Enhanced recombination empowers the detection and mapping of Quantitative Trait Loci.

Modern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.

Vitamin D Deficiency and Gingival Enlargement: A Case Report.

The occurrence of vitamin D insufficiency is rising constantly, and most pediatric patients are below the required levels. Individuals with vitamin D deficiency are more susceptible to inflammatory diseases because it reduces their immunity. The role of vitamin D deficiency in gingival enlargement has been reported in the literature. In this case report, we are describing a case in which a vitamin D supplement has resolved the gingival enlargement significantly without any invasive procedure. A 12-year-old boy reported a chief complaint of swollen gums in the upper and lower front teeth region. On clinical examination, there was minor surface plaque and calculus along with the formation of pseudopockets, but there was no clinical attachment loss. The patient has been advised to undergo laboratory tests for a complete blood profile, including a vitamin assessment. The patient reported after two and a half months with a gingivectomy on the first quadrant at a private clinic. They reported back to us because they didn't want the same trauma from surgery again and wanted a more conservative treatment option. So, on the basis of the reassessment of reports, vitamin D deficiency was confirmed, and treatment was started with 60,000 thousand I/U of vitamin D supplement weekly and advised for sunlight exposure with minimal clothing. There was a significant decrease in enlargement observed after the six-month follow-up period. Vitamin D supplements can be a more conservative treatment option for gingival enlargement of unknown etiology.

Health-Seeking Behavior and its Determinants for Different Noncommunicable Diseases in Elderly.

Steady improvement in quality of life has led to increased survival time of elderly, especially of those with noncommunicable diseases. A study about their health-seeking behavior (HSB) and its determinants is essential for provision of comprehensive care and facilitate policy development. A cross-sectional study was conducted among 350 elderly participants over 60 years of age in an urbanized village of Delhi. Data was collected using a semi-structured questionnaire, and detailed general and systemic examination was done. Tests of significance were applied to assess the HSB with various demographic, clinico-social, and economic variables. A total of 87.4% of the study population was suffering from at least one noncommunicable disease (NCD). Majority of the study subjects' NCDs (72.66%) were diagnosed while getting treatment for a symptom of the disease. Also, 52.94% of the participants had inappropriate HSB. HSB was significantly associated with gender, age, duration of illness, and importantly with modifiable variables like the level of literacy, distance of preferred health facility, and presence of multimorbidity. HSB was found to be inappropriate in over half of the participants. Policymaking should focus on modifiable variables like education, distance of health facility, and multimorbidity, especially for commonly ignored diseases like osteoarthritis and diabetes.

Reliable genotyping of recombinant genomes using a robust hidden Markov model.

Meiotic recombination is an essential mechanism during sexual reproduction and includes the exchange of chromosome segments between homologous chromosomes. New allelic combinations are transmitted to the new generation, introducing novel genetic variation in the offspring genomes. With the improvement of high-throughput whole-genome sequencing technologies, large numbers of recombinant individuals can now be sequenced with low sequencing depth at low costs, necessitating computational methods for reconstructing their haplotypes. The main challenge is the uncertainty in haplotype calling that arises from the low information content of a single genomic position. Straightforward sliding window-based approaches are difficult to tune and fail to place recombination breakpoints precisely. Hidden Markov model (HMM)-based approaches, on the other hand, tend to over-segment the genome. Here, we present RTIGER, an HMM-based model that exploits in a mathematically precise way the fact that true chromosome segments typically have a certain minimum length. We further separate the task of identifying the correct haplotype sequence from the accurate placement of haplotype borders, thereby maximizing the accuracy of border positions. By comparing segmentations based on simulated data with known underlying haplotypes, we highlight the reasons for RTIGER outperforming traditional segmentation approaches. We then analyze the meiotic recombination pattern of segregants of 2 Arabidopsis (Arabidopsis thaliana) accessions and a previously described hyper-recombining mutant. RTIGER is available as an R package with an efficient Julia implementation of the core algorithm.

How to Perform Discriminant Analysis in Medical Research? Explained with Illustrations.

Discriminant function analysis is the statistical analysis used to analyze data when the dependent variable or outcome is categorical and independent variable or predictor variable is parametric. It is a parametric technique to determine which weightings of quantitative variables or predictors best discriminates between two or more than two categories of dependent variables and does so better than chance. Discriminant analysis is used to find out the accuracy of a given classification system in predicting the sample into a particular group. Discriminant analysis includes the development of discriminant functions for each sample and deriving a cutoff score that is used for classifying the samples into different groups. Discriminant function analysis is a statistical analysis used to find out the accuracy of a given classification system or predictor variables. This article explains the basic assumptions, uses, and necessary requirements of discriminant analysis with a real-life clinical example. Whenever a new classification system is introduced, discriminant function analysis can be used to find out the accuracy with which the classification is able to differentiate a particular sample into different groups. Thus, it is a very useful tool in medical research where classification is required.

plotsr: visualizing structural similarities and rearrangements between multiple genomes.

SUMMARY: Third-generation genome sequencing technologies have led to a sharp increase in the number of high-quality genome assemblies. This allows the comparison of multiple assembled genomes of individual species and demands new tools for visualizing their structural properties. Here, we present plotsr, an efficient tool to visualize structural similarities and rearrangements between genomes. It can be used to compare genomes on chromosome level or to zoom in on any selected region. In addition, plotsr can augment the visualization with regional identifiers (e.g. genes or genomic markers) or histogram tracks for continuous features (e.g. GC content or polymorphism density). AVAILABILITY AND IMPLEMENTATION: plotsr is implemented as a python package and uses the standard matplotlib library for plotting. It is freely available under the MIT license at GitHub (https://github.com/schneebergerlab/plotsr) and bioconda (https://anaconda.org/bioconda/plotsr). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar.

Potato is the most widely produced tuber crop worldwide. However, reconstructing the four haplotypes of its autotetraploid genome remained an unsolved challenge. Here, we report the 3.1 Gb haplotype-resolved (at 99.6% precision), chromosome-scale assembly of the potato cultivar 'Otava' based on high-quality long reads, single-cell sequencing of 717 pollen genomes and Hi-C data. Unexpectedly, ~50% of the genome was identical-by-descent due to recent inbreeding, which was contrasted by highly abundant structural rearrangements involving ~20% of the genome. Among 38,214 genes, only 54% were present in all four haplotypes with an average of 3.2 copies per gene. Taking the leaf transcriptome as an example, 11% of the genes were differently expressed in at least one haplotype, where 25% of them were likely regulated through allele-specific DNA methylation. Our work sheds light on the recent breeding history of potato, the functional organization of its tetraploid genome and has the potential to strengthen the future of genomics-assisted breeding.

COVID-19 vaccine confidence project for perinatal women (CCPP)-Development of a stepped-care model to address COVID-19 vaccine hesitancy in low and middle-income countries.

Background: COVID-19 vaccine hesitancy (CVH) is common among perinatal women in low and middle-income countries (LMICs), but it is often unaddressed. This could be due to a lack of feasible, scalable, and acceptable interventions and models for CVH in LMICs. Our study aimed to develop a CVH intervention model that can be implemented in LMICs using existing human healthcare resources. Methods: A literature review was conducted on aspects of vaccine hesitancy, pre-existing interventions, and models for addressing vaccine hesitancy (COVID-19 and non-COVID-19). The lead authors (RR and PKuk) formed a team consisting of vaccinators, experts, and stakeholders. Members shared their perspectives and proposals for various models and interventions that could be implemented in LMICs. A CVH intervention model was developed using a logic model, a WHO implementation toolkit, experts' feedback, and consensus. Results: A consensus was reached to develop a COVID-19 Vaccine Confidence Project for Perinatal Women (CCPP), which is a primary health care worker (HCWs)-based stepped-care model. The CCPP model includes HCW training, integration into ongoing COVID-19 vaccination programs, CVH screening, CVH intervention, and referral services suitable for implementation in LMICs. Conclusion: The CCPP project/model provides a practical approach that can help in the early detection and management of CVH. The model can be tailored to different healthcare settings to improve COVID-19 vaccine uptake among perinatal women in LMICs.

Efficacy and Safety of Thalidomide in Patients With Transfusion-Dependent Thalassemia.

OBJECTIVE: To assess the efficacy and safety of thalidomide in children with transfusion-dependent thalassemia. METHODS: This prospective, single center, open-label study enrolled children aged 12-18 years, and who received thalidomide for a duration of 6 months at a starting dose of 2-3 mg/kg/day. Efficacy was assessed by reduction in transfusion requirement and rate of fall of hemoglobin. Efficacy was classified as major, moderate and minimal/no response depending on the reduction in transfusion requirement. Safety was assessed by adverse effects related to thalidomide. RESULTS: 37 children [mean (SD) age, 14.7 (1.8) years were included. Rate of fall of hemoglobin reduced from a mean of 1.0 (0.24) g/week pre-thalidomide therapy to 0.58 (0.26) g/week after 6 months of thalidomide (P<0.001). 19 children (51.3%) had major response and 12 (32.4%) had moderate response. In 13.5% and 32.4% children response was observed within the first and second month of therapy, respectively. 15 (40.5%) children remained transfusion - free for a median (IQR) time of 6 (3-10) weeks of thalidomide therapy. Mean serum ferritin (SD) decreased from 1758.9 (835.1) to 1549.6(1016.9) (P<0.001). Mean HbF (SD) showed an increase from 2.95(2.6) to 49.2(33.3) (P<0.001). In 32 children, 47 adverse events were observed. Common adverse events were constipation and neutropenia (mostly mild). CONCLUSIONS: Thalidomide resulted in major/moderate response in majority of children with transfusion-dependent thalassemia with satisfactory adverse effect profile.

Using the longest run subsequence problem within homology-based scaffolding.

Genome assembly is one of the most important problems in computational genomics. Here, we suggest addressing an issue that arises in homology-based scaffolding, that is, when linking and ordering contigs to obtain larger pseudo-chromosomes by means of a second incomplete assembly of a related species. The idea is to use alignments of binned regions in one contig to find the most homologous contig in the other assembly. We show that ordering the contigs of the other assembly can be expressed by a new string problem, the longest run subsequence problem (LRS). We show that LRS is NP-hard and present reduction rules and two algorithmic approaches that, together, are able to solve large instances of LRS to provable optimality. All data used in the experiments as well as our source code are freely available. We demonstrate its usefulness within an existing larger scaffolding approach by solving realistic instances resulting from partial Arabidopsis thaliana assemblies in short computation time.

Hypertension during pregnancy and risk of stillbirth: challenges in a developing country.

OBJECTIVE: To explore the factors associated with hypertensive disorders of pregnancy (HDP) in women experiencing stillbirth (SB). MATERIAL AND METHODS: It was an observational, analytical case-control study, done as part of the World Health Organization South-East Asian Region Office SB project. The epidemiological profile, history, clinical features, and investigations of stillborn cases having HDP were noted, an equal number of age and parity matched subjects with HDP having livebirth were taken as controls. RESULTS: Out of 46,816 deliveries, 1239 (26.2/1000) were stillborn. The maternal causes contributed 374/1239 (30.2%) stillbirths according to the CODAC classification, HDP was the most common maternal cause (304/1239, 24.9%). Subjects with HDP were included as cases. On comparing the cases with controls it was found that significantly more number of cases had inadequate antenatal visits (p < .001, OR -4.8), two or more abortions (p < .001, OR -1.9), early onset of hypertension (p < .0001, OR -5.6) and complications such as fetal growth restriction (FGR) (p < .001, OR -2.3) and abruption (p < .001, OR -4.0). Women with preeclampsia were less likely to have SB compared to those with gestational or chronic hypertension (p = .0001, OR -2.3). The birth weight/placental weight ratio of more than eight had the highest odds ratio regarding contribution to SB among hypertensive women (p = .0001 OR -6.7). CONCLUSION: In women with HDP, adequate antenatal care would lead to the prevention of complications such as anemia, abruption, and FGR, and thus prevent SB. The high BW/PW ratio is a potential marker of risk of SB.

Gamete binning: chromosome-level and haplotype-resolved genome assembly enabled by high-throughput single-cell sequencing of gamete genomes.

Generating chromosome-level, haplotype-resolved assemblies of heterozygous genomes remains challenging. To address this, we developed gamete binning, a method based on single-cell sequencing of haploid gametes enabling separation of the whole-genome sequencing reads into haplotype-specific reads sets. After assembling the reads of each haplotype, the contigs are scaffolded to chromosome level using a genetic map derived from the gametes. We assemble the two genomes of a diploid apricot tree based on whole-genome sequencing of 445 individual pollen grains. The two haplotype assemblies (N50: 25.5 and 25.8 Mb) feature a haplotyping precision of greater than 99% and are accurately scaffolded to chromosome-level.

An Aptamer Linked Immobilized Sorbent Assay (ALISA) to Detect Circulatory IFN-α, an Inflammatory Protein among Tuberculosis Patients.

Dysregulation of IFN-α is the basis for pathogenesis of autoimmune as well as infectious diseases. Identifying inflammatory signatures in peripheral blood of patients is an approach for monitoring active infection. Hence, estimation of type I IFNs as an inflammatory biomarker to scrutinize disease status after treatment is useful. Accordingly, an Aptamer Linked Immobilized Sorbent Assay (ALISA) for the detection of IFN-α in serum samples was developed. Sixteen aptamers were screened for their ability to bind IFN-α. Aptamer IFNα-3 exhibited specificity for IFN-α with no cross-reactivity with interferons ß and γ and human serum albumin. The disassociation constant (Kd) was determined to be 3.96 ± 0.36 nM, and the limit of detection was ∼2 ng. The characterized IFNα-3 aptamer was used in ALISA to screen tuberculosis (TB) patients' sera. An elevated IFN-α level in sera derived from untreated TB patients (median = 0.31), compared to nontuberculous household contacts (median = 0.13) and healthy volunteers (median = 0.12), and further a decline in IFN-α level among treated patients (median = 0.13) were seen. The ALISA assay facilitates direct estimation of inflammatory protein(s) in circulation unlike mRNA estimation by real time PCR. Designing of aptamers similar to the IFNα-3 aptamer provides a novel approach to assess other inflammatory protein(s) in patients before, during, and after completion of treatment and would denote clinical improvement in successfully treated patients.

Adverse effects with intravenous methotrexate in children with acute lymphoblastic leukemia/lymphoma: a retrospective study.

Methotrexate (MTX) forms the backbone of maintenance cycles in childhood acute lymphoblastic leukemia (ALL) chemotherapy, including interim maintenance. There is sufficient published data describing toxicities of high dose MTX (HD-MTX), but toxicities with escalating doses of MTX (Capizzi regimen) is not well documented. Capizzi regimen is thought to be relatively safe; we contend that even low escalating doses of MTX have significant toxicities. Our study intends to characterise such events with Capizzi MTX in comparison to that seen with HD-MTX. The retrospective study was conducted at a tertiary care centre of North India. We looked for the presence of six main toxicities: febrile neutropenia, thrombocytopenia, mucositis, hepatic toxicity, renal toxicity and skin toxicity from the clinical records of children with newly diagnosed acute lymphoblastic leukemia and lymphoma (intermediate and high risk disease), treated at our centre from November 2013 to July 2018. Intermediate risk ALL (IR-ALL) received Capizzi MTX, whereas high risk ALL (HR-ALL/T-NHL), received HD-MTX. Both these regimens do not use L-asparaginase. A total of 237 cycles of Capizzi escalating MTX and 151 cycles of HD-MTX (B cell: 3 gm/m2 and T cell ALL/T-NHL: 5 gm/m2) during interim maintenance were studied in 93 children. Fifty-four (54) children were of IR (all B cell ALL) and 39 of HR-ALL (21 B-ALL, 18 T-ALL/T-NHL). The combined incidence of toxicities, were similar between the two groups: 68/237 cycles (28.7%) of Capizzi MTX and 45/151 cycles (29.8%) of HD-MTX (P = 0.815). However, mucositis was more commonly witnessed in the later group at 22/151 cycle (14.6%) versus 13/237 cycles (5.5%) in Capizzi MTX (P = 0.002). Nephrotoxicity and skin toxicity was seen only in the HD-MTX group. There was no difference in the severity of toxicity, graded using NCI CTCAE v 5.0, between the two groups. There was no mortality directly attributable to methotrexate toxicity (Grade V toxicity). Serum MTX levels were available in 69/151 (45.7%) cycles of HD-MTX and showed no association with toxicity in this group. Also, there was no difference in the incidence of combined toxicities between groups with (19/69 cycles) or without (26/82 cycles) available serum MTX levels in the HR group (P = 0.577). Male gender, lower baseline ANC and lower BMI had significant association with toxicity. Methotrexate related toxicity is common with both Capizzi and HD-MTX schedule in childhood ALL with a correlation of lower BMI, baseline ANC and male gender. However, it is possible to administer Capizzi as well as HD-MTX in lower middle income countries, with manageable toxicity. Further studies will be required to substantiate our findings and determine the predictors of such events.

Impact and prognosis of the expression of IFN-α among tuberculosis patients.

Mycobacterium tuberculosis (M.tb) infection stimulates the release of cytokines, including interferons (IFNs). IFNs are initiators, regulators, and effectors of innate and adaptive immunity. Accordingly, the expression levels of Type I (α, ß) and II (γ) IFNs, among untreated tuberculosis (TB) patients and household contacts (HHC) clinically free of TB was assessed. A total of 264 individuals (TB patients-123; HHC-86; laboratory volunteers-55; Treated TB patients-36) were enrolled for this study. IFN-α mRNA expression levels predominated compared to IFN-γ and IFN-ß among untreated TB patients. IFN-α transcripts were ~3.5 folds higher in TB patients compared to HHC, (p<0.0001). High expression of IFN-α was seen among 46% (56/ 123) of the TB patients and 26%, (22/86) of HHCs. The expression levels of IFN-α correlated with that of IFN transcriptional release factor 7 (IRF) (p<0.0001). In contrast, an inverse relationship exists between PGE2 and IFN-α expression levels; high IFN-α expressers were associated with low levels of PGE2 and vice-versa (Spearman's rho = -0.563; p<0.0001). In-vitro, IFN-α failed to restrict the replication of intracellular M.tb. The anti-mycobacterial activity of IFN-γ was compromised in the presence of IFN-α, but not by IFN-ß. The expression of IFN-α and ß diminished or is absent, among successfully treated TB patients. These observations suggest the utility of assessment of Type I IFNs expression levels as a prognostic marker to monitor tuberculosis patient response to chemotherapy because changes in Type I IFNs expression are expected to precede the clearance and /reduction in bacterial load.

SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies.

Genomic differences range from single nucleotide differences to complex structural variations. Current methods typically annotate sequence differences ranging from SNPs to large indels accurately but do not unravel the full complexity of structural rearrangements, including inversions, translocations, and duplications, where highly similar sequence changes in location, orientation, or copy number. Here, we present SyRI, a pairwise whole-genome comparison tool for chromosome-level assemblies. SyRI starts by finding rearranged regions and then searches for differences in the sequences, which are distinguished for residing in syntenic or rearranged regions. This distinction is important as rearranged regions are inherited differently compared to syntenic regions.

Determinants of Expenditure on Diabetes Care: A Community Based Longitudinal Study in aResettlement Colony of Delhi.

INTRODUCTION: Diabetes is one of the expensive diseases due to its chronic nature and gradual involvement of multiple organs, Moreover loss of economic productivity further enhances the cost of care. Several factors were reported to have impact on overall economic burden in diabetic patients. So, the present study aims to determine influence of various socio-demographic and clinical factors on expenditure of diabetes care among patients residing in resettlement colony of East Delhi. METHODOLOGY: A community based one year longitudinal study was conducted in Kalyanpuri area of East Delhi. All the diabetes patients aged 25 years and who were the permanent residents of Kalyanpuri, attending the Diabetic Clinic of a government hospital in November-December 2014 were selected for the study. A pre-tested semi-structured interview schedule was used as study tool. Each subject was followed up 3 monthly from January to December 2015. RESULTS: Data of 150 study subjects was analyzed. Out of 150 subjects 45(30 %) were male and 105 (70%) female. Overall mean age of study subjects was 53 ± 10 years Among socio-demographic factors, Expenditure on diabetes care showed significant association with male gender and among clinical factors, longer duration since diagnosis, use of Insulin with Oral Hypoglycemic drugs, hospitalization and utilization of private care has shown positive association with expenditure on diabetes care. CONCLUSION: The present study concludes that there is need of better provisioning of services for diabetes care in government health facilities to cater needs of growing diabetic population..

Gallstone disease and its correlates among patients attending teaching hospital of North India.

BACKGROUND: Gallstone disease (GSD) represents a significant burden for healthcare systems worldwide and is one of the most common disorders among patients presenting to emergency rooms with abdominal discomfort. AIM AND OBJECTIVE: This study was aimed to find correlates (demographic, dietary, and behavioral) of GSD in patients attending teaching hospital in North India. METHODOLOGY: A case-control study was conducted from January 2013 to December 2013 among subjects attending outpatient department of general surgery in a teaching hospital. Data collection for both cases and controls was done on a self-designed pretested "interview schedule" which assessed the sociodemographic profile, personal history, medical history, comorbidities, physical examination including anthropometry, and dietary intakes. To measure the strength of association, odds ratio (OR) was calculated. Binary logistic regression was used for multivariate analysis. RESULTS: A total of 120 cases and the same number of age- and sex-matched controls were included in final analysis. In binary logistic regression, maximum association was seen with physical inactivity [OR 3.93, confidence interval (CI): 1.98-7.78] followed by current consumption of smokeless tobacco (OR 3.65, CI: 1.65-8.09), current smoking (OR 2.69, CI: 1.13-6.37), nonvegetarian diet (OR 3.10, CI: 1.65-5.83), and fat intake (OR 2.14, CI: 1.14-4.02). Current alcohol consumption (OR 0.90, CI: 0.41-1.98), high waist-hip ratio (OR 1.54, CI: 0.67-3.56), and intake of fruits and green leafy vegetables (OR 1.86, CI: 0.61-5.61) were not significantly associated with GSD. CONCLUSION: Physical inactivity, smokeless tobacco, nonvegetarian diet, current smoking, high fat intake, and family history were found to be risk factors for the development of GSD.

Gallstone disease and quantitative analysis of independent biochemical parameters: Study in a tertiary care hospital of India.

INTRODUCTION: Gallstone disease (GSD) represents a significant burden for health-care systems worldwide and is one of the most common disorders among patients presenting to emergency rooms with abdominal discomfort. This study was aimed to know the association of biochemical parameters and development of GSD. MATERIALS AND METHODS: We conducted a case-control study from January 2013 to December 2013 among patients attending outpatient department of general surgery at a tertiary care hospital of Delhi, North India; we recruited 120 cases and the same number of controls in the study. Data collection for both cases and controls was done on a self-designed pretested interview schedule and all the patients were investigated for relevant biochemical parameters. Logistic regression was used for univariate and multivariate analyses to find out different correlates and analyze the independent effects of these correlates on GSD. RESULTS: We have included 120 cases and the same number of age- and sex-matched controls in the study and final analysis. Almost 70% of the cases were females and 30% were males. In multivariate logistic regression, we have found maximum significant association with serum total cholesterol (odds ratio [OR]: 1.013, confidence interval [CI]: 1.003-1.024, P = 0.041) followed by low-density lipoprotein (LDL) cholesterol (OR: 1.017, CI: 1.001-1.034, P = 0.04) and triglycerides (OR: 1.009, CI: 1.001-1.018, P = 0.049). Fasting blood sugar (P = 0.18), high-density lipoprotein cholesterol (P = 0.07), aspartate transaminase (P = 0.21), alanine transaminase (P = 0.29), and total bilirubin (P = 0.13) were not found to be independently associated with GSD. CONCLUSION: Biochemical parameters such as plasma total cholesterol, triglycerides, and LDL cholesterol level were independently associated with GSD.