Mutagenesis In Cells Research Articles

The Zinc and Iron Binuclear Transport Center of ZupT, a ZIP Transporter from Escherichia coli

ZupT fromEscherichia coliis a member of the Zrt-/Irt-like Protein (ZIP) transporter family, which is responsible for zinc uptake during zinc-sufficient conditions. ZIP transporters have been shown to transport different divalent metal ions including zinc, iron, manganese, and cadmium. In this study, we show that ZupT has an asymmetric binuclear metal center in the transmembrane domain; one metal-binding site, M1, binds zinc, cadmium, and iron, while the other, M2, binds iron only and with higher affinity than M1. Using site-specific mutagenesis and transport activity measurements in whole cells and proteoliposomes, we show that zinc is transported from M1, while iron is transported from M2. The two sites share a common bridging ligand, a conserved glutamate residue. M1 and M2 have ligands from highly conserved motifs in transmembrane domains 4 and 5. Additionally, M2 has a ligand from transmembrane domain 6, a glutamate residue, which is conserved in the gufA subfamily of ZIP transporters, including ZupT and the human ZIP11. Unlike cadmium, iron transport from M2 does not inhibit the zinc transport activity but slightly stimulates it. This stimulation of activity is mediated through the bridging carboxylate ligand. The binuclear zinc-iron binding center in ZupT has likely evolved to enable the transport of essential metals from two different sites without competition; a similar mechanism of metal transport is likely to be found in the gufA subfamily of ZIP transporter proteins.

Precancerous liver diseases do not cause increased mutagenesis in liver stem cells

Inflammatory liver disease increases the risk of developing primary liver cancer. The mechanism through which liver disease induces tumorigenesis remains unclear, but is thought to occur via increased mutagenesis. Here, we performed whole-genome sequencing on clonally expanded single liver stem cells cultured as intrahepatic cholangiocyte organoids (ICOs) from patients with alcoholic cirrhosis, non-alcoholic steatohepatitis (NASH), and primary sclerosing cholangitis (PSC). Surprisingly, we find that these precancerous liver disease conditions do not result in a detectable increased accumulation of mutations, nor altered mutation types in individual liver stem cells. This finding contrasts with the mutational load and typical mutational signatures reported for liver tumors, and argues against the hypothesis that liver disease drives tumorigenesis via a direct mechanism of induced mutagenesis. Disease conditions in the liver may thus act through indirect mechanisms to drive the transition from healthy to cancerous cells, such as changes to the microenvironment that favor the outgrowth of precancerous cells.

Is micronucleus assay in oral exfoliated cells a suitable tool for biomonitoring children exposed to environmental pollutants? A systematic review.

The aim of this review was to evaluate if micronucleus assay in oral exfoliated cells is a suitable tool for biomonitoring children exposed to environmental pollutants. Through the electronic databases PubMed/Medline, Scopus, and Web of Science, all published studies until April 2021 that examined the relationship between exposure to environmental pollutants and micronucleus frequency in oral cells were searched. All relevant articles using a combination of the following keywords-"children," "micronucleus," "oral cells," and "environmental pollution"-were considered. A total of 20 papers met the criteria for inclusion in the systematic review. The results regarding the cytogenetic damage induced by environmental pollutants are conflicting. Some authors have demonstrated that environmental pollution induces mutagenesis in oral cells while others did not. Following the parameters of the Project for Effective Public Health Practices (EPHPP) and after extensive reading of all the articles included, a total of 12 articles had moderate and strong scores and 8 had a classification considered weak. Taken together, this review was able to demonstrate the association between micronucleus frequency and exposure to environmental pollutants in oral exfoliated cells of children.

Does Panoramic X-ray Induce Cytogenetic Damage to Oral Cells? A Systematic Review With Meta-analysis.

The aim of this review was to evaluate the scientific literature regarding the cytogenetic damage in oral exfoliated cells of adult patients submitted to panoramic X-ray. An extensive search of the literature was conducted on PubMed, Scopus and Web of Science databases for all studies published until April 2021 using combinations of the following keywords: "panoramic X-ray," "DNA damage," "genetic damage", "genotoxicity", "mutagenicity", cytotoxicity", "buccal cells", "oral mucosa", "tongue", "gingiva", "micronucleus assay", according to the PRISMA guidelines. All clinical studies in English language were included in the study. A total of 10 studies were identified. As expected, the results regarding the cytogenetic damage induced by panoramic X-ray are conflicting. Some authors have demonstrated that panoramic X-ray induces mutagenesis in oral cells, whereas others did not. After reviewing the 10 studies, two were classified as strong, four were considered moderate, and four were considered weak, according to the quality assessment components of the Effective Public Health Practice Project (EPHPP). Meta-analysis data revealed a negative response related to mutagenicity in oral cells by panoramic X-ray. Taken together, this review failed to demonstrate the association between micronucleus frequency and panoramic X-ray.

The evolution of hematopoietic cells under cancer therapy

Chemotherapies may increase mutagenesis of healthy cells and change the selective pressures in tissues, thus influencing their evolution. However, their contributions to the mutation burden and clonal expansions of healthy somatic tissues are not clear. Here, exploiting the mutational footprint of some chemotherapies, we explore their influence on the evolution of hematopoietic cells. Cells of Acute Myeloid Leukemia (AML) secondary to treatment with platinum-based drugs show the mutational footprint of these drugs, indicating that non-malignant blood cells receive chemotherapy mutations. No trace of the 5-fluorouracil (5FU) mutational signature is found in AMLs secondary to exposure to 5FU, suggesting that cells establishing the leukemia could be quiescent during treatment. Using the platinum-based mutational signature as a barcode, we determine that the clonal expansion originating the secondary AMLs begins after the start of the cytotoxic treatment. Its absence in clonal hematopoiesis cases is consistent with the start of the clonal expansion predating the exposure to platinum-based drugs.

Abstract 39: APOBEC3A drives acquired resistance to targeted therapies in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes or simply provides the selective pressure necessary for the outgrowth of drug resistant clones remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy induced A3A mutagenesis either by gene deletion or RNAi mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non small cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ammal Abbasi, Naveed Nikpour, Adam Langenbucher, Wenjia Su, Marcello Stanzione, Lecia V. Sequist, Rémi Buisson, Michael S. Lawrence, Aaron N. Hata. APOBEC3A drives acquired resistance to targeted therapies in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 39.

Pathways and signatures of mutagenesis at targeted DNA nicks.

Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.

Loss of the abasic site sensor HMCES is synthetic lethal with the activity of the APOBEC3A cytosine deaminase in cancer cells.

Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.

Effects of Interleukin-1β in Glycinergic Transmission at the Central Amygdala

Interleukin-1β (IL-1β) is an important cytokine that modulates peripheral and central pain sensitization at the spinal level. Among its effects, it increases spinal cord excitability by reducing inhibitory Glycinergic and GABAergic neurotransmission. In the brain, IL-1β is released by glial cells in regions associated with pain processing during neuropathic pain. It also has important roles in neuroinflammation and in regulating NMDA receptor activity required for learning and memory. The modulation of glycine-mediated inhibitory activity via IL-1β may play a critical role in the perception of different levels of pain. The central nucleus of the amygdala (CeA) participates in receiving and processing pain information. Interestingly, this nucleus is enriched in the regulatory auxiliary glycine receptor (GlyR) β subunit (βGlyR); however, no studies have evaluated the effect of IL-1β on glycinergic neurotransmission in the brain. Hence, we hypothesized that IL-1β may modulate GlyR-mediated inhibitory activity via interactions with the βGlyR subunit. Our results show that the application of IL-1β (10 ng/ml) to CeA brain slices has a biphasic effect; transiently increases and then reduces sIPSC amplitude of CeA glycinergic currents. Additionally, we performed molecular docking, site-directed mutagenesis, and whole-cell voltage-clamp electrophysiological experiments in HEK cells transfected with GlyRs containing different GlyR subunits. These data indicate that IL-1β modulates GlyR activity by establishing hydrogen bonds with at least one key amino acid residue located in the back of the loop C at the ECD domain of the βGlyR subunit. The present results suggest that IL-1β in the CeA controls glycinergic neurotransmission, possibly via interactions with the βGlyR subunit. This effect could be relevant for understanding how IL-1β released by glia modulates central processing of pain, learning and memory, and is involved in neuroinflammation.

Andrographolide, an Antioxidant, Counteracts Paraquat- Induced Mutagenesis in Mammalian Cells.

Paraquat (1,1'-dimethyl, 4,4'-bipyridinium dichloride; PQ), a commonly used herbicide worldwide, is both toxic and mutagenic. The mutagenic effect of PQ stems from its ability to redox-cycle, generating oxidative stress and subsequently oxidative DNA damage, which miscodes when replication is attempted. Andrographolide (AP1), the major constituent in the leaves of the herbaceous plant Andrographis paniculata, is a diterpenoid with reported antioxidant activity. The present study employed the mammalian cell line AS52 to investigate the protective effect of AP1 against PQ-induced mutagenesis. AP1 induced cytotoxicity in AS52 cells in a dose-dependent manner (IC50 = 15.7 µM), which allowed the selection of a non-lethal dose for the mutagenesis studies. While PQ was mutagenic in AS52 cells as evidenced by the increased levels of 6-TGr mutants, AP1 by itself did not increase the mutation frequency. However, co-treatment with AP1 (1-5 µM) or the antioxidant N-acetylcysteine (2 mM) almost completely counteracted the mutagenicity of PQ (10-100 µM) in AS52 cells. Taken together, these findings suggest that AP1, and likely by extension, A. paniculata extracts, are effective antioxidants that can protect against PQ-induced mutations, and thus could be a promising alternative treatment for PQ poisoning.

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Mutational signatures associated with exposure to carcinogenic microplastic compounds bisphenol A and styrene oxide.

Microplastic pollutants in oceans and food chains are concerning to public health. Common plasticizing compounds Bisphenol-A (BPA) and Styrene-7,8-Oxide (SO) are now labeled as carcinogens. We show that BPA and SO cause deoxyribonucleic acid damage and mutagenesis in human cells, and analyze the genome-wide point mutation and genomic rearrangement patterns associated with BPA and SO exposure. A subset of the single- and doublet base substitutions shows mutagenesis near or at guanine, consistent with these compounds’ preferences to form guanosine adducts. Presence of other mutational signatures suggest additional mutagenesis probably due to complex effects of BPA and SO on diverse cellular processes. Analyzing data for 19 cancer cohorts, we find that tumors of digestive and urinary organs show relatively high similarity in mutational profiles, and the burden of such mutations increases with age. Even within the same cancer type, proportions of corresponding mutational patterns vary among the cohorts from different countries, as does the amount of microplastic waste in ocean waters. BPA and SO are relatively mild mutagens, and other environmental agents can also potentially generate similar, complex mutational patterns in cancer genomes. Nonetheless, our findings call for systematic evaluation of public health consequences of microplastic exposure worldwide.

Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Cytogenetic Biomonitoring in Buccal Mucosa Cells of COVID-19 Patients: Preliminary Findings.

COVID-19 may lead to progressive respiratory failure as a consequence of alveolar damage, resulting in death. The aim of this study was to evaluate cytogenetic damage in oral cells of COVID-19 patients by micronucleus assay. A total of 11 COVID-19 patients aged 40.7±9.3 years (5 men and 6 women) were included in this study. For the control group, a total of 15 participants not infected with SARS-CoV-2 virus were included. The mean age was 41.6±6.2 years (5 men and 10 women). The results showed statistically significant differences (p<0.05) in micronucleated buccal mucosa cells of COVID-19 patients. In addittion, a statistically significant increase in karyolysis and karrhyorexis (p<0.05) was observed in COVID-19 patients compared to control. SARS-CoV-2 virus can induce mutagenesis and cytotoxicity in oral cells.

CRISPR/Cas9 in epigenetics studies of health and disease.

Epigenetics is the heritable phenotypic changes without altering the genotype. Epigenetic processes are such as histone methylation, acetylation, ubiquitination, sumoylation, phosphorylation, ADP ribosylation, DNA methylation and non-coding RNAs interactions associated with structural changes in chromatin. The change of structure is either open chromatin for "active" state or closed chromatin for "inactive" state, that regulates important biological phenomenon like chromatin condensation, gene expression, DNA repair, cellular development, differentiation and homeostasis, etc. However, dysregulation of epigenetic patterns causes diseases like cancer, diabetes, neurological disorder, infectious diseases, autoimmunity etc. Besides, the most important clinical uses of Epigenetics studies are i. identification of disease biomarkers and ii. development of their therapeutics. Epigenetic therapies include epi-drugs, combinatorial therapy, nanocarriers, plant-derived products that are being used for changing the epigenetic pattern to reverse gene expression. However, the developed epi- drugs cause off-target gene and transposable elements activation; promote mutagenesis and carcinogenesis in normal cells, are the major hurdles regarding their clinical use. Therefore, advanced epigenetic therapeutics are required to develop target-specific epigenetic modifications to reverse gene expression pattern. CRISPR-Cas9 (Clustered Regularly Interspaced Palindrome Repeats-associated protein 9) system-mediated gene activation mechanism paves new methods of target-specific epigenetic therapeutics to cure diseases. In this chapter, we discuss how CRISPR/Cas9 and dCas9 have recently been engineered for epigenome editing. Different strategies have been discussed used for epigenome editing based on their efficacy and complexity. Last but not least we have discussed the limitations, different uses of CRISPR/Cas9 and dCas9 in the area of genetic engineering.

Secretory phospholipase A2-X (Pla2g10) is a novel progesterone receptor target gene exclusively induced in uterine luminal epithelium for uterine receptivity in mice

BackgroundAberration of estrogen (E2) and/or progesterone (P4) signaling pathways affects expression of their target genes, which may lead to failure of embryo implantation and following pregnancy. Although many target genes of progesterone receptors (PRs) have been identified in uterine stroma, only a few PR targets have been reported in the epithelium. Secretory phospholipase A2-(PLA2)-X, a member of the PLA2 family that releases arachidonic acids for the synthesis of prostaglandins that are important for embryo implantation, is dysregulated in the endometrium of patients suffering from repeated implantation failure. However, it is not clear whether sPLA2-X is directly regulated by ovarian steroid hormones for embryo implantation in the uterus.ResultP4 induced the Pla2g10 encoding of secretory PLA2-X in the apical region of uterine LE of ovariectomized mice via PR in both time- and dose-dependent manners, whereas E2 significantly inhibited it. This finding is consistent with the higher expression of Pla2g10 at the diestrus stage, when P4 is elevated during the estrous cycle, and at P4-treated delayed implantation. The level of Pla2g10 on day 4 of pregnancy (day 4) was dramatically decreased on day 5, when PRs are absent in the LE. Luciferase assays of mutagenesis in uterine epithelial cells demonstrated that four putative PR response elements in a Pla2g10 promoter region are transcriptionally active for Pla2g10. Intrauterine delivery of small interfering RNA for Pla2g10 on day 3 significantly reduced the number of implantation sites, reinforcing the critical function(s) of Pla2g10 for uterine receptivity in mice.ConclusionsPla2g10 is a novel PR target gene whose expression is exclusively localized in the apical region of the uterine LE for uterine receptivity for embryo implantation in mice.

Mutagenicity of 7-ketocholesterol in CHO cells: The role of lipid peroxidation

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 μM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

Genetic code expansion in mammalian cells: A plasmid system comparison

Genetic code expansion with unnatural amino acids (UAAs) has significantly broadened the chemical repertoire of proteins. Applications of this method in mammalian cells include probing of molecular interactions, conditional control of biological processes, and new strategies for therapeutics and vaccines. A number of methods have been developed for transient UAA mutagenesis in mammalian cells, each with unique features and advantages. All have in common a need to deliver genes encoding additional protein biosynthetic machinery (an orthogonal tRNA/tRNA synthetase pair) and a gene for the protein of interest. In this study, we present a comparative evaluation of select plasmid-based genetic code expansion systems and a detailed analysis of suppression efficiency with different UAAs and in different cell lines.

Novel ALDH5A1 variants and genotype: Phenotype correlation in SSADH deficiency.

To determine genotype-phenotype correlation in succinic semialdehyde dehydrogenase (SSADH) deficiency. ALDH5A1 variants were studied with phenotype correlation in the SSADH natural history study. Assignment of gene variant pathogenicity was based on in silico testing and in vitro enzyme activity after site-directed mutagenesis and expression in HEK293 cells. Phenotypic scoring used a Clinical Severity Score (CSS) designed for the natural history study. Twenty-four patients were enrolled (10 male, 14 female, median age 8.2 years). There were 24 ALDH5A1 variants, including 7 novel pathogenic variants: 2 missense, 3 splice site, and 2 frameshift. Four previously reported variants were identified in >5% of unrelated families. There was a correlation with age and presence (p = 0.003) and severity (p = 0.002) of epilepsy and with obsessive-compulsive disorder (OCD) (p = 0.016). The median IQ score was 53 (Q25-Q75, 49-61). There was no overall correlation between the gene variants and the CSS, although a novel missense variant was associated with the mildest phenotype by CSS in the only patient with a normal IQ, whereas a previously reported variant was consistently associated with the most severe phenotype. Seven novel pathogenic and one previously unpublished benign ALDH5A1 variants were detected. There is an age-dependent association with worsening of epilepsy and presence of OCD in SSADH deficiency. Overall, there does not appear to be a correlation between genotype and phenotypic severity in this cohort of 24 patients. We did find a suspected correlation between a novel pathogenic missense variant and high functionality, and a previously reported pathogenic missense variant and maximal severity.