MSACL 2025 Abstract

UPLC-MS/MS Analysis Confirming the Pathogenicity of a Variant of Unknown Significance in a Patient With GNPTAB-Related Disorder

Xinying Hong (1), Cara Skraban (2), Natalie Burrill (2), Rebecca C Ahrens-nicklas (2)
(1) Department of Pathology and Laboratory Medicine and (2) Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA, USA

Xinying Hong, PhD (Presenter) The Children’s Hospital of Philadelphia

Presenter Bio: Dr. Xinying Hong is a board-certified clinical biochemical geneticist and Assistant Professor of Pathology and Laboratory Medicine at the Children's Hospital of Philadelphia, where she also serves as Assistant Director of the Metabolic and Advanced Diagnostic Laboratory. She earned her PhD in Chemistry from the University of Washington and completed her clinical training at CHOP. Dr. Hong’s research focuses on the discovery and clinical application of novel biomarkers to improve the diagnosis, prognosis, and treatment monitoring of inborn errors of metabolism, with a particular emphasis on lysosomal storage disorders.

Relevant Financial Disclosures (within past 24 months, reported on May 31, 2026) No relevant financial relationship(s) to disclose.

CASE DESCRIPTION:
The patient is a 4-month-old female. The prenatal history was significant for a poor fetal growth. The patient was found postnatally on DOL 10 to have a large perimembranous ventricular septal defect (VSD), mild anterior deviation without pulmonary stenosis, and no right ventricular outflow tract obstruction. VSD repair was performed. She had poor weight gain. Later, she was incidentally found to have a right humerus fracture, prompting a skeletal survey and additional X-rays, which showed fractures involving multiple body regions. CT showed subdural collections overlying the frontal lobes. Concerns for non-accidental trauma were raised. Genetics was consulted to evaluate for possible genetic etiologies of her heart defect, multiple fractures, poor growth, and subdural hematoma. Rapid analysis of the genome was performed and revealed two variants in the gene GNPTAB, one classified as pathogenic and one as a variant of uncertain significance (VUS).

BACKGROUND:
GNPTAB-related disorder is a recessively inherited lysosomal storage disorder that is caused by the deficiency of N-acetylglucosame-1-phosphotransferase (GlcNAcPTase). GlcNAcPTase is part of the mannose-6-phosphate system, which is essential to target most of the lysosomal enzymes to the lysosome. Without the Man-6P system, the newly synthesized lysosomal enzymes are missorted into extracellular space and their respective substrates accumulate in the lysosome, resulting in the characteristic “inclusion cells”, along with the bone, connective tissue, and neurological symptoms typically observed in affected patients. The level of residual enzyme activity determines the severity of the disease. Complete deficiency of GlcNAcPTase typically results in mucolipidosis (ML) II, which is the most severe end of the spectrum. ML II patients present at birth and typically progresses to death in childhood. Patients with the mildest form of the condition, ML III α/β, typically present around 3 years of age with morbidity around early to mid-adulthood. Phenotypes intermediate between ML II and ML IIIα/β are often characterized by growth failure in infancy, resembling ML II, and neuromotor and speech development resembling ML IIIα/β.

The patient is not presenting with the classic neonatal presentation consistent with Mucolipidosis II but may have a milder form of the condition. Osteopenia, fractures and failure to thrive can be seen in the condition. Congenital heart disease and subdural hemorrhages are not common, but possible. Overall, it was unclear if the patient has GNPTAB-related disorder. Further biochemical testing was initiated.

MS METHOD AND RESULTS:
A highly multiplexed panel targeting more than urinary 30 glycosaminoglycan (GAG) and glycoprotein-derived mono and oligosaccharides was employed. These biomarkers are highly disease-specific, since the non-reducing end reflect the underlying enzyme deficiency. The reducing end of the mono and oligosaccharides were derivatized with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP). The excess, unreacted PMP was removed by liquid-liquid extraction. The derivatized analytes were quantified by UPLC-MS/MS.

Patients with GNPTAB-related disorder would demonstrate disturbance of more than one lysosomal enzyme. From our previous experience, galactosylated and sialylated oligosaccharides are the most sensitive biomarker for GNPTAB-related disorder in this panel. Other biomarkers can be seen elevated in the severe ML II.

In our proband, the galactosylated and sialylated oligosaccharides were moderately elevated, while other analytes were within normal limits. This is consistent with a dual deficiency of beta-galactosidase and sialidase. Such findings can be observed in galactosialidosis and GNPTAB-related disorder. Given the genetic testing result, this is consistent with GNPTAB-related disorder and confirmed the pathogenicity of the variant of unknown significance.

DISCUSSION AND CONCLUSION:
Our case highlights the critical role of biochemical testing in the context of widespread genetic testing, since more and more VUS are being discovered. Biochemical testing complements genetic testing in delineating the pathogenicity of novel variants. In our case, it was important to profile biomarkers from multiple conditions that eventually confirmed the diagnosis. LC-MS/MS analysis is a powerful technique in biochemical testing, offering superior quantification capabilities and multiplexing potential.