A Novel Frameshift Mutation Associated with Hurler's Syndrome: A Case Report

Abstract

Mucopolysaccharidosis 1 (MPS1) is a rare inherited lysosomal storage disorder resulting from the absence or reduction of lysosomal alpha-l-iduronidase due to mutations in the IDUA gene. Three major clinical manifestations have been established including Hurler's or severe type (OMIM 607914), Hurler–Scheie or intermediate type (MIM 607914) and Scheie's or attenuated type (MIM 607016). In the present study, a patient whose disease was diagnosed by biochemical and enzymatic assay was studied in our laboratory. Molecular analysis implemented by PCR-sequencing of all 14 exons and exon–intron junctions confirmed a novel deleterious mutation in a homozygous state. The result of this study has broadened the genotypic spectrum of MPS1 patients, assisting in a more effective approach for carrier testing and counseling.

Introduction

Mucopolysaccharidosis type 1 (MPS1) is a genetic disorder caused by mutations in both alleles of IDUA gene, resulting in accumulation of mucopolysaccharides (heparan sulfate and deramatan sulfate) within the lysosome in various tissues. The IDUA gene, which is localized on the 4p16.3 chromosomal locus, contains 14 exons with an approximate length of 19 kb. The gene transcript is a mature mRNA of 2.3 kb that encodes a 653-amino acid peptide. Two distinct regions of the IDUA gene have been recognized: one which covers a sequence of 18 kb including the first two exons and a long intron of 13 kb and another 4.5 kb segment which spans the remaining 12 exons. ^{1 2 3}

The most common signs of MPS1 include skeletal deformation, respiratory distress, hepatosplenomegaly, hernia, coarse facial features, and short statue. With regards to the onset of symptoms and the severity of the disease, MPS1 is classically categorized into three major subtypes ^{4 5 6} : (1) the severe subtype, in which the symptoms of the disease are diagnosed up to 12 months, lifespan is limited to the first decade of life, and patients present central nervous system involvement; (2) the intermediate subtype in which physical features exhibited by individuals are extremely heterogeneous, patients demonstrate delayed progression of the disease with little or no intellectual dysfunction compared to those affected by the severe form; (3) the attenuated form of the disease, in which symptoms usually begin after 5 years of age, cognitive function is normal, and patients survive to adulthood. ^{7 8 9 10}

Although combinations of different mutant alleles in the locus of the gene broaden the spectrum of clinical phenotypes, intragenic polymorphism appearing throughout the gene may also render the pathogenic sequence changes more or less deleterious through alteration of mutant allele expression. ^{6 10}

More than 200 diverse disease-causing mutations have been described, including missense and nonsense mutations, small deletions and insertions and splice site mutations. These mutations have been scattered across the IDUA gene ( http://www.hgmd.org / ).

Identification of pathogenic mutations in pedigrees affected with MPS1 could provide a beneficial tool to physicians so as to better ascertainment of the disease for appropriate therapeutic selection, prenatal diagnosis, newborn screening, and carrier screening.

Our study was aimed to examine mutational characteristics of IDUA gene to recognize mutant alleles in a girl with MPS1 and determine the relationship between the genotype and clinical phenotype.

Materials and Methods

Patient Description

The case was referred to the special medical center with suspected clinical symptoms for MPS1. The patient was 4 years old, an offspring of healthy consanguineous parents. The mother of the patient was also experienced in utero fatality in her first pregnancy. Her co-twin sister died around 3 weeks after birth. The history of the family showed that the cousin of the patient had the same condition and died at the age of 7 years because of pulmonary infection. According to her physician, she showed normal development until 9 months of age when delayed development appeared. Then she was referred to pediatric neurologist for suspected psychomotor retardation and hypotonia. She also has experienced repeated upper respiratory tract infections, requiring the patient to be admitted to the hospital for the recovery.

At the time of referral, her weight was 19 kg and her length was 105 cm with an upper segment–lower segment ratio of 1.1. Her physical features were characterized by short stature, umbilical hernia, hypotonia, short neck, joint stiffness, large tongue, thick lips, corneal opacity, and short stubby fingers. The facial features included enlarged head, coarse face with flattened nasal dorsum and frontal bossing. Skeletal survey showed dysostosis multiplex ( Fig. 1 ) including thick calvarium, coax valga, increased acetabular angle with broad iliac wings, thoracolumbar kyphosis, odontoid hypoplasia, and short and thick ulna and radius with irregular epiphyseal ends. The patient also had obstructive respiratory problems, hepatosplenomegaly and severe cognitive dysfunction. Unfortunately, the patient's parents were not available for participation in this study. Based on symptoms and biochemical tests, genetic testing for the IDUA gene was suggested.

Fig. 1.

Radiographs of the patient with mucopolysaccharidosis type 1. ( A ) Radiograph of pelvis shows abnormal acetabular angles with flaring of iliac wings. ( B ) Radiograph of the right hand illustrates hypoplastic distal ulna and radius along with proximal tapering of metacarpals.

Genotyping

Prior to testing, the family of the patient had given written consent for blood sampling and subsequent molecular studies. DNA genomic was obtained from peripheral white blood cells by QIAamp DNA mini kit (Cat.#463000) under the manufacturer instructions. DNA sequence of the IDUA gene was initially obtained from NCBI (National Center for Biotechnology Information; reference sequence: NC_000004.12). Nine primer pairs for PCR amplification and sequencing were designed with the help of primer-3 software ( http://bioinfo.ut.ee/primer3-0.4.0/ ), spanning all 14 exons and exon–intron junctions. PCR was conducted in a reaction volume of 25 µL consisting of 100 ng template DNA, 2.5 mM of MgCl ₂ , 200 mM of dNTPs, 10 pmol of each primer, and 1.5 U of taq polymerase. PCR products (5 µL) were then loaded on 1.5% agarose gel with a 100-bp marker and visualized under UV (ultraviolet) light. The resultant products of PCR-amplification were eight fragments of different sizes including exons 1, 2, 3–4, 5–6, 7–8, 9–10, 11, 12, and 13–14. Thermal cycling conditions and primer pairs designed for PCR are shown in Table 1 . Amplified PCR fragments were sequenced on ABI 3100 Genetic Analyzer (Applied Biosystems, United States) and then the analysis data software, Finch TV ( https://digitalworldbiology.com/FinchTV ; Geospiza Inc., Seattle, Washington, United States) was applied to view the chromatogram sequence of IDUA obtained from sequencing. The amino acid query sequence was then compared against a reference sequence on NCBI (reference sequence NM_000203.3).

Table 1. Forward and reverse sequence, amplicon length, annealing temperature, and PCR condition used in this study.

Exon	Primers (5′ to 3′)	Product size (bp)	Annealing temperature (°C)	PCR conditions
Exon 1	F-GAGTCATCGGTCCTCAGAGC R-AGGACCCACCCACAAACAC	554	62	Denaturation 95°C 20 min, 30 cycles at 95°C 1 min, 61°C 1 min, 72°C 50 s, final extension 72°C 10 min
Exon 2	F-CGCTGCCAGCCATGCTGAGGCTCG R-CCTCCCATCTGTGCCTCTGTAAGG	253	58.3	Denaturation 95°C 20 min, 32 cycles at 95°C 30 s, 61.9°C at 30 s, 72°C 1.10 s, final extension 72°C 5 min
Exon 3–4	F-GGGTTATTTTCCAAGGGGAAG R-CCAACCTATCCCTTGTCACC	691	58.5
Exon 5–6	F-ATGCAGACGCCCTTCATC R-CCTGCTCCAGGATGGAGA	641	59
Exon 7–8	F-CCACGACGGTACCAACTTCT R-TCCCCTTGGTGAAGGAGTC	831	59
Exon 9	F-CTGGGGACTCCTTCACCAAG R-CAGGTAGCGCGTGACGTA	443	61.2
Exon 10	F-GTAAGCCGGGGTTCCAGG R-CGGTCCTCAGGGTTCTCC	386	61.9
Exon 11	F-TGTGGGTGGGAGGTGGAG R-GAGGGAAGGGCTGTGATGG	302	65.7	Denaturation at 95°C 5 min, 35 cycles at 95°C 1 min, 62°C 1 min, 72°C 50 s, final extension 72°C 5 min
Exon 12	F-ACAGTGTGTGGGGTGAAGG R-TTGCTGGTGCACGTGTGT	354	63.3	As exon 2
Exons 13–14	5-CCTAGGGGACATGAGATGGA-3 5-CTCCAGCTGGGTCCTCATC-3	839	58.5	As exon 2

In Silico Analysis

All variations in coding regions and exon–intron junctions were identified. We sifted through listed mutations stored on the online databases HGMD (Human Gene Mutation Database: www.hgmd.cf.ac.uk/ ), 1,000 genomes ( http://1000genomes.org ), ExAC ( http://exac.broadinstitute.org/variant/4-995534-AG-A ; Exome Aggregation Consortium), and ClinVar ( https://www.ncbi.nlm.nih.gov/clinvar/ ) to filter out known putative mutations in the IDUA gene. MutationTaster ( www.mutationtaster.org ) and SIFT ( http://sift.jcvi.org/ ; Sorting Intolerant from Tolerant), PROVEAN ( http://provean.jcvi.org/index.php ; Protein Variation Effect Analyzer), and DDIG-in ( http://sparks-lab.org/yueyang/server/ddig/ ; Detecting disease-causing genetic variations due to indels) were employed to predict the potential effect of a detected alterations on the functionality of a protein. We visualized the effect of this mutation on the structure of protein by Swiss Model ( https://swissmodel.expasy.org/ ).

Results

The patient was diagnosed to have MPS1 according to her clinical features and genetic testing. The measurement of urinary glycosaminoglycans (GAGs) confirmed extra level of GAGs excretion and analysis of lysosomal enzyme from dried blood yielded the following results in Table 2 . As the alpha-l-iduronidase activity was below its reference range, genetic test was performed to confirm the diagnosis. All exonic regions (exons 1–14), as well as exon–intron junctions were screened to find the genetic problems, upon which a 2-bp deletion in exon 5 c.474-475delCT ( Fig. 2 ) and 11 nonpathogenic variations were characterized ( Table 3 ). The patient presented with severe form of disease (Hurler's syndrome). One year after initiation of enzyme replacement therapy (ERT), all clinical symptoms of the patient persisted or deteriorated except for improvement in upper airway obstruction.

Table 2. The results of blood and urinary tests in the patient.

Parameters	Results	Reference range
Alpha-L-iduronidase	0.00	0.04–0.26 nmol/21 h punch
Iduronate-sulfatase	0.05	0.02–0.25 nmol/21 h punch
Arylsulfatase B	1.25	0.2–1.41 nmol/21 h punch
Beta-galactosidase	0.72	0.5–3.2 nmol/21 h punch
Urinary GAG measurement	57.8	9.5–25.7 mg GAG/mmol creatine

Fig. 2.

Partial chromatogram of exon 5 illustrating nucleotide sequence of the patient carrying homozygous deletion mutation c.474-475delCT against normal control.

Table 3. Sequence variations identified in the IDUA gene .

Location	Nuc. change¤	AA change¤	Evaluation
Exon 1	c.99T > G (homo)	p.H33Q	SNP site
Exon 3	c.314G > A (homo)	p.R105Q	SNP site
Exon 4	c.474-475delCT	p.S159Gfs*81	Deleterious
Exon 5	c.543T > C (homo)	p.N181N	SNP site
Intron 5	c.590-8C > T (homo)	–	SNP site
Exon 7	c.942G > C (homo)	p.A314A	SNP site
Exon 8	c.1081G > A (homo)	p.A361T	SNP site
Exon 8	c.1164G > C (homo)	p.T388T	SNP site
Exon 8	c.1190-10dupC (homo)	–	Likely neutral
Exon 9	c.1230C > G (homo)	p.T140T	SNP site
Exon 9	c.1360G.A (homo)	p.V454I	SNP site
Exon 10	c.1467C.T (homo)	p.R489R	SNP site

Abbreviations: AA, amino acid; dup, duplication; fs, frameshift; homo, homozygous; Nuc, nucleotide; SNP, single nucleotide polymorphism.

¤reference number NM_000203.4 and NP_000194.2.

Discussion

Since the cloning of the IDUA gene, more than 200 disease-associated mutations have been documented. Although the majority of these mutations are unique to a single family, a few regionally-specific mutations have been reported. In Europe, p.W402* and p.Q70* are the two most common mutations with different frequency in each country. The p.W402* has high incidence in Central Europe, the United States, and the United Kingdom, ^{11 12 13 14 15} while p.Q70* showed a considerable increase in Eastern and Northern European, contributing to 80% of entire mutant alleles in Finland and its frequency decrease from North to South Europe. ^{11 12 16} Two types of approved therapies for MPS1 patients include ERT and hematopoietic stem cell transplantation (HSCT). ERT is an intravenous infusion of laronosidase utilized to treat the attenuated form of MPS1. Since the enzyme does not infiltrate the blood–brain barrier, it is considered not to be effective in the CNS of patients related to Hurler's syndrome. ^{17 18} HSRT provides donor cells that allow the endogenous production of defective enzyme and has shown to mitigate CNS involvement if it is performed at the early stages of the disease. ^{19 20} We report here a small deletion (c.474-475delCT p.S159Gfs*81) in exon 4 of the IDUA gene that causes a shift in the reading frame. The new reading frame begins at codon 159 and terminates 80 codons downstream of the deletion site and is highly likely to create a truncated protein. Alpha-L-iduronidase protein contains three different domains including a TIM barrel, a b-sandwich with the first β -strand and type III fibronectin-like domain ²¹ ( Fig. 3A ). Protein modeling shows that mutated enzyme loses half of the active site that seems to be critical to the catalytic mechanism and substrate coordination ( Fig. 3B ). This impairment in the structure of the enzyme causes complete loss of enzyme activity, thus resulting in the development of the severe form of the disease. A combination of in silico analysis tools were employed to confirm the impact of this mutation on the protein function. This variation predicted to be deleterious by PROVEAN and disease causing by DDIG-in. SIFT and Mutation taster also called c.474-475delCT damaging and disease causing. According to in silico analysis done by MutationTaster, c.474-475delCT also activates NMD (nonsense-mediated decay pathway) pathway, a process through which mRNAs with premature termination codon are targeted for degradation. ²² In fact, NMD pathway regulates the phenotypic consequences of heritable diseases that are caused because of the presence of the premature translation termination codons (PTCs). ^{23 24} To date, there have been a few studies analyzing causative variations in the IDUA gene in Iranian population. However, existing studies could not provide a comprehensive picture of mutations related to MPS1 because of small sample sizes. ^{25 26} Therefore, further studies are required to characterize the genetic profile of MPS1 in Iranian patients.

Fig. 3.

Structure analysis for alpha-L-iduronidase enzyme; ( A ) visualization of normal enzyme and the important residues for binding of the substrate (red); ( B ) structure of mutant alpha-L-iduronidase showing partial loss of TIM barrel, complete loss of the other two domains ( β -sandwich and the Ig-like) and removing of the residues responsible for substrate binding. Ig, immunoglobulin.

Conclusion

In conclusion, our finding has expanded the mutational spectrum of Iranian patients with MPS1, paving way to accurate prognosis of the disease and more effective genetic counseling offered to such patients and their at risk family members.