Factors Influencing Willingness to Undergo Lung Cancer Screening in the Future: A Cross-Sectional Study of Japanese University Students.

Lung cancer (LC) is currently the leading cause of cancer deaths in Japan. Early detection through lung cancer screening (LCS) is important for reducing mortality. Therefore, exploring the factors affecting willingness to undergo LCS, particularly among young people, is important. This study aimed to elucidate the inclination toward LCS and its determining factors among Japanese university students. This cross-sectional study, involving 10,969 Japanese university students, was conducted in April 2023. A Pearson's chi-square test and a binomial logistic regression analysis were used to analyze factors related to the dependent variable, willingness to undergo LCS in the future. Out of the 6779 participants (61.8%) involved in this study, 6504 (95.9%) provided valid responses, and 4609 (70.9%) expressed a willingness to undergo LCS in the future. Analysis revealed current smoking as a barrier to future willingness to undergo LCS. Other barriers included postponing the age of screening, anxiety about the screening content, and concerns about the possibility of having cancer after screening. Addressing barriers, such as current smoking and anxiety about screening, that prevent young people from undergoing LCS in the future is crucial. Therefore, universities should provide opportunities to educate students about LCS and explore various educational methods.

Association between the experience of exertional heat illness (EHI) and living conditions of collegiate student athletes.

Exertional heatstroke (EHS), a severe form of exertional heat illness (EHI), is the third leading cause of death in athletes; thus, early detection and prevention of EHI can help prevent EHS, which is a life-threatening condition. This study aimed to clarify the association between the cognizance of experiencing EHI and living conditions and specific EHI symptoms among collegiate athletes. This study was conducted in October 2022 by administering a questionnaire to 237 male collegiate athletes. Of the 215 (90.7%) respondents, 197 (91.6%) provided valid responses; among them, 88 (44.7%) responded they had experienced EHI, while 109 (55.3%) had not. A history of medical examinations due to EHI, having experienced headaches during summer activities, and having read the EHI manual were factors indicating cognizance of EHI. The number of times meals containing a staple food, main dish, and side dish were eaten in a day was a factor in preventing EHI. Early detection of EHI is important for its prevention, and it is important that athletes themselves have knowledge of symptoms and can correctly self-diagnose EHI. Emphasizing the potential of a well-balanced dietary intake has the potential to prevent EHI is crucial.

University students' living conditions during the COVID-19 pandemic and predictors of their subjective health views: A cross-sectional survey.

This study aimed to explore the factors influencing subjective health views based on the living conditions and concerns of university students during the coronavirus infection 2019 (COVID-19) pandemic. From March to April 2021, a questionnaire survey was administered to 8,547 Japanese university students, and logistic regression analysis was used to explore factors related to subjective health views. The results showed that satisfaction with quality of sleep (OR = 2.651, 95% Cl 2.370-2.966,p < 0.001), satisfaction with university life (OR = 2.486, 95%Cl 2.215-2.789, p < 0.001), satisfaction with diet (OR = 1.849, 95% CI: 1.496-2.285, p < 0.001), regular exercise (OR = 1.759, 95% CI: 1.594-1.941, p < 0.001), consciousness of nutritional balance (OR = 1.276, 95% CI: 1.147-1.420,p < 0.001), eating breakfast every day (OR = 1.247, 95% CI: 1.121-1.387, p < 0.001), and consuming soft drinks at least once a week (OR = 0.865, 95% CI: 0.755-0.966, p = 0.010) were positive factors for subjective views of health. On the other hand, anxiety about whether the necessary credits can be obtained (OR = 0.885, 95% CI: 0.799-0.980, p = 0.019), infection from minimal outings (OR = 0.881, 95% CI: 0.794-0.976, p = 0.016) building and maintaining friendships on campus (OR = 0.867, 95% CI: 0.767-0.980, p = 0.023), and being able to continue working (OR = 0.713, 95% CI: 0.640-0.795, p < 0.001) were identified as negative factors. To ensure a healthy university life during the COVID-19 pandemic or future pandemic, supports tailored to students' living conditions and measures to address their anxieties are required.

Nicotine Dependence among College Students Uninterested in Smoking Cessation during the COVID-19 Pandemic: A Cross-Sectional Survey.

This study investigated nicotine dependence among Japanese university students who had reached the smoking age (20 years or older) by the time of the coronavirus disease 2019 (COVID-19) pandemic and examined factors that encourage early smoking cessation. Social dependence on nicotine was evaluated using the Kano Total Social Nicotine Dependence Level (KTSND), and physiological dependence was evaluated using the Fagerström Nicotine Dependence Index (FTND). Of the 356 college students who smoked (4.4% of the total), 182 (51.1%) stated that they were not interested in quitting. Furthermore, 124 (68.1%) of those with no interest in quitting smoking were aware that smoking is a high-risk factor for COVID-19, and 58 (31.9%) were unaware. The group not aware of this risk had significantly higher KTSND scores than the group aware of it. The examination of cigarette type that indicated the users of non-conventional cigarette products and dual-user groups scored significantly higher than the cigarette group on FTND items. Overall, the smokers scored above the normal range for social nicotine dependence, suggesting the need to reduce nicotine dependence to encourage college students who continue to smoke to quit smoking.

Factors affecting health-related quality of life among firefighters during the COVID-19 pandemic: A single-center study.

During the coronavirus disease 2019 (COVID-19) outbreak, firefighters have been working in an environment that is both physically and mentally taxing. This study aimed to investigate factors affecting health-related quality of life (HRQOL) among firefighters in Japan during the COVID-19 pandemic. A total of 227 firefighters from a single firefighting organization were surveyed in June 2021, during the fourth infection spread period of COVID-19 in Japan. Regression analysis was performed to examine factors affecting HRQOL of firefighters measured with the SF-8. In the present study, factors affecting HRQOL among firefighters during the COVID-19 pandemic were lack of sleep, physical abnormalities due to infection control measures, exercise habits, living with family members, and history of suspected COVID-19 infection. The present findings may help develop support services for first responders, including firefighters during the COVID-19 pandemic.

Willingness and Predictors of Bystander CPR Intervention in the COVID-19 Pandemic: A Survey of Freshmen Enrolled in a Japanese University.

The coronavirus disease 2019 (COVID-19) pandemic has decreased bystander cardiopulmonary resuscitation (BCPR) intervention rates. The purpose of this study was to elucidate the willingness of university freshmen to provide BCPR during the COVID-19 pandemic and the predictors thereof. A cross-sectional survey of 2789 newly enrolled university students was conducted after the end of the sixth wave of the COVID-19 epidemic in Japan; predictors of willingness to provide BCPR were assessed by regression analysis. Of the 2534 participants 1525 (60.2%) were willing to intervene and provide BCPR during the COVID-19 pandemic. Hesitancy due to the anxiety that CPR intervention might result in poor prognosis was a negative predictor of willingness. In contrast, anxiety about the possibility of infection during CPR intervention did not show a negative impact. On the other hand, interest in CPR and willingness to participate in a course, confidence in CPR skills, awareness of automated external defibrillation, and knowledge of CPR during the COVID-19 pandemic, were also positive predictors. This study suggests that the barrier to willingness to intervene with BCPR during a COVID-19 pandemic is not fear of infection, but rather hesitation due to the possibility of poor prognosis from the intervention. The significance of conducting this study during the COVID-19 epidemic is great, and there is an urgent need for measures to overcome hesitation regarding BCPR.

Prevalence of SARS-CoV-2 antibodies among university athletic club members: A cross-sectional survey.

School-based coronavirus disease 2019 (COVID-19) testing is an important part of a comprehensive prevention strategy in public health. To assess the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in a university athletic club community with repeated occurrences of SARS-CoV-2 infections, we conducted a cross-sectional survey for asymptomatic antibody prevalence using a SARS-CoV-2 rapid antibody test kit. On January 26, 2021 we administered questionnaires to determine their history of contact with infected individuals and took blood samples from 129 undergraduates. The prevalence of SARS-CoV-2 antibodies among the subjects was 3.9%. Only 6.2% of the participants reported close contact with infected individuals. In this study, we clarified the prevalence of asymptomatic SARS-CoV-2 antibodies in university athletic clubs where SARS-CoV-2 infections had repeatedly occurred, which will be helpful in discussing how to identify and prevent the transmission of infections within university athletic club communities.

Evaluation of Japanese university students' perception of smoking, interest in quitting, and smoking behavior: An examination and public health challenges during the COVID-19 pandemic.

This study examined college students' perceptions of the association between smoking and novel coronavirus disease 2019 (COVID-19), changes in smoking behavior, and interest in quitting categorized by smoking device, to identify public health challenges. A questionnaire survey was conducted among 8,547 students in a Japanese university in March and April 2021. In response to "Awareness of the increased risk of COVID-19 infection due to smoking and the tendency to develop severe disease", current smokers (70.2%) were more aware of the risk than non-smokers (49.8%) (p < 0.001), with no significant difference according to smoking device (p = 0.213). "Interest in quitting smoking" (p = 0.323), and "Changes in smoking behavior during the COVID-19 pandemic" (p = 0.146) did not differ by smoking device. However, approximately 50% of the respondents answered that they were not interested in quitting smoking, while two-thirds reported that the number of cigarettes they smoked did not change during the pandemic. During the COVID-19 pandemic, college students were found to be less interested in quitting and not likely to change their smoking behavior, despite the knowledge of the increased risk of COVID-19 transmission and severity of disease from smoking, regardless of smoking device.

Antibody response of smokers to the COVID-19 vaccination: Evaluation based on cigarette dependence.

Smokers may have lower antibody titers after vaccination with a coronavirus disease 2019 (COVID-19) mRNA vaccine. However, to the best of our knowledge, no study has evaluated antibody titers after COVID-19 vaccination based on the level of smokers' cigarette dependence. In this study, we measured the level of serum anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) spike protein receptor-binding domain (S-RBD) immunoglobulin-G (IgG) by enzyme linked immunosorbent assay of 55 actively smoking Japanese social workers (firefighters, paramedics, and rescue workers) who had received two doses of the BNT162b2 vaccine. Further, we assessed their cigarette dependence using the Fagerstrom Test for Nicotine Dependence (FTND), measured their serum cotinine levels, and tested for their correlation with anti-RBD IgG levels. Serum anti-SARS-CoV-2 S-RBD protein IgG levels after BNT162b2 vaccination showed a significant negative correlation with FTND (ρ = -0.426, p = 0.001). In addition, serum cotinine level showed a significant positive correlation with FTND (ρ = 0.470, p = 0.000). However, no significant negative correlation was noted between serum cotinine and serum anti-SARS-CoV-2 S-RBD protein IgG levels (ρ = -0.156, p = 0.256). Our results suggest that smokers with strong cigarette dependence have inadequate anti-SARS-CoV-2 S-RBD protein IgG levels after COVID-19 mRNA vaccination.

A Survey of Living Conditions and Psychological Distress in Japanese University Freshmen during the COVID-19 Pandemic.

Since the novel coronavirus disease 2019 (COVID-19) pandemic, educational institutions have implemented measures such as school closures, raising concerns regarding the increase in psychological distress among university students. The purpose of this study is to identify factors that have influenced psychological distress among college freshmen during the COVID-19 pandemic. A questionnaire survey was conducted at the conclusion of the sixth wave of COVID-19 in Japan. Psychological distress was measured using the six-item Kessler Psychological Distress Scale (K6). Factors affecting psychological distress were calculated using regression analysis. Of the 2536 participants, 1841 (72.6%) reported having no psychological distress, while 695 (27.4%) reported having psychological distress. Factors that were identified to contribute to psychological distress were lack of sleep, weight gain or loss, worsening of interpersonal relationships, and physical symptoms and illnesses. A willingness to join an athletic club and having an environment in which it is easy to discuss worries and anxieties with others were factors that were identified to hinder psychological distress. It is necessary for universities to offer enhanced supports for physical and interpersonal activities. Additionally, it is imperative to encourage students to look after their physical health and to actively utilize university-based consultation systems.

Effects of Heat-Not-Burn Cigarette Smoking on the Secretion of Saliva and Its Innate Immune System Components.

Saliva and salivary antimicrobial proteins play important roles in the innate immunity, which prevents infections of orally invading bacteria and viruses. In this study, we compared the secretion rates of salivary lactoferrin (Lac) and lysozyme (Lys) in heat-not-burn (HNB) cigarette smokers and non-smokers. The analysis population for this study included 212 members of the fire department, including 32 HNB cigarette smokers, 17 paper cigarette smokers, 14 combined HNB and paper cigarette smokers, and 149 non-smokers. Salivary Lac and Lys concentrations were assessed using enzyme immunoassay. Saliva secretion was significantly lower among HNB cigarette smokers (p < 0.01) than among non-smokers. Accompanying this result, salivary Lac and Lys secretion rates were significantly lower among smokers, particularly HNB cigarette smokers, than among non-smokers (all p < 0.01). Our findings suggest a possible adverse effect of HNB cigarette on the amount of Lac and Lys released into the oral cavity.

Japanese patients with mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: In vitro functional analysis of five novel HMGCS2 mutations.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) deficiency is a metabolic disorder caused by mutations in the HMGCS2 gene. The present study describes the identification of four cases of HMGCS2 deficiency in Japan. Hepatomegaly and severe metabolic acidosis were observed in all cases. Fatty liver was identified in three cases, which suggested the unavailability of fatty acids. All patients presented with a high C2/C0 ratio, suggesting that the fatty acid oxidation pathway was normal during metabolic crisis. Genetic analyses revealed five rare, novel variants (p.G219E, p.M235T, p.V253A, p.S392L and p.R500C) in HMGCS2. To confirm their pathogenicity, a eukaryotic expression system and a bacterial expression system was adopted that was successfully used to obtain affinity-purified HMGCS2 protein with measurable activity. Purified M235T, S392L and R500C proteins did not retain any residual activity, whilst the V253A variant showed some residual enzymatic activity. Judging from the transient expression experiment in 293T cells, the G219E variant appeared to be unstable. In conclusion, the present study identified five novel variants of HMGCS2 that were indicated to be pathogenic in four patients affected by HMGCS2 deficiency.

Development of Specific Fluorogenic Substrates for Human ß-N-Acetyl-D-hexosaminidase A for Cell-Based Assays.

Inhibitors of human ß-N-acetyl-D-hexosaminidase (hHEX) A and human O-GlcNAcase (hOGA) reportedly play roles in multiple diseases, suggesting their potential for pharmacological chaperone (PC) therapy of Sandhoff disease (SD) and Tay-Sachs disease (TSD), as lysosomal storage diseases, and Alzheimer's disease and progressive supranuclear palsy, respectively. In particular, hHEXA inhibitors as PCs have been shown to successfully enhance hHEXA levels, leading to the chronic form of SD and TSD. In the diagnosis of enzyme deficiencies in SD and TSD, artificial hHEXA substrates based on 4-methylumbelliferone as a fluorophore are available and generally used; however, they do not have sufficient performance to screen for potential inhibitors for a PC therapy from compound libraries. Further, there are currently few fluorogenic substrates for hHEXA suitable for such requirements and there are no substrates ideal for cell-based inhibitor screening. Here, we clarified the difference in enzyme active site structure between hHEXA and hOGA from their tertiary structures. To develop lysosome-localized hHEXA-specific fluorogenic substrates based on the difference in their active site structures, our developed quinone methide cleavage substrate design platform was applied for the molecular design of substrates. Thereafter, we synthesized via the shortest route and evaluated novel three-color fluorogenic substrates for hHEXA that exhibited excellent specificity and sensitivity in three human cell lines. The designed substrates represent the first-in-a class of new substrates that can be utilized to screen hHEXA inhibitors in adherent human cultured cells.

Deficiency of 3-hydroxybutyrate dehydrogenase (BDH1) in mice causes low ketone body levels and fatty liver during fasting.

d-3-Hydroxy-n-butyrate dehydrogenase (BDH1; EC 1.1.1.30), encoded by BDH1, catalyzes the reversible reduction of acetoacetate (AcAc) to 3-hydroxybutyrate (3HB). BDH1 is the last enzyme of hepatic ketogenesis and the first enzyme of ketolysis. The hereditary deficiency of BDH1 has not yet been described in humans. To define the features of BDH1 deficiency in a mammalian model, we generated Bdh1-deficient mice (Bdh1 KO mice). Under normal housing conditions, with unrestricted access to food, Bdh1 KO mice showed normal growth, appearance, behavior, and fertility. In contrast, fasting produced marked differences from controls. Although Bdh1 KO mice survive fasting for at least 48 hours, blood 3HB levels remained very low in Bdh1 KO mice, and despite AcAc levels moderately higher than in controls, total ketone body levels in Bdh1 KO mice were significantly lower than in wild-type (WT) mice after 16, 24, and 48 hours fasting. Hepatic fat content at 24 hours of fasting was greater in Bdh1 KO than in WT mice. Systemic BDH1 deficiency was well tolerated under normal fed conditions but manifested during fasting with a marked increase in AcAc/3HB ratio and hepatic steatosis, indicating the importance of ketogenesis for lipid energy balance in the liver.

Recent advances in understanding beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency (OMIM #203750, *607809) is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. This disorder is clinically characterized by intermittent ketoacidotic crises under ketogenic stresses. In addition to a previous 26-case series, four series of T2-deficient patients were recently reported from different regions. In these series, most T2-deficient patients developed their first ketoacidotic crises between the ages of 6 months and 3 years. Most patients experienced less than three metabolic crises. Newborn screening (NBS) for T2 deficiency is performed in some countries but some T2-deficient patients have been missed by NBS. Therefore, T2 deficiency should be considered in patients with severe metabolic acidosis, even in regions where NBS for T2 deficiency is performed. Neurological manifestations, especially extrapyramidal manifestations, can occur as sequelae to severe metabolic acidosis; however, this can also occur in patients without any apparent metabolic crisis or before the onset of metabolic crisis.

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells.

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.

Intronic antisense Alu elements have a negative splicing effect on the inclusion of adjacent downstream exons.

Alu elements occupy 10% of the human genome. However, although they contribute to genomic and transcriptomic diversity, their function is still not fully understood. We hypothesized that intronic Alu elements may contribute to alternative splicing. We therefore examined their effect on splicing using minigene constructs including exon 9-exon 11 inclusive of ACAT1 with truncated introns 9 and 10. These constructs contained a suboptimal splice acceptor site for intron 9. Insertion of AluY-partial AluSz6-AluSx, originally located in ACAT1 intron 5, in an antisense direction within intron 9 had a negative effect on exon 10 inclusion. This effect was additive with that of an exonic splicing enhancer mutation in exon 10, and was canceled by the substitution of G for C at the first nucleotide of exon 10 which optimized the splice acceptor site of intron 9. A sense AluY-partial AluSz6-AluSx insertion had no effect on exon 10 inclusion, and one antisense AluSx insertion had a similar effect to antisense AluY-partial AluSz6-AluSx insertion. The shorter the distance between the antisense Alu element and exon 10, the greater the negative effect on exon 10 inclusion. This distance effect was more evident for suboptimal than optimal splice sites. Based on our data, we propose that intronic antisense Alu elements contribute to alternative splicing and transcriptomic diversity in some genes, especially when splice acceptor sites are suboptimal.

Heterozygous carriers of succinyl-CoA:3-oxoacid CoA transferase deficiency can develop severe ketoacidosis.

Succinyl-CoA:3-oxoacid CoA transferase (SCOT, gene symbol OXCT1) deficiency is an autosomal recessive disorder in ketone body utilization that results in severe recurrent ketoacidotic episodes in infancy, including neonatal periods. More than 30 patients with this disorder have been reported and to our knowledge, their heterozygous parents and siblings have had no apparent ketoacidotic episodes. Over 5 years (2008-2012), we investigated several patients that presented with severe ketoacidosis and identified a heterozygous OXCT1 mutation in four of these cases (Case1 p.R281C, Case2 p.T435N, Case3 p.W213*, Case4 c.493delG). To confirm their heterozygous state, we performed a multiplex ligation-dependent probe amplification analysis on the OXCT1 gene which excluded the presence of large deletions or insertions in another allele. A sequencing analysis of subcloned full-length SCOT cDNA showed that wild-type cDNA clones were present at reasonable rates to mutant cDNA clones. Over the following 2 years (2013-2014), we analyzed OXCT1 mutations in six more patients presenting with severe ketoacidosis (blood pH ≦7.25 and total ketone body ≧10 mmol/L) with non-specific urinary organic acid profiles. Of these, a heterozygous OXCT1 mutation was found in two cases (Case5 p.G391D, Case6 p.R281C). Moreover, transient expression analysis revealed R281C and T435N mutants to be temperature-sensitive. This characteristic may be important because most patients developed ketoacidosis during infections. Our data indicate that heterozygous carriers of OXCT1 mutations can develop severe ketoacidotic episodes in conjunction with ketogenic stresses.

Single-nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in the ACAT1 gene.

BACKGROUND: ß-ketothiolase (T2, gene symbol ACAT1) deficiency is an autosomal recessive disorder, affecting isoleucine and ketone body metabolism. We encountered a patient (GK03) with T2 deficiency whose T2 mRNA level was <10% of the control, but in whom a previous routine cDNA analysis had failed to find any mutations. Genomic PCR-direct sequencing showed homozygosity for c.941-9T>A in the polypyrimidine stretch at the splice acceptor site of intron 9 of ACAT1. Initially, we regarded this variant as not being disease-causing by a method of predicting the effect of splicing using in silico tools. However, based on other findings of exon 10 splicing, we eventually hypothesized that this mutation causes exon 10 skipping. METHODS: cDNA analysis was performed using GK03's fibroblasts treated with/without cycloheximide (CHX), since exon 10 skipping caused a frameshift and nonsense-mediated mRNA decay (NMD). Minigene splicing experiment was done to confirm aberrant splicing. RESULTS: cDNA analysis using fibroblasts cultured with cycloheximide indeed showed the occurrence of exon 10 skipping. A minigene splicing experiment clearly showed that the c.941-9T>A mutant resulted in transcripts with exon 10 skipping. There are few reports describing that single-nucleotide substitutions in polypyrimidine stretches of splice acceptor sites cause aberrant splicing. CONCLUSION: We showed that c.941-9T>A induces aberrant splicing in the ACAT1 gene. Our ability to predict the effects of mutations on splicing using in silico tools is still limited. cDNA analysis and minigene splicing experiments remain useful alternatives to reveal splice defects.

A novel mutation (c.121­13T>A) in the polypyrimidine tract of the splice acceptor site of intron 2 causes exon 3 skipping in mitochondrial acetoacetyl-CoA thiolase gene.

Mitochondrial acetoacetyl-CoA thiolase (T2) (gene symbol: ACAT1) deficiency is an autosomal recessive disorder affecting isoleucine catabolism and ketone body utilization. In this study, mutational analysis of an Indian T2-deficient patient revealed a homozygous mutation (c.121­13T>A) located at the polypyrimidine tract of the splice acceptor site of intron 2, and exon 3 skipping was identified by cDNA analysis using cycloheximide. We made three mutant constructs (c.121­13T>A, T>C, and T>G substitutions) followed by making a wild-type minigene construct that included an ACAT1 segment from exon 2 to 4 for a splicing experiment. The minigene splicing experiment demonstrated that exon 3 skipping was induced not only by c.121­13T>A mutation, but also by the other two substitutions. It was difficult to predict the effect of these mutations on splicing using in silico tools, as predictions of different tools were inconsistent with each other. The minigene splicing experiment remains the most reliable method to unravel splicing abnormalities.