Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function.

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.

HACE1 negatively regulates neuroinflammation through ubiquitylating and degrading Rac1 in Parkinson's disease models.

Neuroinflammation plays an important role in neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease. HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) is a tumor suppressor. Recent evidence suggests that HACE1 may be involved in oxidative stress responses. Due to the critical role of ROS in neuroinflammation, we speculated that HACE1 might participate in neuroinflammation and related neurodegenerative diseases, such as PD. In this study, we investigated the role of HACE1 in neuroinflammation of PD models. We showed that HACE1 knockdown exacerbated LPS-induced neuroinflammation in BV2 microglial cells in vitro through suppressing ubiquitination and degradation of activated Rac1, an NADPH oxidase subunit. Furthermore, we showed that HACE1 exerted vital neuronal protection through increasing Rac1 activity and stability in LPS-treated SH-SY5Y cells, as HACE1 knockdown leading to lower tolerance to LPS challenge. In MPTP-induced acute PD mouse model, HACE1 knockdown exacerbated motor deficits by activating Rac1. Finally, mutant α-synuclein (A53T)-overexpressing mice, a chronic PD mouse model, exhibited age-dependent reduction of HACE1 levels in the midbrain and striatum, implicating that HACE1 participated in PD pathological progression. This study for the first time demonstrates that HACE1 is a negative regulator of neuroinflammation and involved in the PD pathogenesis by regulating Rac1 activity. The data support HACE1 as a potential target for PD and other neurodegenerative diseases.

SUMO1-modified DNA methyltransferase 1 induces DNA hypermethylation of VWC2 in the development of colorectal cancer.

Aberrant DNA methylation of genes is closely linked to many aspects of tumor development. This study focuses on the effect of DNA hypermethylation of von Willebrand factor C domain containing 2 (VWC2) on colorectal cancer (CRC) progression and the underpinning mechanism. According to data in the bioinformatic systems, VWC2 had the highest degree of DNA methylation in colonic adenocarcinoma, and it showed DNA hypermethylation in rectal adenocarcinoma as well. CRC and the para-tumorous tissues were collected from 86 patients. VWC2 was expressed at low levels in CRC samples and inversely correlated with tumor stage and tumor biomarker expression. DNA hypermethylation and reduced expression of VWC2 were also detected in CRC cell lines HCT-116 and HT29. VWC2 overexpression suppressed the malignant growth of cells in vitro and in vivo. Co-immunoprecipitation and western blot assays showed that small ubiquitin-like modifier 1 (SUMO1) mediated SUMOylation of DNA methyltransferase 1 (DNMT1) and strengthened its protein stability, which promoted DNA methylation and suppression of the VWC2 gene. In summary, this study demonstrates that SUMO1-mediated activation of DNMT1 induces DNA methylation and downregulation of VWC2 in CRC to augment cancer development.

Long non-coding RNA GAS5 overexpression inhibits M2-like polarization of tumour-associated macrophages in SMCC-7721 cells by promoting PTEN expression.

Tumour-associated macrophage (TAM) polarization is associated with hepatocellular carcinoma but the molecular mechanism of this polarization is still unknown. Peripheral blood mononuclear cells were induced to differentiate into M0, M1 and M2 macrophages and TAMs. TAMs were transfected with pcDNA3.1-GAS5, pcDNA3.1-NC, si-GAS5, si-PTEN or si-Ctrl. A human liver cancer cell line (SMCC-7721) was incubated with the modified TAM supernatant. Quantitative real-time PCR and Western blot were performed to detect gene and protein expression. The cell proliferation and invasion properties of the SMCC-7721 cells were detected by MTT and Transwell assays. GAS5 is up-regulated in M1 macrophages and down-regulated in M2 macrophages and TAMs. GAS5 overexpression promoted M1-like polarization of TAMs and inhibited M2-like polarization of TAMs. Moreover, GAS5 promoted the expression of PTEN in TAMs. PTEN-silenced TAM supernatant treatment promoted cell proliferative and invasive properties of the SMCC-7721 cells and diminished the effect of GAS5-overexpressed TAM supernatant on the cell proliferation and invasion by SMCC-7721 cells. Our results demostrared that GAS5 overexpression inhibited M2-like polarization of TAMs by enhancing PTEN expression, thereby inhibiting cell proliferation and invasion by SMCC-7721 cells. Thus, our results suggest that GAS5 may be a new therapeutic target for HCC treatment.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

Transition to next generation analysis of the whole mitochondrial genome: a summary of molecular defects.

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed "Next-Generation Sequencing" (NGS) technology offers a robust high-throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS-NGS cohort of 391 patients, 21 mutation-positive cases (5.4%) have been identified. The mtWGS-NGS provides a one-step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

Biochemical analysis of human POLG2 variants associated with mitochondrial disease.

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.

SURF1-associated Leigh syndrome: a case series and novel mutations.

Leigh syndrome (LS) is a mitochondrial disease that typically presents in infancy with subacute neurodegenerative encephalopathy. It is genetically heterogeneous, but mutations in the complex IV assembly genes, particularly SURF1, are an important cause. In this study, SURF1 gene was sequenced in 590 patients with clinical suspicion of LS, complex IV deficiency, or clinical features of mitochondrial disorders. We identified 21 patients with clinical features of LS who are either homozygous or compound heterozygous for SURF1 mutations. Twenty-two different mutations were identified, including 13 novel mutations. Of the 42 mutant alleles, 36 (86%) are null mutations (frameshift, splicing, or nonsense) and 6 (14%) are missense. We have also reviewed the previously reported SURF1 mutations and observed a clustering of mutation in exon 8 of SURF1, suggesting a vital function for this region. Although mutations in SURF1 have been mainly associated with typical LS, five of the patients in this report had an atypical course of LS. There is no definite genotype-phenotype correlation; however, frameshift mutations resulting in protein truncation closer to the C-terminus may carry a better prognosis.

An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory's experience.

PURPOSE: The mitochondrial genome is highly polymorphic. A unique feature of deleterious mitochondrial DNA (mtDNA) mutations is heteroplasmy. Genetic background and variable penetrance also play roles in the pathogenicity for a mtDNA variant. Clinicians are increasingly interested in requesting mtDNA testing. However, interpretation of uncharacterized mtDNA variants is a great challenge. We suggest a stepwise interpretation procedure for clinical service. METHODS: We describe the algorithms used to interpret novel and rare mtDNA variants. mtDNA databases and in silico predictive algorithms are used to evaluate the pathogenic potential of novel and/or rare mtDNA variants. RESULTS: mtDNA variants can be classified into three categories: benign variants, unclassified variants, and deleterious mutations based on database search and in silico prediction. Targeted DNA sequence analysis of matrilineal relatives, heteroplasmy quantification, and functional studies are useful to classify mtDNA variants. CONCLUSION: Clinical significance of a novel or rare variant is critical in the diagnosis of the disease and counseling of the family. Based on the results from clinical, biochemical, and molecular genetic studies of multiple family members, proper interpretation of mtDNA variants is important for clinical laboratories and for patient care.

Targeted array CGH as a valuable molecular diagnostic approach: experience in the diagnosis of mitochondrial and metabolic disorders.

Oligonucleotide array-based comparative genomic hybridization (aCGH) targeted to coding exons of genes of interest has been proven to be a valuable diagnostic tool to complement with Sanger sequencing for the detection of large deletions/duplications. We have developed a custom designed oligonucleotide aCGH platform for this purpose. This array platform provides tiled coverage of the entire mitochondrial genome and high-density coverage of a set of nuclear genes involving mitochondrial and metabolic disorders and can be used to evaluate large deletions in targeted genes. A total of 1280 DNA samples from patients suspected of having mitochondrial or metabolic disorders were evaluated using this targeted aCGH. We detected 40 (3%) pathogenic large deletions in unrelated individuals, including 6 in genes responsible for mitochondrial DNA (mtDNA) depletion syndromes, 23 in urea cycle genes, 11 in metabolic and related genes. Deletion breakpoints have been confirmed in 31 cases by PCR and sequencing. The possible deletion mechanism has been discussed. These results illustrate the successful utilization of targeted aCGH to detect large deletions in nuclear and mitochondrial genomes. This technology is particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing. For female carriers of X-linked disorders, if sequencing analysis does not detect point mutations, targeted aCGH should be considered for the detection of large heterozygous deletions.

Impact of restricted marital practices on genetic variation in an endogamous Gujarati group.

Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries-old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with groups outside the gol. Gujarati Patels practice this form of "exogamic endogamy." We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non-Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y-chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low-level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine-scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation.

Successful treatment of an enormous rectal mucosa-associated lymphoid tissue lymphoma by endoscopic full-thickness resection: A case report.

BACKGROUND: Colorectal mucosa-associated lymphoid tissue lymphoma (MALToma), a rare kind of nongastric MALToma, lacks consensus on its endoscopic features and standard therapies. According to previous studies on the clinical characteristics and outcomes of colorectal MALToma, endoscopic resection remains a good therapeutic strategy. CASE SUMMARY: A 71-year-old woman suffered intermittent hematochezia for 1 mo, accompanied with abdominal pains but without weight loss, fever, chills or fatigue. Colonoscopy showed a massive hemispheric mass with rough and hyperemic mucosa in the lower rectum. Narrow-band imaging magnifying endoscopy detected some branching abnormal blood vessels and disappearance of glandular structure, which was similar with the tree-like appearance sign in gastric MALToma. Endoscopic ultrasonography revealed the lesion to be hypoechoic, boundary-defined, and echo uniform inside, originating from the muscularis propria. Abdominal enhanced computed tomography (CT) demonstrated a soft tissue mass with defined boundary. No enlarged superficial lymph nodes were detected by B-mode ultrasound. C13-urea breath test and serum Helicobacter pylori antibody were both negative. The patient underwent endoscopic full-thickness resection. Postoperative pathological analysis indicated colorectal MALToma. The patient remained asymptomatic after discharge, and follow-up positron emission tomography-CT and colonoscopy showed no residual lesion, remnants or lymph node metastasis. CONCLUSION: This case provides new information on the specific endoscopic features of colorectal MALToma and an alternative treatment for patients.

Comprehensive analysis of the differential expression and prognostic value of COL1A2 in colon adenocarcinoma.

BACKGROUND: Colon adenocarcinoma (COAD) is a highly heterogeneous disease, which is the second most common cancer in females and third in males. Collagen type I alpha 2 (COL1A2) has been documented to be involved in the carcinogenesis of multiple tumors; however, the expression and prognostic significance of COL1A2 and its underlying mechanism in COAD remains unclarified. MATERIALS AND METHODS: The general profile of COL1A2, its expression pattern, and prognostic value were systematically assessed through various bioinformatics tools. The protein level of COL1A2 was verified in COAD patients using immunohistochemistry analysis. In addition, enrichment analyses were performed to explore the possible regulatory pathways of COL1A2 in COAD. RESULTS: The mRNA and protein levels of COL1A2 were significantly increased in COAD than that in normal tissues (P < 0.05). The COL1A2 expression tended to increase along with cancer stages and nodal metastasis status in COAD, while the promoter methylation levels of COL1A2 might negatively related to its mRNA expression. Survival analysis showed that COL1A2 was a reliable predictor for distinguishing the status of disease-specific survival (DSS), overall survival (OS), and progression-free survival (PFS), and might serve as a robust independent prognostic biomarker for DSS and OS in COAD patients (P < 0.05). The enrichment analysis showed focal adhesion as the most possible regulatory pathway by COL1A2. CONCLUSION: Collectively, COL1A2 functioned as an independent prognostic biomarker and might be a potential therapeutic target in COAD.

TLR2 Potentiates SR-Marco-Mediated Neuroinflammation by Interacting with the SRCR Domain.

Microglial activation-induced neuroinflammation is critical in the pathogenesis of neurodegenerative diseases. Activated microglia are regulated mainly by innate pattern recognition receptors (PRRs) on their surface, of which macrophage receptor with collagenous structure (Marco) is a well-characterized scavenger receptor constitutively expressed on specific subsets of macrophages, including microglia. Increasing evidence has shown that Marco is involved in the pathogenesis of a range of inflammatory processes. However, research on the role of Marco in regulating neuroinflammation has reported conflicting results. In the present study, we examined the role Marco played in triggering neuroinflammation and its underlying mechanisms. The results demonstrated that silencing the Marco gene resulted in a significantly reduced neuroinflammatory response and vice versa. α-Syn stimulation in Marco overexpressing cells induced a pronounced inflammatory response, suggesting that Marco alone could trigger an inflammatory response. We also found that TLR2 significantly promoted Marco-mediated neuroinflammation, indicating TLR2 was an important co-receptor of Marco. Knocking down the TLR2 gene in microglia and mouse substantia nigra resulted in decreased expression of Marco. Subsequent mechanistic studies showed that deleting the SRCR domain of Marco resulted in disruption of the inflammatory response and the interaction between TLR2 and Marco. This suggested that TLR2 binds directly to the SRCR domain of Marco and regulates Marco-mediated neuroinflammation. In summary, this investigation revealed that TLR2 could potentiate Marco-mediated neuroinflammation by interacting with the SRCR domain of Marco, providing a new target for inhibiting neuroinflammation in neurodegenerative diseases.

Molecular spectrum of SLC22A5 (OCTN2) gene mutations detected in 143 subjects evaluated for systemic carnitine deficiency.

Systemic primary carnitine deficiency (CDSP) is caused by recessive mutations in the SLC22A5 (OCTN2) gene encoding the plasmalemmal carnitine transporter and characterized by hypoketotic hypoglycemia, and skeletal and cardiac myopathy. The entire coding regions of the OCTN2 gene were sequenced in 143 unrelated subjects suspected of having CDSP. In 70 unrelated infants evaluated because of abnormal newborn screening (NBS) results, 48 were found to have at least 1 mutation/unclassified missense variant. Twenty-eight of 33 mothers whose infants had abnormal NBS results were found to carry at least 1 mutation/unclassified missense variant, including 11 asymptomatic mothers who had 2 mutations. Therefore, sequencing of the OCTN2 gene is recommended for infants with abnormal NBS results and for their mothers. Conversely, 52 unrelated subjects were tested due to clinical indications other than abnormal NBS and only 14 of them were found to have at least one mutation/unclassified variant. Custom designed oligonucleotide array CGH analysis revealed a heterozygous approximately 1.6 Mb deletion encompassing the entire OCTN2 gene in one subject who was apparently homozygous for the c.680G>A (p.R227H) mutation. Thus, copy number abnormalities at the OCTN2 locus should be considered if by sequencing, an apparently homozygous mutation or only one mutant allele is identified.

Maternal systemic primary carnitine deficiency uncovered by newborn screening: clinical, biochemical, and molecular aspects.

BACKGROUND: Systemic primary carnitine deficiency is an autosomal recessive disorder of the carnitine cycle caused by mutations in the SLC22A5 gene that encodes the carnitine transporter, organic cation transporter. Systemic primary carnitine deficiency typically presents in childhood with either metabolic decompensation or cardiomyopathy. We report five families in which low free carnitine levels in the infants' newborn screening have led to the diagnosis of maternal systemic primary carnitine deficiency. METHODS: Blood samples from the infants and /or their family members were used to extract the DNA. The entire coding regions of the SLC22A5 gene were sequenced. The clinical data were obtained from the referring metabolic specialists. RESULT: Sequencing the SLC22A5 gene allowed molecular confirmation with identification of three novel mutations: c.1195C>T (p.R399W), c.1324_1325GC>AT (p.A442I), and c.43G>T (p.G15W). All infants were asymptomatic at the time of diagnosis, and one was found to have systemic primary carnitine deficiency. Three mothers are asymptomatic, one had decreased stamina during pregnancy, and one has mild fatigability and developed preeclampsia. DISCUSSION: These findings provide further evidence that systemic primary carnitine deficiency presents with a broad clinical spectrum from a metabolic decompensation in infancy to an asymptomatic adult. The maternal systemic primary carnitine deficiency was uncovered by the newborn screening results supporting the previous notion that newborn screening can identify some of the maternal inborn errors of metabolism. It also emphasizes the importance of maternal evaluation after identification of a low free carnitine level in the newborn screening.

MPV17-associated hepatocerebral mitochondrial DNA depletion syndrome: new patients and novel mutations.

Mitochondrial DNA depletion syndromes are autosomal recessive diseases characterized by a severe decrease in mitochondrial DNA content leading to dysfunction of the affected organ. They are phenotypically heterogeneous and classified as myopathic, encephalomyopathic, or hepatocerebral. The latter group has been associated with mutations in TWINKLE,POLG1, DGUOK genes and recently with mutations in the MPV17 gene. MPV17 encodes a mitochondrial inner membrane protein and plays an as yet poorly understood role in mitochondrial DNA maintenance. Mutations in the MPV17 gene have been reported in patients who came to medical attention during infancy with liver failure, hypoglycemia, failure-to-thrive and neurological symptoms. In addition, a homozygous p.R50Q mutation has been identified in patients with Navajo neurohepatopathy. To date, 13 different mutations in 21 patients have been reported. We report eight new patients with seven novel mutations, including four missense mutations (c.262A>G (p.K88E), c.280G>C (p.G94R), c.293C>T (p.P98L), and c.485C>A (p.A162D)), one in-frame deletion (c.271_273del3 (p.L91del)), one splice site substitution (c.186+2T>C), and one insertion (c.22_23insC). The p.R50Q mutation, which occurs in a CpG dinucleotide, is the most common MPV17 mutation and, to date, has only been found in the homozygous state. Clinically, patients homozygous for p.R50Q or compound heterozygous for the p.G94R and p.P98L mutations have a better prognosis, with all the other mutations associated with early death if not treated by liver transplantation. Localizing the mutations within the predicted MPV17 protein structure reveals clustering of mutations in the region of the putative protein kinase C phosphorylation site.

Milder clinical course of Type IV 3-methylglutaconic aciduria due to a novel mutation in TMEM70.

Mitochondrial disorders are a large and genetically heterogeneous group of disorders posing a significant diagnostic challenge. Only approximately 10-20% of patients have identifiable alterations in their mitochondrial DNA (mtDNA). The remaining ~80-90% of affected patients likely harbor mutations in nuclear genes, most of which are still poorly characterized, and therefore not amenable to efficient screening using currently available molecular methods. Here we present a patient, who has been followed since birth after presenting with neonatal hyperammonemia, lactic acidosis, Reye-like syndrome episodes, and ventricular tachyarrhythmia. Initial biochemical work-up revealed hyperalaninemia, normal plasma glutamine, mild orotic aciduria and significant amounts of urinary 3-methylglutaconic (3-MGC) and 3-methylglutaric (3-MGA) acids. Muscle biopsy demonstrated the presence of ragged-red fibers and non-specific structural abnormalities of mitochondria. The activities of respiratory chain enzymes (complexes I-IV) showed no deficiency. Mutational analysis of the entire mitochondrial genome did not reveal deleterious point mutations or large deletions. Long-term follow-up was significant for a later-onset hypertrophic cardiomyopathy, muscle weakness, and exercise intolerance. Although she had frequent episodes of Reye-like episodes in infancy and early childhood, mostly triggered by illnesses, these symptoms improved significantly with the onset of puberty. In the light of recent reports linking cases of type IV 3-methylglutaconic aciduria (3-MGCA) and hypertrophic cardiomyopathy to mutations in TMEM70, we proceeded with sequencing analysis of this gene. We identified one previously reported splice site mutation, c.317-2A>G and a novel mutation c.494G>A (p.G165D) in an evolutionarily conserved region predicted to be deleterious. This variant was not identified in 100 chromosomes of healthy control subjects and 200 chromosomes of patients with cardiomyopathies. Western blotting using a polyclonal antibody against ATP5J, subunit F6 of ATP synthase, on patient's skin fibroblasts showed undetectable amount of the ATP5J protein. In comparison to the previously reported cases, we note that our patient had normal growth parameters and cognitive development, absence of structural heart and urinary tract defects, no dysmorphic features, improvement of symptoms with age, and persistence of hypertrophic cardiomyopathy.

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

Two mtDNA mutations 14487T>C (M63V, ND6) and 12297T>C (tRNA Leu) in a Leigh syndrome family.

Mitochondrial cytopathies are characterized by a large variability of clinical phenotypes and severity. The 14487T>C mutation in mtDNA has been recently described to be associated with Leigh syndrome. The 12297T>C mutation has been described in isolated dilated cardiomyopathy patients. Here, we report a family with multiple members who harbor both mutations, with only a few individuals who are affected with Leigh syndrome. Mitochondrial whole genome sequencing analysis in the proband's muscle specimen detected two nearly homoplasmic mutations: 14487T>C (M63V in ND6) and 12297T>C in the tRNA (Leu) (CUN) gene. These two mutations were also detected in the blood, urine sediments, hair follicles, and buccal swab samples of all matrilineal relatives tested. All individuals tested were nearly homoplasmic for the 12297T>C mutation, but had variable degrees of heteroplasmy for 14487T>C. We also screened for the frequency of these two mutations. Of 268 patients with Leigh or Leigh-like disease, one case was found to harbor the 14487T>C mutation (0.3%), and one had the 12297T>C mutation (0.3%). Neither mutation was detected in the 88 patients meeting MELAS syndrome criteria nor in the 56 patients with respiratory chain complex I or I+III deficiency. In conclusion, the 14487T>C mutation appears as the primary etiology of Leigh syndrome in this family, demonstrating the high level of heteroplasmy needed for a clinically significant phenotype with this mutation. The 12297T>C mutation was not associated with dilated cardiomyopathy for the family members who were clinically evaluated and who were shown by testing to be nearly homoplasmic for that mutation.