Molecular basis of mucopolysaccharidosis IVA (Morquio A syndrome): A review and classification of GALNS gene variants and reporting of 68 novel variants

Abstract Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N‐acetylgalactosamine‐6‐sulfatase (GALNS) gene. We collected, analyzed, and uniformly summarized all published GALNS gene variants, thus updating the previous mutation review (published in 2014). In addition, new variants were communicated by seven reference laboratories in Europe, the Middle East, Latin America, Asia, and the United States. All data were analyzed to determine common alleles, geographic distribution, level of homozygosity, and genotype‐phenotype correlation. Moreover, variants were classified according to their pathogenicity as suggested by ACMG. Including those previously published, we assembled 446 unique variants, among which 68 were novel, from 1190 subjects (including newborn screening positive subjects). Variants' distribution was missense (65.0%), followed by nonsense (8.1%), splicing (7.2%), small frameshift deletions(del)/insertions(ins) (7.0%), intronic (4.0%), and large del/ins and complex rearrangements (3.8%). Half (50.4%) of the subjects were homozygous, 37.1% were compound heterozygous, and 10.7% had only one variant detected. The novel variants underwent in silico analysis to evaluate their pathogenicity. All variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) to make them publicly available. Mutation updates are essential for the correct molecular diagnoses, genetic counseling, prenatal and preimplantation diagnosis, and disease management.


| INTRODUCTION
Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome; MIM# 253000) is an autosomal recessive lysosomal storage disorder caused by mutations in the GALNS gene, which encodes for the enzyme Nacetylgalactosamine-6-sulfatase (GALNS; EC 3.1.6.4). Importantly, reduced or totally absent GALNS enzyme activity leads to impaired degradation of the glycosaminoglycans (GAGs) chondroitin-6-sulfate (C6S) and keratan sulfate (KS) and their subsequent accumulation in tissues (Khan et al., 2017;Matalon et al., 1974). C6S and KS are mainly produced in cartilage and are stored primarily in the lysosomes and extracellular matrix of this tissue, leading to skeletal and connective tissue abnormalities (Khan et al., 2017;. MPS IVA is a rare disease, with an estimated prevalence varying from 1 in 71,000 births in the United Arab Emirates, to 1 in 323,000 births in Denmark, and to 1 in 1,872,000 births in Malaysia (Leadley et al., 2014). The clinical presentation of MPS IVA disease shows a spectrum of phenotypes ranging from a classical, rapidly progressing early-onset form to a nonclassical, slowly progressing, late-onset form. An intermediate slowly progressing form with early-onset has also been identified (Lee et al., 2012;Tüysüz et al., 2019). The classical disease phenotype typically presents in the first year of life with systemic bone dysplasia, short trunk dwarfism, spinal cord compression, cervical instability, joint laxity, pulmonary compromise, abdominal hernia, and corneal opacification (Galimberti et al., 2018;Hendriksz et al., 2013;Lin et al., 2014;Peracha et al., 2018). If untreated, these symptoms lead to death typically in the second decade (Lavery & Hendriksz, 2015;Lin et al., 2020). In nonclassical forms, symptoms may not appear or be recognized until later in childhood, or even until early adulthood (Galimberti et al., 2018;Montaño et al., 2007;Tüysüz et al., 2019) and may include minor skeletal abnormalities, such as a less pronounced short stature (Moisan et al., 2020).
Unfortunately, due to the rarity of the disease, the difficult differential diagnosis, and the clinical heterogeneity (Peracha et al., 2018), it may take months or even years from symptom onset to the diagnosis (Galimberti et al., 2018;Hendriksz et al., 2013;Rigoldi et al., 2018).
Enzyme replacement therapy (ERT) with recombinant human GALNS (elosulfase alpha) is currently the only approved disease-specific treatment option for patients with MPS IVA  and can improve endurance, respiratory function, and quality of life Hendriksz et al., 2016Hendriksz et al., , 2018. Moreover, early intervention with ERT may improve bone growth (Akyol et al., 2019).
Thus, timely diagnosis and intervention may optimize treatment outcomes and reduce mortality.
The classical diagnostic approach starts with suspicion of MPS IVA, often based on clinical signs and skeletal radiographs. With the introduction of pilot or routine newborn screening (NBS) programs, presymptomatic neonates can also precociously come to light, due to low GALNS enzymatic activity in dried blood spots Lin et al., 2020). In both approaches, MPS IVA diagnosis is confirmed by the enzyme assay of GALNS activity in leukocytes or fibroblasts (Hendriksz et al., 2013;Peracha et al., 2018) followed by molecular analysis.
Standard DNA sequencing is routinely used as the first-level molecular analysis to detect causative variants in GALNS exons and in their flanking sequences, thus allowing confirmation of diagnoses based on biochemical analyses and aiding in genetic counseling (Filocamo et al., 2018). With advances in next-generation sequencing (NGS) and the availability of gene panels for groups of diseases or symptoms, molecular diagnosis may sometimes precede enzyme testing in the diagnostic pathway. However, in some cases, both Sanger sequencing and NGS approaches might fail to identify the pathogenic alleles (Caciotti et al., 2018). In a few cases, additional approaches are implemented to verify the presence of genetic alterations commonly not detected by first-level analyses (i.e., genetic rearrangements, deep intronic alterations, etc.) (Caciotti et al., 2015(Caciotti et al., , 2018. The GALNS gene (Ensembl ID: ENSG00000141012), located on chromosome 16q24.3, contains 14 exons and is approximately 43 kb in length (Ensembl, 2020). A previous review of MPS IVA variants identified 277 published unique alterations, most of them being ZANETTI ET AL. missense variants distributed throughout the coding sequence and at flanking splice sites . Introns containing Alu repetitive elements can result in recombination events, potentially leading to large deletions (up to 8.0 kb) and/or rearrangements (Hori et al., 1995). In this study, we uniformly collected and summarized all published GALNS gene variants, updating the previous mutation review . In addition, previously undescribed genotypes were communicated by seven reference laboratories in Europe, the Middle East, Latin America, Asia, and the United States.
When possible, data were analyzed to determine the most common alleles, geographic distribution, levels of homozygosity, and genotypephenotype correlation. Variants were classified according to their pathogenicity as suggested by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/ AMP) (Richards et al., 2015). A summary of all GALNS variants so far identified will aid in the interpretation of molecular results and help to confirm the diagnosis in patients with suspected MPS IVA. Including those previously published, we collected 446 unique variants, among which 68 were novel, from a total of 1190 subjects. Novel variants were further analyzed by in silico tools to predict their potential pathogenicity to aid clinical interpretation.

| Editorial policies and ethical considerations
All participants (or parents/guardians) who provided samples for genetic testing in this study gave their informed consent for biochemical and molecular analyses.

| Literature search and collection of novel variants
A literature search was performed in PubMed and Google using the search terms "GALNS variants" and "GALNS mutations." Publications were filtered from the last GALNS mutation update  to December 2020. Additional variants were retrieved from the Human Gene Mutation Database (HGMD) Professional 2020.1. Results were limited to studies in humans. Each publication meeting the search criteria was screened by two reviewers for information regarding GALNS

| Sequencing
Details regarding DNA extraction, target amplification/enrichment, Sanger sequencing, and NGS are available in the supplementary materials. Primer sequences will be made available upon request. sequence (GRCh38.p13 genome build). When discrepancies were found, corrections were made accordingly. All misreported variants and other discrepancies were recorded.
Overall, including those previously published, 446 unique variants were collected from 1190 genotyped subjects. The current update provides an additional 169 unique variants to the 277 previously reported (Table S2). Five of the 169 unique variants were reported only at the protein level (nucleotide alterations were not provided in the reporting papers). Annotation checks of variants revealed several misreported variants that were corrected accordingly, when possible (Table S3).
Of the 654 genotypes identified in the current update, 448 (68.5%) were collected from the literature and 206 (31.5%) were reported by communications from laboratories (Table 1). On the whole, 53 patients were related to family members with the same variant; 48 were sibling pairs, two patients were twins, and one group of three patients were siblings. In addition, the genotypes of 43 neonates (13 from literature and 30 from laboratories) that tested positive at the NBS were collected. All of these ultimately resulted in the aggregation of 2323 alleles for which at least one alteration of the GALNS gene was reported, from 1190 individuals diagnosed with MPS IVA or who tested positive to the NBS (Tables S4 and S5). The total count of 2323 alleles also comprises 82 gene alterations reported in the original papers as tabular lists without information about their occurrence in patients .
Due to this lack of information, these instances were not included in the list of genotypes.
Of the identified individuals, 600 (50.4%) were homozygous for F I G U R E 1 Distribution of subjects' zygosity. Percentage distribution of zygosity of the collected genotypes (n = 1190). "Other" refers to subjects in which >2 variants were described additional analyses to search for large deletions/duplications and/or gross rearrangements (Bochernitsan et al., 2018;Jezela-Stanek et al., 2019;Leong et al., 2019;Szklanny et al., 2018;Tapiero-Rodriguez et al., 2018;Tüysüz et al., 2019). Notably, three subjects with two different homozygous variants were described previously (Bunge et al., 1997;Tomatsu, Filocamo, et al., 2004) and in this study. Six subjects (two of whom were siblings) carrying three distinct variants were reported previously (Cozma et al., 2015;Morrone, Tylee, et al., 2014;Tulebayeva et al., 2020), and in this study; segregation analysis was not available in any of these cases. In agreement with the previous report , most unique variants here reported were missense (65.0%), followed by nonsense (8.1%), splice site variants (7.2%), and small frameshift deletions or insertions (7.0%). All other variant types each had a frequency ≤4% (Figure 2). At the time of the previous update, few large deletions or insertions had been reported, potentially due to underdetection . Here, we report 15 subjects with 17 unique large deletions, insertions, or complex rearrangements.

| Most frequently reported GALNS alleles
Commonly reported variants occurred throughout the length of the GALNS gene with no particular "hotspot" regions for variation ( Figure 3).
The 10 most commonly reported variants collectively occurred in 625 alleles and accounted for only 26.9% of all alleles (Table 2) (Table 2). These three alleles were also the most frequent in both the 2014 update      (Tomatsu, Dieter, et al., 2004) and was suggested to be a founder mutation (Bochernitsan et al., 2018

| Newly identified variants
The genotypes of 206 subjects were collected from seven laboratories (Table S5), among which 68 novel genetic alterations were reported (
The huge number of variants falling in the "uncertain significance" class is consistent with the high genetic heterogeneity shown by the GALNS gene and it is also strongly influenced by the absence of robust functional evidence for most variants (i.e., in vitro evaluation of enzymatic activity, in vivo activity for homozygotes, etc.).
All classified variants and their associated pathogenic evidence were submitted to ClinVar, where they can be retrieved by the fol-

| Variants detected by newborn screening programs
A total of 43 neonates identified through pilot NBS studies, evaluating GALNS enzyme activity in dried blood spots (DBS), were included in the present analysis (

| FUTURE PROSPECTS
The results of the first two NBS pilot programs recently reported Scott et al., 2020) may encourage the expansion of NBS to wider populations, thus allowing early identification of affected children and, hence, timely intervention with enzyme replacement therapy. However, a careful long-term follow-up of positive cases must be guaranteed, as well as a deeper analysis of potential pseudo-deficiency alleles. When novel variants are identified, which occurs quite commonly with NBS, we recommend pursuing the in vitro analysis of variant expression, besides enzyme activity confirmation in blood cells or fibroblasts and measurement of specific biomarkers, such as KS or C6S, with sensitive methods.
Moreover, to exclude pseudo-deficiency, a close clinical evaluation of siblings and a search for the same variant(s) in the family should also be performed. All of these analyses would help to understand more precociously the potential pathogenic significance of the variant(s).
The review of the literature since 2014 and the data from the contributing laboratories shows a greater use of NGS in the diagnosis of MPS IVA, with targeted gene panels used more frequently than whole-exome sequencing. Several diagnoses of MPS IVA were made using large panels, including hundreds or even thousands of genes addressing specific groups of disorders (i.e., inborn errors of metabolism) or clinical signs (i.e., dysmorphology and skeletal dysplasia, disorders with orodental involvement, etc.). A few cases were diagnosed by whole-exome sequencing or by second-or third-level analyses aiming to detect gross deletions/ rearrangements, copy number variations, or deep intronic variants.
It is clear that the choice of molecular methods applied strictly depends on the query to be solved as well as on the technology available. It is likely that the use of NGS technologies will continue to expand given the anticipated reductions in cost and will help to molecularly define a higher number of suspects. Given the het- Sanofi, and has received honoraria from Biogen, BioMarin Pharmaceutical