Background

Mucopolysaccharidoses IVA (MPS IVA) or Morquio A syndrome is an autosomal recessive lysosomal storage disorder caused by mutations in GALNS gene on chromosome 16q24. This gene encodes N-acetylgalactosamine-6-sulfatase enzyme. In MPS IVA, lysosomal degradation of keratan sulphate and chondroitin-6-sulphate is impaired (Akyol et al. 2019). Its clinical feature includes short stature, skeletal and joint abnormalities including genu valgum, joint hypermobility, hip dislocation and dysplasia, dental anomalies, corneal clouding, hearing loss, pectus carinatum, spinal cord compression, spinal instability and thoracolumbar kyphoscoliosis. MPS IVA has variable severity, but patients with the severe phenotype usually die in their second or third decade of life. Respiratory impairment, cardiovascular disease and spinal cord instability are the main cause of morbidity and mortality (Akyol et al. 2019; Bertolin et al. 2021; Diaz-Ordoñez et al. 2022).

Hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) are two main treatments for MPS which slow the progression of this disease and improve quality of life (Taylor et al. 2019).

In HSCT, the donor leukocytes go to the host tissues and supply the endogenous enzyme, while in the ERT (elosulfase alpha for MPS IVA) which is a lifelong therapy, exogenous enzymes are transported to host tissues. Although ERT can improve some clinical symptoms of the patients, it cannot pass through the blood–brain barrier and so cannot improve neurological implications. While in HSCT, donor-derived hematopoietic cells can pass through the blood–brain barrier and differentiate into microglia (Aldenhoven et al. 2008; Qu et al. 2022). Even if the ERT is started at an early stage, its effect is limited on skeletal dysplasia in MPS IVA patients. Although according to some studies HSCT may affect on bone, there is no evidence to improve bone growth if the HSCT starts after 4 years of age (Akyol et al. 2019; Sawamoto et al. 2020; Wang et al. 2016; Yabe et al. 2016). Also there are risks of graft versus host disease (GVHD) and rejection in HSCT and it needs a donor who is HLA matched to the patient (Sawamoto et al. 2020).

Preimplantation genetic diagnosis (PGD) makes it possible to select the healthy embryos before implantation in families with known disease causing mutations. PGD was applied for human more than 3 decades ago for the first time (Handyside et al. 1990; Verlinsky et al. 1990). Since then, PGD has been used for diagnosis of many genetic disorders such as hearing impairment, alpha and beta thalassaemia, Steroid-Resistant Nephrotic Syndrome, Neurofibromatosis, Neurodegenerative disorders, inherited heart diseases, etc., in the embryos (Chen et al. 2021; He et al. 2021; Khordadpoor Deilamani and Akbari 2019a, b; Merker et al. 2015; Sciorio et al. 2021; Yeates et al. 2022).

PGD has been also applied for HLA typing. The HLA (human leukocyte antigen) genes located on chromosome 6, encode antigen presenting proteins on the cell surface and have a main role in the immune system performance. HLA complex is responsible for GVHD and rejection after HSCT and organ or tissue transplantation (Alelign et al. 2018; Mori et al. 2021; Wiebe and Nickerson 2020). Due to high diversity of HLAs, the probability that two unrelated persons be HLA matched is very low and HLA-identical siblings provide the best opportunity to achieve a successful transplantation. When there is no HLA matched donor for a patient, PGD can be offered for the therapeutic purpose (Fernández et al. 2014; Kakourou et al. 2019).

Here we report the first use of PGD for both MPS IVA and HLA typing in a family with an affected son in order to select and transfer the unaffected embryos that are HLA matched with the patient too.

Methods

Patients

A couple (20-year-old female and 34-year-old male) who were distant relatives and having a 3-year-old son affected with MPS IVA were referred to the PGD center of Tehran Medical Genetics Laboratory. The homozygous mutation of c.319G>A (p.Ala107Thr) in exon 3 of GALNS gene had been found in the affected son by whole exome sequencing (WES) in the same laboratory. The family members were HLA typed with SSP (using Olerup SSP HLA Typing Kits) and/ or NGS method (Using AllType-NGS kit from One Lambda Inc. Thermofischer) and no one had matched HLA with the patient.

After PGD counseling, the informed written consent was obtained from the patient's parents. The local ethics committee approved this study.

In vitro fertilization

The In Vitro Fertilization procedure was performed at Avecina IVF center. Nine embryos were produced. Cells were biopsied from each embryo, washed in PBS and were transferred to our PGD center in 2.5 µl of PBS.

Whole genome amplification

Picoplex WGA kit (Rubicon Genomics) was used to perform whole genome amplification (WGA) on blastomeres as well as positive and negative control samples in 3 steps of cell lysis/DNA extraction, preamplification and amplification. Then QIAquick PCR purification kit from QIAGEN was used to purify the WGA products.

Cycle sequencing and haplotype analysis

Two different tests including Sanger sequencing of exon 3 of GALNS gene and linkage analysis by STR markers were used to assess the transmission of c.319G>A (p.Ala107Thr) mutation in GALNS gene to the embryos. Six dinucleotide short tandem repeats (STR) markers (rs58441312, rs1555521601, rs56406234, rs373372968, D16S3026 and D16S3121) encompassing the GALNS gene were included in linkage analysis.

Also a panel of 11 STR markers located inside the HLA locus was used to test the HLA haplotypes (Table 1). Forward primers of the STR markers were labeled with FAM or HEX fluorescent dyes.

Table 1 The primers for STR markers flanking the GALNS gene or in HLA loci
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The markers were amplified by several multiplex PCR and the fragment separation was done using Applied Biosystems 3130 Genetic Analyzer, Hi-Di formamide and GS 500 Liz size standard. The GeneMarker software was used to analyze the data.

Results

Sanger sequencing confirmed the c.319G>A (p.Ala107Thr) mutation which was detected by the whole exome sequencing in GALNS gene (Fig. 1). The segregation of the mutation was also confirmed in the family.

Fig. 1
figure 1

Homozygous mutation of c.319G>A (p.Ala107Thr) in GALNS gene in the Patient

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HLA typing showed that the patient’s father was homozygous for HLA loci except for the HLA-DPB1 (DPB1*02:01:02/DPB1*04:01:01). Based on ImMunoGeneTics/HLA (IMGT/HLA) website (DPB1 T-Cell Epitope Algorithm v1.0 2012), these HLA-DPB1 alleles were permissive mismatch. So the embryos had the chance to inherit any paternal HLA-DPB1 allele for HSCT if they are matched with the donor for HLA-A, B, C, DRB1, and DQB1 loci (10/10) (Table 2).

Table 2 HLA types in the patient and his parents
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In pre-PGD the haplotypes of the STR markers which were linked to the mutated GALNS allele as well as HLA loci were inferred by investigating the affected son, his parents and some other family members.

For PGD, the biopsied cells of nine embryos were received from the IVF center.

The WGA products were then used in order to determine the mutation status of the embryos. The embryos that had at least one normal allele in GALNS gene were tested with STR markers in HLA loci (Figs. 2 and 3). The summarized results are presented as follows (Table 3).

Fig. 2
figure 2

Haplotype analysis for STR markers flanking the GALNS gene and sequencing results of GALNS mutation

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Fig. 3
figure 3

Haplotype analysis for STR markers in the HLA loci

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Table 3 The summary of the PGD testing results for MPS IVA and HLA typing
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Embryos 3 and 7 were recommended to be transferred. The first transfer was unsuccessful. The conception was successful at the second transfer. Prenatal diagnosis was done at 12th week of gestation and confirmed the conception of a girl who was heterozygous for normal allele and has matched-HLA with the affected son. The fetus is not born yet. After its birth HSCT will be performed from the Umbilical cord blood.

Discussion

MPS IVA is a lysosomal storage disease which can be fatal in patients with severe phenotypes (Sawamoto et al. 2020; Wang et al. 2016; Yabe et al. 2016).

The first successful case report of HSCT for MPS IVA was reported in 2014 (Chinen et al. 2014).

In 2020, HSCT was suggested as a useful treatment option for MPS IVA patients that could improve pulmonary performance, density of bone mineral and daily life activities and could reduce the need to surgeries in these patients. However HSCT has the risk of mortality and morbidity more than ERT (Sawamoto et al. 2020).

In 2022 Qu et al. reported that the lysosomal enzyme restored to the normal level after HSCT in patients with MPS IVA and VI. Also the functions of respiratory and nervous systems were improved (Qu et al. 2022).

PGD for Morquio syndrome was done previously in order to prevent the birth of new affected child (Qubbaj et al. 2008). However, there is no report of PGD with the purpose of treatment for this disorder. Here we report the first application of PGD for MPS IVA and HLA typing for future HSCT treatment for the affected child in a family. SSP and NGS-based HLA typing revealed there is no appropriate donor in this family. In the HLA registry there were some HLA matched donor oversees. However the patient's parents could not accept the risk of any morbidity or mortality of HSCT from a non-relative donor to their son and requested for the PGD test.

The ESHRE guideline recommended using two upstream and two downstream markers that are linked to the disease causing gene in indirect mutation analysis to reduce the risk of Allele dropout (ADO) and no diagnosis results in PGD. Also for HLA typing at least one STR marker located upstream of HLA-A, one between HLA-A and HLA-B, one between HLA-B and HLA-DRA, one between HLA-DRA and HLA-DQB1 and one downstream of HLA-DQB1 should be used to be able to assess the recombination in the HLA region. Applying two markers makes the test more powerful (Harton et al. 2011).

We used 3 upstream and 3 downstream STR markers flanking the GALNS gene and totally eleven markers between HLA loci. Allele dropout (ADO) and Locus dropout (LDO) were observed in different loci (Fig. 2 and 3). Also, due to incomplete adenylation in PCR, analysis of rs58441312 marker which is an AT repeat was difficult in most samples especially in WGA products. However, using sufficient number of STR markers in combination with Sanger sequencing made it possible to determine the genotypes of embryos.

Embryos 3, 6, 7 and 8 had at least one normal allele. These embryos as well as embryo 4 for which the zygosity of the mutation was not determined in the first try, were tested for STR markers in HLA loci which resulted in determining two HLA matched embryos (embryos 3 and 7).

The second transfer of the embryos resulted in pregnancy. Prenatal diagnosis in 12th week of gestation confirmed the conception of a heterozygous and HLA matched female which was also normal for the copy number of chromosomes 13, 18, 21, and X based on the QF-PCR Method.

As far as moral issues regarding PGD for HSCT is concerned, it is argued that since performing HSCT from an existing child to cure a sibling is acceptable, so PGD for HSCT is moral too (Pennings et al. 2002). Also when deciding to have children for less important reasons such as improving the marriage of disconnected couples is acceptable, wishing to conceive a child with PGD for HLA typing to treat another child is not morally wrong (Nickel and Kamani 2018). When there is no proper treatment except HSCT, the HSCT has high success rate, there is no HLA matched donor or HSCT from HLA matched sibling has advantage in comparison with nonrelative donors and HSCT can wait at least 9–12 months for the birth of the donor child, the conditions are appropriate for PGD for HLA typing (Pennings et al. 2002).

This report of PGD for MPS IVA and HLA may accelerate the setup of this test in other laboratories especially in countries with high frequency of consanguineous marriages and higher risk of genetic disorders such as MPS IVA.

Conclusions

In conclusion, despite the complexity of the procedure, PGD for MPS IVA and HLA typing can be considered as an option for curing the MPS IVA patients with HSCT. This should be considered especially for cases with no HLA matched donor.