Atropisomeric meroterpenoids with rare triketone-phloroglucinol-terpene hybrids from Baeckea frutescens.

Baefrutones A-F (1-6), six new meroterpenoids with rare triketone-phloroglucinol-monoterpene/sesquiterpene frameworks, together with their biosynthetically related intermediate (±)-baeckenon B (7), were isolated from the aerial part of Baeckea frutescens under the guidance of HPLC-Q/TOF-MS2 investigation. Compounds 1-4 represent the first examples of natural meroterpenoids existing as four pairs of inseparable diastereomeric atropisomers (2 : 1, 1H NMR integration) caused by the restricted rotation around the C-6-C-7-C-1' bonds arising from the intramolecular hydrogen bond between C-1 carbonyl and 2'-OH. The discovery of these architectures not only largely enriched the chemodiversity of the meroterpenoid and atropisomer library, but also might be exciting and challenging for asymmetric organic synthesis. Their structures and absolute configurations were established by extensive spectroscopic analysis, X-ray diffraction, and ECD calculations. Compounds 5 and 6 were biomimetically synthesized from 7 and ß-caryophyllene via a regioselective oxidative hetero-Diels-Alder reaction, thus providing access to the construction of the 6/6/9/4 tetracyclic ring system. The anti-inflammatory activities of these meroterpenoids were also discussed.

[Development of an 18 X-InDel multiplex PCR system].

OBJECTIVE: To investigate Insertion/Deletion (InDel) polymorphism on the X chromosome and to screen 18 InDel loci for the Chinese Han population as a forensic DNA typing system auxiliary. METHODS: Eighteen X-InDel markers were selected using the Human Genome Browser and dbSNP database. Multiplex PCR primer pairs of selected X-InDel markers were designed using Primer 3 software and divided into 3 groups according to the amplified fragment length, labeled by FAM, HEX and TAMRA fluorescence dye, respectively. The population genetics research and comparative analysis of Chinese Han nationality and 4 main minorities, the Hui, Wei, Mongol, and Tibetan nationalities, were investigated with the system. RESULTS: A new multiplex genotyping system, named InDel X-18PLEX, was successfully developed and validated, consisted of 18 X-InDel markers on the X chromosome and 1 Amelogenin gender marker. No deviation from Hardy-Weinberg equilibrium expectations was detected in the distribution of genotypes in the 5 investigated ethnic groups. However, there was significant difference between their distributions. From the investigation of Han nationality, high female (0.9999994) and male (0.999 88) overall discrimination power values were obtained, as well as high overall mean exclusion chance values in trios (0.999 992) and in duos (0.99). CONCLUSION: InDel X-18PLEX meets the requirements as a forensic DNA complementary kit, providing effective supplementary analytical tools for difficult cases.

[Forensic application of 30 InDel loci in Han and She nationalities of Eastern China].

OBJECTIVE: To evaluate the forensic application value of 30 insertion/deletion (InDel) loci included in Investigator DIPplex Kit in Han and She nationalities of Eastern China. METHODS: A total of 565 unrelated individuals in Han nationality and 119 ones in She nationality of Eastern China were investigated using Investigator DIPplex Kit. Allele frequencies, population genetics parameters of the 30 InDel loci were statistically calculated. RESULTS: In Han nationality, the mean Ho was 0.413 3, the mean DP was 0.551 1, the mean PIC was 0.320 0. And in She nationality, the mean Ho was 0.389 6, the mean DP was 0.543 3, the mean PIC was 0.310 0. No deviation from Hardy-Weinberg equilibrium was observed in Han and She nationalities (P > 0.05). CONCLUSION: The 30 loci in Investigator DIPplex Kit show good genetic diversity in Han and She nationalities, and could be used as a supplemental tool for some special paternity cases.

Messenger RNA profiling for forensic body fluid identification: research and applications.

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

[Progress in InDel as a new generation of genetic marker].

As forensic DNA typing experienced three generations of genetic marker researching stage, short tandem repeat (STR) has been widely used in forensic identification as a mature tool. Further exploration of the human genome led to the discovery of polymorphism markers of single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel). InDel, which combines the desirable characteristics of previous genetic markers as a new type of genetic marker, has got extensive concern in fields like medical molecular biology and forensic biology. This paper generally reviews the history of research and the corresponding results of InDel along the line of time axis as well as the different aims of these research focusing on the progress in the multiple amplification system with several InDel as the genetic marker (autosomal or X chromosome) in forensic biology and anthropology. Finally, the direction of research in this field and the problems to be solved have been put forward.

[Typing and polymorphism analysis of 16 STR loci on X chromosome].

OBJECTIVE: To develop a PCR-based X-STR kit for typing of 16 X-STR loci and investigate the polymorphisms of the X-STR markers. METHODS: Sixteen STR loci (GATA 165B12, DXS101, GATA 172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA 31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132) located on X chromosome were selected. The primers for multiplex PCR were designed by Primer Premier 5.0 software and labeled by four fluorescences (FAM, HEX, TAMRA and ROX). The developed multiplex PCR system was used for investigating the polymorphisms of the X-STR markers in Han populations. RESULTS: The 16-plex amplification system named IDtyper X-16 was successfully developed and validated. Among the 16 X-STR loci, DXS7133 and DXS7423 were found to be moderately polymorphic and the other 14 X-STR markers were highly polymorphic (P1C > 0.5, H > 0.5). The cumulative discrimination power in females and in males were 0.999 999 999 999 97 and 0.999 999 993 respectively in Han population. The combined power of exclusion in trios and in duos were 0.999 999 93 and 0.999990, respectively. CONCLUSION: The IDtyper X-16 kit is highly valuable in forensic science and is suitable for paternity testing in disputed cases.

[Algorithms of likelihood ratio for discriminating full sibling from half sibling].

OBJECTIVE: To derive the formulae for likelihood ratio calculation in discriminating full sibling from half sibling with single-parent participation or without parent participation. METHODS: Null hypothesis and alternative hypothesis were established for discriminating full sibling from half sibling in two circumstances: two children with single-parent and without parent participation. Conditional probabilities of the genetic evidentiary under null and alternative hypotheses were calculated according to the Bayesian theory. The likelihood ratios were established with the conditional probability under alternative hypothesis division that under null hypothesis, followed with simplification. All the formulae were validated in a real case. RESULTS: While mother or fathers' genetic information available in differentiating full sibling from half sibling, 14 different genotype combinations could be shared by the two detected children at a given locus and the likelihood ratio could be calculated with 5 different formulae respectively. While both parents' genetic information unavailable, 11 different genotype combinations could be shared and the likelihood ratio could be calculated with 7 different formulae respectively. It was validated in a real case that the power of the likelihood ratio method developed for discriminating full sibling from half sibling with single-parent participation was higher than that of the ratio of full sibling index over half sibling index. CONCLUSION: The formulae of likelihood ratio developed are useful for discriminating full sibling from half sibling with single-parent participation or without parent participation.

[The clinical evaluation of piperacillin and sulbactam sodium in treatment of respiratory, urinary tracts and other infections in 579 patients].

OBJECTIVE: To evaluate the clinical efficacy and safety of piperacillin and sulbactam sodium combinations in the treatment of common infections. METHODS: This was a multi-centre, prospective and open study. All subjects from 57 wards caught common infection like respiratory (RTI) or urinary diseases (UTI). The dosages of piperacillin and sulbactam sodium combinations 2.5 g injection were determined according to indications: for adult, 2.5 g or 5 g per time, 2 time/day; for severe or obstinate infection, 2.5 g or 5 g per time, 3 time/day. General information, clinical response pre- and post-treatment, infected locus, drug recipe and protocol, prognosis and adverse reaction were recorded. RESULTS: Data of 579 cases were collected with 388 males and 191 females. The average age was (66.8 ± 17.0) years. There were 500 patients who were suffering with RTI, with 362 cases of pneumonia, 102 of acute exacerbation of chronic bronchitis, and 36 of other infections. There were 50 cases with UTI, with 31 of simple urinary tract infection, and 19 of complex urinary tract infection. In addition, there were 9 cases of combined RTI and UTI, and 20 of other infections including peritonitis. The average duration of anti-microbial for RTI and UTI was (8.65 ± 3.78) days and (7.45 ± 3.46) days respectively with the total efficacy rate was 92.6% and 98.0% respectively for RTI and UTI. The incidence of adverse events was only 0.86% (5 cases), including nausea, rash, itching, ALT elevation and suspected drug induced fever in each one. CONCLUSION: Piperacillin and sulbactam sodium compound had high clinical efficacy and safety in the treatment of common infections including RTI and UTI.

[A primary study of criterion for source identification of gastrointestinal tumor sample with Identifiler multiplex system].

OBJECTIVE: To investigate the criterion for source identification of gastrointestinal tumor based on the number of identical allele (IAn) and the number of matched STR locus with 2 identical alleles (A2) in Identifiler system. METHODS: One hundred and five pairs of gastrointestinal tumor samples and homologous normal samples (TN group) were genotyped with Identifiler system. The numbers of STR locus with genotypic alteration (STRGA) in each tumor were determined by comparing the genotype of the matched STR loci in each pair of samples. According to the limited distribution of IAn and A2, 16 different values of IAn was substituted into the published discriminant functions to obtain the cut-off values of IAn and A2 for source identification of tumor sample. Indices including sensitivity (SEN), specificity (SPE), accuracy (AC), positive predictive value (PPV) and negative predictive value (NPV) for distinguishing tumor from an unrelated individual or a full sibling of the patient were calculated. Concordance of the identification results based on the determined criteria and the definite facts were statistically tested with Kappa index. RESULTS: The total frequency of STRGA was 5.46%. There were 31.43% of the 105 tumor samples carried at least one STR locus with STRGA mutation. According to the Fisher discrimination rules, criteria I (IAn>or=23 and A2>or=8) and criteriall (IAn>or=26 and A2>or=11) meet the requirements of distinguishing tumor sample from an unrelated individual or a full sibling of the patient with tumor, respectively. SEN=0.971 0, PPV=1.000 0, PPV=0.891 9 and Kappa=0.923 5, when the criteria were used to determine the specified relatives. CONCLUSION: Criteria I and criteria II were powerful for distinguishing tumor sample from an unrelated individual or a full sibling of the patient with tumor, respectively, when the Identifiler system was adopted for source identification of gastrointestinal tumor sample.

[A primary study for criteria of full sibling determination with Identifiler system].

OBJECTIVE: To investigate the criteria of the number of identical allele (IAn) and the number of matched STR locus with 2 identical alleles (A2) for full sibling (FS) determination with Identifiler system. METHODS: According to the limited distribution of IAn. and A2, all of the 31 potential values of IAn. were substituted into the published discriminant functions to obtain the cut-off values of IAn and A2 for FS determination, and then 4 different criteria were determined to distinguish 280 FS pairs from 2283 individual pairs, respectively, which had been genotyped with Identifiler system. Cumulative full sibling index (CFSI) of the samples were calculated with ITO method, and 4 different criteria of CFSI (>1, > or =5, > or =20 and > or =100) were also utilized to determinate FS, respectively. Indices including sensitivity (SEN), specificity (SPE), accuracy(AC), positive predictive value(PPV) and negative predictive value (NPV) of the 8 different criteria for FS determination were calculated, respectively. Concordance of FS determination between the criteria based on IAn and A2 and that of CFSI were statistically tested with Kappa index. RESULTS: All the individual pairs, which meet the requirement of (I) IAn > or =15 and A2 > or =4, or (II) IAn > or =16 and A2 > or =3, or (III) IAn > or = 17 and A2 > or =3, or (IV) IAn > or =18 and A2 > or =3, could been concluded as FS. AC, SPE and NPV of the 4 criteria mentioned above and the 4 criteria of CFSI were all over 0.9500 in FS determination. Indices between criterion II and CFSI > or =5, criterion III and CFSI > or =20, criterion IV and CFSI > or =100 were similar with each other and the Kappa indexes of the 3 groups were 0.9049, 0.9204 and 0.9083, respectively. PPV and NPV of criterion III and CFSI > or =20 were all over 0.9500. CONCLUSION: The criterion of IAn > or =17 and A2 > or =3 was feasible and efficient for FS determination with Identifiler system, power of which was similar with the criterion of CFSI > or =20.

[Universal algoritihms for paternity index in trios and its extended application].

OBJECTIVE: To introduce an universal algorithm for kinship index between a baby and a random person with biologic mother reference. METHODS: Based on the formulas of paternity index in trios (PIT), common factors shared in these formulas were deduced following reconstructions of these formulas with the common factors. Universal algorithms for other common kinship indices, such as grandparental index (GI), half sibling index (HSI), avuncular index (AI) and first cousin index (CI1st), were investigated according to avuncular index rule and the coefficient of relationship (r). RESULTS: The common factor shared in the formulas for PI(T) calculation was 1 plus reciprocal of the frequency of the allele with identity by state between the alleged father and the detected baby. Two general formulas for PI(T), GI, AI, HSI and CI1st with biologic mother reference were successfully established with the common factor and r value. CONCLUSION: The calculation was simplified with the universal algorithms for common kinship indices between random person and the baby with biologic mother reference and the batch arithmetic operation with the universal algorithms can be easily realized with programming.

[Differences of DNA methylation profiles in monozygotic twins' blood samples].

OBJECTIVE: To evaluate the potential usefulness of DNA methylation in individual discrimination of monozygotic twins by investigating the differences of DNA methylation profiles in monozygotic twins' blood samples. METHODS: Blood samples from 22 pairs of monozygotic twins were obtained with informed consent. Genomic DNA extracts were bisulfite treated followed by detection with Infinium HumanMethylation27 BeadChip Assays(Illumina, USA). Epigenetic distances between each pair of monozygotic twins and each pair of unrelated individuals of same gender were calculated with Euclidean distance algorithms. Distribution of epigenetic distance in monozygotic twin group was statistically compared with that in unrelated individuals. RESULTS: Difference of epigenetic distance between male and female pairs was not statistically significant in unrelated individual group or in monozygotic twin group (P = 0.0695 and 0.4825, respectively). Epigenetic distance of monozygotic twins was significantly lower than that of unrelated individual pair of same gender (Median: 6.02 vs 7.20, P = 0.0002). However, all the epigenetic distance in monozygotic twin group or in unrelated individuals were significantly higher than 4.00 (P < 0.000 1). CONCLUSION: DNA methylation profiles of monozygotic twin's blood samples were significantly different with each other, which was similar to that in unrelated individuals of same gender. These results indicated that DNA methylation was a useful biomarker in individual discrimination of monozygotic twins.

[Establishment of universal algorithms for commonly used kinship indices between two individuals].

OBJECTIVE: To establish universal algorithms for commonly used kinship indices between two individuals. METHODS: Based on the formulas of paternity index in duos(PID), full sibling index(FSI), half sibling index (HSI), avuncular index (AI), grandparental index (GI) and first cousin index (CI1st) deduced from ITO method, the common factors, 1 plus reciprocal of the frequency of the allele with identity by state between the two individuals, shared in these formulas were abstracted with induction method, following with reconstruction of these formulas with the common factor and the coefficient of relationship (r). RESULTS: A universal algorithm for PI(D), HSI, AI, GI and CI1st, was developed with the common factor and r value according to the heterozygosity of the two individuals. Meanwhile, a group of two formulas for FSI calculation was also established according to the individuals' heterozygosity. CONCLUSION: The universal algorithms for the 6 types of kinship indices are practical in corresponding kinship determination and the batch arithmetic operation with the universal algorithms can be easily programmed.

[Identification of sibling sisters using STR and SNP].

OBJECTIVE: The methods for identification of sibling sisters were explored with detection of genetic markers on autochromosome and X-chromosome. METHODS: Genomic DNA of the sibling sisters were extracted, and 15 STRs on autochromosome and 17 STRs on X-chromosome were genotyped by Sinofiler kit, Mentype Argus X-8 kit and in-house kit of X-STRs, respectively. 11 X-SNPs were genotyped with TaqMan technology. RESULTS: Full sibling relationship of the test samples were confirmed by calculating full sibling index of STRs in autochromosome, which were also supported by the detection of 1-2 same alleles at each locus on X-chromosome. CONCLUSION: In identification of full sibling sister, not only STRs on autochromosome but also polymorphism genetic markers in X-chromosome can be utilized.

[InDel_typer30: a multiplex PCR system for DNA identification among five Chinese populations].

OBJECTIVE: To develop a multiplex PCR system, using insertion/deletion (InDel) polymorphism markers, for forensic DNA identification among Han, Hui, Uighur, Mongolian and Tibetan populations in China. METHODS: Highly polymorphic InDel markers from human autosomes were selected using the Human Genome Browser in Galaxy system and dbSNP database. Multiplex PCR primer pairs of selected InDel markers were designed using Primer 3 software. The multiplex PCR system was developed using a five fluorescence dye labeling system. Genetic polymorphisms of selected InDel markers were investigated using the multiplex PCR system among five populations in China. RESULTS: A new multiplex genotyping system, named InDel_typer30, was successfully developed and validated in this study. The InDel_typer30 system consisted of 30 highly polymorphic InDel markers and 1 Amelogenin gender marker. The average expected heterozygosity of the 30 InDel markers was 0.464, 0.460, 0.453, 0.466 and 0.469 for the Han, Hui, Uighur, Mongolian and Tibetan populations, respectively. The average discrimination power was 0.595, 0.585, 0.586, 0.589 and 0.595 for the Han, Hui, Uighur, Mongolian and Tibetan populations, respectively. The cumulative discrimination power (CDP) were all above 0.999 999 999 996 for the 5 populations. CONCLUSION: InDel_typer30 was a useful forensic DNA identification tool for human identification among Han, Hui, Uighur, Mongolian and Tibetan populations in China.

[Effect of urinary trypsin inhibitor on DNA genotyping in urine samples].

OBJECTIVE: To study the effect of urinary trypsin inhibitor (UTI) on STR genotyping with urinary samples. METHODS: Midstream urine samples of 5 male and 5 female volunteers were collected respectively, sub-packaged, added with different concentration of UTI and stored at -80 degrees C. Genomic DNA was extracted from those urinary samples, of which STR profiles were genotyped with IdentifilerTM kit at 8 different time points. Results of genotyping in urinary samples were compared with those of the homogenous blood control samples and the successful rate of genotyping in different group of urinary samples treated with UTI was determined. RESULTS: Fifteen STR loci included in Identifiler system were all detected in control blood samples and urinary samples stored for 1 day. STR locus loss was observed and all 15 STR loci disappeared in female urinary samples untreated with UTI while those storage periods prolonged to 3 and 9 days, respectively. However, all 15 STR loci could be detected in female urinary samples treated with UTI and stored for as long as 9 days. No STR loci could be detected in male urinary samples preserved without UTI for 7 days while 9 STR loci detected preserved with UTI for 9 days. There was no significant difference among the average detection ratios of STR loci in female urinary samples treated with UTI at concentrations of 0.2, 0.4 or 0.6 microg/mL and stored for 30 days, mean of which was as high as 0.8400 +/- 0.0423, statistically higher than that in male urinary samples (0.1600 +/- 0.0423). CONCLUSION: Detection rate of STR loci in urinary samples preserved with UTI was increased significantly, which results in prolonging the storage periods of urinary samples for personal identification.

[Application of multiple polymorphism genetic markers in determination of half sibling sharing a same mother].

OBJECTIVE: Determination strategies for half sibling sharing a same mother were investigated through the detection of autosomal and X-chromosomal STR (X-STR) loci and polymorphisms on hypervariable (HV) region of mitochondrial DNA (mtDNA). METHODS: Genomic DNA were extracted from blood stain samples of the 3 full siblings and one dubious half sibling sharing the same mother with them. Fifteen autosomal STR loci were genotyped by Sinofiler kit, and 19 X-STR loci were genotyped by Mentype Argus X-8 kit and 16 plex in-house system. Polymorphisms of mtDNA HV-I and HV-II were also detected with sequencing technology. RESULTS: Full sibling relationship between the dubious half sibling and each of the 3 full siblings were excluded based on the results of autosomal STR genotyping and calculation of full sibling index (FSI) and half sibling index (HIS). Results of sequencing for mtDNA HV-I and HV-II showed that all of the 4 samples came from a same maternal line. X-STR genotyping results determined that the dubious half sibling shared a same mother with the 3 full siblings. CONCLUSION: It is reliable to combine three different genotyping technologies including autosomal STR, X-STR and sequencing of mtDNA HV-I and HV-II for determination of half sibling sharing a same mother.

[Source identification of gastric cancer tissue with discriminatory analysis].

OBJECTIVE: To evaluate discriminatory analysis on source identification of gastric cancer tissue based on the number of matched STR locus or identical allele. METHODS: Twenty two pairs of fresh gastric cancer tissue and homologous normal tissue were genotyped with Identifiler kit. Frequencies of STR genotypic alteration (STR(GA)), the number of matched STR locus without identical allele (A0), with 1 identical allele (A1), or with 2 identical alleles (A2) and the number of total identical alleles (IAn) were calculated with counting method. A1, A2 and IAn were evaluated with Fisher discriminant functions to determine the source of each gastric cancer tissue. Effectiveness of the identification of gastric cancer tissue was evaluated with error rate. RESULTS: The total frequency STR(GA) was 3.03% (95% CI: 1.46%-4.88%). There were 31.38% (95% CI: 13.86%-54.87%) of gastric cancer samples carried at least one STR locus with STRGA. It was confirmed by the Fisher discriminant functions that each of the 22 gastric cancer tissue samples came from its homologous normal tissue with an error rate of 0.00%. CONCLUSION: Frequency of STRGA in gastric cancer tissue was high. Fisher discriminant functions based on the number of identical alleles or matched STR loci could be a feasible method for source identification of body for gastric cancer tissue samples.

[X-sNP genotyping using the TaqMan probe technology].

OBJECTIVE: To develop a rapid, accurate and economical real time fluorescence PCR method with TaqMan probe technology to detect the X chromosome single nucleotide polymorphism (X-SNP). METHODS: TaqMan probes and polymerase chain reaction primers were respectively designed according to the 13 X-SNP. Then, the X-SNP were genotyped after the amplification by real time fluorescence PCR. RESULTS: All the loci follow the Hardy-Weinberg equilibrium. The polymorphic information content for 13 distinct loci varied between 0.3497 and 0.3750 while the heterozygosity ranged from 0.4537 to 0.5021. A real time fluorescent PCR method based on TaqMan probe was successfully developed and the results were accordant with those analyzed by DNA sequencing of the 13 X-SNP. CONCLUSION: The allele specific real time fluorescence PCR based on TaqMan probe is a sensitive, simple technology and suitable for rapid analysis of XSNP. All the loci show highly polymorphic and may be potential in forensic genetics.

Novel mitochondrial DNA mutations associated with Chinese familial hypertrophic cardiomyopathy.

1. Hypertrophic cardiomyopathy (HCM) is a genetic disorder that has a complex set of symptoms and potentially devastating consequences. Increasing evidence indicates that mitochondrial DNA (mtDNA) mutations are responsible for the development of HCM, but the mtDNA mutations appear to differ considerably among different populations and regions. 2. In the present study, three families with HCM were found and investigated: one in Shandong province and two in the Chongqing region of China. The entire mtDNA genome from the 18 affected and 66 unaffected family members was sequenced directly and the mtDNA mutations were determined. 3. The frequency of haplogroup M10 was significantly higher in family members with HCM (HCM group) than in unaffected family members (normal group). Three mtDNA mutations were found with a significantly higher frequency in affected individuals than in unaffected family individuals, namely G7697A in the cytochrome c oxidase subunit II gene (P < 0.0001; odds ratio (OR) 227.5; 95% confidence interval (CI) 23.6­2194.8) and T12477C (P = 0.0037; OR 5.6; 95% CI 1.8­17.6) and G13135A in the NADH dehydrogenase 5 gene (P < 0.0001; OR 26.0; 95% CI 6.9­98.3), suggesting that these mutations are probably associated with susceptibility to HCM. In addition, mitochondrial Complex I activity was markedly decreased in the HCM group, suggesting that these mutations most likely affect mitochondrial respiratory function. 4. In conclusion, the results of the present study imply that mtDNA mutations G7697A, T12477C and G13135A are genetic factors that indicate a susceptibility to HCM and that could be used for the large-scale screening of genetic markers as well as the early diagnosis of HCM.