BMC Med Genomics. 2024 Jun 3;17(1):151. doi: 10.1186/s12920-024-01910-x

PMID: 38831290

Mengni Yi, Pinquan Shen, Huiwen Zhang

Highlights: This study investigates the clinical and molecular characteristics of 13 patients with mild Mucopolysaccharidosis type IVA (MPS IVA) to address the issue of delayed diagnosis. The research identified five novel GALNS variants, including a critical deep intronic variant (c.121-210 C>T) that appeared synonymous (p.G43G) but was proven to cause aberrant splicing. While the median age of symptom onset was 6.8 years, the diagnosis was delayed by an average of 5.6 years. The study highlights that deep intronic variants should be investigated when WES results are negative and emphasizes the role of orthopedists in recognizing early signs like waddling gait and flat feet.

Background: Mucopolysaccharidosis IVA (MPS IVA), or Morquio A syndrome, is an autosomal recessive lysosomal storage disorder caused by GALNS enzyme deficiency, leading to keratan sulfate accumulation and systemic bone dysplasia. While severe forms are identified early, mild phenotypes often present with atypical symptoms, leading to significant diagnostic delays. Identifying the genetic basis in mild cases is challenging, as some alleles remain unidentified in standard screenings due to deep intronic locations.

Objective and methods: The study aimed to characterize the clinical features and genetic variants of 13 mild MPS IVA patients from six unrelated families to facilitate early diagnosis. Diagnosis was confirmed via GALNS enzyme activity testing in leukocytes. Molecular analysis involved Sanger sequencing, Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS). A specific focus was placed on validating the pathogenicity of a deep intronic variant using functional mRNA analysis.

Material and Methods: Genomic DNA was analyzed using WES and Sanger sequencing. For functional validation of the deep intronic variant (c.121-210 C>T), Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines were established from a patient. To prevent nonsense-mediated mRNA decay (NMD) and stabilize unstable transcripts, cells were treated with cycloheximide. RNA was isolated, reverse-transcribed, and sequenced to observe splicing defects. Variants were classified according to ACMG guidelines.

Results: The study identified widespread musculoskeletal abnormalities, with waddling gait (85%), short neck (69%), and flat feet (62%) being the most common symptoms. Radiologically, all patients showed vertebral body modifications and acetabular dysplasia. Five novel variants were discovered. Notably, the deep intronic variant c.121-210 C>T (NM_000512.5), initially classified as a synonymous p.G43G variant, was proven via cDNA analysis to cause an 11-bp deletion (p.Gly43Aspfs*5), leading to a premature stop codon. This underscores the necessity of checking deep intronic regions in undiagnosed cases.

Keywords: Mucopolysaccharidosis IVA, MPS IVA, GALNS, Mild type, Deep intronic variants