Identification of genetic variants associated with a wide spectrum of phenotypes clinically diagnosed as Sanfilippo and Morquio syndromes using whole genome sequencing

**Background:** Mucopolysaccharidoses (MPS) are lysosomal storage disorders caused by deficiencies in enzymes that degrade glycosaminoglycans, leading to accumulation in multiple organs. MPS III (Sanfilippo syndrome) and MPS IV (Morquio syndrome) are autosomal recessive disorders with overlapping clinical features, making accurate diagnosis challenging without molecular testing. In Pakistan, consanguineous marriages are common, increasing the burden of recessive disorders, yet limited genetic studies have been conducted. This study aimed to clinically characterize and identify causal genetic variants in Pakistani families with suspected Sanfilippo or Morquio syndromes using whole genome sequencing (WGS).

**Methods:** The study was approved by the bioethical committee of Quaid-i-Azam University. Patients were recruited from the outpatient department of Children Hospital, Pakistan Institute of Medical Sciences (PIMS), Islamabad, based on clinical profiles. Peripheral blood samples were collected from 58 individuals (26 affected, 32 unaffected) from 14 families. DNA was extracted using the phenol-chloroform method. WGS was performed, and reads were mapped to the hg38 reference using BWA. Variant prioritization focused on non-synonymous and splice-site variants with minor allele frequency <0.01 in gnomAD, homozygous variants due to consanguinity, and segregation analysis. Variants were classified according to ACMG guidelines. In silico tools (MutationTaster, SIFT, Polyphen-2, CADD, etc.) were used for pathogenicity prediction. Protein modeling was performed using I-TASSER, and evolutionary conservation was assessed with ConSurf.

**Key Results:** Among 14 families, 6 novel and 6 reported variants were identified. For MPS III (6 families, 11 patients, mean age 7.2 years): Family A had a homozygous NAGLU c.2020C>T (p.Arg674Cys) variant (pathogenic). Family B had SGSH c.221C>T (p.Arg74His) (pathogenic). Family C had a novel VWA3B c.2951A>C (p.Glu984Ala) variant (uncertain significance). Family D had SGSH c.364C>T (p.Gly122Arg) (pathogenic). Family E had a novel SGSH c.258A>C (p.Asn86Lys) (uncertain significance). Family F had SGSH c.817G>A (p.Asp273Asn) (likely pathogenic). For MPS IV (8 families, 15 patients, mean age 7.3 years): Family A had a novel GALNS splice site variant c.1365-2A>G (likely pathogenic). Families B, G, and H shared GALNS c.107A>C (p.Leu36Arg) (pathogenic). Family D had GALNS c.1259G>C (p.Pro420Arg) (uncertain significance). Family E had a novel GNPTG c.179G>A (p.Gly60Glu) (uncertain significance). Family F had a novel GALNS splice site variant c.423-1G>A (pathogenic). Family H also had a novel BTD c.1336G>C (p.Asp446His) (pathogenic), indicating dual molecular diagnosis. Family C remained unsolved. None of the variants were found in homozygous state in population databases or in-house controls.

**Clinical Implications:** This study demonstrates the genetic heterogeneity of MPS III and IV in a consanguineous Pakistani population, identifying both known and novel variants. The discovery of variants in VWA3B and GNPTG, genes not previously associated with MPS, expands the phenotypic spectrum and underscores the importance of WGS for accurate diagnosis. The high prevalence of the GALNS p.Leu36Arg variant in three families suggests a founder effect. Early and precise molecular diagnosis is crucial for genetic counseling, carrier testing, prenatal screening, and potential therapeutic interventions (e.g., enzyme replacement therapy for MPS IVA). The study also highlights the limitations of clinical diagnosis alone due to overlapping phenotypes and the need for functional validation of in silico predictions.