Genetic Variants in Mucopolysaccharidosis Type I Patients from Pakistan: Identification of a Novel 14-Nucleotide Deletion and A Highly Prevalent Missense Variant in a Cohort of 10 Families

Rutaba Gul1, Sabika Firasat1*, Mulazim Hussain2, Fatima Akbar1, Kiran Fatima1, Muhammad Farooq Sabar3 and Kiran Afshan1

1Department of Zoology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

2The Children Hospital, Pakistan Institute of Medical Sciences, Islamabad, Pakistan

3Centre of Applied Molecular Biology, University of the Punjab, Lahore, Pakistan

ABSTRACT

Autosomal recessive mucopolysaccharidosis-I (MPS-I), an inborn error of metabolism, in which heparan sulfate and dermatan sulfate accumulate in the cells due to deficiency of enzyme alpha-iduronidase (IDUA) is more common in consanguineous populations. Previously, variants in alpha-iduronidase (IDUA) gene are reported to cause MPS-1 phenotypes. The aim of the present study was to identify genetic variants in the IDUA gene in ten unrelated MPS-1 affected consanguineous local families reporting to the children Hospital, Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan. Blood samples from affected and unaffected family members were collected and sequencing of IDUA gene was performed. In silico analysis of all identified disease-causing variants was done to check their affect on protein structure and function. Clinical examination of all MPS-1 patients showed coarse facial features, skeletal malformation, hernia, corneal clouding, abdominal distention and hepatosplenomegaly. Sequencing of IDUA gene revealed ten missense variations and eight synonymous variations. In silico tools including mutation taster, SIFT, PolyPhen and Provean suggested three variants as disease-causing. Among disease-causing variants, a previously reported missense variant i.e., c.1469T>C causing p.Leu490Pro was identified in 80% of our analyzed families. Furthermore, a novel 14 nucleotide deletion i.e., c.568_581del AACGTCTCCATGAC causing p.Asn190Hisfs*204 and a single nucleotide deletion i.e., c.784delC causing p.His262Thrfs*55 were segregating with MPS-I phenotype. This study reports a previously reported missense variant in 80% of screened families, a novel (c.568_581del AACGTCTCCATGAC ) and a previously reported disease-causing deletion each in affected family.

Article Information

Received 16 April 2022

Revised 06 May 2024

Accepted 12 May 2024

Available online 24 January 2025

(early access)

Published 29 December 2025

Authors’ Contribution

RG: Data curation, formal analysis, investigation, methodology, software, validation, writing original draft, writing review and editing. SF: Conceptualization, formal analysis, funding acquisition, methodology, resources, supervision, validation, visualization, writing original draft, writing review and editing, project administration. MH: Data curation, formal analysis, investigation, validation, writing review and editing. FA: Data curation, investigation, methodology. KF: Investigation, methodology, validation. MFS: Data curation, formal analysis, investigation, methodology, resources, validation. KA: Investigation, resources, validation, writing review and editing.

Key words

DNA sequencing, IDUA gene, MPS-I, Mucopolysaccharidosis-I, Consanguinity

DOI: https://dx.doi.org/10.17582/journal.pjz/20220416090446

* Corresponding author: [email protected]

0030-9923/2026/0001-0325 $ 9.00/0

Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).


INTRODUCTION

Mucopolysaccharidosis-I (MPS-I) (OMIM# 252800) is a lysosomal storage disorder LSD, that results from accumulation of glycosaminoglycans (GAGs) i.e., dermatan sulfate and heparin sulfate in various tissues of the body and leads to failure of multiple organs. MPS-I is inherited as an autosomal recessive disorder affecting 1 in 100,000 live births and is considered as pan-ethnic (Muenzer et al., 2009; Ngiwsara et al., 2018). It is a progressive disorder characterized by coarse facial features, defective hearing, stunted growth, hernias, corneal clouding, hepatosplenomegaly, dysostosis multiplex, sleep apnea, progressive brain abnormalities, cardiac abnormalities and joint contractures (Beck et al., 2014; Ngiwsara et al., 2018; Gul et al., 2019). The disease pathophysiology is not yet understood. Previously MPS-I mice models have shown that the buildup of GAGs causes the deactivation of osteoclastic protease cathepsin K, which further leads to the reduced degradation of cartilage and pathology of bone which is observed in MPS-1 phenotypes (Wilson and Bromme, 2010). The progression in arthropathy is intervened by the storage of glycosaminoglycans (GAGs), which causes elevation in recruitment of cytokine and chemokine (IL-1-beta, TNF-α etc.) which further increases the chondrocyte apoptosis, synovial hyperplasia and joint destruction along with inflammation (Simonaro et al., 2008, 2010).

Variants in IDUA gene (OMIM# 252800) encoding a lysosomal enzyme called alpha-L-iduronidase (EC 3.2.1.76) are reported to cause MPS-1 phenotypes (Hopwood and Morris, 1990; Muenzer, 2011; Beck et al., 2014). IDUA gene is located on chromosome 4p16.3 with 14 exons encoding a 653 residues polypeptide (Scott et al., 1993). Three subtypes of MPS-1 based on disease severity include Hurler (MPS I-H) (OMIM 607014) the most severe neuropathic form with life expectancy of less than 10 years, Hurler-Scheie (MPS I-H/S) (OMIM 607015) intermediate form with mild or no neurological involvement and patients may survive till third decade of life (Ngiwsara et al., 2018), Scheie (MPS I-S) (OMIM 607016) being milder of all with late onset and normal life span with available therapies (Muenzer et al., 2009; Bertola et al., 2011). Diagnosis of MPS-1 is confirmed by assay of enzyme activity and variants analysis of IDUA gene (Ngiwsara et al., 2018). Most common treatment method is enzyme replacement therapy (ERT) (Braulke and Bonifacino, 2009; Muenzer et al., 2009) and hematopoietic stem cell transplantation (HSCT). ERT is also not effective for neuronal conditions due to the inability to cross the blood brain barrier (Scarpa et al., 2017). For severe form of disease i.e., MPS1-H, HSCT should be done before age of two years, for this purpose molecular genetics diagnosis to identify causative variants for each patient is essential but not available in Pakistan (Terlato and Cox, 2003; Afroze and Brown, 2017). However, alternative therapeutic measures i.e., use of pharmacological chaperones and gene therapy are still under investigations (Penati et al., 2017; Pereira et al., 2018). Therefore, genetic diagnosis of MPS-1 cases especially from consanguineous families with expected high prevalence (Afroze and Brown, 2017; Afzal et al., 2018; Gul et al., 2019) will lead to identification of novel pathogenic variants to aid in better understanding of genotype-phenotype correlations and therapeutic interventions.

In this study we aimed to study the genetic basis of the mucopolysaccharidosis type I patients and to reveal their molecular findings and variants for causing the phenotype and to provide the families with genetic counselling.

MATERIALS AND METHODS

Patients recruitment

A total of ten MPS-1 affected unrelated consanguineous families were recruited from The Children Hospital, Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan during the time period from November 2017 to December 2018. Detailed pedigrees were drawn by interviewing family members. All patients were diagnosed initially through phenotypic and radiological findings (X-Ray). Among probands, there are 7 female patients and 3 male patients. Among ten families, seven have more than one affected alive/deceased patients while 3 families have single affected individual (Table I). The blood samples were collected from MPS-1 patients and parents of each patient to confirm the heterozygosity. Blood samples were stored in 10 ml vacutainer tubes (BD vacutainer K2 EDTA 18 mg) prior to DNA extraction. Dry blood spot (DBS) was performed only for the selected patients (MPS-03 and MPS-06) with severe phenotypes through tandem mass spectrometry (TMS), which measured the levels of enzyme in peripheral blood leukocytes.

Amplification of IDUA gene and its nucleotide sequencing

Genomic DNA was extracted by a modified method as described by Kaul et al. (2010). Quantity and purity of extracted DNA was accessed using a µDrop Plate reader (MultiskanTM, Thermo Fisher Scientific, and Waltham, MA, USA). Primers for amplification of all exons of IDUA gene (NM_000203.5) and their flanking splice sites were same as described previously by Gul et al. (2019). DNA of proband of each MPS-1 family was amplified in a 25µl reaction volume using T100 thermal cycler (Bio-Rad, CA, USA) and same reaction composition and cycling profile as described previously (Gul et al. 2019). The amplified PCR products were loaded on the 1.5% agarose gel along with 1kb size ladder to evaluate product size and purified by using DNA purification Kit (Wiz Bio Solutions, Seongnam, Korea). Sequencing was performed using Big Dye Terminator Ready reaction mix (Applied Biosystems, Foster City, CA, USA) following manufacturer instructions on an automated ABI 3100 genetic analyzer. Results were analyzed by BioEdit (version 7.0.5.3). After identification of disease-causing variant/s in each proband, these variants were screened using DNA of available unaffected family members to check segregation with disease phenotype.

In silico analysis

All identified variants listed in Tables I and II were checked for disease-causing potential through mutation taster (http://www.mutationtaster.org/) and their affect on protein structure and function was predicted through different bioinformatics tools like Chimera (version 1.13.1). Sorting intolerance from tolerance (SIFT) (https://sift.bii.a-star.edu.sg/), Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), Provean (http://provean.jcvi.org/), Hope (https://www3.cmbi.umcn.nl/hope/), Mutpred LOF (http://mutpredlof.cs.indiana.edu/), I-Mutant (https://folding.biofold.org/i-mutant/i-mutant2.0.html), Panther (http://www.pantherdb.org/), SNPs and GO (http://snps.biofold.org/snps-and-go/snps-and-go.html), and PHD-SNP (http://snps.biofold.org/phd-snp/phd-snp.html), Mutation assessor (http://mutationassessor.org/r3/). I-Tasser was used to model protein structures affected by frameshift variants identified in this study. 3D structure models for the frameshift variants of IDUA protein were built using I-Tasser (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). I-Tasser employs an integrated approach including comparative modeling, threading, and ab-initio modelling (Roy et al., 2010).

RESULTS

All ten families reported here belonged to Punjab province of Pakistan. Family and clinical details of each proband are listed in Table I. At the time of enrollment, three families (MPS-01, MPS-02 and MPS-05) had single affected individual, four families (MPS-03, MPS-04, MPS-08 and MPS-10) had two patients, whereas family MPS-07, MPS-09 and MPS-06 had three, four and five patients, respectively. Among five cases of MPS-06, four patients died before the age of five years and the only alive patient was six months old. MPS-1 patients of all enrolled families showed disease phenotype i.e., bone deformities, respiratory problems, hepatosplenomegaly, coarse facial features, growth problems, hernia, corneal clouding, joint contractures, and sleeping disorders (Table I). However, neurological problems were detected in MPS-03 patient, cardiac problems were present in MPS-01 and MPS-06 patients, kyphosis/gibbus formation were found in seven cases (MPS-01, 02, 03, 06, 08, 09 and 10) and dysostosis multiplex was present in MPS-04 patient. The age of patients ranged from 6 months to 14 years at the time of enrollment (Table I).

Upon sequencing of all IDUA (NM_000203) gene coding exons and the intron-exon boundaries using DNA samples of probands of all enrolled families, a total of 20 variants were identified (Tables I, II) including thirteen previously reported and seven novel variants. Among these variants, there are two deletions and 18 single nucleotide substitutions. Ten nucleotide substitutions lead to missense variations whereas eight were synonymous variants (Tables I, II). According to mutation taster results, both deletions were disease-causing however, except p. L490P, all missense and synonymous variants were benign variants. We identified a homozygous disease-causing variant in each family (Table I).

NM_000203.5 (IDUA): c.568_581delAACGTCTCCATGAC; p. (Asn190Hisfs*204)

DBS performed indicated the complete absence of enzyme in patient i.e., 0.00 (cut-off values >0.7). Sequence analysis of IDUA gene in family MPS-03, discovered a fourteen-nucleotide deletion segregating with disease phenotype in a consanguineous family (Fig. 1B). This novel deletion i.e., NM_000203.5 (IDUA): c.568_581delAACGTCTCCATGAC; p. (Asn190Hisfs*204) was predicted to cause substitution of asparagine to histidine at 190 position followed by a frameshift with an in frame stop codon leading to a truncated protein of 392 amino acids i.e., Asn190Hisfs*204 as predicted by mutation taster. The longer the deletion is,and more highly conserved region is (Fig. 3A), higher are the chances for the variant to be pathogenic and de novo variant with paternity and maternity confirmed (PS1) is also strong candidate for pathogenicity according to ACMG standards and guidelines. The heterozygosity of the variant was confirmed in parents (Fig. 1B)

 

Table I. Characteristics of MPS I patients and their identified pathogenic variants in IDUA gene.

Family ID

Gender

Parental consanguinity

Age at enrollment

Nucleotide change

Protein change

Status/exon

MPS-01

Male

Yes

10 years

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-02

Female

Yes

14 months

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-03

Male

Yes

1.5 years

c.568_581del

AACGTCTCCATGAC

p.Asn190His

fs*204

Homozygous/5

MPS-04

Female

Yes

6 years

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-05

Female

Yes

19 months

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-06

Female

Yes

6 months

c.784delC

p.His262Thrfs*55

Homozygous/6

MPS-07

Female

Yes

8 years

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-08

Female

Yes

14 years

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-09

Male

Yes

3 years

c.1469T>C

p.Leu490Pro

Homozygous/10

MPS-10

Female

Yes

8.5 years

c.1469T>C

p.Leu490Pro

Homozygous/10

 

Table II. List of identified benign variants, ACMG classification, frequencies and exonic positions in IDUA gene of MPS I patients.

S. No

Nucleic acid change

Amino acid

change

ACMG interpretation

Global minor allele frequency

Exon/ Intron

Accession numbers

Exonic variants

1

c.24C>A

No change

BS1

0.39257

Exon 1

rs11248061

2

c.60G>A

No change

BS1

0.39437

Exon 1

rs10902762

3

c.99T>G

p.His33Gln

BS1

0.10803

Exon 1

rs10794537

4

c.314G>A

p.Arg105Gln

BS1

0.16873

Exon 3

rs3755955

5

c.543T>C

No change

BS1

0.21985

Exon 5

rs6815946

6

c.942G>C

No change

BS1

0.21865

Exon 7

rs6830825

7

c.1164G>C

No change

BS1

0.21865

Exon 8

rs6836258

8

c.1081G>A

p.Ala361Thr

BS1

0.21765

Exon 8

rs6831280

9

c.1230C>G

No change

BS1

0.21785

Exon 9

rs115790973

10

c.1232T>C

p.Val411Ala

N/A

N/A

Exon 9

This report

11

c.1292C>T

p.Ala431Val

N/A

N/A

Exon 9

This report

12

c.1360G>A

p.Val454Ile

BS1

0.21865

Exon 9

rs73066479

13

c.1364C>G

p.Ala455Gly

N/A

N/A

Exon 9

This report

14

c.1654A>T

p.Thr552Ser

N/A

N/A

Exon 12

This report

15

c.1786A>C

p.Ser596Arg

N/A

N/A

Exon 13

This report

Intronic variants

16

g.15984T>C

No change

BS1

0.21885

Intron 9/10

rs115134980

17

g.15989C>G

No change

N/A

N/A

Intron 9/10

This report

 

ACMG, American college of medical genetics and genomics; BS, strongly benign, N/A: not available.

 

 

(Richard et al., 2015). Polyphen-2 in silico tool predicted p.Asn190His variant as probably damaging with score of 1. According to PROVEAN p. Asn190Hisfs*204 variation is deleterious with a probability score of -1197.876 (Cut-off value= -2.5). The effect of p.Asn190His substitution on the structure and function of protein is detected through HOPE and it highlighted that mutant amino acid i.e., asparagine (N) is larger in size than the wild type amino acid histidine (H). Asn is located in a preferred secondary structure i.e., turn while histidine prefers to be in another secondary structure thus destabilized the local conformation. Histidine is highly conserved in the region so its substitution will be probably damaging for the protein. Mutpred-LOF gave a score of 0.56678 (>0.5) confirms the pathogenicity. I-Mutant showed the greater decrease in stability of protein with a score of -2.52 Kcal/mol.

NM_000203.5 (IDUA): c.784delC; p.(His262Thrfs*55)

In family MPS-06, the DBS indicated 0.00 (cut-off values >0.7) values for enzyme measurement in peripheral blood leukocytes. Also, molecular identification revealed a previously reported homozygous deletion of cytosine nucleotide i.e., c.784delC causing a substitution of histidine with threonine at 262 position followed by a frameshift with an in frame stop codon thus causing a shorter protein of 315 amino acids i.e., p. His262Thrfs*55 was segregating with MPS-1 phenotype (Fig. 2). According to Richard et al. (2015) if the small in-frame deletion is in the highly conserved region (Fig. 3B) and is de novo with paternity and maternity confirmed, the variant is strongly pathogenic (PS2) (Richard et al., 2015). In silico analysis through SIFT predicted histidine to threonine substitution at 262 position to affect protein function (probability score of 0.00). Polyphen-2 score for p.His262Thr was 1 and PROVEAN score for p. His262Thrfs*55 was -917.011, respectively predicting this frameshift and in frame stop codon as deleterious to protein function. When the effect of p.His262Thr variant on the structure and function of protein was analyzed through HOPE, it revealed the size of mutant residue is small and hydrophobicity of the mutant amino acid is greater than the wild type. Histidine at position 262 form hydrogen bonds with Glutamic acid at 182 position and this change in amino acid leads to loss of hydrogen bonds in the core and thus disturb the correct folding of protein. Histidine at 262 position is highly conserved so alteration in this residue is probably damaging for protein. Mutpred-LOF score was 0.56907 which confirms the pathogenicity (>0.5 = pathogenic). I-Mutant showed the greater decrease in the stability of protein with a score of -0.81. Both PHD-SNP and SNPandGO confirms the variant p.His262Thr as disease-causing.

 

 

There are three domains of IDUA protein including a TIM barrel domain (residues 42–396), a β-sandwich domain (397–545) and an immunoglobulin-like domain (residues 546–642) (McKusick et al., 1972; Poletto et al., 2018). We checked conservation of amino acid residues for both frameshift variants as shown in fig. 3 which revealed that wild type sequence was conserved among different orthologs of IDUA (Fig. 3). Furthermore, modeling of wild type and both frameshift mutant variants of the IDUA protein identified in MPS-03 and MPS-06 families respectively was performed using I-Tasser (Fig. 4). As both frameshift variants i.e., p.Asn190Hisfs*204 and p.His262Thrfs*55 lead to truncated proteins of 392 and 315 amino acids thus the structural modeling showed loss of two of the three domains of IDUA protein i.e. in the mutant proteins. The enzyme active site predicted for the wild type as well as for both truncated proteins is same at 175 residue. But, the ligand binding sites for the wild type protein were predicted to be twelve in number at residues 53, 91, 181, 182, 264, 299, 304, 305, 306, 349, 352 and 363 but these were predicted to be eleven at 91, 143, 181, 185, 188, 219, 246, 294, 299, 322, 334 residues for p.Asn190Hisfs*204 mutant and only seven at residue number 53, 91, 181, 182, 264, 299 and 304 for the p.His262Thrfs*55 mutant. These predictions showed that both mutant proteins were truncated causing protein structure and function defects.

 

NM_000203.5 (IDUA): c.1469T>C; p. (Leu490Pro)

We identified a high frequency of a previously reported NM_000203.5 (IDUA): c.1469T>C; p. (Leu490Pro) variant in our study on Pakistani MPS-1 families (Table I). Among ten analyzed families, the percentage of p.L490P was 80% (8/10) (MPS-01,02,04,05,07,08,09,10), suggesting that most collected families of MPS-1 have this variant (Gul et al., 2019, 2023). For this variant according to Richard et al (2015), when prevalence increases statistically in affected individuals than controls that variant is also considered as strong candidate for pathogenicity (PS4). P.Leu490Pro is one of the high prevalent variant in patients with MPS-I throughout the world with confirmation of paternity and maternity heterozygosity state. Mutpred score for p.Leu490Pro was 0.564. I-Mutant results showed the greater decrease in the stability of protein with reliability index score of 5 and thermal value DDG is -0.91 Kcal/mol. According to PHD-SNP and SNP and GO analysis this variant is disease-causing with scores of 2 and 0.709, respectively.

DISCUSSION

Inbreeding is customary in Pakistan, which predisposes to increased burden of autosomal recessive diseases in society including LSDs. Among mucopolysaccharidosis, MPS-I with different phenotypic variation is found to be more prevalent worldwide including Pakistani population (Chkioua et al., 2011; Cheema et al., 2017). The ratios of different phenotypes of MPS-1 were reported as Hurler 60.9%, Hurler/Scheie 23%, Sheie 12.9% and 3.2% undetermined by Beck et al. (2014).

Variants in a single gene are responsible for three MPS-1 phenotypes; thus, identification of causative variants for each patient is important to develop genotype-phenotype correlation (De Ru et al., 2012). Consequently, molecular genetics diagnosis leads to therapeutic management as hematopoietic stem cell transplantation is used for patients presenting with severe phenotype i.e., Hurler and show better results with earlier age at treatment (Ghosh et al., 2017). This data necessitates molecular genetic screening of MPS-1 cases especially from consanguineous populations that will not only identify prevalent variants to establish molecular genetics diagnosis testing but will have clinical utility for disease management. For this purpose, the present study was performed to screen IDUA gene in ten MPS-1 segregating unrelated consanguineous Pakistani families (Table I).

Variant’s analysis of IDUA gene confirmed one homozygous disease-causing variant in each of our screened families. Among identified variants, a previously reported missense variant i.e., p.L490P was detected in eight out of ten analyzed families, and it also provide the possibility of this variant to be a founder variant in Pakistani cohort but still more studies are required to confirm its status (Table I) (Tieu et al., 1995). The disease phenotype of all of NM_000203.5 (IDUA): c.1469T>C; p. (Leu490Pro) carrying patients was Hurler-Scheie similar to previously reported Pakistani patients carrying same variant (Arora et al., 2007; Gul et al., 2019, 2023; Zahoor et al., 2019). The substitution of leucine at position 490 with proline may affect enzymatic activity of protein causing an intermediate phenotype (Ghosh et al., 2017). The nature and position of substituted amino acid and also newly inserted amino acid describe the impact of that very substitution on structure of protein (Firasat et al., 2018). According to HOPE, leucine at position 490 is located in helix-loops-helix region (482-508 amino acids residues) of β-sandwich domain (482-508) which is important for interaction with other two domains of enzyme, and its substitution with proline may result in loss of this interaction (Maita et al., 2013; Gul et al., 2019).

Sequencing of IDUA coding sequence in remaining two families revealed, a novel deletion of 14 nucleotides (c. 568_581delAACGTCTCCATGAC) in exon 5 and a previously reported single nucleotide deletion i.e., c.784delC, in exon 6 in family MPS-03 and MPS-06, respectively. According to mutation taster both deletions lead to the frameshift of the coding sequence thus creating a pre-mature stop codon i.e., c. 568_581delAACGTCTCCATGAC leading to p.Asn190Hisfs*204 and c.784delC causing p.His262Thrfs*55 with truncated proteins of 392 and 315 amino acids, respectively. Previously, c.784delC causing p. His262Thrfs*55 was reported by Al-Jasmi et al. (2012) in an MPS-1 patient of Pakistani origin with central nervous system involvement indicating Hurler Phenotype (Al-Jasmi et al., 2012). Here in our study MPS-06 patient carrying same variant also had by birth onset of disease. According to Maquat (2005), all those transcripts which generate pre-mature termination codon at 50-55 base pairs upstream of exon-exon junction complex undergo the process of non-sense mediated decay (NMD) in mammals (Maquat, 2005). Furthermore, IDUA variants which cause premature protein truncations lead to absence of enzyme and severe phenotype (Keeling et al., 2013; Huang et al., 2018). Absence of enzyme was further confirmed by dried blood spot (DBS) tandem mass spectrometry (TMS) for MPS-03 and MPS-06 patients, which gave a value of 0.00 (cut-off values >0.7) for both cases. Thus, both of these variants lead to truncating proteins predicted to be degraded by NMD leading to absence of α-L-iduronidase enzyme and excessive accumulation of GAGs hence showing severe phenotype with by birth onset of disease and neuropathic involvement and in our study patients. Furthermore, asparagines amino acid at 190 position of IDUA protein is among one of the six potential sites i.e., N110, N190, N336, N372, N415 and N451 which are subject to post-translational glycosylation (Scott et al., 1991), thus substitution with histidine at this position may impact protein function as described previously by Maita et al. (2013).

The remaining 17 variants including 9 missense and 8 synonymous were benign variants according to mutation taster (Table II). Previously it is reported that benign variants may also contribute to variable enzyme activity in healthy subjects (Scott et al., 1993) thus may contribute to the phenotypic variability among patients, along with different pathogenic variants. Consequently, including one novel deletions, we described three variants and their genotypic-phenotypic correlation in the Pakistani MPS-1 patients.

CONCLUSION

These results indicate the heterogeneous IDUA variant spectrum in this population. Mutational screening of the IDUA gene facilitates an accurate diagnosis of MPS-1 clinical subtypes, which are necessary for selection of treatment options (Poe et al., 2014), provides carrier status, and will contribute to the development of novel therapeutic approaches.

DeclarationS

Acknowledgment

We thank patients and their families for their participation in this study.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not for profit sectors.

IRB approval and Ethical statement

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional/ethical review boards and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethical Committee Quaid-i-Azam University, Islamabad (BEC-FBS-QAU2019-198).

Data transparency

All data and materials in this study comply with the field standards.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

Patients signed informed consent regarding publishing their data and photographs (if required).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Submission declaration and verification

This description has not been published previously or under consideration anywhere else. This work is approved by all authors and responsible authorities. If accepted, this work will not be published anywhere else in any form without written consent of the copy right holder.

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Afroze, B. and Brown, N., 2017. Ethical issues in managing Lysosomal storage disorders in children in low and middle income countries. Pak. J. med. Sci., 33: 1036. https://doi.org/10.12669/pjms.334.12975

Afzal, R., Firasat, S., Kaul, H., Ahmed, B., Siddiqui, S.N., Zafar, S.N., Shahzadi, M. and Afshan, K., 2019. Mutational analysis of the CYP1B1 gene in Pakistani primary congenital glaucoma patients: Identification of four known and a novel causative variant at the 3′ splice acceptor site of intron 2. Congenit. Anom., 59: 152-161. https://doi.org/10.1111/cga.12312

Al-Jasmi, F.A., Tawfig, N., Berniah, A., Ali, B.R., Taleb, M., Hertecant, J.L., Bastaki, F. and Souid, A.K., 2012. Prevalence and novel mutations of lysosomal storage disorders in United Arab Emirates. J. Inherited Metab. Dis., 10: 1-9. https://doi.org/10.1007/8904_2012_182

Arora, R.S., Mercer, J., Thornley, M., Tylee, K. and Wraith, J.E., 2007. Enzyme replacement therapy in 12 patients with MPS I–H/S with homozygous p. Leu490Pro mutation. J. Inherit. Metab. Dis., 30: 821-821. https://doi.org/10.1007/s10545-007-0551-9

Beck, M., Arn, P., Giugliani, R., Muenzer, J., Okuyama, T., Taylor, J. and Fallet, S., 2014. The natural history of MPS I: Global perspectives from the MPS I registry. Genet. Med., 16: 759-765. https://doi.org/10.1038/gim.2014.25

Bertola, F., Filocamo, M., Casati, G., Mort, M., Rosano, C., Tylki-Szymanska, A., Tüysüz, B., Gabrielli, O., Grossi, S., Scarpa, M. and Parenti, G., 2011. IDUA mutational profiling of a cohort of 102 European patients with mucopolysaccharidosis type I: Identification and characterization of 35 novel α-L-iduronidase (IDUA) alleles. Hum. Mutat., 32: 2189-2210. https://doi.org/10.1002/humu.21479

Braulke, T. and Bonifacino, J.S., 2009. Sorting of lysosomal proteins. Biochim. biophys. Acta Mol. Cell Res., 1793: 605-614. https://doi.org/10.1016/j.bbamcr.2008.10.016

Cheema, H.A., Malik, H.S., Hashmi, M.A., Fayyaz, Z., Mushtaq, I. and Shahzadi, N., 2017. Mucopolysaccharidoses clinical spectrum and frequency of different types. J. Coll. Physiol. Surg. Pak., 27: 80-83.

Chkioua, L., Khedhiri, S., Turkia, H.B., Chahed, H., Ferchichi, S., Dridi, M.F.B., Laradi, S. and Miled, A., 2011. Hurler disease (mucopolysaccharidosis type IH): clinical features and consanguinity in Tunisian population. Diagn. Pathol., 6: 1-7. https://doi.org/10.1186/1746-1596-6-113

De Ru, M.H., Teunissen, Q.G., Van Der Lee, J.H., Beck, M., Bodamer, O.A., Clarke, L.A., Hollak, C.E., Lin, S.P., Rojas, M.V.M., Pastores, G.M. and Raiman, J.A., 2012. Capturing phenotypic heterogeneity in MPS I: Results of an international consensus procedure. Orphanet. J. Rare Dis., 7: 1-9. https://doi.org/10.1186/1750-1172-7-22

Firasat, S., Kaul, H., Ashfaq, U.A. and Idrees, S., 2018. In silico analysis of five missense mutations in CYP1B1 gene in Pakistani families affected with primary congenital glaucoma. Int. Opthamol., 38: 807-814. https://doi.org/10.1007/s10792-017-0508-4

Ghosh, A., Mercer, J., Mackinnon, S., Yue, W.W., Church, H., Beesley, C.E., Broomfield, A., Jones, S.A. and Tylee, K., 2017. IDUA mutational profile and genotype–phenotype relationships in UK patients with mucopolysaccharidosis type I. Hum. Mutat., 38: 1555-1568. https://doi.org/10.1002/humu.23301

Gul, R., Firasat, S., Hussain, M., Afshan, K. and Nawaz, D., 2019. IDUA gene mutations in mucopolysaccharidosis type-1 patients from two Pakistani inbred families. Congenit. Anom., 60: 1-2. https://doi.org/10.1111/cga.12354

Gul, R., Firasat, S., Schubert, M., Ullah, A., Peña, E., Thuesen, A.C., Hussain, M., Staeger, F.F., Gjesing, A.P., Albrechtsen, A. and Hansen, T., 2023. Identifying the genetic causes of phenotypically diagnosed Pakistani mucopolysaccharidoses patients by whole genome sequencing. Front. Genet., 14: 1128850. https://doi.org/10.3389/fgene.2023.1128850

Hopwood, J.J. and Morris, C.P., 1990. The mucopolysaccharidoses: Diagnosis, molecular genetics and treatment. Mol. Biol. Med., 7: 381-404.

Huang, L., Low, A., Damle, S.S., Keenan, M.M., Kuntz, S., Murray, S.F., Monia, B.P. and Guo, S., 2018. Antisense suppression of the nonsense mediated decay factor Upf3b as a potential treatment for diseases caused by nonsense mutations. Genom. Biol., 19: 1-16. https://doi.org/10.1186/s13059-017-1386-9

Kaul, H., Riazuddin, S.A., Shahid, M., Kousar, S., Butt, N.H., Zafar, A.U., Khan, S.N., Husnain, T., Akram, J., Hejtmancik, J.F. and Riazuddin, S., 2010. Autosomal recessive congenital cataract linked to EPHA2 in a consanguineous Pakistani family. Mol. Vis., 16: 511.

Keeling, K.M., Wang, D., Dai, Y., Murugesan, S., Chenna, B., Clark, J., Belakhov, V., Kandasamy, J., Velu, S.E., Baasov, T. and Bedwell, D.M., 2013. Attenuation of nonsense-mediated mRNA decay enhances in vivo nonsense suppression. PLoS One, 8: 60478. https://doi.org/10.1371/journal.pone.0060478

Maita, N., Tsukimura, T., Taniguchi, T., Saito, S., Ohno, K., Taniguchi, H. and Sakuraba, H., 2013. Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module. Proc. natl. Acad. Sci., 110: 14628-14633. https://doi.org/10.1073/pnas.1306939110

Maquat, L.E., 2005. Nonsense-mediated mRNA decay in mammals. J. Cell. Sci., 118: 1773-1776. https://doi.org/10.1242/jcs.01701

McKusick, V.A., Howell, R.R., Hussels, I.E., Neufeld, E.F. and Stevenson, R.E., 1972. Allelism, non-allelism and genetic compounds among the mucopolysaccharidoses: Corrective factors in nosology, genetics and therapy. Trans. Assoc. Am. Physiol., 85: 151-171.

Muenzer, J., 2011. Overview of the mucopolysaccharidoses. Rheumatology, 50: 4-12. https://doi.org/10.1093/rheumatology/ker394

Muenzer, J., Wraith, J.E. and Clarke, L.A., 2009. Mucopolysaccharidosis I: management and treatment guidelines. Pediatrics, 123: 19-29. https://doi.org/10.1542/peds.2008-0416

Ngiwsara, L., Ketudat-Cairns, J.R., Sawangareetrakul, P., Charoenwattanasatien, R., Champattanachai, V., Kuptanon, C., Pangkanon, S., Tim-Aroon, T., Wattanasirichaigoon, D. and Svasti, J., 2018. p. X654R IDUA variant among Thai individuals with intermediate mucopolysaccharidosis type I and its residual activity as demonstrated in COS-7 cells. Annls Hum. Genet., 82: 150-157. https://doi.org/10.1111/ahg.12236

Penati, R., Fumagalli, F., Calbi, V., Bernardo, M.E. and Aiuti, A., 2017. Gene therapy for lysosomal storage disorders: Recent advances for metachromatic leukodystrophy and mucopolysaccaridosis I. J. Inherit. Metab. Dis., 40: 543-554. https://doi.org/10.1007/s10545-017-0052-4

Pereira, D.M., Valentão, P. and Andrade, P.B., 2018. Tuning protein folding in lysosomal storage diseases: The chemistry behind pharmacological chaperones. Chem. Sci., 9: 1740-1752. https://doi.org/10.1039/C7SC04712F

Poe, M.D., Chagnon, S.L. and Escolar, M.L., 2014. Early treatment is associated with improved cognition in Hurler syndrome. Annls Neurol., 76: 747-753. https://doi.org/10.1002/ana.24246

Poletto, E., Pasqualim, G., Giugliani, R., Matte, U. and Baldo, G., 2018. Worldwide distribution of common IDUA pathogenic variants. Clin. Genet., 94: 95-102. https://doi.org/10.1111/cge.13224

Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W., Hegde, M., Lyon, E., Spector, E. and Voelkerding, K., 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med., 17: 405-423. https://doi.org/10.1038/gim.2015.30

Roy, A., Kucukural, A. and Zhang, Y., 2010. I-TASSER: A unified platform for automated protein structure and function prediction. Nat. Protoc., 5: 725-738. https://doi.org/10.1038/nprot.2010.5

Scarpa, M., Orchard, P.J., Schulz, A., Dickson, P.I., Haskins, M.E., Escolar, M.L. and Giugliani, R., 2017. Treatment of brain disease in the mucopolysaccharidoses. Mol. Genet. Metab., 122: 25-34. https://doi.org/10.1016/j.ymgme.2017.10.007

Scott, H.V., Nelson, P., Litjens, T., Hopwood, J. and Morris, C.P., 1993. Multiple polymorphisms within the α-L-iduronidase gene (IDUA): implications for a role in modification of MPS-I disease phenotype. Hum. mol. Genet., 2: 1471-1473. https://doi.org/10.1093/hmg/2.9.1471

Scott, H.S., Anson, D.S., Orsborn, A.M., Nelson, P.V., Clements, P.R., Morris, C.P. and Hopwood, J.J., 1991. Human alpha-L-iduronidase: cDNA isolation and expression. Proc. natl. Acad. Sci., 88: 9695-9699. https://doi.org/10.1073/pnas.88.21.9695

Simonaro, C.M., D’Angelo, M., He, X., Eliyahu, E., Shtraizent, N., Haskins, M.E. and Schuchman, E.H., 2008. Mechanism of glycosaminoglycan-mediated bone and joint disease: Implications for the mucopolysaccharidoses and other connective tissue diseases. Am. J. clin. Pathol., 172: 112-122. https://doi.org/10.2353/ajpath.2008.070564

Simonaro, C.M., Ge, Y., Eliyahu, E., He, X., Jepsen, K.J. and Schuchman, E.H., 2010. Involvement of the Toll-like receptor 4 pathway and use of TNF-α antagonists for treatment of the mucopolysaccharidoses. Proc. natl. Acad. Sci., 107: 222-227. https://doi.org/10.1073/pnas.0912937107

Terlato, N.J. and Cox, G.F., 2003. Can mucopolysaccharidosis type I disease severity be predicted based on a patient’s genotype? A comprehensive review of the literature. Genet. Med., 5: 286-294. https://doi.org/10.1097/01.GIM.0000078027.83236.49

Tieu, P.T., Bach, G., Matynia, A., Hwang, M. and Neufeld, E.F., 1995. Four novel mutations underlying mild or intermediate forms of α‐L‐iduronidase deficiency (MPS IS and MPS IH/S). Hum. Mutat., 6: 55-59. https://doi.org/10.1002/humu.1380060111

Wilson, S. and Brömme, D., 2010. Potential role of cathepsin K in the pathophysiology of mucopolysaccharidoses. J. Ped. Rehab. Med., 2: 139-146. https://doi.org/10.3233/PRM-2010-0116

World Medical Association, 2013. World medical association declaration of Helsinki: Ethical principles for medical research involving human subjects. J. Am. Med. Assoc., 310: 2191-2194. https://doi.org/10.1001/jama.2013.281053

Zahoor, M.Y., Cheema, H.A., Ijaz, S., Anjum, M.N., Ramzan, K. and Bhinder, M.A., 2019. Mapping of IDUA gene variants in Pakistani patients with mucopolysaccharidosis type 1. Annls Pediatr. Endocrinol. Metab., 32: 1221-1227. https://doi.org/10.1515/jpem-2019-0188