trigger for the development of Korean specific mtGenome data followed by numerous future studies.

Case studies of two families with MIDD and MELAS: heteroplasmy level in m. 3243A>G mutation and the first report on m.3271T>C mutation in Colombia / Dos familias con midd y melas: nivel de heteroplasmia de la mutación m. 3243a>g y primer reporte de la mutación m.3271t>c en colombia

MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) and MIDD syndrome (maternally inherited diabetes and deafness) are mitochondrial diseases caused in most cases by the same mutation m.3243A> G, which affects the gene MT-TL1. The cases of two families with MELAS are presented here. In the first case, the m.3243A>G mutation was detected and the heteroplasmy level in blood, urine and oral mucosa were determined, finding a great phenotypic variability: the patient had higher heteroplasmy in the three tissues compared against oligosymptomatic relatives, and the mother had high blood sugar levels and hearing loss, suggesting a phenotype near to MIDD. In the second family, the m.3271T>C mutation was detected, which constitutes the first case reported in Colombia. These findings suggest that MIDD and MELAS, often described as distinct entities, are part of the same entity with variable expressivity partially depending on heteroplasmy.

El síndrome MELAS (encefalomiopatía mitocondrial, acidosis láctica y episodios similares a isquemia cerebral) y el síndrome MIDD (diabetes y sordera de herencia materna) son enfermedades mitocondriales producidas en la mayor parte de los casos por una misma mutación: la m.3243A>G que afecta al gen MT-TL1. Se presentan los casos de dos familias con MELAS. En la primera se detecta la mutación m.3243A>G y se determina el nivel de heteroplasmia en sangre, orina y mucosa oral, con lo que se evidencia una gran variabilidad fenotípica: la paciente tenía una mayor heteroplasmia en los tres tejidos en comparación con sus familiares oligosintomáticos y la madre tenía una glicemia elevada e hipoacusia, sugiriendo un fenotipo cercano al MIDD. En la segunda familia se detecta la mutación m.3271T>C, siendo el primer caso reportado en Colombia. Estos hallazgos sugieren que el MIDD y el MELAS, descritos frecuentemente como entidades distintas, hacen parte de una misma entidad con expresividad variable dependiendo en parte de la heteroplasmia.

Association of clinical features with mitochondrial DNA 3243 A to G mutation heteroplasmy levels in patients with maternally inherited diabetes and deafness / 中华内分泌代谢杂志

Objective To summarize the clinical phenotype profiles and mitochondrial DNA mutation in maternally inherited diabetes and deafness ( MIDD ) , and to improve the diagnosis and treatment of this disease in clinical practice. Methods Sixteen patients with MIDD in six families from Peking Union Medical College from 2007 to Dec 2014 were confirmed as carrying the mitochondrial ( mt) DNA 3243 A to G mutation. Sanger sequencing was used to detect the mt DNA 3243 A to G mutation. The peak height G/A ratio was used to determine mutation heteroplasmy levels. Results The patients with early onset of diabetes (35. 0 ± 14. 6 years), deafness, normal or lower body mass index ( BMI) , and maternal hereditary tendency suggested the diagnosis of MIDD. The peak height G/A ratio was significantly different according to the onset age of MIDD [≤25 years (61. 6 ± 20. 17)%;25-45 years (16.59±8.64)%;>45 years(6.37±0.59)%;P<0.01]. The peak height G/A ratio was negatively correlated with the onset age of MIDD(r=-0. 785,P=0. 001). Conclusion Early onset of diabetes with deafness, normal/lower BMI, and maternal hereditary tendency strongly suggests the diagnosis of MIDD. The peak height G/A ratio might provide a simple prediction regarding the onset age and severity of MIDD.

Genetics of Mitochondrial Myopathies

Mitochondrion is an intracellular organelle with its own genome. Its function in cellular metabolism is indispensable that mitochondrial dysfunction gives rise to multisystemic failure. The manifestation is most prominent with tissues of high energy demand such as muscle and nerve. Mitochondrial myopathies occur not only by mutations in mitochondrial genome, but also by defects in nuclear genes or secondarily by toxic insult on mitochondrial replication. Currently curative treatment modality does not exist and symptomatic treatment remains mainstay. Administration of L-arginine holds great promise according to the recent reports. Advances in mitochondrial RNA import might enable a new therapeutic strategy.

Comportamiento de la mutación mtDNA A3243G en dos familas antioqueñas de pacientes diagnosticados con el síndrome MELAS / Behavior of the mtDNA mutation A3243 in two antioquian families of patients with melas syndrome

Introducción: mutaciones en mtDNA causan citopatias mitocondriales, la más común de ellases el síndrome MELAS; la transición A3243G en tRNA de leucina (tRNALeu) se presenta en 80% depacientes. La heteroplasmia, observada en citopatias mitocondriales, consiste en coexistenciade moléculas mutadas y normales en una célula, situación en la cual, dependiendo de su cantidad,afecta su función con expresión clínica variable.Objetivo: evaluar el comportamiento de la cantidad de heteroplasmia de la mutación 3243G ensu expresión clínica y en la dependencia de variantes nucleares.Pacientes y métodos: se buscaron mutaciones en el gen que codifica para el tRNA de leucinapor secuencia y por PCR-RFLP en 34 pacientes, y se tamizó en familiares de los portadores de lamutación. Se tipificaron cuatro Specific Population Allele (SPA) en pacientes y familiares con lamutación A3243G.Resultados: se halló la mutación A3243G en el tRNALeu en dos pacientes, luego de tamizar lamutación A3243G en ambas familias se evaluó la cantidad de mtDNA mutado (MDNA),encontrando que los casos índices de ambas familias presentaron la mayor cantidad de MDNA;en la primera familia se detectó la mutación en 15 miembros que presentaron diversos síntomas.En la segunda familia se detectó la mutación en un miembro con parálisis cerebral, en dos conmigraña y en uno asintomático.Conclusiones: la severidad de los síntomas se correlaciona con la cantidad de MDNA, se encontróademás correlación entre mtDNA mutado (MDNA) y el Índice de Ancestría Amerindio en cadaindividuo (IAA), indicando una posible influencia del contexto nuclear amerindio en lasegregación y replicación mitocondrial.

Mitochondrial DNA mutations cause mitochondrialcytopathies. Among them Mitochondrial Encephalomyopathy,Lactic Acidosis, and Stroke-like episodes(MELA) is the commonest. The transition 3243A>G inthe Leucine tRNA is present in 80% of the patients.Heteroplasmy is observed in mitochondrialcytopathies, characterized by the coexistence of mutantand wild type molecules in a cell. Depending on thelevel of heteroplasmy, function and clinicalmanifestations might result affected.Objective: To test the degree of heteroplasmy of themutation 3243G on its expression (syntoms) andnuclear-variants dependence.Patients and methods: Mutations in the tRNALeu genewere sought in 34 patients by sequencing and PCR-RFLP.Four SPA (specific population alleles) were typed inpatients and their relatives carrying the mutation3243A>G.Results: The mutation 3243A>G in the Leucine tRNAgene was found in two patients. This mutation wasscreened in their relatives and the amount of mutantDNA (MDNA) was assessed. The index cases presentedwith the higher amounts of MDNA in both families. Infamily one, the mutation was detected in 14members, three of which presented with short stature,one with hearing loss, one with type 2 diabetes, 8 withmigraine and one healthy individual. In family two themutation was detected in one member with brainparalysis, two with migraine and one healthyindividual.Conclusions: Severity of the symptoms in patientsaffected with MELAS is correlated with the amount ofMDNA. Furthermore, it was found a correlationbetween MDNA and IAA, suggesting a possible effect ofamerind nuclear ontext in The mitochondrialsegregation and replication.

Heteroplasmy of Mitochondrial DNA Shown in Korean and Mongolian / 체질인류학회지

As characterization of mitochondrial DNA (mtDNA) shows maternal inheritance and exists as more than thousands copies per cell, it is widely used for population genetics and forensic scientific field. However, mitochondrial DNA study has difficulties because heteroplasmy of mtDNA is being reported from coding and control region. In this study, we have analyzed 200 samples to examine heteroplasmy in mitochondrial DNA of Korean and Mongolian. The control region and coding region in mtDNA of blood from Koreans and Mongolians were analyzed with PCR amplication and sequencing. As a result, several heteroplasmy was observed from total 10 positions including 5 positions in coding region and 5 positions in control region, respectively. Moreover, it showed more than one heteroplasmy in coding region from 6 samples in Korean and 17 samples in Mongolian. Interestingly, heteroplasmy at 5178 position was shown in 6 samples among 23 samples. Considering that the position is important for deciding haplogroup D, we suggest that additional analysis on 4883 position needs for correct haplogrouping. Beside, we also found heteroplasmy in the other positions of 204, 4853, or 16249. Therefore, we suggest that it is required of combinatory analysis on several key nucleotide positions to obtain good results when determining mitochondrial haplogroups.

Heteroplasmy Pattern Within D-loop Region of Mitochondrial DNA in Koreans / 대한법의학회지

Mitochondrial DNA (mtDNA) sequencing has been validated as a useful tool in forensics. However, there are several aspects that need to be considered in order for it to be used as firm evidence in a courtroom. Heteroplasmy is one of those. Heteroplasmy means the status that different mtDNA populations exist in a single individual, tissue, cell or even mitochondrion. As the underlying phenomenon of heteroplasmy and polymorphism is similar, care must be taken in analyzing the data from the samples with heteroplasmy. Heteroplasmy can reinforce the use of mtDNA however if we know a lot about it. For this much has to be revealed. In this we screened 494 unrelated Korean using DGGE for the D-loop to determine how frequent heteroplasmy is, and the heteroplasmy pattern was confirmed through sequencing. This paper discussed the results of a comparison with other reports along with several aspects of heteroplasmy.

Intra-individual Difference of Length Heteroplasmy in Blood and Hair Shaft Mitochondrial DNA / 대한법의학회지

To observe mtDNA length heteroplasmy in a homoploymeric cytosine tract of the mitochondrial HV2 region, we carried out size-based separation of PCR products, which was produced by using primers designed to minimize the stutter production. Blood and hair shaft samples were collected from 25 individuals. The result showed significant qualitative/quantitative peak pattern variations among blood and hair shaft mtDNA profiles. Based on the results of this study, an exclusion depended solely on differences in length of the major C-tract variant could thus be an erroneous interpretation. Therefore, differences in the number of cytosine or qualitative/quantitative peak pattern variations in the C-tract of the mtDNA HV2 region cannot be used alone to support an interpretation of exclusion.

DGGE assay for heteroplasmy in mitochondrial DNA / 대한법의학회지

Heteroplasmy in the hypervariable region 1 of the mtDNA was screened using DGGE. Blood samples from 150 unrelated Koreans were used. Appropriate condition was set up using mixed samples with previously known HV1 sequences. This method is capable of detecting heteroplasmic proportion less than 5 %. Multiple bands pattern meaning heteroplasmy was observed in 41 individuals (27.3%). The majority of heteroplasmic variants could not be detected by direct sequencing of PCR products. For the distribution pattern of heteroplasmy in different tissues within one individual, 14 autopsy cases were screened. Out of these, two were heteroplasmic, but there was no difference between various tissues in the heteroplasmic DGGE band pattern.

Interpretation of C tract in the D-Loop of Mitochondrial DNA / 대한법의학회지

The D-loop of mitochondrial DNA contain C tracts(homopoymeric cytosines) in the positions of 16183 -16193, 303 -315, 568 -573 of GI 1944628 of NCBI. When the sequence of these positions are analyzed, the sequencing reactions or their signals after the C tract were abruptly decreased and cannot clarify the nucleotide signals in many cases. It is hard to characterize of the C tract. To characterize the C tract, we tried to analyze the sequence of clone and amp-length polymorphism with silver stain after amplification of small fragments including C tract of 16183 -16193 and 303-315.These results were compared with the results of characterization of nucleotides after separation of each nucleotide signal from the electropherogram which were increased in the Y axis to intensify the small overlapped signals using CHROMAS program. All methods we tried showed similar pattern of heteroplasmies of homopolymeric cytosines. Conclusively, it is not necessary to try resequencing reaction after cloning or amp-length polymorphism using silver stain. Intensification of the small overlapped signals in Y axis and separation of each signal is enough method to characterize the C tract.