Rapid genotyping of inversion variants in Mucopolysaccharidosis type II using long-range PCR: A case report

Abstract

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disease caused by a deficiency in iduronate-2-sulfatase (IDS), leading to the accumulation of dermatan sulfate and heparan sulfate in lysosomes. Traditionally, genotyping of the IDS gene has been conducted through exome sequencing, which fails to detect inversion variants. Consequently, when no pathogenic variants are detected in exons, additional PCR-based analysis is required. Herein, we introduce a rapid genotyping technique method using long-range PCR for MPS II patients. We successfully identified an inversion variant and confirmed the sequences of the inversion regions. We also confirmed that the pathogenic variant in the patient originated de novo. These findings suggest that long-range PCR genotyping can identify inversion variants more rapidly compared to the previous PCR-based methods, making it a valuable tool for newborn screening (NBS) and genetic diagnosis.

Highlights

• •Inversion variants in the intron area are developed in patients with MPS II.
• •Long-range PCR successfully could detect an inversion variant in MPS II.
• •Long-range PCR and next-generation sequencing identify inversion variants.

1. Introduction

Mucopolysaccharidosis II (Hunter syndrome, MPS II) is a lysosomal storage disease caused by pathogenic variants in the IDS gene coding iduronate-2-sulfatase (IDS), which is located on the long arm of the X chromosome (Xq28). The gene spans 28.3 kb and comprises nine exons [1,2]. Previous studies have indicated that the frequency of MPS II ranges from 1 in 100,0001 to 70,000 births, with higher frequencies reported in East Asian countries, including Japan, than those reported in European countries [3,4].

Patients with MPS II exhibit various symptoms, such as characteristic facial features, short stature, hearing loss, hepatosplenomegaly, and cardiomyopathy. There are two phenotypes of MPS II: (1) the severe type, characterized by neurodevelopmental delays, accounts for two-thirds of all MPS II cases, and (2) the moderate type, which lacks developmental delays. Severe cases often result in death within the first decade due to recurrent respiratory infections; however, the long-term outlook for MPS II has improved with the introduction of enzyme replacement therapy (ERT) [5]. Despite these advances, improving neurodevelopmental outcomes in patients with severe MPS II remains challenging due to the inability of enzyme products to cross the blood-brain barrier. The recently approved ERT drug in Japan, Izcargo^Ⓡ (pabinafusp alfa), can penetrate the parenchyma and enhance neurodevelopmental outcomes [6].

Pathogenic variants of the IDS gene can lead to the accumulation of dermatan sulfate and heparan sulfate in lysosomes due to a defect in IDS. IDSP1, the pseudogene of IDS, is located 19.7 kb telomerically and in the opposite orientation to the functional IDS. IDSP1 spans 1418 bp and includes two exons [7]. The sequence of IDSP1 shares homology with exons 2 and 3 and introns 2, 3, and 7 of IDS; these highly homologous regions facilitate various types of recombination, including inversion variants [8]. Reports of IDSP1-related inversion variants have indicated that the breakpoint predominantly occurs in intron 7 of IDS (Table 1).

Table 1.

Phenotypes of patients with MPS II with inversion variants.

Phenotypes (n)	Breakpoint	References
Severe (6/6)	Intron 7	Bunge (1998) [21]
Severe (1/1)	Intron 7	Alves (2006) [18]
Severe (4/4)	Intron 7	Zhang (2011) [13]
Severe (1/2), Other (1⁎/2)	Intron 7	Tajima (2013) [20]
Severe (13/13)	Intron 7	Brusius-Facchin (2014) [14]
Severe (3/3)	NA	Amartino (2014) [12]
Severe (2/3), Mild (1/3)	NA	Alcántara-Ortigoza (2016) [9]
Attenuated (1/1)	Intron 7	Lin (2019) [15]
Total: Severe: 30/33 (90.9 %), Mild: 1/33 (3.0 %), Attenuated: 1/33 (3.0 %)

^⁎ERT started during the presymptomatic period; NA: not available.

The most frequent genotypes of MPS II include nonsense and missense variants, frameshift variants, and partial and complete IDS deletions, along with IDSP1-related inversion variants [9]. The prevalence of inversion variants among MPS II patients ranges from 1.5 % to 12.6 % [[10], [11], [12], [13], [14]], and among infants with suspected MPS II referred from NBS, it has been reported at 0.7 % (1/140) [15]. In most cases, these inversion variants result in a severe MPS II phenotype (Table 1). Although MPS II genotyping is predominantly performed using exome sequencing—which can detect nonsense, missense, and frameshift variants, as well as partial and complete IDS deletions—it fails to identify inversion variants. In this study, we investigated a case of MPS II potentially involving an inversion variant and successfully identified it using long-range PCR of IDS. We describe here the rapid genotyping technique for inversion variants using long-range PCR and characterizing the inversion regions in a patient with MPS II.

2. Materials and methods

2.1. Study patient

Our patient was a four-year-old boy born by emergency caesarean section due to unreassuring fetal status at 38 weeks gestation, weighing 2132 g. Aside from low birth weight, there were no complications, and he was discharged on the ninth day after birth. The patient's parents are a healthy, non-consanguineous Japanese couple.

The patient was born in an area where NBS for MPS II has not been implemented; hence, he was not screened. Initially, his development was typical: he could hold up his head at three months, sit independently at seven months, and walk alone by one year. Although he began babbling during this period, his speech decreased over time, and developmental delays were noted at his 18-month check-up. At two years old, he spoke only a few words, prompting a medical consultation to investigate his developmental delay.

The patient was diagnosed with hepatosplenomegaly, oar-shaped rib deformation, and extensive Mongolian spots upon physical examinations. His developmental quotient (DQ) was 50 according to the Kyoto Scale of Psychological Developmental 2001, and he was diagnosed with autism spectrum disorder. Head MRI scans revealed honeycomb-like vacuoles in the perivascular space. Biochemical analysis indicated IDS enzyme activity in his blood was below the detectable range (cut-off value: 5 pmol/h/disc), and his urinary uronic acid was 167 mg/g creatinine (reference standard value: 56.3 mg/g creatinine, as indicated by the laboratory [SRL, Inc., Tokyo, Japan]). Additionally, his urinary levels of dermatan sulfate and heparan sulfate were remarkably elevated. Based on these findings, the physician suspected MPS II and requested a genetic examination from the Kazusa DNA Research Institute (Chiba, Japan). However, no pathogenic variants were detected in the targeted IDS gene sequencing of exons and adjacent introns; therefore, no further genetic diagnosis of MPS II was performed. At three years old, after moving houses, the patient visited our hospital, where MPS II was suspected during his initial physical examination. His DQ has declined to 30 on the Kyoto Scale of Psychological Development 2001. To confirm the genetic diagnosis, we conducted comprehensive sequencing of the entire IDS gene using long-range PCR and next-generation sequencing (NGS) [16].

2.2. Long-range PCR to detect inversion variant

The conditions for long-range PCR, including primer sequences, are detailed in our previous report [16]. Briefly, three regions of IDS were amplified using long-range PCR with three primer pairs (Fig. 1, Supplemental data 1) designed using Primer-3 (v.0.4.0; https://bioinfo.ut.ee/primer3-0.4.0/) based on parameters outlined by Togi et al. [17]. Primers M2-1f and M2-6r targeted regions outside exons 9 and 8, respectively; primers M2-10f and M2-15r targeted introns 8 and 5, respectively; and primers M2-19f and M2-33r were positioned in intron 5 and outside of exon 1, respectively. Most inversion variants typically occur between intron 7 of IDS and a homologous region in IDSP1 (Fig. 1, Table 1). Consequently, the annealing region of M2-15r shifted in the opposite direction, preventing the amplification of the PCR product M2-10f/M2-15r (A-2) (Fig. 1B).

Fig. 1.

Long-range PCR of IDS.

The top panel displays the gene structures of IDS and IDSP1 in a healthy control. The second top panel shows the gene structure of a patient with inversion variants between IDS and IDSP1. The striped box marks the genomic region of intron 7 in IDS and its homologous region in IDSP1. The wavy pattern box marks the genomic region of intron 2 in IDS and its homologous region in IDSP1. Insert A presents the results of long-range PCR: in the healthy control, PCR products A-1, −2, and A-3 were obtained; however, A-2 was not amplified in the patient. Insert B illustrates the PCR results for the inversion region: in the patient, the PCR products containing the inversion region, B-2 and B-4, indicated by red arrows and circles, were amplified. These products were not amplified in the health control.

PCR conditions were as follows: 94 °C for 2 min, followed by 30 cycles of 98 °C for 10 s and 64.3 °C for 30 s, and a final extension at 68 °C for 13 min and 36 s using KOD FX (Toyobo, Osaka, Japan) and a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA).

2.3. Confirmation of inversion variant

Two primer pairs (IDS8-F/IDS7s-R and IDSP1rev-R/IDSP1rev-F) were used to confirm the inversion variant. Primers IDS8-F and IDS7s-R were located in exons 8 and 7 of IDS, respectively, whereas primers IDSP1rev-R and IDSP1rev-F were placed outside exon 2 and 1937 bp downstream of exon 3 of IDSP1, respectively (Fig. 1). In healthy control, PCR products B-1 and B-3 were amplified using IDS8-F/IDS7s-R and IDSP1rev-R/IDSP1rev-F, respectively. In the patient, the annealing regions of IDS7s-R and IDSP1rev-R shifted in opposite directions, resulting in the non-amplification of PCR products B-1 and B-3. In contrast, PCR products IDS8-F/IDSP1rev-R (B-2) and IDS7s-R/IDSP1rev-F (B-4) were successfully amplified (Fig. 1C).

PCR conditions were as follows: 94 °C for 2 min, followed by 30 cycles at 98 °C for 30 s and 64.3 °C for 30 s, and a final extension at 68 °C for 3 min and 30 s using KOD FX and a Veriti Thermal Cycler. PCR products were purified via Sanger sequencing using an Agencourt AMP XP PCR Purification Kit (Beckman Coulter, Brea, CA, USA) according to manufacturer instructions and quantified using a Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA) with a Qubit 2.0 Fluorometer (Life Technologies).

2.4. Sanger sequencing

The PCR products of B-2 and B-4, containing the inversion regions, were sequenced by Sanger sequencing at an external contract lab, Eurofins Genomics K.K. (Tokyo, Japan), using the primers shown in Supplemental data 1, Supplemental data 2.

2.5. Ethics

This study was approved by the Ethics Committee of Kumamoto University (approval no. 1537). Written informed consent was obtained from the parents or legal guardians of the newborn.

3. Results

In the long-range PCR of the patient, the PCR products of M2-1f/M2-6r (A-1) and M2-19f/M2-33r (A-3) were amplified, whereas the product of M2-10f/M2-15r (A-2) was not amplified (Fig. 1A). Considering this result and the previous exome sequencing performed at another institution, which failed to detect the pathogenic variant, we suspected an inversion variant between intron 7 of IDS and IDSP1. To confirm the inversion variant, we analyzed four combinations of primers: IDS8-F/IDS7s-R, IDSP1rev-R/IDSP1rev-F, IDS8-F/IDSP1rev-R, and IDS7s-R/IDSP1rev-F. As depicted in Fig. 1C, PCR products of IDS8-F/IDS7s-R (B-1) and IDSP1rev-R/IDSP1rev-F (B-3) were amplified in healthy controls but not in the patient. In contrast, PCR products IDS8-F/IDSP1rev-R (B-2) and IDS7s-R/IDSP1rev-F (B-4) were amplified in the patient but not in healthy controls, suggesting the presence of an inversion variant between intron 7 of IDS and IDSP1.

Subsequently, we sequenced the B-2 and B-4 products to confirm the inversion region as between intron 7 of IDS and its homologous region, intron 3 of IDSP1 (Supplemental data 2). We could not determine a definite breakpoint because of the high homology between intron 7 of IDS and intron 3 of IDSP1.

Moreover, we tested the patient's mother for the same variant and found no inversion variant in IDS. leading to the conclusion that this variant was a de novo pathogenic variant (Supplemental data 3).

Since the patient was clinically diagnosed with MPS II, we initiated treatment with pabinafusp alfa. Although developmental delays have been observed, ERT has shown benefits, including a reduction in the frequency of respiratory infections.

4. Discussion

In this report, we present a patient with MPS II who was definitively diagnosed through rapid genotyping of an inversion variant using long-range PCR and NGS techniques. The phenotypes associated with the inversion variant of MPS II typically manifest as severe, characterized by low IDS enzyme activity. The breakpoint for this inversion variant occurs between intron 7 of IDS and intron 3 of IDSP1, areas noted for their high homology (Table 1). This inversion spans approximately 37 kbp, including the promoter region of exon 1 of IDS; however, a stop codon emerges at 24–26 bp downstream (nineth codon) of exon 7 (Supplemental date 2b, Supplemental data 4), and no poly-A sequences were found. Consequently, while some mRNA lengths may be transcribed, they are generally too immature to produce functional enzyme proteins, leading to a lack of mature IDS protein expression in most cases, as these inversion variants are null variants.

The genotyping for infants suspected of having MPS II was mostly performed using exome sequencing. If no pathogenic variant was detected in the exons and adjacent intronic sequences, further PCR-based analysis, such as restriction fragment length polymorphism, was conducted to detect inversion variants [15,[18], [19], [20], [21]]. We have developed and implemented a method combining long-range PCR and NGS techniques, differing from traditional exome sequencing, to directly detect the abnormal intron 7, such as the inversion variant of intron 7. Our method offers advantages over previous approaches, as the current NBS system quickly and easily detects individuals with the inversion variant of intron 7. Moreover, even without NGS technology, the long-range PCR technique alone can detect this inversion variant, as shown in this study. To our knowledge, this is the first report identifying the sequence of the inversion region in IDS. Recently, new approaches using gene editing technology are being actively developed, and MPS II is a candidate disease for such interventions [22,23]. Genetic information from patients is crucial for designing gene-based therapy treatment strategies.

Moreover, maternal genetic analysis indicated a high probability of a de novo variant. While MPS II is often clinically diagnosed using biomarkers like elevated urinary glycosaminoglycans without determination of the specific variant type, identifying the variant in the maternal gene is pertinent for genetic counseling and assessing the risk of inheritance in siblings [20].

5. Conclusion

We have demonstrated that our long-range PCR technique can successfully detect an inversion variant in a patient with MPS II, a variant that was undetectable by exome sequencing. The combination of long-range PCR and NGS allows for rapid and efficient identification of the inversion variant in intron 7. These techniques prove to be practical within the current NBS, enabling the accurate and swift detection of infants with MPS II.

The following are the supplementary data related to this article.

Supplemental data 1

The sequence of primers used in this study

Supplemental data 2

Sanger sequencing of inversion regions and the sequence of inversion regions

Schematic representation of the inversion regions, B-2 and B-4. The striped box marks the genomic region of intron 7 in IDS and its homologous region in IDSP1, whereas the wavy pattern box denotes the genomic region of intron 2 in IDS and its homologous region in IDSP1. The green triangle marks the location of the sequencing primer, and the red arrow indicates the sequenced area.

Supplemental data 2a presents the sequence of B-2, and Supplemental data 2b shows the sequence of B-4. In B-2, the green, light blue, earth color, and yellow shadings represent exon 8 of IDS, exon 3 of IDSP1, exon 2 of IDSP1, and the primers, respectively. In B-4, the green and yellow shadings indicate exon 7 of IDS and the primers, respectively.

Supplemental data 3

PCR confirmation of inversion mutation

The electrophoretic photo shows the PCR patterns of the patient and his mother for B-1, B-2, B-3, and B-4. Schematic representations of these segments are shown in Fig. 1.

Supplemental data 4

Long-range PCR of the inversion region

The top panel shows the gene structure of the inversion region, with a black triangle showing the primer locations for long-range PCR. The sequences of the primers are provided on the lower right panel. The electrophoretic image demonstrates the long-range PCR patterns for the patient and a healthy control, highlighting that in the patient, two products were amplified, containing the inversion regions of the centromeric and telomeric areas, respectively.

Funding

This study was supported in part by a Health and Labor Sciences Research Grant for Research on Rare and Intractable Diseases from the Ministry of Health, Labor, and Welfare, Japan (grant number JPMH23FC1033); a Grant-in-Aid for Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development (AMED; grant numbers JP22gk0110050h0003, JP23ek0109636s0301); and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Japan Society for the Promotion of Science [JSPS] KAKENHI: grant number JP20K08207).

CRediT authorship contribution statement

Yusuke Hattori: Writing – review & editing, Writing – original draft, Validation, Project administration, Methodology, Data curation. Jun Kido: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization. Keishin Sugawara: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Formal analysis. Takaaki Sawada: Validation, Investigation, Data curation. Shirou Matsumoto: Validation. Kimitoshi Nakamura: Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.