Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication.

Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

Serum level and tumor tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B: a potential biomarker for colorectal cancer.

BACKGROUND: This study explored the applicability of serum level and tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B (RRM2B) as reliable biomarkers for colorectal cancer (CRC) progression and metastasis. METHODS AND RESULTS: The present descriptive-analytic cohort study was conducted on 50 newly diagnosed CRC patients (stage II, III) and 50 healthy individuals. The new cases had not received any therapeutic intervention and underwent surgery immediately after the initial diagnosis. Tumorous tissues and marginal healthy tissues (as control) were excised to determine the mRNA tissue expression of RRM2B by Real-Time PCR. Serum RRM2B protein was measured using an ELISA method once in the control group. In the patients, serum RRM2B protein was evaluated before, 1 and 3 months after surgery. The tumor metastasis node (TMN) classification system and liver metastasis were evaluated in CRC patients. The results showed significantly lower RRM2B serum levels in 1 and 3 months after surgery compared with the pre-surgery condition (P = 0.014, P < 0.001 respectively). The mean RRM2B gene expression was 51% lower in tumor tissue than its adjacent normal tissue (P < 0.001). No significant relationship was found between serum level of RRM2B and tumor staging and metastasis in patients before surgery (P = 0.373, P = 0.189), 1 month after surgery (P = 0.960, P = 0.088), and 3 months after surgery (P = 0.407, P = 0.724). RRM2B expression in tumor tissue is not associated with tumor staging and metastasis (P = 0.254, P = 0.721). CONCLUSION: These data suggest measuring serum protein level of RRM2B could have a role in CRC progression, although this study should be considered preliminary due to small sample size and short follow-up duration.

Importance of Family History in the Era of Exome Analysis: A Report of a Family with Multiple Concurrent Genetic Diseases.

Multiple familial diseases in a single patient often present with overlapping symptomatology that confers difficulty in delineating a clinical diagnosis. Pedigree analysis has been a long-standing practice in the field of medical genetics to discover familial diseases. In recent years, whole exome sequencing (WES) has proven to be a useful tool for aiding physicians in diagnosing and understanding disease etiology. This report shows that pedigree analysis and WES are co-dependent processes in establishing diagnoses in a family with 4 different genetic disorders: Birt-Hogg-Dubé Syndrome, RRM2B-related mitochondrial disease, CDC73-related primary hyperparathyroidism, and familial prostate cancer.

Genetic Modification of Huntington Disease Acts Early in the Prediagnosis Phase.

Age at onset of Huntington disease, an inherited neurodegenerative disorder, is influenced by the size of the disease-causing CAG trinucleotide repeat expansion in HTT and by genetic modifier loci on chromosomes 8 and 15. Stratifying by modifier genotype, we have examined putamen volume, total motor score (TMS), and symbol digit modalities test (SDMT) scores, both at study entry and longitudinally, in normal controls and CAG-expansion carriers who were enrolled prior to the emergence of manifest HD in the PREDICT-HD study. The modifiers, which included onset-hastening and onset-delaying alleles on chromosome 15 and an onset-hastening allele on chromosome 8, revealed no major effect in controls but distinct patterns of modification in prediagnosis HD subjects. Putamen volume at study entry showed evidence of reciprocal modification by the chromosome 15 alleles, but the rate of loss of putamen volume was modified only by the deleterious chromosome 15 allele. By contrast, both alleles modified the rate of change of the SDMT score, but neither had an effect on the TMS. The influence of the chromosome 8 modifier was evident only in the rate of TMS increase. The data indicate that (1) modification of pathogenesis can occur early in the prediagnosis phase, (2) the modifier loci act in genetic interaction with the HD mutation rather than through independent additive effects, and (3) HD subclinical phenotypes are differentially influenced by each modifier, implying distinct effects in different cells or tissues. Together, these findings indicate the potential benefit of using genetic modifier strategies for dissecting the prediagnosis pathogenic process in HD.

Isozymes of P5C reductase (PYCR) in human diseases: focus on cancer.

Δ1-Pyrroline-5-carboxylate (P5C) reductase (PYCR or P5CR) catalyzes the conversion of P5C to L-proline (Pro) with concomitant oxidation of a cofactor, NADPH or NADH. Mammalian PYCR have been studied since 1950' and currently three isozymes of human PYCR, 1, 2, and L, have been identified and characterized and their roles in genetic diseases and cancer biology have been keenly investigated. These three isozymes are encoded by three different genes localized at three different chromosomes, and catalyze NAD(P)H-dependent reduction of P5C to Pro important for the transfer of oxidizing potential across the mitochondrion and cell. The review summarizes the current understanding of these three human PYCR isozymes and their roles in diseases with a focus on cancer.

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool.

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Renal dysfunction, rod-cone dystrophy, and sensorineural hearing loss caused by a mutation in RRM2B.

More than two decades ago, a recessive syndromic phenotype affecting kidneys, eyes, and ears, was first described in the endogamous Afrikaner population of South Africa. Using whole-exome sequencing of DNA from two affected siblings (and their carrier parents), we identified the novel RRM2B c.786G>T variant as a plausible disease-causing mutation. The RRM2B gene is involved in mitochondrial integrity, and the observed change was not previously reported in any genomic database. The subsequent screening revealed the variant in two newly presenting unrelated patients, as well as two patients in our registry with rod-cone dystrophy, hearing loss, and Fanconi-type renal disease. All patients with the c.786G>T variant share an identical 1.5 Mb haplotype around this gene, suggesting a founder effect in the Afrikaner population. We present ultrastructural evidence of mitochondrial impairment in one patient, to support our thesis that this RRM2B variant is associated with the renal, ophthalmological, and auditory phenotype.

TP53 mutations and relevance of expression of TP53 pathway genes in paediatric acute myeloid leukaemia.

Limited data are available on the incidence and impact of TP53 alterations and TP53 pathway deregulation in paediatric acute myeloid leukaemia (AML). We analysed TP53 alterations in bone marrow samples of 229 patients with de novo paediatric AML, and detected heterozygous missense exon mutations in two patients (1%) and 17p deletions of the TP53 gene in four patients (2%). These patients more frequently had complex karyotype (50% vs. 4%, P = 0·002) or adverse cytogenetic abnormalities, including complex karyotype (67% vs. 17%, P = 0·013), compared to TP53 wild-type. Differential expression of TP53 pathway genes was associated with poor survival, indicating a role for TP53 regulators and effector genes.

Refinement of the deletion in 8q22.2-q22.3: the minimum deletion size at 8q22.3 related to intellectual disability and epilepsy.

Kuechler et al. [2011] reported five patients with interstitial deletions in 8q22.2-q22.3 who had intellectual disability, epilepsy, and dysmorphic features. We report on a new patient with the smallest overlapping de novo deletion in 8q22.3 and refined the phenotype. The proposita was an 8-year-old girl, who developed seizures at 10 months, and her epileptic seizure became severe and difficult to control with antiepileptic drugs. She also exhibited developmental delay and walked alone at 24 months. She was referred to us for evaluation for developmental delay and epilepsy at the age of 8 years. She had intellectual disability (IQ 37 at 7 years) and autistic behavior, and spoke two word sentences at 8 years. She had mild dysmorphic features, including telecanthus and thick vermilion of the lips. Array comparative genomic hybridization detected a 1.36 Mb deletion in 8q22.3 that encompassed RRM2B and NCALD, which encode the small subunit of p53-inducible ribonucleotide reductase and neurocalcin delta in the neuronal calcium sensor family of calcium-binding proteins, respectively. The minimum overlapping region between the present and previously reported patients is considered to be a critical region for the phenotype of the deletion in 8q22.3. We suggest that the deletion in 8q22.3 may represent a clinically recognizable condition, which is characterized by intellectual disability and epilepsy.

Ribonucleotide reductase inhibition improves the symptoms of a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200 ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200 ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.

Case Report: A novel RRM2B variant in a Chinese infant with mitochondrial DNA depletion syndrome and collective analyses of RRM2B variants for disease etiology.

Background: There are few reports of infantile mitochondrial DNA depletion syndrome (MDDS) caused by variants in RRM2B and the correlation between genotype and phenotype has rarely been analyzed in detail. This study investigated an infantile patient with MDDS, from clinical characteristics to genetic causes. Methods: Routine physical examinations, laboratory assays, which included gas chromatography-mass spectrometry of blood and urine, and MRI scans were performed to obtain an exact diagnosis. Whole-exome sequencing was used to pinpoint the abnormal gene and bioinformatic analyses were performed on the identified variant. Results: The case presented with progressive neurologic deterioration, failure to thrive, respiratory distress and lactic acidosis. Sequencing revealed that the patient had a homozygous novel missense variant, c.155T>C (p.Ile52Thr), in exon 2 of the RRM2B gene. Multiple lines of bioinformatic evidence suggested that this was a likely detrimental variant. In addition, reported RRM2B variants were compiled from the relevant literature to analyze disease etiology. We found a distinctive distribution of genotypes across disease manifestations of different severity. Pathogenic alleles of RRM2B were significantly enriched in MDDS cases. Conclusion: The novel variant is a likely genetic cause of MDDS. It expands our understanding of the pathogenic variant spectrum and the contribution of the RRM2B gene to the disease spectrum of MDDS.

A novel RRM2B mutation associated with mitochondrial DNA depletion syndrome.

Mitochondrial DNA (mtDNA) depletion syndromes are disorders characterized by infantile-onset, severe progression, and the drastic loss of mtDNA content in affected tissues. In a patient who showed severe hypotonia, proximal tubulopathy and sensorineural hearing loss after birth, we observed severe mtDNA depletion and impaired respiratory chain activity in muscle due to heterozygous variants c.686G > T and c.551-2A > G in RRM2B, encoding the p53R2 subunit of the ribonucleotide reductase.

Novel and recurrent nuclear gene variations in a cohort of Chinese progressive external ophthalmoplegia patients with multiple mtDNA deletions.

OBJECTIVES: This study aimed to investigate the clinical and genetic spectrum in Chinese patients with multiple mtDNA deletions presenting with autosomal-inherited mitochondrial progressive external ophthalmoplegia (PEO). METHODS: Long-range polymerase chain reaction and massively parallel sequencing of the mitochondrial genome were performed to detect deletions in muscle mtDNA of 274 unrelated families. Then, targeted next generation sequencing was used to detect nuclear gene variations in patients with multiple mtDNA deletions. RESULTS: A total of 40 Chinese PEO patients (10 males and 30 females) from 20 families were found to have multiple mtDNA deletions in this study, and the median age at onset was 35 (1-70) years. PEO and positive family history were the two prominent features of these patients, and ataxia, neuropathy, and hypogonadism were also present as onset symptoms in some patients. Fifteen of 20 probands with multiple mtDNA deletions were identified to carry nuclear gene variants; eight (40.0%) probands had variants within POLG, two (10.0%) within TWNK, two (10.0%) within RRM2B, two (10.0%) within TK2, and one (5.0%) within POLG2. A total of 24 variants were found in these five nuclear genes, of which 19 were novel. The causal nuclear genetic factors in five pedigrees remain undetermined. CONCLUSIONS: The POLG gene is the most common disease-causing gene in this group of PEO patients with multiple mtDNA deletions. While inherited PEO is the most prominent symptoms in these patients, genotypic and phenotypic heterogeneity still exist, for example in onset age, initial symptoms, and accompanying manifestations.

RRM2B Is Frequently Amplified Across Multiple Tumor Types: Implications for DNA Repair, Cellular Survival, and Cancer Therapy.

RRM2B plays a crucial role in DNA replication, repair and oxidative stress. While germline RRM2B mutations have been implicated in mitochondrial disorders, its relevance to cancer has not been established. Here, using TCGA studies, we investigated RRM2B alterations in cancer. We found that RRM2B is highly amplified in multiple tumor types, particularly in MYC-amplified tumors, and is associated with increased RRM2B mRNA expression. We also observed that the chromosomal region 8q22.3-8q24, is amplified in multiple tumors, and includes RRM2B, MYC along with several other cancer-associated genes. An analysis of genes within this 8q-amplicon showed that cancers that have both RRM2B-amplified along with MYC have a distinct pattern of amplification compared to cancers that are unaltered or those that have amplifications in RRM2B or MYC only. Investigation of curated biological interactions revealed that gene products of the amplified 8q22.3-8q24 region have important roles in DNA repair, DNA damage response, oxygen sensing, and apoptosis pathways and interact functionally. Notably, RRM2B-amplified cancers are characterized by mutation signatures of defective DNA repair and oxidative stress, and at least RRM2B-amplified breast cancers are associated with poor clinical outcome. These data suggest alterations in RR2MB and possibly the interacting 8q-proteins could have a profound effect on regulatory pathways such as DNA repair and cellular survival, highlighting therapeutic opportunities in these cancers.

Saccharomyces cerevisiae as a Tool for Studying Mutations in Nuclear Genes Involved in Diseases Caused by Mitochondrial DNA Instability.

Mitochondrial DNA (mtDNA) maintenance is critical for oxidative phosphorylation (OXPHOS) since some subunits of the respiratory chain complexes are mitochondrially encoded. Pathological mutations in nuclear genes involved in the mtDNA metabolism may result in a quantitative decrease in mtDNA levels, referred to as mtDNA depletion, or in qualitative defects in mtDNA, especially in multiple deletions. Since, in the last decade, most of the novel mutations have been identified through whole-exome sequencing, it is crucial to confirm the pathogenicity by functional analysis in the appropriate model systems. Among these, the yeast Saccharomyces cerevisiae has proved to be a good model for studying mutations associated with mtDNA instability. This review focuses on the use of yeast for evaluating the pathogenicity of mutations in six genes, MPV17/SYM1, MRM2/MRM2, OPA1/MGM1, POLG/MIP1, RRM2B/RNR2, and SLC25A4/AAC2, all associated with mtDNA depletion or multiple deletions. We highlight the techniques used to construct a specific model and to measure the mtDNA instability as well as the main results obtained. We then report the contribution that yeast has given in understanding the pathogenic mechanisms of the mutant variants, in finding the genetic suppressors of the mitochondrial defects and in the discovery of molecules able to improve the mtDNA stability.

miR-942 promotes proliferation and metastasis of hepatocellular carcinoma cells by inhibiting RRM2B.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. MicroRNA-942 (miR-942) plays a critical role in promoting proliferation and metastasis of cancer cells and is associated with poor prognosis in some types of cancers. However, the prognostic value of miR-942 and its functional role in HCC remain unclear. MATERIALS AND METHODS: Real-time PCR (RT-PCR) was used to detect the expression of miR-942 in HCC tissues and adjacent normal liver tissues. Next, the correlations between miR-942 expression and clinicopathological parameters including the survival rate were analyzed. Interaction between miR-942 and ribonucleotide reductase regulatory TP53 inducible subunit M2B (RRM2B) was determined by RT-PCR, Western blot and luciferase assay. The biological influence of miR-942 on HCC cell lines was studied using CCK-8 assay, colony formation assay and transwell assay in vitro. Western blot and RT-PCR were used to analyze the change of downstream genes after miR-942 mimics transfection. RESULTS: miR-942 was significantly up-regulated in HCC. Its high expression was associated with serum alanine transaminase level (P=0.0350), tumor size (P=0.0195), T stage (P=0.0045) and lymphatic metastasis (P=0.0013). High expression of miR-942 was associated with shorter overall survival and disease-free survival time of HCC patients. RRM2B was validated as a target gene of miR-942. miR-942 mimics markedly promoted the malignant phenotypes of Huh7 and MHCC97H cell lines, while its inhibitor had the opposite effect. miR-942 can regulate the downstream genes of RRM2B including Egr-1 and PTEN, markers of epithelial-mesenchymal transition and matrix metalloproteinases. CONCLUSION: miR-942 may serve as a potential biomarker for HCC and its inhibitor may be a therapeutic agent for the treatment of this deadly disease.

A homozygous variant in RRM2B is associated with severe metabolic acidosis and early neonatal death.

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.

Congenital cochlear deafness in mitochondrial diseases related to RRM2B and SERAC1 gene defects. A study of the mitochondrial patients of the CMHI hospital in Warsaw, Poland.

OBJECTIVES: Although hearing loss is a well-known symptom of mitochondria-related disorders, it is not clear how often it is a congenital and cochlear impairment. The Newborn Hearing Screening Program (NHSP) enables to distinguish congenital cochlear deafness from an acquired hearing deficit. The initial aim of the study was to research the frequency of the congenital cochlear hearing loss among patients with various gene defects resulting in mitochondrial disorders. The research process brought on an additional gain: basing on our preliminary study group of 80 patients, in 12 patients altogether we identified two defected genes responsible for mitochondrial disorders, whose carriers did not pass the NHSP. Finally, these patients were diagnosed with the congenital cochlear deafness. MATERIAL AND METHODS: The results of the NHSP in the patients with mitochondrial disorders diagnosed in our tertiary reference center were analyzed. Only the cases with confirmed mutations were qualified for the study group. The NHSP database included 80 patients with mutations in 31 different genes: 25 nuclear-encoded and 6 mtDNA-encoded. We searched the literature for the presence of a congenital hearing impairment (CHI) in mitochondrial disorders caused by changes in 278 already known genes. RESULTS: For 68 patients from the study group the NHSP test indicated a proper cochlear function and thus suggested normal hearing. For 12 mitochondrial patients, the NHSP test indicated the requirement for the further audiological diagnosis, and finally CHI was confirmed in 8 of them. This latter subset included patients with pathogenic variants in RRM2B and SERAC1, known as "deafness-causing genes". Contrary to our initial expectations, the patients carrying mutations in other "deafness-causing genes": MPV17, POLG, COX10, as well as other mitochondria-related genes, all reported in literature, did not indicate any CHI following the NHSP test. CONCLUSION: Our study indicates that the cochlear CHI is a phenotypic feature of the RRM2B and SERAC1 related defects. The diagnosis of the CHI following the NHSP allows to early distinguish those defects from other mitochondria-related disorders in which the NHSP test result is correct. Wider studies are needed to assess the significance of this observation.