Human papillomavirus (HPV) integration signature in cervical lesions: identification of MACROD2 gene as HPV hot spot integration site.

BACKGROUND: High-risk HPV is clearly associated with cervical cancer. Integration of HPV DNA into the host genome is considered a key event in driving cervical carcinogenesis. However, the mechanism on how HR-HPV integration influences the host genome structure has remained enigmatic. METHODS: In our study, 25 DNA samples including 11 from fresh-frozen cervical carcinomas and 14 from fresh-frozen high-grade squamous intraepithelial lesion (HSILs) were detected using the method of HPV capture combined with next generation sequencing. RESULTS: We calculated the frequency in each viral gene or region and found that breakpoints were prone to occur in L1 and L2 instead of E2 in the cervical cancer (P = 0.0004 and P = 5.15 × 10-40) and HSIL group (P = 2.1 × 10-32 and P = 7.06 × 10-13). The results revealed that HPV16 showed a strong tendency toward intronic region (P = 5.02 × 10-64) but a subtle tendency toward intergenic region (P = 0.04). The most frequent integration site was in the MACROD2 gene (introns 2, 4, 5, 6, 8 and 9), which in MACROD2 functional domain. CONCLUSION: Our results revealed that MACROD2 is HPV hot spot integration site in cervical lesions, and its deficiency alter DNA repair and sensitivity to DNA damage thought impaired PARP1 activity resulting in chromosome instability.

Intragenic Deletion in MACROD2: A Family with Complex Phenotypes Including Microcephaly, Intellectual Disability, Polydactyly, Renal and Pancreatic Malformations.

Diagnosing a complex genetic syndrome and correctly assigning the concomitant phenotypic traits to a well-defined clinical form is often a medical challenge. In this work, we report the analysis of a family with complex phenotypes, including microcephaly, intellectual disability, dysmorphic features, and polydactyly in the proband, with the aim of adding new aspects for obtaining a clear diagnosis. We performed array-comparative genomic hybridization and quantitative reverse transcriptase PCR (qRT-PCR) analyses. We identified a deletion of chromosome 20p12.1 involving the macrodomain containing 2/mono-ADP ribosylhydrolase 2 gene (MACROD2) in several members of the family. This gene is actually not associated with a specific syndrome but with congenital anomalies of multiple organs. qRT-PCR showed higher levels of a MACROD2 mRNA isoform in the individuals carrying the deletion. Our results, together with other data reported in the literature, support the hypothesis that the deletion in MACROD2 can affect correct embryonic development and that the presence of another associated event, such as epigenetic modifications at the MACROD2 locus, can influence the level of severity of the pathology.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers.

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

Crosstalk between Gut Microbiota and Epigenetic Markers in Obesity Development: Relationship between Ruminococcus, BMI, and MACROD2/SEL1L2 Methylation.

Changes in gut microbiota composition and in epigenetic mechanisms have been proposed to play important roles in energy homeostasis, and the onset and development of obesity. However, the crosstalk between epigenetic markers and the gut microbiome in obesity remains unclear. The main objective of this study was to establish a link between the gut microbiota and DNA methylation patterns in subjects with obesity by identifying differentially methylated DNA regions (DMRs) that could be potentially regulated by the gut microbiota. DNA methylation and bacterial DNA sequencing analysis were performed on 342 subjects with a BMI between 18 and 40 kg/m2. DNA methylation analyses identified a total of 2648 DMRs associated with BMI, while ten bacterial genera were associated with BMI. Interestingly, only the abundance of Ruminococcus was associated with one BMI-related DMR, which is located between the MACROD2/SEL1L2 genes. The Ruminococcus abundance negatively correlated with BMI, while the hypermethylated DMR was associated with reduced MACROD2 protein levels in serum. Additionally, the mediation test showed that 19% of the effect of Ruminococcus abundance on BMI is mediated by the methylation of the MACROD2/SEL1L2 DMR. These findings support the hypothesis that a crosstalk between gut microbiota and epigenetic markers may be contributing to obesity development.

Association of the MACROD2 rs6110695 A>G polymorphism with an increasing WBC count in a Korean population.

INTRODUCTION: We aimed to find a novel candidate gene related to the white blood cell (WBC) count in a Korean population. Since WBC count has been reported to have a relation to the risk of chronic diseases according to previous literature, WBC level prediction can be helpful for managing future risk of chronic disease development. In this aspect, a gene newly found in the present study is expected to be utilized as a tool for judging an individual's WBC level. METHODS: Based on the 153 study participants' genotype data produced by the Korean Chip. The mono-adenosine diphosphate ribosylhydrolase 2 (MACROD2) rs6110695 A>G polymorphism had a significant strong association with WBC count, thus, the MACROD2 gene emerged as a novel candidate gene for WBC count. To verify the effects of the single-nucleotide polymorphisms on WBC count, the participants were grouped according to the rs6110695 AA and AG genotypes. RESULTS: WBC to apolipoprotein A-I ratio, WBC count, granulocyte to lymphocyte ratio, monocyte to platelet ratio, and interferon-γ level were significantly higher in the AG genotype group than in the AA genotype group. Through the receiver operating characteristic curve analysis, the rs6110695 AA and AG genotypes were discriminated by the optimal WBC count cutoff value of 5.450. As expected, the results in the participants having a WBC count over 5.450 were similar to the AG genotype group. CONCLUSIONS: We revealed that the MACROD2 rs6110695 AG genotype has an association with increasing WBC count. Since, as previous literature described, WBC count is one of the main risk factors for chronic diseases, WBC count measurement in individuals with the rs6110695 AG genotype that was found in the present study may help manage future chronic disease risk.

Peripheral Expression of MACROD2 Gene Is Reduced Among a Sample of Turkish Children with Autism Spectrum Disorder.

Background: Genomic variations in mono-ADP ribosylhydrolase 2 (MACROD2) have been associated with autism spectrum disorder (ASD) in recent genome-wide studies and case reports. In this study, we aimed to evaluate the MACROD2 expression profile in patients with ASD. Methods: The study group included 100 children with a DSM-5 diagnosis of ASD, and the control group consisted of 105 healthy controls. Blood samples were obtained from all participants in this study, and the gene expression level was determined using quantitative reverse transcription PCR (RT-qPCR). Statistical analysis was performed with R 3.4.0 and Statistical Program for Social Sciences (SPSS for Windows, 21.0). Results: The mean ages of the participants in the study and control groups were 9.22 ± 3.62 and 9.27 ± 3.86 years, respectively. There was no significant difference concerning gender (P = .944) and age (P = .914) between the 2 groups. MACROD2 gene expression was found to be decreased in the study group compared to the control group (study group = 5.73, control group = 89.56; fold change =-3.967; P < .001). While the level of MACROD2 expression was not correlated with the ASD severity, it was associated with the severity of the hyperactivity/impulsivity symptoms (P = .008). Conclusions: This is the first study in the literature investigating the peripheral expression of the MACROD2 gene. We showed that the expression level of MACROD2 was decreased in patients with ASD when compared to the control group. As the relationship between the MACROD2 gene expression profile and ASD remains to be further investigated, this study may provide an insight for further studies.

Association of MACROD2 gene variants with obesity and physical activity in a Korean population.

BACKGROUND: Obesity is a serious and common complex disease caused by the influence of genetic and environmental factors. Therefore, we aimed to evaluate the effect of genetic variants on obesity and the possibility of preventing obesity through physical activity using association analysis. METHODS: This study analyzed the association between obesity and variants in the MACROD2 gene in the Korean association resource (KARE) cohort using logistic regression analysis. Linear regression analysis was performed for obesity-related phenotype traits including body mass index (BMI), body fat percentage (BFP), abdominal fat percentage (AbFP), and the waist-to-hip ratio (WHR). The level of physical activity was analyzed by dividing the participants into two groups according to the cutoff of one hour or more of daily intense activity. RESULTS: As a result, rs6079275 in the MACROD2 gene had the highest significance in obesity and phenotypic characteristics. Minor allele carriers (CC, CG) of rs6079275 decreased the obesity risk (OR = 0.57, 95% CI = 0.40-0.82, p = 2.34 × 10-3 ) and showed a tendency to decrease the risk of BMI (ß = -0.312, p = 8.99 × 10-4 ), BFP (ß = -0.482, p = 4.19 × 10-3 ) and AbFP (ß = -0.0051, p = 5.96 × 10-4 ). In addition, the participants with the minor allele (C) of rs6079275 had a reduced obesity risk with high physical activity (OR = 0.23, 95% CI: 0.14-0.93, p = 0.036). CONCLUSIONS: This study demonstrated that variants in the MACROD2 gene were correlated with obesity, phenotypic traits, and physical activity in the Korean population. Therefore, we suggest the possibility of preventing obesity by identifying this genetic variation and the interactive effect of lifestyle in Koreans.

Behavioural Characterisation of Macrod1 and Macrod2 Knockout Mice.

Adenosine diphosphate ribosylation (ADP-ribosylation; ADPr), the addition of ADP-ribose moieties onto proteins and nucleic acids, is a highly conserved modification involved in a wide range of cellular functions, from viral defence, DNA damage response (DDR), metabolism, carcinogenesis and neurobiology. Here we study MACROD1 and MACROD2 (mono-ADP-ribosylhydrolases 1 and 2), two of the least well-understood ADPr-mono-hydrolases. MACROD1 has been reported to be largely localized to the mitochondria, while the MACROD2 genomic locus has been associated with various neurological conditions such as autism, attention deficit hyperactivity disorder (ADHD) and schizophrenia; yet the potential significance of disrupting these proteins in the context of mammalian behaviour is unknown. Therefore, here we analysed both Macrod1 and Macrod2 gene knockout (KO) mouse models in a battery of well-defined, spontaneous behavioural testing paradigms. Loss of Macrod1 resulted in a female-specific motor-coordination defect, whereas Macrod2 disruption was associated with hyperactivity that became more pronounced with age, in combination with a bradykinesia-like gait. These data reveal new insights into the importance of ADPr-mono-hydrolases in aspects of behaviour associated with both mitochondrial and neuropsychiatric disorders.

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis.

Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell's response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a 'macrodomain' harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

Split hand/foot malformation associated with 20p12.1 deletion: A case report.

Split hand/foot malformation (SHFM) or ectrodactyly is a rare congenital disorder affecting limb development characterized by clinical and genetic heterogeneity. SHFM is usually inherited as an autosomal dominant trait with incomplete penetrance. Isolated and syndromic forms are described. The extent of associated malformations is highly variable and multiple syndromes with clinical and genetic overlap have been described. We report here a 28 year-old man presenting with SHFM, sparse hair and widespread freckles. Array-CGH identified a 450 kb de novo 20p12.1 microdeletion encompassing three exons (exon 6 to 8) of MACROD2. Although MACROD2 mutations have not been associated with limb malformation until now, it is located next to KIF16B, which is involved in fibroblast growth factor receptor (FGFR) signaling. Additionally, the deletion encompassed a histone modification H3K27ac mark, known as a provider of quantitative readout of promoter and enhancer activity during human limb development. Altogether, these findings suggest that the 20p12.1 CNV is causative of SHFM in the present case through disturbance of regulatory elements functioning.

Copy Number Variation Pattern for Discriminating MACROD2 States of Colorectal Cancer Subtypes.

Copy number variation (CNV) is a common structural variation pattern of DNA, and it features a higher mutation rate than single-nucleotide polymorphisms (SNPs) and affects a larger fragment of genomes. CNV is related with the genesis of complex diseases and can thus be used as a strategy to identify novel cancer-predisposing markers or mechanisms. In particular, the frequent deletions of mono-ADP-ribosylhydrolase 2 (MACROD2) locus in human colorectal cancer (CRC) alters DNA repair and the sensitivity to DNA damage and results in chromosomal instability. The relationship between CNV and cancer has not been explained. In this study, on the basis of the genome variation profiling by the SNP array from 651 CRC primary tumors, we computationally analyzed the CNV data to select crucial SNP sites with the most relevance to three different states of MACROD2 (heterozygous deletion, homozygous deletion, and normal state), suggesting that these CNVs may play functional roles in CRC tumorigenesis. Our study can shed new insights into the genesis of cancer based on CNV, providing reference for clinical diagnosis, and treatment prognosis of CRC.

MACROD2 expression predicts response to 5-FU-based chemotherapy in stage III colon cancer.

BACKGROUND: Colorectal cancer (CRC) is caused by genetic aberrations. MACROD2 is commonly involved in somatic focal DNA copy number losses, in more than one-third of CRCs. In this study, we aimed to investigate the association of MACROD2 protein expression with clinical outcome in stage II and stage III colon cancer. METHODS: Tissue microarrays (TMA) containing formalin-fixed paraffin-embedded tissue cores from 386 clinically well-annotated primary stage II and III colon cancers were stained by immunohistochemistry and evaluated for MACROD2 protein expression. Disease-free survival (DFS) analysis was performed to estimate association with clinical outcome. RESULTS: Loss of nuclear MACROD2 protein expression in epithelial neoplastic cells of stage III microsatellite stable (MSS) colon cancers was associated with poor DFS within the subgroup of 59 patients who received 5-fluorouracil (5-FU)-based adjuvant chemotherapy (p=0.005; HR=3.8, 95% CI 1.4-10.0). CONCLUSION: These data indicate that low nuclear expression of MACROD2 is associated with poor prognosis of patients with stage III MSS primary colon cancer who were treated with 5-FU-based adjuvant chemotherapy.

Mono-ADP-Ribosylhydrolase MACROD2 Is Dispensable for Murine Responses to Metabolic and Genotoxic Insults.

ADP-ribosylation is an important post-translational protein modification that regulates diverse biological processes, controlled by dedicated transferases, and hydrolases. Disruption in the gene encoding for MACROD2, a mono-ADP-ribosylhydrolase, has been associated to the Kabuki syndrome, a pediatric congenital disorder characterized by facial anomalies, and mental retardation. Non-coding and structural mutations/variations in MACROD2 have been associated to psychiatric disorders, to obesity, and to cancer. Mechanistically, it has been recently shown that frequent deletions of the MACROD2 alter DNA repair and sensitivity to DNA damage, resulting in chromosome instability, and colorectal tumorigenesis. Whether MACROD2 deletion sensitizes the organism to metabolic and tumorigenic stressors, in absence of other genetic drivers, is unclear. As MACROD2 is ubiquitously expressed in mice, here we generated constitutively whole-body knock-out mice for MACROD2, starting from mouse embryonic stem (ES) cells deleted for the gene using the VelociGene® technology, belonging to the Knockout Mouse Project (KOMP) repository, a NIH initiative. MACROD2 knock-out mice were viable and healthy, indistinguishable from wild type littermates. High-fat diet administration induced obesity, and glucose/insulin intolerance in mice independent of MACROD2 gene deletion. Moreover, sub-lethal irradiation did not indicate a survival or lethality bias in MACROD2 knock-out mice compared to wild type littermates. Altogether, our data point against a sufficient role of MACROD2 deletion in aggravating high-fat induced obesity and DNA damage-associated lethality, in absence of other genetic drivers.

Mono-ADP-Ribosylhydrolase Assays.

Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.

Lack of replication of previous autism spectrum disorder GWAS hits in European populations.

Common variants contribute significantly to the genetics of autism spectrum disorder (ASD), although the identification of individual risk polymorphisms remains still elusive due to their small effect sizes and limited sample sizes available for association studies. During the last decade several genome-wide association studies (GWAS) have enabled the detection of a few plausible risk variants. The three main studies are family-based and pointed at SEMA5A (rs10513025), MACROD2 (rs4141463) and MSNP1 (rs4307059). In our study we attempted to replicate these GWAS hits using a case-control association study in five European populations of ASD patients and gender-matched controls, all Caucasians. Results showed no association of individual variants with ASD in any of the population groups considered or in the combined European sample. We performed a meta-analysis study across five European populations for rs10513025 (1,904 ASD cases and 2,674 controls), seven European populations for rs4141463 (2,855 ASD cases and 36,177 controls) and five European populations for rs4307059 (2,347 ASD cases and 2,764 controls). The results showed an odds ratio (OR) of 1.05 (95% CI = 0.84-1.32) for rs10513025, 1.0002 (95% CI = 0.93-1.08) for rs4141463 and 1.01 (95% CI = 0.92-1.1) for rs4307059, with no significant P-values (rs10513025, P = 0.73; rs4141463, P = 0.95; rs4307059, P = 0.9). No association was found when we considered either only high functioning autism (HFA), genders separately or only multiplex families. Ongoing GWAS projects with larger ASD cohorts will contribute to clarify the role of common variation in the disorder and will likely identify risk variants of modest effect not detected previously. Autism Res 2017, 10: 202-211. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Discovery and Replication of Gene Influences on Brain Structure Using LASSO Regression.

We implemented least absolute shrinkage and selection operator (LASSO) regression to evaluate gene effects in genome-wide association studies (GWAS) of brain images, using an MRI-derived temporal lobe volume measure from 729 subjects scanned as part of the Alzheimer's Disease Neuroimaging Initiative (ADNI). Sparse groups of SNPs in individual genes were selected by LASSO, which identifies efficient sets of variants influencing the data. These SNPs were considered jointly when assessing their association with neuroimaging measures. We discovered 22 genes that passed genome-wide significance for influencing temporal lobe volume. This was a substantially greater number of significant genes compared to those found with standard, univariate GWAS. These top genes are all expressed in the brain and include genes previously related to brain function or neuropsychiatric disorders such as MACROD2, SORCS2, GRIN2B, MAGI2, NPAS3, CLSTN2, GABRG3, NRXN3, PRKAG2, GAS7, RBFOX1, ADARB2, CHD4, and CDH13. The top genes we identified with this method also displayed significant and widespread post hoc effects on voxelwise, tensor-based morphometry (TBM) maps of the temporal lobes. The most significantly associated gene was an autism susceptibility gene known as MACROD2. We were able to successfully replicate the effect of the MACROD2 gene in an independent cohort of 564 young, Australian healthy adult twins and siblings scanned with MRI (mean age: 23.8 ± 2.2 SD years). Our approach powerfully complements univariate techniques in detecting influences of genes on the living brain.