Exome sequencing in Thai patients with familial obesity.

Obesity is a major worldwide health issue, with increasing prevalence in adults and children from developed and developing countries. Obesity causes several chronic diseases, including cardiovascular and respiratory diseases, osteoarthritis, hypertension, stroke, type II diabetes, obstructive sleep apnea, and several types of cancer. Previous genome-wide association studies have identified several genes associated with obesity, including LEP, LEPR, POMC, PCSK1, FTO, MC3R, MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B, SIM1, and TNKS/MSRA. However, most of these variants are found in the intronic or intergenic regions, making it difficult to elucidate the underlying mechanisms. Therefore, in this study, we performed a whole exome sequencing of the protein-coding regions in the total genome (exome) of two obese and one normal subject belonging to the same Thai family to identify the genes responsible for obesity. We identified 709 functional variants that were differentially expressed between obese and normal subjects; of these, 65 were predicted to be deleterious to protein structure or function. The minor allele frequency of 14 of these genes (ALOX5AP, COL9A2, DEFB126, GDPD4, HCRTR1, MLL3, OPLAH, OR4C45, PRIM2, RXFP2, TIGD6, TRPM8, USP49, and ZNF596) was low, indicating causal variants that could be associated with complex traits or diseases. Genotyping revealed HCRTR1, COL9A2, and TRPM8 to be associated with the regulation of feeding behavior and energy expenditure. These genes constituted a network of pathways, including lipid metabolism, signaling transduction, immune, membrane transport, and gene regulation pathways, and seemed to play important roles in obesity.

Mitochondrial DNA polymorphism in substantia nigra.

Inefficiencies in mitochondrial respiration mainly affecting complex I and IV activities, occur with increasing age and have been suggested as a possible etiological factor in age-related neurodegenerative diseases. It has been suggested that this finding may be explained by an accumulation of mtDNA mutations. We hypothesise that some polymorphic mitochondrial genomes encode less efficient respiratory protein subunits and are therefore less tolerant of acquired mutations. If this hypothesis is correct, individuals with 'less efficient' mtDNA genotypes may be predisposed both to more rapid biological aging and to neurodegenerative disease. In this study we investigate the substantia nigra mtDNA composition from 4 elderly individuals (2 non-parkinsonian and 2 with idiopathic Parkinson's disease) to determine whether there is sufficient polymorphism to account for different possible respiratory efficiencies. THe mitochondrial tRNAArg, tRNAHis, tRNAScr, tRNALeu(CUN), ND4L, ND4 and ND5 genes as well as parts of the ND3 and ND6 subunit coding regions were analysed (4221 bp), revealing the presence of multiple deletions and 48 discrete polymorphic sites. These included 23 missense, two tRNA and one nonsense polymorphism. Eight of the missense polymorphisms caused nonconservative amino acid replacements at sites of moderate to high evolutionary constraint. These findings suggest that mtDNA diversity in the ageing brain may account for a range of bioenergetic outcomes. The variation in mtDNA genotype involves both inherited (fixed familial) polymorphism and superimposed acquired mutations.

Leber's hereditary optic neuropathy in Thailand.

PURPOSE: To study the clinical features of Leber's hereditary optic neuropathy (LHON) in Thai patients as compared with patients in the United States, Europe, and other Asian countries. METHODS: The blood mitochondrial DNA of patients from 19 Thai pedigree families was studied for LHON mutation by restriction enzyme analysis. RESULTS: Mitochondrial mutation at nucleotide position 11778 was detected in 37 affected patients and 21 unaffected maternal relatives. Ten of the 19 families were sporadic in transmission. The male preponderance in affected patients was 76%. The onset of visual loss ranged from 6 to 53 years of age (mean = 21.5 years). Of the 31 patients whose eyes were affected bilaterally, 48.4% developed visual loss simultaneously. Unilateral visual loss was found in 2 patients but 1 already had a blind eye resulting from trauma. Onset interval between eyes was up to 12 months (mean = 2.3 months). No associated heart disease or neurological disorder was detected in our pedigrees. Hyperemic disc, retinal telangiectasia, and tortuosity of vessels appeared on ophthalmoscopy in 29% of the patients. Final visual outcome was 0.1, or worse in 82.3%, with a mean follow-up period of 19.5 months. CONCLUSION: The clinical features of LHON in Thai patients are similar to those found in patients harboring the 11778 mutation in the United States, Europe, and Japan. However, although there is a male predominance in all populations studied, this is not so marked in the European and Thai populations.

Mitochondrial genome analysis in Kearns-Sayre syndrome.

We analysed the mitochondrial genome of one patient with chronic and progressive bilateral ophthalmoplegia. This patient also had abnormal EKG showing cardiac conduction defects and pigmentary retinopathy, suggestive of the Kearns-Sayre syndrome. On muscle biopsy, with Gomori trichrome stain, the fibers showed an increase in red-staining material in the intermyofibrillary network and the subsarcolemmal region. On electron microscopy, aggregations of abnormal mitochondria were demonstrated, confirming the diagnosis of mitochondrial myopathy. Analysis of mitochondrial DNA (mtDNA) from the patient and her mother showed no deleted mtDNA.

Molecular examination of GH gene deletion in familial growth hormone deficiency.

The human growth hormone gene (GH gene) from nine members of a family with familial growth hormone deficiency was examined. The patients were diagnosed as having growth hormone deficiency clinically and by response to hormonal treatment. PCR amplification was carried out using total DNA extracted from leukocytes. The flanking regions of the GH gene which are highly homologous were amplified by one pair of primers. PCR products of 1900 bp and 1919 bp were obtained. By using the combination of restriction enzymes BgII, HaeII and SmaI to digest these PCR products, the various sizes of GH gene deletion can be detected. None of the possible deletions was found in these patients and their relatives by either PCR or Southern blot analysis.

Seizures in myoclonic epilepsy with ragged-red fibers detected by DNA analysis: a case report.

A 19-year-old Thai woman presented with progressive ataxia and generalized tonic-clonic seizures. Later on, she developed status epilepticus. Blood was tested by molecular DNA analysis which showed A8344G mitochondrial DNA mutation associated with myoclonic epilepsy with ragged-red fibers (MERRF). We confirmed this finding in other members of this family. This is an interesting case report in Thailand of MERRF identified to have A-->G transition mutation at nucleotide 8344 of mitochondrial tRNA(lys) gene without ragged-red fibers from histopathologic studies of muscle. Molecular genetic analysis in suspicious cases of mitochondrial disorders is necessary for proper management and genetic counseling.

Experience of ApoE study in Thai elderly.

UNLABELLED: The association between ApoE E4 and dementia is reported in Alzheimer's disease and other dementia such as in multi-infarct dementia. OBJECTIVES: To examine the association between apolipoprotein E genotype (ApoE) and dementia in Thai elderly and patients to examine the alleles frequencies of ApoE in a Thai population. MATERIAL AND METHOD: Seventy-eight cases and ninety-four controls from a community based population were recruited. Their ages were all over 50 years. Dementia was diagnosed by DSM IV criteria. Blood was taken and stored for DNA extraction and for restriction enzyme analysis of ApoE genotype. Descriptive analysis and odds ratios from SPSS 9.0 program were used in this study. RESULTS: Alleles frequencies of ApoE E2, E, E4 in normal controls were 0.03, 0.80, 0.17 and alleles frequencies of ApoE E3, E4 in dementia subjects were 0.71 and 0.29, respectively. Odds ratios for dementia risk of apolipoprotein genes were as follows: 0.62 for ApoE E3 and 1.98 for ApoE E4. In this study, forty-two dementia subjects had Alzheimer's disease. Fifty nine point five per cent of Alzheimer's disease subjects carried ApoE E4 (positive predictive value is 0.60). CONCLUSION: Thai elderly carry ApoE genotype distribution similar to that reported in other ethnic groups. Bearing ApoE E4 gene increases the risk of developing dementia. The use of ApoE genotyping can only be a diagnostic adjunct for Alzheimer's disease.

Leber's hereditary optic neuropathy (LHON) with mitochondrial ND4 gene mutation (11778) in a Thai patient.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease, characterized by bilateral optic atrophy predominantly in healthy young males. This disorder has shown to be associated with DNA mutation in mitochondrial genome of the patients. We report here a young man who came to the hospital with subacute visual loss in one eye, followed by the other eye within two months. His echocardiogram was normal. A G-->A base substitution at nucleotide position 11,778 which changes a conserved arginine to histidine at amino acid position 340 of ND4, a protein subunit of respiratory chain enzyme complex I in oxidative phosphorylation system, was detected in his leucocyte mitochondrial genome.

An unusual family with Leber's hereditary optic neuropathy and facioscapulohumeral muscular dystrophy.

We performed an observational prospective analysis to study the clinical characteristics as well as a molecular genetic analysis of 17 members of a Thai family who had visual loss and/or muscle weakness. Their blood mitochondrial DNA were examined for the presence of the G11778A Leber's hereditary optic neuropathy (LHON) mutation. Facioscapulohumeral muscular dystrophy (FSHD) DNA analysis was performed in four members who had visual loss. Of 17 family members, the eight members who had the 11778 LHON mutation were all from branch 'a'. Three of these eight members had FSHD with a 17-27-kb deletion of a tandem repeat in the 4q35 subtelomere, and two had been clinically diagnosed as FSHD. Four of six examined members in branch 'b' showed muscular dystrophy clinically diagnosed as FSHD. No correlation of blood DNA analysis between LHON and FSHD in affected members was found. We describe the first family with FSHD and G11778A LHON in which a mutation in mitochondrial DNA at nucleotide position 11778 of branch 'a' was found to be the origin of the mutation.

Tissue segregation of a heteroplasmic mtDNA mutation in MERRF (myoclonic epilepsy with ragged red fibers) encephalomyopathy.

The distribution of the causal 8344A-->G mtDNA mutation has been examined in six tissues of a patient with myoclonic epilepsy with ragged red fibers (MERRF), to study the developmental genetics of this type of mitochondrial disorder, and to determine the pathophysiological importance of the mtDNA heteroplasmy generally observed in such patients. Heteroplasmy of the mtDNA was observed in all six tissues (cerebellum, cerebrum, pancreas, liver, muscle, and heart) suggesting that, whereas the mtDNA mutation is relatively new, the mutated population must have existed before the formation of the three primary embryonic layers. The tissue distribution reveals significant variations in the ratio between the mutated and the normal mtDNA species, indicating the randomness of mtDNA segregation during developmental cell division and differentiation events. The result suggests the existence of tissue-specific nuclear factor(s) that determines the expression of the 8344A-->G mutation in various tissues; in MERRF syndrome, expression is mainly in the central nervous system.

Mitochondrial DNA sequence analysis in congenital myotonic dystrophy.

Mitochondrial genetic modifying factors have been suspected in several autosomally inherited diseases. The congenital variant of myotonic dystrophy, in which there is striking maternal inheritance pattern, is a likely candidate disease. To investigate this possibility, we sequenced completely the mitochondrial genome in 2 patients with congenital myotonic dystrophy. Comparison of the two sequences with control data failed to reveal a specific nucleotide variant or length variant in this disease. We conclude that a mitochondrial genetic modifying factor is not present in congenital myotonic dystrophy.

Significance of mitochondrial DNA deletions in myotonic dystrophy.

Mitochondrial DNA (mtDNA) deletions have been noted in small quantities in a handful of atypical cases of myotonic dystrophy and there are clinical and pathological parallels between this autosomal dominant disease and certain mitochondrial myopathies where such deletions are well recognised. We studied 20 individuals from typical pedigrees of myotonic dystrophy (of whom 19 were clinically affected) with Southern blot analysis, and 2 of the affected individuals with PCR analysis of mtDNA, but were unable to demonstrate the previously noted deletions in any quantity by either method. We conclude that especially in view of known naturally occurring large scale and minor length variants in mtDNA, these previous findings are of dubious relevance to the disease.

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base.

A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.

A tRNA(Lys) mutation in the mtDNA is the causal genetic lesion underlying myoclonic epilepsy and ragged-red fiber (MERRF) syndrome.

Skeletal muscle mtDNA of three patients with mitochondrial encephalomyopathy, characterized clinically by myoclonic epilepsy and ragged-red fiber (MERRF) syndrome, has been sequenced to determine the underlying molecular defect(s). An A-to-G substitution of nt 8344 in the tRNA(Lys) gene, a substitution suggested to be associated with MERRF encephalomyopathy, was detected in these patients. Abnormal patterns of mitochondrial translation products were observed in the skeletal muscle of patients, consistent with the expected consequential defect in protein synthesis. The genealogical studies of the three patients, as well as mtDNA from one published MERRF patient and from nine other normal and disease controls, revealed that the tRNA(Lys) mutations in the MERRF patients have arisen independently. These observations provided evidence that the base substitution is a causal mutation for MERRF.

Heterogeneity in the phenotypic expression of the mutation in the mitochondrial tRNA(Leu) (UUR) gene generally associated with the MELAS subset of mitochondrial encephalomyopathies.

BACKGROUND: Point mutations in the mitochondrial (mt) genome underlie a number of neurological disorders. Some are well defined including the myoclonus epilepsy ragged red fibre syndrome (MERRF) and the mitochondrial encephalopathy lactic acidosis stroke like episode syndrome (MELAS). However, other clinical phenotypes are less distinctive and mitochondrial studies are often included in the workup in complex neurological syndromes of uncertain aetiology. AIMS: We investigated 27 consecutive patients with varied clinical phenotypes referred to our laboratory for mtDNA studies to determine the incidence of recognised point mutations in a patient group with a range of phenotypes including many where mt disease was possible but did not fall into a classical syndrome. METHODS: The recognised point mutations were detected by amplification of the appropriate DNA fragment by PCR followed by restriction-endonuclease digestion of the normal and mutant species. RESULTS: The A-G base substitution mutation at nucleotide (nt)3243 in the tRNA(Leu) gene of mtDNA which is present in the majority of cases of MELAS syndrome, was detected in four cases, only one of whom had typical MELAS symptoms. Their clinical manifestations ranged from mild deafness to a mixture of chronic progressive external ophthalmoplegia symptoms (CPEO) and stroke like episodes. The nt3243 mutation was also identified in one of seven mtDNA deletion negative CPEO cases. CONCLUSIONS: The presentation of the mtDNA mutation at nt3243 appears therefore to be quite variable with some mild phenotypes as well as severe phenotypes observed. In general, the chance of finding a mitochondrial point mutation in a patient with an atypical clinical phenotype is small.

Mitochondrial DNA polymorphism in disease: a possible contributor to respiratory dysfunction.

Intergenomic variation in the human mitochondrial genome was examined in 27 mtDNA sequences using a pairwise analysis technique. Analysis of 16 of these mtDNA sequences from patients with mitochondrial cytopathies indicated a wide range between different mitochondrial genes in the degree of nucleotide variation from the standard Cambridge sequence. Mean complex I polymorphic frequencies in cytopathic (CPEO, MERRF, MELAS and LHON collectively) patients and in LHON patients differed significantly from controls (P < or = 0.05, t). Total mean sequence divergence (mean number of diverging nucleotides between two sequences per 100 bp) over the entire mtDNA coding region was 0.21% for cytopathies (n = 16) as opposed to 0.18% for a control group (n = 4). Within the cytopathy group, the greatest pairwise divergence was observed in ND3 and ND6 subunits of complex I (0.46 and 0.70% respectively) and the magnitude of specific gene divergences differed considerably from those observed for the corresponding genes in the control population. The extent to which the increased variation in ND3 and ND6 is a general phenomenon applicable to all subjects rather than a finding specific to cytopathies cannot be stated with certainty given the small control group. Regardless as to which of these suggestions is correct, the possibility exists that increased nucleotide variation in certain mitochondrial ND subunits may contribute to respiratory inefficiency through a cumulative effect of a series of polymorphisms of minor individual mutagenic potential.

A unique 3.5-kb deletion of the mitochondrial genome in Thai patients with Kearns-Sayre syndrome.

Kearns-Sayre syndrome is one of the neurological diseases caused by a defect in the energy-producing system of mitochondria. Keams-Sayre is known to be associated with a deletion in the mitochondrial genome and is usually detected in muscle biopsies of the patients. In this study, we report the molecular lesion of mitochondrial DNA (mtDNA) in four Thai patients admitted to hospital with encephalomyopathies. The 3.5-kb deletion of mtDNA was detected by Southern analysis, mapped by amplification with five primer pairs covering almost the total mitochondrial genome, and confirmed by PCR primer shift analysis. The deleted position was localized to nt 10208/13765 or nt 10204/13761 spanning the coding area of subunits 3 (ND3), 4L (ND4L), 4 (ND4), and 5 (ND5) of respiratory chain enzyme complex I and the tRNA genes for histidine, serine, leucine, and arginine. The sequence flanking the deletion was a 4-bp repeat of TCCC. All four patients have exactly the same 3558-bp mtDNA deletion; this is the only deleted position in their mtDNA but is different from those reported in the literature. The deletion seems to be found only in Thai patients, although they present with different clinical manifestations and none of them is not related.

A new disease-related mutation for mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) syndrome affects the ND4 subunit of the respiratory complex I.

The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A----G base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNA(Leu), was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A----G base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A----G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.

Rapid and noninvasive screening of patients with mitochondrial myopathy.

In recent years, several point mutations in the mitochondrial genome have been associated with human disease. PCR Polymerase Chain Reaction/restriction endonuclease based techniques provide a reliable method for screening large numbers of specimens for many of the reported mutations. Muscle tissue usually carries the mutations and has been used in earlier studies. We describe a technique for analysis of mtDNA derived from hair follicles for a range of mutations. Both the 3243 A-->G MELAS and 8344 A-->G MERRF mutations were detected in mtDNA from hair follicles. In patients where both muscle and hair were screened, the mutation load was apparently higher in muscle. Furthermore, in patients positive for a given mutation, all the hair follicles analysed were shown to harbour the mutation, although the proportion of wild type to mutant mtDNA was found to somewhat vary. The advantages of this method are (1: six hair follicles provide sufficient mtDNA for analysis of at least 20 different mutations, and (2: specimen collection and transport to a central laboratory are easier than for other tissues. Our studies show that hair follicles constitute a reliable specimen for mitochondrial mutation screening at a diagnostic level.