A3243G Point Mutation Research Articles

#4044 FOCAL SEGMENTAL GLOMERULOSCLEROSIS DUE TO A3243G POINT MUTATION IN THE MTDNA CODING FOR TRNALEU(UUR)

Abstract Background and Aims Mitochondrial Diseases represent a heterogeneous set of maternally inherited diseases that arise by mutations either of the mitochondrial DNA (mtDNA) or in genes of nuclear DNA (nDNA) linked to the cell mitochondrial cross-talk. Many mutations in mtDNA that code for different genes are linked to a wide spectrum of kidney manifestations ranging from focal segmental glomerulosclerosis (FSGS), tubule-interstitial disease (TIN), nephrotic syndrome (NS), and proximal or distal tubulopathy. Renal manifestations of mtDNA disorders are poorly recognized in clinical practice and often misdiagnosed for other conditions like FSGS, Alport Syndrome, steroid-resistant nephrotic syndrome. The guanine for adenine point mutation (A–>G) at position 3243 in the mtDNA has emerged as the most common mutation found in patient with FSGS often accompained by diabetes mellitus and deafness. Focal Segmental Glomerulosclerosis, (FSGS) is the prevalent histologic finding in patients with mtDNA point mutations. Epidemiologic studies have shown that about 1% of diabetic populations in Europe and Japan have this mutation. Method A 34-year-old man presented for evaluation with recently diagnosed Type II diabetes mellitus and serum creatinine of 3.9 mg/dL with nephrotic range proteinuria (3.2 gr/die). His family history included Type II diabetes mellitus, chronic kidney disease, coronary artery disease, and hypertension. The patient medical history included hypertension, dyslipidemia, and sensorineural hearing loss which had initially developed during adolescence. Serum creatinine was reportedly elevated years before this presentation, and urinalysis showed mild proteinuria with no microhematuria, white cells, or casts. Results On evaluation his blood pressure was 150/90 mmHg, presented mild peripheral leg edema. Laboratory results showed serum creatinine 3.4 mg/dl (eGFR 23 ml/min), HbA1c of 7.2%, albumin 2.8 gr/dL. Urinalysis showed protein (3+), glucose (+1) and no hematuria. Urinary protein-creatinine ratio was 3 mg/g. Serum C3 and C4, immunoglobulins, liver enzymes were normal; autoimmunity tested negative (ANCA, Anti-PLA2r, Anti nuclear antobodies). A kidney biopsy was performed. Light microscopy revealed glomeruli with segmental obliteration of glomerular capillaries by collagenous sclerosis (Fig. 1). Both the Jones methenamine silver stain and Masson's trichrome stain (Fig. 1B, C) showed segmental collagen deposition and sclerosis of glomerular capillaries. Tubular atrophy and fibrosis were also present. Immunofluorescence microscopy was negative. A diagnosis of not otherwise specified focal segmental glomerulosclerosis (NOS FSGS) was made. Interestingly electron microscopy showed mitochondrial abnormalities in the tubular cells. In particular, the cytoplasm of a tubular cell appears to be whiffed by mitochondria of enlarged size and altered shape (Fig. 2). Mithocondria appeared dysmorphic with extensive reduction of mitochondrial cristae (Fig. 2 arrows). The patient was screened for mtDNA mutations and guanine for adenine substitution (A–>G) at position 3243 was detected. The patient received a diagnosis of chronic kidney disease with histological features of FSGS due to an A3243G point mutation in the mtDNA with associated diabetes mellitus and sensorineural deafness. Conclusion This case underscores the role of electron microscopy in identifying mithocondrial related nephropathies. Even more important is to evaluate mtDNA mutations in cases with familial FSGS, sensorineural hearing loss, cardiomyopathy and diabetes.

The Spectrum of Maculopathy in Mitochondrial DNA A3243G Mutation: A Case Series of Six Patients.

The mitochondrial DNA (mtDNA) A3243G point mutation encompasses a heterogenous group of disorders including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD), and, rarely, chronic progressive external ophthalmoplegia (CPEO). Regardless of the clinical phenotype, a specific retinopathy has been associated with the presence of this mitochondrial DNA mutation. We present six female patients exhibiting retinopathy of the A3243G point mutation at various stages. Six female patients (37 - 70 years old) with the A3243G point mutation (four MELAS, one MIDD, and oneCPEO) exhibited a maculopathy. Visual acuity ranged from 1/60 to 10/10. Visual field abnormalities varied from minimal decreased sensitivity to absolute central scotomas. They all exhibited, at various degrees, a characteristic pattern of perimacular and peripapillary retinal pigment epithelium (RPE) alterations, with mottled dys-autofluorescence and RPE atrophy and deposits on OCT. The level of visual impairment depended on the foveal involvement and the extension of RPE atrophy. The severity of the maculopathy was not related to age. In the only long-term follow-up (15 years), evolution was slowly progressive. A single mtDNA point mutation at locus 3243 can result in a variety of clinical presentations (MELAS, MIDD, or CPEO). Ocular involvement may manifest as a perimacular/peripapillary RPE atrophy/deposit, which can variably impact central visual function (from asymptomatic to legal blindness). The discovery of such a maculopathy should prompt the ophthalmologist to complete the personal and family history, namely, asking for the presence of diabetes mellitus and/or deafness.

MITOCHONDRIAL DISEASES (Posters): P.183MELAS and reversible cerebral vasoconstriction

A 22-year-old woman was admitted due to fever and severe headache for 10 days and altered mental status and generalized tonic-clonic seizures for 8 days. She had a short stature and hairy body surface, and her mother passed away about ten years ago due to ``encephalitis''. Laboratory data showed elevated lactate in both peripheral blood and CSF. Brain MRI using diffusion weighted imaging revealed hyperintense lesions located in the whole right temporal and occipital lobes and pulvinar of thalamus. MR angiography revealed segmental stenoses at the right P1 and M2-3 junction. Muscle biopsy showed typical RRF and SSV. A genetic study revealed a mitochondrial DNA A3243G point mutation. The patient's clinical symptoms and MRI/MR angiography (MRA) findings improved after the administration of L-arginine, L-carnitine, lipoic acid, vitamin B and dexamethasone. Therefore we speculate the possible pathophysiology just like RCVS (reversible cerebaral vasoconstriction syndrome) involving in the stroke-like episodes of MELAS.

Mitochondrial A3243G mutation results in corneal endothelial polymegathism.

The mitochondrial DNA point mutation A3243G leads to a spectrum of syndromes ranging from MIDD to MELAS. Ocular manifestations include pattern macular dystrophy and concentric perifoveal atrophy. Given the high metabolic demand of corneal endothelial cells, we performed specular biomicroscopy analysis in patients harboring the mitochondrial DNA point mutation A3243G to assess for the associated presence of corneal endothelial abnormalities. We present a case series with participants from two institutions. Patients diagnosed with macular dystrophy associated with MIDD or MELAS, and the mitochondrial DNA point mutation A3243G were recruited. Exclusion criteria included a prior diagnosis, or a positive family history, of endothelial corneal dystrophy. Slit-lamp corneal examination and specular biomicroscopy were performed. Corneal endothelial cell count, cell size and polymegathism, and central corneal thickness were assessed. Patients diagnosed with MIDD or MELAS based on clinical history and examination were genetically tested for the mitochondrial DNA point mutation A3243G using pyrosequencing. Five patients (two male and three female participants) from five different families, and with different ethnic backgrounds, met the inclusion criteria. Their ages ranged from 41 to 60years. Corneal endothelial changes observed using slit-lamp examination were primarily mild to rare guttata. Specular biomicroscopy displayed mainly polymegathism associated with guttata. The average endothelial cell count was 2358±456 cells per mm2, the average endothelial cell size was 442±103μm2 and the average central corneal thickness (CCT) was 551±33μm. These values were similar to that of the average population. The average coefficient of variation (COV), an index of heterogeneity in cell size, was 42.0 ± 4.1%. When compared to the average population, the average COV was significantly higher than predicted for the patients' age. None of the patients had signs of corneal edema. One patient had a pre-Descemet's opacity. In patients with the mitochondrial DNA point mutation A3243G, corneal endothelial polymegathism is present. This is mainly associated with mild guttata. The findings of corneal endothelial cell polymegathism may be a biomarker of mitochondrial disease, specifically in patients with the mitochondrial DNA A3243G mutation.

Renal involvement in MELAS syndrome – a series of 5 cases and review of the literature

Renal dysfunction is increasingly recognized as a potential clinical feature of mitochondrial cytopathies such as mitochondrial encephalomyopathy, lacticacidosis and stroke-like episodes (MELAS) syndrome. Five cases of MELAS syndrome with renal involvement from 4 unrelated families are presented in this case series. Three of the 5 patients had a history of maternally-inherited diabetes and/or deafness. Focal and segmental glomerulosclerosis and arteriolar hyaline thickening were the most striking findings on renal biopsy. In addition to clinical presentation with the typical symptoms of MELAS syndrome, genetic testing in these patients identified the A3243G point mutation in the tRNALeu gene of the mitochondrial DNA (mtDNA). The diagnosis of MELAS syndrome was thus considered to be unequivocal. The incidence of kidney disease in MELAS syndrome may be underestimated although a study is required to investigate this hypothesis. As the A3243G mtDNA mutation leads to a progressive adult-onset form of focal segmental glomerulosclerosis (FSGS), screening for the MELAS A3243G mtDNA mutation should therefore be performed especially in patients with maternally-inherited diabetes or hearing loss presenting with FSGS.

P.17.14 Myohistological mitochondrial changes in patients with mtDNA singular deletions, multiple deletions and A3243G point mutation

Chronic progredient external opthalmoplegia (CPEO) and the MELAS syndrome are the most common disorders that are associated with mitochondrial changes. CPEO is either associated with singular or multiple mtDNA deletions, whereas, MELAS is usually caused by the A3243G point mutation on mtDNA. The basic diagnostic criteria for these diseases are histopathological changes in muscle biopsy. So far, a systematic study comparing myopathological changes in these three groups has not yet been reported. We analysed muscle biopsies from 29 patients (16 males, 13 females) with mitochondrial diseases to investigate whether the genetic background influences the incidence of histopathological changes. 10 patients had singular mtDNA deletions and 11 patients had multiple deletions due to POLG1 nuclear mutations. Additionally, 8 patients harboured the MELAS A3243G point mutation. Freeze-dried sections of muscle biopsies were stained for histochemical reactions with Gomori trichrome, COX/SDH and SDH stainings. Furthermore, immunohistological reaction with COX antibody was also carried out. In all three groups, the COX-negative fibers were observed most frequently. The frequency of RRF was about a third of the COX-negative fibers. Dark blue COX-negative fibers were significantly higher than the hyperreactive fibers in SDH staining. The parallel analysis of individual pathological fibres showed that, in all three groups, all COX negative fibers had pathological changes also in other stainings and immunohistology. The results confirm that the COX/SDH double staining is the most sensitive method in the diagnosis compared to the other two stainings. Nevertheless, these changes could have been occurred due to the high overlap between the three groups in each individual cases.

MELAS and Reversible Vasoconstriction of the Major Cerebral Arteries

A 26-year-old woman was admitted due to an altered mental status and generalized tonic-clonic seizures. She had experienced chronic migraine-like headaches, progressive bilateral hearing loss, a short stature and nephrotic syndrome. Laboratory data showed elevated lactate and pyruvate levels. Brain MRI using diffusion-weighted imaging revealed a hyperintense lesion in the left temporal lobe. MR angiography revealed segmental stenosis at the C1 and M1-2 junction. A genetic study revealed a mitochondrial DNA A3243G point mutation. The patient's clinical symptoms and MRI/MR angiography (MRA) findings improved within four weeks. We herein discuss the possible pathophysiology involving both stroke-like episodes and reversible vasoconstriction.

Human Amniotic Epithelial Cells are Reprogrammed More Efficiently by Induced Pluripotency than Adult Fibroblasts

Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents.

Pulmonary hypertension in a child with mitochondrial A3243G point mutation

Mitochondrial diseases are a group of disorders caused by pathologic dysfunction of the mitochondrial respiratory chain that present with a wide range of clinical expression. Cardiorespiratory complications have previously been described in association with mitochondrial disease; however, pulmonary hypertension has rarely been reported. Pulmonary hypertension is characterized by elevated pulmonary arterial pressure and secondary right ventricular failure. It is a life-threatening condition with a poor prognosis if untreated. We report a case of 3-year-4-month-old boy who had mitochondrial A3243G point mutation with pulmonary hypertension. The unusual features of our case strengthen the concepts of pulmonary hypertension should be considered as another potential manifestation of mitochondrial disease.

The search for mitochondrial tRNALeu(UUR) A3243G mutation among type 2 diabetes mellitus patients in the Nigerian population

The study aimed to compare the incidence of the pathogenic point mutation A3243G in the gene tRNA Leu(UUR) indicating sub-type 2 diabetes mellitus conducted within the Nigerian population with that reported in other populations. 112 patients diagnosed with type 2 diabetes (T2D) mellitus according to the World Health Organization criteria were selected based on family history and re-evaluated for associated disorders from the diabetic clinics in the Northern part of Nigeria. The mtDNA of these patients was extracted and the tRNA Leu(UUR) gene screened for A3243G by PCR-RFLP method. Probands with maternal history were further investigated for other mutations using PCR-sequencing methods. None of the 112 patients were found to carry the A3243G mutation in the mitochondrial tRNA Leu(UUR) gene in the homoplasmic or in the heteroplasmic form. However, C3254T was identified in two of our patients. This mutation was reported to be associated with gestational diabetes and linked with population from sub-Saharan Africa. The A3243G mutation in mitochondrial tRNA Leu(UUR) is not a frequent cause of maternal diabetes in the Nigerian population contrary to other reported populations. However, further screening of an enlarged selected study group is necessary to fully determine the prevalence of this mutation in this population. This further search will help to fully appreciate the prevalence of maternal inheritance and diabetic deafness (MIDD) as extensively reported in other populations. Key words: Maternal diabetes, mitochondrial gene, maternal Inheritance and diabetic deafness, Nigeria, sub-Saharan Africa.

Disruption of Endothelial Tight Junctions in a Patient with Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes (MELAS)

An electron microscopic study revealed disruption of capillary endothelial tight junctions (TJs) in both biopsied muscle, taken at 5 years and 1 month of age, and the autopsied brain, taken at 13 years and 6 months of age, in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and mitochondrial DNA (mtDNA) point mutation A3243G. This endothelial barrier disruption might result in vasogenic edema and systemic lactic acidosis, possibly the critical pathology of MELAS.

Mitochondrial DNA mutation analysis in 97 Chinese patients with mitochondrial cephalomyopathy

To investigate the characteristics of mitochondrial DNA mutations and genotype-phenotype correlations in mitochondrial encephalomyopathies. Biopsy of skeletal muscle and collection of peripheral blood samples were conducted among 97 patients with mitochondrial encephalomyopathies. Southern blotting, PCR-RFLP and direct sequencing of PCR products were performed to search large scale deletions, and common and uncommon pathological point mutations in the muscle and/or blood mtDNA. Seventy-seven patients were identified to be with mitochondrial DNA mutations, including single large deletion (n = 21), multiple large-scale deletions (n = 4), A3243G point mutation (n = 43), A8344G point mutation (n = 6), T8993G mutation (n = 1), T8993C mutation (n = 1), and T3271C mutation (n = 1). Total mtDNA sequencing revealed 4 different novel point mutations in four unrelated patients with isolated mitochondrial myopathy. The type and frequency of mtDNA mutations in this series of Chinese mitochondrial encephalopathies are consistent with those reported abroad, Although there is some association between the genotype and phenotype, heterogeneity in phenotype and genotype is also a prominent feature seen in this series of patients, especially those with A3243G mutation.

Hyperkalemia in familial mitochondrial cytopathy

To contribute to understanding the pathogenesis of hyperkalemia that often occurs in patients with diabetes. We describe 3 familial cases of mitochondrial diabetes mellitus. The mitochondrial A3243G point mutation was confirmed in a mother and her 2 children. We examined their clinical features and pathological findings, and assessed heteroplasmy of mutant mitochondria DNA (mtDNA) by molecular analysis. The second son had spontaneous hyperkalemia and hyporeninemic hypoaldosteronism. Histopathological examination revealed severe tubulointerstitial and vascular changes around the juxtaglomerular apparatus. The mother only showed intermittent hyperkalemia concurrently with the aggravation of heart failure, and the pathological changes in her kidneys were mild. Heteroplasmy was more severe in the second son than in the mother. Heteroplasmy of mitochondrial cytopathy combined with diabetes mellitus led to abnormalities resembling those seen in Type IV renal tubular acidosis.

Characterisation of the macular dystrophy in patients with the A3243G mitochondrial DNA point mutation with fundus autofluorescence

Introduction:The mitochondrial DNA A3243G point mutation is associated with a wide variety of systemic manifestations including a macular dystrophy. The characteristics of fundus autofluorescence (AF) in these patients are distinctive...

A Case of MELAS Syndrome Manifested by Insulin-deficient Diabetes Mellitus

MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) syndrome is characterized by stroke-like episodes before the age of 40, encephalopathy, seizures, dementia and lactic acidosis, and is caused by mutations in mitochondrial DNA. Diabetes mellitus and cardiac involvement are also frequently seen in MELAS syndrome. It is a classic mitochondrial disorder that shows a slow, chronic, progressive course, and presents with multiple organ involvement including the central nervous system, skeletal muscle, eye, cardiac muscle and gastrointestinal system. Here, we report a case of 33 year-old woman with insulin-deficient diabetes mellitus and sensorineural hearing loss experiencing repeated stroke-like episodes. MELAS syndrome with an A3243G point mutation was confirmed by mitochondrial DNA sequencing analysis. (J Korean Endocr Soc 23:444~449, 2008) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

MELAS Masquerading as a Systemic Vasculitis

Mitochondrial encephalomyopathy, lactic acidosis, and stroke (MELAS) is a mitochondrial genetic disorder caused by a point mutation, resulting in the substitution of guanine for adenine at nucleotide 3243 (A3243G). It is a multisystem disorder with variable manifestations and typically presents between the first and third decades of life. It should be suspected if a patient exhibits stroke-like episodes before age 40, encephalopathy characterized by seizures, dementia, or both, and lactic acidosis, ragged-red fibers in muscle, or both. We present the case of a 26-year-old white man suspected with primary central nervous system vasculitis admitted to our facility with profound constipation from severe intestinal dysmotility. Although his gastrointestinal and neurologic symptoms did not meet criteria for a specific vasculitic syndrome, his symptoms and blood test abnormalities were concerning for such a process. MELAS was included in our differential diagnosis because his symptoms failed to fit a defined vasculitic process. When genetic testing documented the presence of the point mutation A3243G, his diagnosis was changed. This case illustrates the importance of considering a mitochondrial genetic disorder in the differential diagnosis of patients who present to Rheumatologists with suspected unusual or atypical vasculitic symptoms.

A Follow-up Study in a Taiwanese Family with Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke-like Episodes Syndrome

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome is often associated with A3243G point mutation of mitochondrial DNA (mtDNA). We previously described a MELAS family characterized by harboring an additional approximately 260 bp tandem duplication in the D-loop and a novel C3093G point mutation in the 16S rRNA gene of mtDNA in the proband. We studied the clinical progression and fluctuation of mtDNA mutations in this Taiwanese MELAS family. We followed up the clinical course in all members of this family (1 proband, her mother and 3 sons) for 12 years. Mutations of mtDNA in serial muscle biopsies of the proband and blood samples and hair follicles taken at different time points from the members of this family were analyzed. The proband developed repeated stroke-like episodes, chronic intestinal pseudo-obstruction, polyneuropathy, progressive renal failure and dilated cardiomyopathy with heart failure. During the follow-up period, the mother and one of the siblings of the proband developed stroke-like episodes at age 62 and 12, respectively. There was no significant difference in the proportions of mtDNA with A3243G mutation among five serial muscle biopsies of the proband. In one carrier (I-2), the proportion of A3243G mutated mtDNA in blood cells was slightly increased with disease progression. This study underlines the importance of early detection of extraneuromuscular symptoms in the members of a family with MELAS syndrome by adequate follow-up. The age of onset of stroke-like episode in MELAS syndrome may be as late as 62 years. We suggest that the manifestations of MELAS syndrome in this family might be associated with the additional approximately 260 bp tandem duplication in the D-loop region and the coexistence of C3093G mutation in the 16S rRNA gene with the A3243G mutation of mtDNA.

Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function

Mitochondrial DNA (mtDNA) mutations cause several diseases, including mitochondrial inherited diabetes and deafness (MIDD), typically associated with the mtDNA A3243G point mutation on tRNALeu gene. The common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta cell functions. However, this assumption has not yet been tested. We used clonal osteosarcoma cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients and containing either exclusively wild-type or mutated (A3243G) mtDNA. According to the importance of mitochondrial metabolism in beta cells, we studied the impact of the mutation on key parameters by comparing stimulation of these cybrids by the main insulin secretagogue glucose and the mitochondrial substrate pyruvate. Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways leading to a high glycolytic rate (2.8-fold increase), increased lactate production (2.5-fold), and reduced glucose oxidation (-83%). We also observed impaired NADH responses (-56%), negligible mitochondrial membrane potential, and reduced, only transient ATP generation. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell calcium handling with increased cytosolic loads (1.4-fold higher), and elevated reactive oxygen species (2.6-fold increase) under glucose deprivation. The present study demonstrates that the mtDNA A3243G mutation impairs crucial metabolic events required for proper cell functions, such as coupling of glucose recognition to insulin secretion.

Detecting of mtDNA Mutations at Position A3243G and G3316A in Pa-tients with type 2 Diabetes Mellitus in Wenzhou

To investigate the frequencies of mitochondria DNA (mtDNA) tRNA(Leu (UUR)) point mutation A3243G and NADH dehydronase subunit 1(ND1) gene point mutation G3316A in Wenzhou area of Zhejiang Province, and to explore the correlation between these mutations and the clinical manifestations in patients with type 2 mellitus diabetes(T2DM). Two hundreds and forty-four unrelated patients with T2DM and 156 healthy subjects without family history of T2DM were enrolled in Wenzhou area in this study and screened for the point mutations mentioned above with polymerase chain reaction (PCR) and restricted fragment length polymorphism(RFLP) analysis. The heterogeneous mutations were confirmed with DNA sequencing and denaturing high performance liquid chromatography (DHPLC) following T-A cloning of PCR products. The percentage of A3243G mutation in group of patients with T2DM and control were 0.410% and 0.0% (1/244 vs 0/156), respectively; however, there's not any significant difference between these two groups in frequency of A3243G mutation (P>0.05). G3316A mutation was detected in 4 of 244 cases with T2DM (1.639%) and 2 of 156 healthy controls (1.282%), showing that there's also no statistic difference between these two groups in frequency of G3316A mutation (P>0.05). It's shown that the frequency of mtDNA tRNA(Leu (UUR)) A3243G mutation is fairly low in patients with T2DM in Wenzhou area. Thus it's reasonable to assume that this mutation may not be involved in the development and progression of T2DM. Furthermore, it's demonstrated that the rate of G3316A mutation of mtDNA ND1 gene is rare in patients with T2DM in Wenzhou area and this mutation also happened in healthy control. It's suggested that G3316A mutation is just a gene polymorphism of mtDNA and not related to the pathogenesis of T2DM.

Another observation with VATER association and a complex IV respiratory chain deficiency

The VATER association of vertebral anomalies (V), anal atresia (A), esophageal atresia and/or tracheo-esophageal fistula (TE), radial and renal anomalies (R) is a common congenital association of unknown origin with probably heterogeneous causes. Here, we report on a girl presenting with pre- and postnatal growth retardation, esophageal atresia, vertebral and costal anomalies and a unilateral radial defect, consistent with the diagnosis of VATER association. In the first month of life, she presented with failure to thrive, severe episodes of hypoglycemia, liver dysfunction and high levels of lactate, which prompted us to perform metabolic screening. A complex IV respiratory chain deficiency (RCD) was diagnosed on a liver biopsy. The respiratory chain defect was not observed in skin fibroblasts. No mtDNA point mutation or deletion was identified. The girl is now 9 years old and has a normal mental development but persistent feeding difficulties and moderate hyperlactatemia. To our knowledge, this is the second report of VATER association with mitochondrial disorder. In a previous report, a VACTERL association was observed in a girl with the mitochondrial A3243G point mutation. The association of VATER phenotype with a mitochondrial disorder may be coincidental but could also suggest that the presence of multiple malformations is the result of the antenatal expression of RCD.