Enzymatic Testing for Mucopolysaccharidosis Type I in Kuwaiti Newborns: A Pilot Study Toward Newborn Screening

1. Introduction

Mucopolysaccharidoses type I (MPSI, Hurler Syndrome) is an autosomal recessive subtype of lysosomal storage disorders (LSD), caused by the build-up of glycosaminoglycans (GAG) in the lysosomes secondary to deficiency or absence of α-L-iduronidase (IDUA) enzyme, encoded by IDUA gene. The IDUA enzyme is essential for the breakdown of two GAG, heparan sulphate and dermatan sulphate(1–3). The accumulation of lysosomal GAG would result in disturbance of cellular homeostasis leading to progressive cellular damage eventually resulting in a progressive debilitating multisystem disease with variable severity, involving musculoskeletal, cardiac, respiratory, and central nervous systems with poor prognosis in untreated individuals leading to death in the first decade (1,4,5). MPSI has been classified into three different forms based on severity and age of onset: Hurler (severe), Scheie (mild) and Hurler/Scheie (intermediate), with no biochemical differences among them and tendency to be described as having either “severe” or “attenuated” form of MPSI (1,6–8). Infants are typically born with no identifiable clinical symptoms, and based on the severity of the disease, they will be symptomatic within the first three years of life (1,9). MPSI is a pan ethnic disorder affecting males and females equally with incidence of 0.54-1.84 cases per 100,000 newborns (8). Prevalence of severe form is estimated to be 1:100,000 newborns, and attenuated form is estimated 1:500,000 newborns (1). However, due to frequent misdiagnosis and un derdiagnosis of diseases, especially the milder forms, it is difficult to determine the true frequency of disease in general population (1,2). Although it is believed that the incidence of inherited genetic disorders in general is more common in Arab countries compared to the other parts of the world due to the high consanguinity rates, there is no reliable data regarding the incidence and prevalence of MPSI in the Middle East and North Africa (MENA) region (10–13). Since the incidence of MPS1 in Kuwait is unknown, this pilot study was carried out to explore the possibility of including it in the national newborn screening program and thus aiding in assessment of its actual incidence and an early diagnosis.

The available treatment options for MPSI include hematopoietic stem cell transplantation (HSCT) and/or enzyme replacement therapy (ERT) (laronidase), which are more effective when administered before the presence of clinical manifestations (1,6,14–18). Many factors have stimulated the efforts to incorporate LSD in the newborn screening (NBS), especially MPSI: 1). The availability of new promising therapies for MPSI and other LSD, 2). The development of new screening tests using dried blood spots (DBS), 3). Early diagnosis and initiation of treatment are known to improve the outcome (1,9,19–21). Like many rare disorders, early diagnosis of MPSI is a challenge. IDUA enzyme activity and GAG accumulation can be measured at birth in DBS and has been adopted by various NBS programs enabling MPSI detection before the appearance of clinical signs and symptoms (9,19,20). Therefore, MPSI NBS was added to the recommended uniform screening panel (RUSP) in the United States in 2016 to be included in the list of disorders that are screened in different states’ universal NBS programs (5,22).

MPSI was initially screened with a single-tier approach using IDUA enzyme activity resulted in extremely low positive predictive values due to the low cutoff and pseudodeficiency of IDUA (19). However, a second-tier biomarker was needed for precision and improved positive predictive values, which included measurement of multiple glycosaminoglycans (GAGs) in DBS using MS/MS (9,19). This two-tier screening is suggested to be the gold standard for diagnosing MPSI patients in newborns (9). It is anticipated that subsequent evaluation would include further biomarker studies, thorough clinical assessment, and molecular genetic testing.

This study was undertaken with the aim of estimating the frequency of MPSI in Kuwait and to explore the possibility of including it in the expanded national NBS program especially because of the availability of therapeutic measures (23).

2. Methods

2.1. DBS Collection Protocol and Screening Population

All Kuwaiti neonates born at Farwaniya Hospital (FH), one of the main six governmental hospitals in Kuwait between December 2021 and December 2022, were included in this study and screened for MPSI. Following the national NBS protocol that was previously described (23), 618 DBS samples were collected within 48-72 hours of life but on a separate Whatman 903 filter card from which other NBS tests were performed. The DBS cards were then gathered, stored at room temperature, and were sent to an analytical laboratory for analysis (ARCHIMED Life Laboratory, Vienna, Austria.

2.2. Ethical Approval

This research study was approved by the Ethical Committee Board at the Ministry of Health, Kuwait as well as the Kuwait University College of Medicine’s Institutional Review Board, following the declaration of Helsinki. The study number is MK02/21. Written informed consent for the research study and publication data was obtained from the parents of each newborn.

2.3. Analytical Methods

The screening consisted of quantitating IDUA enzyme activity together with enzyme activities for iduronate-2-sulfatase, alpha-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase, arylsulfatase B and beta-glucuronidase in DBS by tandem mass spectrometry (MS/MS) as previously described (24–27). The cut-off value of IDUA activity is >1.5 μmol/L/h. Blood GAGs quantification was not performed as a second tier-testing.

2.4. Molecular Testing

A follow-up genetic analysis of IDUA gene using PCR amplification and sequencing of coding exons 2-14 (exon 10 partly) and flanking intronic regions has been performed on the cases with diminished IDUA enzyme activity as second-tier testing. Genomic DNA was isolated from the same filter card used for enzymatic analysis.

3. Results

A total of 618 newborns, including 331 (54%) males and 287 (46%) females, were screened between December 2021 and December 2022. All the newborns were Kuwaiti descendants. Twenty of them (9 males; 11 females) had deficient IDUA enzyme activity ≤ 1.5 μmol/L/h, ranging from 0.4-1.5 μmol/L/h. However, all had negative genetic testing for IDUA resulting in a false-positive rate of 0.03% (Table 1). None of these newborns were heterozygous for variants in IDUA. In addition, we diagnosed one female baby with MPSI, who belonged to FH residential area, but the parents chose to deliver in a private hospital instead of FH and thus was not screened at birth. However, she was diagnosed with MPSI at age three-month. That being included, MPSI is estimated to be about 0.3% among tested females and 0.2% of all screened cases in Kuwait, which is a relatively high incidence rate compared to the rest of the world. The delay in analysis of DBS has ranged from 1-week to 10-week-2-day due to delay in shipping the samples to the laboratory for analysis. Turn-around time for the genetic testing for final confirmation ranged from 2 to 11-week (Table 1).

Detailed clinical and diagnostic information of the confirmed Positive Case

The single confirmed MPSI presented at age 3-month-15-day and was diagnosed at that time. MPSI diagnosis was confirmed via both biochemical and genetic testing; enzymatic testing showed reduced IDUA activity at 0.1 μmol/L/h (Cut-off >1.5) and genetic testing revealed a previously reported pathogenic homozygous nonsense variant in IDUA (c.1882C>T; p.Arg628Ter). Her clinical examination was notable for facial coarse features, macrocephaly (head circumference >90^th percentile) and extensive Mongolian macules over the back, lower and upper extremities. Imaging was remarkable for mild hepatosplenomegaly and dysostosis multiplex; echocardiogram showed mildly thickened anterior mitral leaflet, and aortic valve. She was noticed to have extensive Mongolian macules, noisy breathing, snoring and recurrent upper respiratory tract infections since the age 1-month-old requiring courses of oral antibiotics, inhaled and systemic steroids and inhaled bronchodilators, prescribed by pediatrician and pediatric pulmonologist. She was started on ERT (laronidase) at age 4-months and underwent successful HSCT at age 9-month. She is currently an 18-month-old, doing well and developing adequately.

4. Discussion

Kuwait is a small country located in the Arabian Peninsula, with a population of 4.45 million people of which 1.45 million are Kuwaiti citizens. The vast majority of healthcare services in Kuwait is distributed among six main governmental general hospitals and sixteen private hospitals. Despite the free healthcare services provided by the governmental hospitals, Kuwaiti citizens prefer seeking medical management through the private medical center to reduce the wait time and the convenience of appointments’ schedule (28). All Kuwaiti citizens and foreigners have a mandatory Kuwait civil identification card (ID) which allows access to certain governmental services including the governmental hospitals. So, each of the six governmental hospitals are assigned to certain residential area, which is documented in the ID of everyone living in Kuwait.

Newborn screening in Kuwait has been expanded in 2014 to include 22 endocrine and metabolic disorders with the goal to early detect and treat certain medical conditions to improve the outcome in a cost-effective manner (29). MPSI is currently not included in the Kuwait national NBS program. Disease prevalence and the frequency of carriers in Kuwaiti population are unknown. We have conducted this pilot study in FH, one of the main six governmental hospitals, to evaluate the incidence of the disease in Kuwait and its carrier frequency, as a first step toward including it in the national NBS program.

In this study, 618 Kuwaiti newborns delivered at FH, were screened for reduced IDUA enzyme activity using DBS, followed by DNA sequencing of the IDUA gene. We identified 20 neonates with reduced IDUA enzyme activity but negative for biallelic pathogenic variants in IDUA gene as second tier testing. None of them were carriers for the disease and unfortunately, total carrier frequency of IDUA variant could not be measured in Kuwaiti newborns.

The reduced IDUA activity has resulted in false rate of 0.03% (Table 1) and could be attributed to several factors: 1. the prolonged transfer time to the laboratory for analysis and thus exposure to the heat, 2. pseudodeficiency, or 3. a heterozygous carrier for a pathogenic variant for IDUA gene.

We have identified one affected female baby with MPSI at age 3-month-old within the study period that belonged to the study site (FH) but was from recruitment in the study as she was born at a private hospital. Her diagnosis was established based on reduced IDUA activity and positive genetic testing for homozygous pathogenic nonsense variant in IDUA (c.1882C>T; p.Arg628Ter), resulting in an incidence of 0.2%. This variant has been associated with severe phenotype of MPSI (Hurler syndrome). It has been frequently reported in homozygous form in affected Kuwaiti individuals and is considered as a founder variant in Kuwait (30,31). The variant (c.1882C>T; p.Arg628Ter) was identified in 14 out of 291 individuals with severe MPSI from Kuwait, accounting for 4.8% of alleles in all individuals (31).

This variant has been previously reported as compound heterozygous with another pathogenic variant in IDUA or in homozygous state. There is a close genotype-phenotype correlation among individuals with MPSI (1,32). However, it is well known that the combinations of two null alleles are associated with more severe phenotype, while at least one missense or intronic variant is associated with a milder disease due to some residual enzyme activity. Homozygosity or compound heterozygosity of this nonsense variant (p.Arg286Ter) has been consistently associated with severe phenotype (Hurler syndrome) due to the premature protein truncation of about 50 C-terminal residues resulting in nonfunctional protein (31–37). However, there are two reported individuals with MPSI, one with the same homozygous variant p.Arg286Ter and the other is compound heterozygous for p.Arg268Ter/p.Gln70Ter, both reported with attenuated phenotype (Hurler Scheie), although the phenotype of second case described is consistent with severe disease (Hurler) (38,39).

However, when it is compound heterozygous with a missense variant, it has been associated with attenuated phenotype (Hurler Scheie) or the severe form (Hurler) (40,41). This variant has been previously reported in individuals with MPSI of Middle Eastern origin (Turkey, Tunisia, Saudi Arabia, and Kuwait) (31,35,36,41,42). The early diagnosis of this case has led to early initiation of treatment (ERT) around age 4-month, followed by HSCT at age 9-month with better outcome. This is the first study in Kuwait and the MENA region determining the incidence of MPSI.

Further screening of all Kuwait babies delivered at the other governmental and private hospitals is required to obtain a more accurate incidence of MPSI in Kuwait and facilitate the implementation of a nationwide screening program for MPSI. The results reported in this study support including MPSI in the national NBS program given the severity of the founder mutation and the effectiveness of early management. If MPSI is to be included in the NBS in Kuwait, we recommend obtaining IDUA enzyme activity in DBS, followed by full DNA sequencing of the IDUA gene as a second-tier testing. This would not only help in achieving complete genotype of IDUA in our population but may also confirm that the nonsense variant (c.1882C>T; p.Arg628Ter) is the most common variant in Kuwaiti population. It will also aid in estimating the carrier frequency for this variant in the Kuwaiti population. Furthermore, it would also allow establishing genotype-phenotype associations and thus improvement in making appropriate therapeutic decisions.

5. Conclusion

This is the first study in Kuwait and MENA region evaluating the incidence and genotype of MPSI via screening asymptomatic newborns. Although none of the 618 screened newborns were confirmed to be affected, we have identified and confirmed MPSI that would have been detected if delivered at FH, resulting in an estimated incidence of MPSI of 0.2% of all screened cases in Kuwait, which is a relatively high incidence rate compared to the rest of the world. Larger cohort is required to have an accurate estimate of the incidence of MPSI in Kuwaiti population and thus enforce incorporating it in the national NBS program because the effective therapeutic measures are available.