Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

Evidence for a shift in placental HLA-G allelic dominance and the HLA-G isoform profile during a healthy pregnancy and pre-eclampsia

Human leukocyte antigen (HLA)-G is a non-classical class Ib major expressed by placental trophoblast cells plays a central role in establishing tolerance to the semi-allogeneic fetus and in placentation. HLA-G exists in different soluble or membrane-bound isoforms. Pre-eclampsia, a major cause of fetal and maternal morbidity and mortality, has been linked to insufficient placentation and an altered immune response in pregnancy, including altered HLA-G expression. The 14 bp insertion/deletion polymorphism in the 3′ untranslated region of the gene and the isoform profile may affect HLA-G expression. The aim of the current pilot study was to characterize the expression patterns of HLAG mRNA, protein and isoform profile in uncomplicated term pregnancies and in cases of pre-eclampsia. Maternal sHLA-G mRNA and protein levels was slightly reduced in pre-eclampsia. No difference was found for placental blood, and no correlation between peripheral and placental sHLA-G levels was found. We observed no association between neither fetal nor maternal HLA-G 14 bp insertion/deletion genotypes and pre-eclampsia, nor a significant difference in isoform profiles. However, in HLA-G 14 bp insertion/deletion heterozygous placental samples, we observed abundant HLA-G1 14 bp insertion allele expression in the term placentae, which is contrary to previous findings in first trimester trophoblast. Increased HLA-G1 14 bp insertion allele expression in the placenta was associated with reduced levels of placental sHLA-G and an altered isoform profile with increased relative levels of HLA-G1 and -G5 and reduced levels of HLA-G3. The results indicate that an allelic shift in heterozygous individuals could represent a novel regulatory pathway.

speck, First Identified in Drosophila melanogaster in 1910, Is Encoded by the Arylalkalamine N-Acetyltransferase (AANAT1) Gene

The pigmentation mutation speck is a commonly used recombination marker characterized by a darkly pigmented region at the wing hinge. Identified in 1910 by Thomas Hunt Morgan, speck was characterized by Sturtevant as the most “workable” mutant in the rightmost region of the second chromosome and eventually localized to 2-107.0 and 60C1-2. Though the first speck mutation was isolated over 110 years ago, speck is still not associated with any gene. Here, as part of an undergraduate-led research effort, we show that speck is encoded by the Arylalkylamine N-acetyltransferase 1 (AANAT1) gene. Both alleles from the Morgan lab contain a retrotransposon in exon 1 of the RB transcript of the AANAT1 gene. We have also identified a new insertion allele and generated multiple deletion alleles in AANAT1 that all give a strong speck phenotype. In addition, expression of AANAT1 RNAi constructs either ubiquitously or in the dorsal portion of the developing wing generates a similar speck phenotype. We find that speck alleles have additional phenotypes, including ectopic pigmentation in the posterior pupal case, leg joints, cuticular sutures and overall body color. We propose that the acetylated dopamine generated by AANAT1 decreases the dopamine pool available for melanin production. When AANAT1 function is decreased, the excess dopamine enters the melanin pathway to generate the speck phenotype.

speck, first identified in Drosophila melanogaster in 1910, is encoded by the Arylalkalamine N-acetyltransferase (AANAT1) gene

The pigmentation mutation speck is a commonly used recombination marker characterized by a darkly pigmented region at the wing hinge. Identified in 1910 by Thomas Hunt Morgan, speck was characterized by Sturtevant as the most 'workable' mutant in the rightmost region of the second chromosome and eventually localized to 2-107.0 and 60C1-2. Though the first speck mutation was isolated over 115 years ago, speck is still not associated with any gene. Here, as part of an undergraduate-led research effort, we show that speck is encoded by the Arylalkylamine N-acetyltransferase 1 (AANAT1) gene. Both alleles from the Morgan lab contain a retrotransposon in exon 1 of the RB transcript of the AANAT1 gene. We have also identified a new insertion allele and generated multiple deletion alleles in AANAT1 that all give a strong speck phenotype. In addition, expression of AANAT1 RNAi constructs either ubiquitously or in the dorsal portion of the developing wing generates a similar speck phenotype. We find that speck alleles have additional phenotypes, including ectopic pigmentation in the posterior pupal case, leg joints, cuticular sutures and overall body color. We propose that the acetylated dopamine generated by AANAT1 decreases the dopamine pool available for melanin production. When AANAT1 function is decreased, the excess dopamine enters the melanin pathway to generate the speck phenotype.

Association between an insertion/deletion polymorphism in the interleukin-1α gene and the risk of colorectal cancer in a Chinese population

Background: Previous studies have reported that polymorphisms in the interleukin-1 gene may be involved in tumorigenesis and tumor progression. Aim: The purpose of the present study was to evaluate whether an insertion/deletion polymorphism, rs3783553, located in the miR-122 target gene interleukin-1α, was associated with the risk of colorectal cancer. Methods: Genomic DNA was extracted from peripheral venous blood of 382 patients with colorectal cancer and 433 controls, and the polymorphism was genotyped using a polymerase chain reaction assay. Results: Significantly decreased colorectal cancer risk was observed to be associated with the interleukin-1α rs3783553 insertion/insertion genotype ( P=0.0001; OR=0.41; 95% CI 0.26, 0.65) and the insertion allele ( P<0.001; OR=0.68; 95% CI 0.55, 0.83). Stratification analysis based on clinical and pathological features also revealed that the “TTCA” insertion allele of rs3783553 contributes to slow the progression of colorectal cancer. Conclusion: These results suggest that the rs3783553 polymorphism could be a useful genetic marker to predict the size/extent of colorectal cancer.

Association of cystathionine beta-synthase polymorphisms and aneurysmal subarachnoid hemorrhage

OBJECTIVECystathionine β-synthase (CBS) is involved in homocysteine and hydrogen sulfide (H2S) metabolism. Both products have been implicated in the pathophysiology of cerebrovascular diseases. The impact of CBS polymorphisms on aneurysmal subarachnoid hemorrhage (aSAH) and its clinical sequelae is poorly understood.METHODSBlood samples from all patients enrolled in the CARAS (Cerebral Aneurysm Renin Angiotensin System) study were used for genetic evaluation. The CARAS study prospectively enrolled aSAH patients at 2 academic institutions in the United States from 2012 to 2015. Common CBS polymorphisms were detected using 5′exonuclease genotyping assays. Analysis of associations between CBS polymorphisms and aSAH was performed.RESULTSSamples from 149 aSAH patients and 50 controls were available for analysis. In multivariate logistic regression analysis, the insertion allele of the 844ins68 CBS insertion polymorphism showed a dominant effect on aSAH. The GG genotype of the CBS G/A single nucleotide polymorphism (rs234706) was independently associated with unfavorable functional outcome (modified Rankin Scale Score 3–6) at discharge and last follow-up, but not clinical vasospasm or delayed cerebral ischemia (DCI).CONCLUSIONSThe insertion allele of the 844ins68 CBS insertion polymorphism was independently associated with aSAH while the GG genotype of rs234706 was associated with an unfavorable outcome both at discharge and last follow-up. Increased CBS activity may exert its neuroprotective effects through alteration of H2S levels, and independent of clinical vasospasm and DCI.

The Allele Frequencies of Three Polymorphisms in Genes Involved in Homocysteine Metabolism in a Group of Unrelated Healthy Singaporeans

The cystathionineβ-synthase (CBS) 844ins68 polymorphism, methionine synthase (MS) A2756G SNP, and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T SNP are associated with homocysteine (Hcy) level in humans. Elevated Hcy level is considered a risk factor for atherosclerotic diseases among Asian populations. Therefore, the three polymorphisms may vary the risk for developing such diseases in Singaporeans. In this study, the three polymorphisms were determined in a group of unrelated healthy Singaporeans (273 Chinese, 127 Indians, and 156 Malays). Regarding allele frequencies, Indians had the highest frequencies of the CBS insertion allele (2.0%) and the MS 2756G allele (26.4%), while Chinese had the highestMTHFR677T allele frequency (27.5%). In addition, theMTHFR677T allele was found significantly lower in Chinese males than in their female counterparts. As the CBS insertion allele was suggested to be associated with lower Hcy level, whereas the MS 2756G allele and theMTHFRT/T genotype were related to higher Hcy level, the MS A/G or G/G genotype and theMTHFRT/T genotype were considered double genetic risk factors for elevated Hcy level. The frequency of such double genetic risk was 0.7% (4 subjects) in the total population consisting of 3 Chinese (1.1%) and 1 Malays (0.6%). NoMTHFRT/T genotype was found in Indians. Such results suggested that Chinese could have higher Hcy levels than Malays while the situation for Indians was complicated. Since human Hcy levels are also affected by environmental factors, further studies are required to better evaluate the association between these three polymorphisms and Hcy levels and/or disease susceptibilities in Singaporeans.