This prospective, single-center, two-group observational study evaluates a non-invasive multi-parameter diagnostic panel for metabolic dysfunction-associated steatotic liver disease (MASLD) in children with obesity. A total of 180 children aged 8 to 18 years with a body mass index at or above the 85th percentile for age and sex are planned for enrollment at a single tertiary pediatric center. Each participant attends a single study visit comprising a fasting venous blood sample for serum biomarkers (cytokeratin-18 M30 and M65, fibroblast growth factor 21, retinol-binding protein 4, insulin-like growth factor binding protein 7, adiponectin, leptin, insulin, and routine biochemistry), abdominal ultrasonography with two-dimensional shear wave elastography, and genotyping of three MASLD-associated variants (PNPLA3 rs738409, TM6SF2 rs58542926, HSD17B13 rs72613567). Participants are classified as MASLD-positive or MASLD-negative according to a guideline-based composite reference standard consisting of ultrasonographic steatosis grading and cardiometabolic risk factor criteria, assessed independently of the candidate index tests. The primary objective is to determine the discriminative performance, expressed as the area under the receiver operating characteristic curve, of a LASSO-regularized logistic regression model combining biomarker, elastography, and genetic predictors. No therapeutic intervention is assigned by the study protocol. Reporting will follow the STARD 2015 statement.
Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease of childhood and is closely associated with obesity. Liver biopsy, the histological reference standard, is not ethically acceptable as a routine screening tool in children because it requires general anaesthesia and carries procedural risk and sampling error. The currently used non-invasive screening tools, alanine aminotransferase and ultrasonography, have limited diagnostic accuracy when used alone. An accurate, non-invasive diagnostic approach for pediatric MASLD is therefore needed. This single-center, prospective, two-group, exploratory pilot diagnostic classification study is conducted at Kayseri City Hospital, Kayseri, Türkiye. Children aged 8 to 18 years with a body mass index at or above the 85th percentile for age and sex, according to Turkish national growth references, are screened consecutively in the pediatric endocrinology outpatient clinic. Enrollment of 180 participants is planned, balanced by sex. Each participant attends a single study visit of approximately three hours. After a 12-hour fast, a single venous blood sample is obtained for routine biochemistry, insulin, and the serum biomarker panel; serum and plasma aliquots are stored at -80 °C until batched analysis by enzyme-linked immunosorbent assay in duplicate with blinded internal controls. Abdominal ultrasonography is performed for hepatic steatosis grading, and liver stiffness is measured by two-dimensional shear wave elastography. A separate whole-blood sample is used for DNA isolation and genotyping of three MASLD-associated variants, from which a three-variant polygenic risk score is derived. Anthropometric measurements including waist circumference percentile, blood pressure, pubertal staging, and questionnaire-based nutritional and physical activity assessment are recorded at the same visit. No therapeutic intervention is assigned by the study protocol. Participants are classified as MASLD-positive or MASLD-negative using a composite reference standard based on ultrasonographic steatosis grading together with cardiometabolic risk factor criteria, in accordance with current pediatric guidelines. To avoid incorporation bias, two-dimensional shear wave elastography is used only as a candidate predictor and does not contribute to the reference standard; reference standard assessment is performed blinded to biomarker and genotype results. The analysis develops a LASSO-regularized logistic regression model combining serum biomarkers, liver stiffness, and the polygenic risk score, with internal validation by bootstrap resampling. Model discrimination is compared with alanine aminotransferase alone and with ultrasonography alone. Reporting will follow the STARD 2015 statement.
Study Type
OBSERVATIONAL
Enrollment
180
All participants undergo the same set of index tests at a single study visit: a fasting venous blood sample for serum cytokeratin-18 M30 and M65, fibroblast growth factor 21, retinol-binding protein 4, insulin-like growth factor binding protein 7, adiponectin, leptin, insulin and routine biochemistry measured by enzyme-linked immunosorbent assay and standard laboratory methods; abdominal ultrasonography with two-dimensional shear wave elastography for liver stiffness; and genotyping of PNPLA3 rs738409, TM6SF2 rs58542926 and HSD17B13 rs72613567 for derivation of a three-variant polygenic risk score. These are observational measurements; no therapeutic intervention is administered.
Kayseri City Hospital
Kayseri, Turkey (Türkiye)
Diagnostic Performance of the LASSO-Regularized Multi-Parameter Panel for MASLD
Discriminative performance of a LASSO-regularized logistic regression model combining serum biomarkers (cytokeratin-18 M30, cytokeratin-18 M65, fibroblast growth factor 21, retinol-binding protein 4, insulin-like growth factor binding protein 7), homeostatic model assessment of insulin resistance, liver stiffness measured by two-dimensional shear wave elastography, and a three-variant polygenic risk score, for classifying participants against the composite reference standard for MASLD. Metric: area under the receiver operating characteristic curve with bootstrap-derived 95% confidence interval.
Time frame: Through study completion, an average of 12 months
Comparative Discrimination of the Panel Versus Alanine Aminotransferase Alone and Ultrasonography Alone
Comparison of the area under the receiver operating characteristic curve of the multi-parameter panel with that of alanine aminotransferase alone and of ultrasonography alone for classification against the composite reference standard, using the DeLong test for correlated curves.
Time frame: Through study completion, an average of 12 months
Serum Biomarker Concentrations in MASLD-Positive Versus MASLD-Negative Children With Obesity
Comparison of serum concentrations of cytokeratin-18 M30 and M65, fibroblast growth factor 21, retinol-binding protein 4, insulin-like growth factor binding protein 7, adiponectin and leptin between participants classified as MASLD-positive and MASLD-negative. Units of measure as reported by the respective enzyme-linked immunosorbent assays.
Time frame: Day 1 (single study visit)
Incremental Discriminative Value of Serum IGFBP7
Incremental contribution of serum insulin-like growth factor binding protein 7 to the classification model, evaluated by change in the area under the receiver operating characteristic curve, the net reclassification improvement, and the integrated discrimination improvement.
Time frame: Through study completion, an average of 12 months
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