A Mouse Systems Genetics Approach Reveals Common and Uncommon Genetic Modifiers of Hepatic Lysosomal Enzyme Activities and Glycosphingolipids.

Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.

Fetal hemoglobin regulating genetic variants identified in homozygous (HbSS) and heterozygous (HbSA) subjects from South Mexico.

Hemoglobin S is caused by a nucleotide change in HBB gene (HBB:c.20A>T, p.Glu6Val), is presented in diverse forms: simple carriers (HbSA), homozygotes (HbSS) also known as sickle cell anemia, and compound heterozygotes with other ß-hemoglobinopathies. It is worldwide distributed, in Mexico, is frequently observed in the southern states Guerrero, Oaxaca and Chiapas. Elevated fetal hemoglobin (HbF) is associated with mild phenotype; single-nucleotide variants (SNVs) in modifier genes, such as BCL11A, HBG2, HBBP1 pseudogene and HBS1L-MYB intergenic region, upregulate HbF synthesis. The aim of this study was to identify HbF regulating genetic variants in HbSS and HbSA Mexican subjects. We studied 39 individuals (HbSS = 24, 61%, HbSA = 15, 39%) from Chiapas (67%) and Guerrero (33%), peripheral blood was collected in ethylenediamine tetraacetic acid (EDTA) for molecular and hematological studies, DNA was isolated by salting-out technic and genotyping was performed through allelic discrimination by real time polymerase chain reaction (RT-PCR) using Taqman® probes for 15 SNV (in BCL11A: rs6706648, rs7557939, rs4671393, rs11886868, rs766432, rs7599488, rs1427407; HBS1L-MYB: rs28384513, rs7776054, rs9399137, rs4895441, rs9402686, rs1320963; HBG2: rs7482144; and HBBP1: rs10128556). The obtained data were analyzed using IMB SPSS v.22.0 software. All minor alleles were observed in frequencies over 0.05, the most frequent was rs9402686 (0.82), while the less frequent was rs101028556 (0.08). In HbSS group, the mean fetal hemoglobin was 11.9 ± 5.9% and was significantly elevated in BCL11A rs11886868 wildtype homozygotes and in carriers of HBS1L-MYB intergenic region rs7776054 (p = 0.04 and p = 0.03, respectively). In conclusion, in HbSS Mexican patients, two SNVs were observed related to increased HbF; BCL11A rs11886868 and HBS1L-MYB rs7776054.

Sickle cell anemia (SCA) is one of the most common types of hemoglobinopathies in people of African ancestry, it is caused by homozygosity of HbS mutation (HBB:c.20A>T). It is known that fetal hemoglobin plays a key role in decreasing HbS polymerization which damages the erythrocyte structure and is responsible for the characteristic hemolytic crises endured by these patients. Single-nucleotide variant (SNV) in genes that regulate fetal hemoglobin (HbF) after birth have been associated with its increment, thus ameliorating the hematologic phenotype of this pathology and other ß-hemoglobinopathies. Therefore, in this study, we identified, for the first time in Mexican patients with SCA (HbSS) and HbS carriers (HbSA), the presence of 15 SNVs on BCL11A, HBS1L-MYB and HBG2; all HbSS patients had anemia and elevated HbF; 2 variants were related to increased HbF rs11688888C of BCL11A and rs7776054G of HBSIL-MYB; and finally, all minor alleles were found at a frequency higher than 0.05.

Identification of genetic modifiers of murine hepatic ß-glucocerebrosidase activity.

The acid ß-glucocerebrosidase (GCase) enzyme cleaves glucosylceramide into glucose and ceramide. Loss of function variants in the gene encoding for GCase can lead to Gaucher disease and Parkinson's disease. Therapeutic strategies aimed at increasing GCase activity by targeting a modulating factor are attractive and poorly explored. To identify genetic modifiers, we measured hepatic GCase activity in 27 inbred mouse strains. A genome-wide association study (GWAS) using GCase activity as a trait identified several candidate modifier genes, including Dmrtc2 and Arhgef1 (p=2.1x10-7), and Grik5 (p=2.1x10-7). Bayesian integration of the gene mapping with transcriptomics was used to build integrative networks. The analysis uncovered additional candidate GCase regulators, highlighting modules of the acute phase response (p=1.01x10-8), acute inflammatory response (p=1.01x10-8), fatty acid beta-oxidation (p=7.43x10-5), among others. Our study revealed previously unknown candidate modulators of GCase activity, which may facilitate the design of therapies for diseases with GCase dysfunction.

Systems Biology Approaches Reveal Potential Phenotype-Modifier Genes in Neurofibromatosis Type 1.

Neurofibromatosis type (NF1) is a syndrome characterized by varied symptoms, ranging from mild to more aggressive phenotypes. The variation is not explained only by genetic and epigenetic changes in the NF1 gene and the concept of phenotype-modifier genes in extensively discussed in an attempt to explain this variability. Many datasets and tools are already available to explore the relationship between genetic variation and disease, including systems biology and expression data. To suggest potential NF1 modifier genes, we selected proteins related to NF1 phenotype and NF1 gene ontologies. Protein-protein interaction (PPI) networks were assembled, and network statistics were obtained by using forward and reverse genetics strategies. We also evaluated the heterogeneous networks comprising the phenotype ontologies selected, gene expression data, and the PPI network. Finally, the hypothesized phenotype-modifier genes were verified by a random-walk mathematical model. The network statistics analyses combined with the forward and reverse genetics strategies, and the assembly of heterogeneous networks, resulted in ten potential phenotype-modifier genes: AKT1, BRAF, EGFR, LIMK1, PAK1, PTEN, RAF1, SDC2, SMARCA4, and VCP. Mathematical models using the random-walk approach suggested SDC2 and VCP as the main candidate genes for phenotype-modifiers.

Modeling Parkinson's Disease Heterogeneity to Accelerate Precision Medicine.

A mechanistic understanding of the diverse clinical manifestations of Parkinson's disease (PD) and variable patient response to treatments is lacking. Genetically diverse PD model organisms can be used to map modifier genes and understand clinically relevant phenotypes of varying severity. This strategy can accelerate the pace of discoveries for precision medicine purposes.

Phenotypic severity in a family with MEND syndrome is directly associated with the accumulation of potentially functional variants of cholesterol homeostasis genes.

BACKGROUND: Male EBP disorder with neurologic defects (MEND) syndrome is an X-linked disease caused by hypomorphic mutations in the EBP (emopamil-binding protein) gene. Modifier genes may explain the clinical variability among individuals who share a primary mutation. METHODS: We studied four males (Patient 1 to Patient 4) exhibiting a descending degree of phenotypic severity from a family with MEND syndrome. To identify candidate modifier genes that explain the phenotypic variability, variants of homeostasis cholesterol genes identified by whole-exome sequencing (WES) were ranked according to the predicted magnitude of their effect through an in-house scoring system. RESULTS: Twenty-seven from 105 missense variants found in 45 genes of the four exomes were considered significant (-5 to -9 scores). We found a direct genotype-phenotype association based on the differential accumulation of potentially functional gene variants among males. Patient 1 exhibited 17 variants, both Patients 2 and 3 exhibited nine variants, and Patient 4 exhibited only five variants. CONCLUSION: We conclude that APOA5 (rs3135506), ABCA1 (rs9282541), and APOB (rs679899 and rs12714225) are the most relevant candidate modifier genes in this family. Relative accumulation of the deficiencies associated with variants of these genes along with other lesser deficiencies in other genes appears to explain the variable expressivity in MEND syndrome.

Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data.

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

SLC23A2-05 (rs4987219) and KRAS-LCS6 (rs61764370) polymorphisms in patients with squamous cell carcinoma of the head and neck.

Cancer is a genetic disease that is highly influenced by environmental factors. To determine the risk factors of squamous cell carcinoma of the head and neck, two polymorphisms, solute carrier family 23 member 2 (SLC23A2-05 [rs4987219]) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-LCS6 (rs61764370), and environmental factors, including smoking and alcohol consumption, were studied in a population. The present study included 165 males diagnosed with squamous cell carcinoma of the head and neck. The control group consisted of 230 healthy male subjects without cancer or a family history of cancer. The SLC23A2-05 and KRAS-LCS6 polymorphisms were analyzed by polymerase chain reaction followed by enzymatic digestion. All patients and healthy subjects were assessed with regard to their smoking habit and alcohol consumption as these are considered to be risk factors for cancer. The statistical analysis was performed using logistic regression, Fisher's exact and χ2 tests. Additional analyses were performed using the programs, multi-factor dimensionality reduction (MDR; version 2.0) and MDR permutation test (version 0.4.7), which consider all variables as risk factors simultaneously. The results of the present study demonstrate that the SLC23A2-05 and KRAS-LCS6 polymorphisms are not a risk factor for squamous cell carcinoma of the head and neck. In the same samples, the association of alcohol consumption (P<0.001) and smoking habit (P<0.001) with cancer presence was positive when each variable was considered individually. Concerning the environmental factors, a positive association of smoking habit and alcohol consumption with cancer, although not with ethnicity (ratio, 1.0244; testing balance accuracy, 0.8733; P<0.001) was identified using the MDR tool, which analyzed the variables and polymorphism genotypes simultaneously. In conclusion, in the present study, squamous cell carcinoma of the head and neck was highly affected by environmental factors when compared with the affect of SLC23A2-05 and KRAS-LCS6 polymorphisms.

Normal ATXN3 Allele but Not CHIP Polymorphisms Modulates Age at Onset in Machado-Joseph Disease.

BACKGROUND: Age at onset (AO) in Machado-Joseph disease (MJD) is closely associated with the length of the CAG repeat at the mutant ATXN3 allele, but there are other intervening factors. Experimental evidence indicates that the normal ATXN3 allele and the C-terminal heat shock protein 70 (Hsp70)-interacting protein (CHIP) may be genetic modifiers of AO in MJD. METHODS: To investigate this hypothesis, we determined the length of normal and expanded CAG repeats at the ATXN3 gene in 210 unrelated patients with MJD. In addition, we genotyped five single nucleotide polymorphisms (SNPs) within the CHIP gene. We first compared the frequencies of the different genotypes in two subgroups of patients who were highly discordant for AO after correction for the length of the expanded CAG allele. The possible modifier effect of each gene was then evaluated in a stepwise multiple linear regression model. RESULTS: AO was associated with the length of the expanded CAG allele (r(2) = 0.596, p < 0.001). Frequencies of the normal CAG repeats at the ATXN3 gene and of CHIP polymorphisms did not differ significantly between groups with highly discordant ages at onset. However, addition of the normal allele improved the model fit for prediction of AO (r(2) = 0.604, p = 0.014). Indeed, we found that the normal CAG allele at ATXN3 had a positive independent effect on AO. CONCLUSION: The normal CAG repeat at the ATXN3 gene has a small but significant influence on AO of MJD.

DNA damage and related modifier genes in Italian Cystic fibrosis patients

Cystic Fibrosis (CF) is an autosomal recessive multisystemic disorder showing a highly heterogeneous phenotype, even among siblings carrying identical CFTR mutations. Moreover, oxidative stress is of central importance in the pathogenesis of cystic fibrosis. The present study seeks to value the presence of oxidative damage in CF patients and the possible modifier effect of repair and glutathione-S-transferase genes. We analysed the presence of DNA damage in leukocytes of 63 CF patients at an Italian CF centre and 63 controls, through the alkaline Comet assay to detect DNA strand breaks. Furthermore, controls and 93 CF subjects were genotyped for 5 genes by RFLP-PCR (XRCC1,0GG1,GSTP1) and PCR assay (GSTM1, GSTT1). No difference in Comet assay values was observed comparing controls to CF patients, although CF subjects showed slightly higher mean values. The crude Odds-Ratio (OR) was higher than one for XRCC1 and GSTP1 genotypes and liver status and for XRCC1 and OGG1 genotypes and pancreatic insufficiency, but in all cases the p-values were not significant. In this case-control study, neither DNA damage ñor gene polymorphisms seem to influence CF manifestation.