Results

In this study, 302 MPS patients were recruited, of whom 13 were found to be related to at least one other patient based on an extensive review of their documents and pedigrees and were therefore excluded from further analyses. As shown in Figure 1, among the remaining 289 unrelated families, 106 (36.3%) had positive results in previous genetic tests (Group A), while 183 (63.7%) had no prior tests or no positive results (Group B). Using ACMG guidelines, we reviewed previously reported variants in Group A patients to verify causal variants and determine their pathogenicity. This approach confirmed the pathogenicity of the reported variants in 105 patients, while disproved a variant in MPS-69 (c.314G>A in IDUA ) due to its population frequency of 15.72% and 13.74% in gnomAD Exomes and Genomes respectively, which resulted in moving this patient to Group B. The positive findings in the remaining 105 Group A patients were reviewed with regard to the test setting, the applied technique, and the variant characteristics. As a result, patients that were studied in a research setting, or had INDEL variants identified by WES, were investigated by Sanger verification. Among them, all variants except two (in MPS-215 and MPS-237) were confirmed. In MPS-215, who had the clinical diagnosis of MPS II, Sanger verification did not confirm the previously reported variants, but revealed a known in-frame hemizygous deletion (c.121_123del3, p.Asp35Gly1) in IDS . Sanger sequencing did not confirm c.1104_1105delCT in the second patient (MPS-237), in whom, biallelic variants c.1104_1105delCT and c.656G>A had previously been reported by another lab using Whole Exome Sequencing. Therefore, MPS-237 was allocated to Group B and the NGS test was repeated using the IEM panel, which again showed the same variants in the patient. Reappearance of the same findings led us to believe that Sanger analysis had failed to call c.1104_1105delCT, likely as a result of allelic dropout in a GC rich region. Among the reported variants, there were two CNVs
In Group B, a total of 185 patients went through target capture NGS using the Ion Ampliseq IEM panel, consisting of 594 genes, and high-quality sequencing results were obtained. On average, 463,693,908 bases were called, 2,557,476 reads were mapped, and a mean depth of coverage of 304X over the targeted regions were achieved. Moreover, 87.2% of all the bases were covered by 20 or more reads, and an average uniformity of 83.3% was obtained per sample, indicating that the coverage of target regions was evenly distributed.
NGS analysis of Group B patients discovered causal MPS variants in 124/185 (67.02%), and non-MPS IEM variants in 5/185 (2.7%) patients. Details of the non-MPS IEMs are shown in Table 1. For the remaining 55 negative patients, IGV was employed to manually review the NGS sequence data of the clinically suggested MPS genes. This approach showed sufficient depth of coverage for the exons of the corresponding genes in 10 patients, low to zero depth of coverage in 41 patients, and non-annotated variants in 4 patients. The breakdown of the corresponding genes for 41 patients with low-depth coverage is as follows: exon 1 ofARSB in 13 patients, exon 1 of GALNS in 16 patients, exon 1 of IDUA in 11 patients, and exon 1 of NAGLU in 1 patient. Complementary Sanger sequencing for low coverage regions of the aforementioned genes resulted in discovering causal variants in 26 additional patients (26/41, 63.4%), which in turn improved the diagnostic yield by 14% (26/185) (Figure 1).
Regarding the 4 non-annotated variants, manual IGV review revealed the following variants, which were then confirmed by Sanger analysis: c.1279del in GALNS , g.997263G>A (c.1650+5G>A) in IDUA , c.544G>C inIDUA , and g.43033381A>G (c.1012+4A>G) in HGSNAT. As a result, the diagnostic yield was further improved by 2.2% (4/185), leading to a total improvement of 16.2% (30/185).
The IGV data corresponding to 124 positive MPS patients and 5 non-MPS patients in Group B were manually inspected to determine whether further confirmatory tests were required. Eighty-six variants with depressed allele fraction (<30% of alternate allele), mostly INDELs, were investigated by Sanger verification and/or segregation analysis, which all were confirmed. Overall, among 185 studied patients in Group B, 154 patients (83.2%) were found to harbor putative causal variants in MPS genes, five patients (2.7%) were diagnosed with a non-MPS IEM, while in 26 patients (14.1%) no pathogenic variant could be identified in the studied genes. Hence, the clinical diagnosis of MPS was not genetically confirmed in 16.8% of group B patients. 142 of these 154 patients were clinically assigned an MPS type, among them, the molecular findings were concordant in 97.2% (138/142). The four discordant cases comprised two MPS VI patients with the clinical diagnosis of MPS I (MPS-154) and IV (MPS-32), as well as two MPS IV patients with the clinical diagnosis of MPS I (MPS-213) and VI (MPS-142).
Further analyses on a total of 258 MPS patients (104 confirmed Group A Patients and 154 positive Group B MPS patients) were then carried out regarding pedigree analysis, geographical origin, and the distribution of variants in MPS genes (Table 2). In 258 genetically confirmed patients, 34.9% had MPS IV, 22.9% MPS I, 22.5% MPS VI, 12.9% MPS III, and 7.1% MPS II. In total, 191 unique putatively casual variants in 8 MPS genes were discovered in 258 MPS patients, among them 97.4% (186/191) were pathogenic/likely pathogenic (P/LP), and 2.6% (5/191) were variants of unknown significance (VUS) according to the ACMG/AMP guidelines. Furthermore, according to the public version of the HGMD® database (Stenson et al. 2014), and an intensive literature review, 90/258 patients (34.9%) harbored novel variants with no pathogenicity report, among which, 21/90 (23.3%) had variants sharing their location with a known pathogenic variant, but the nucleotide and/or amino acid changes were different. The remaining 168 of 258 patients (65.1%) possessed known causal variants with pathogenicity report.
Three patients had two homozygous variants in their corresponding MPS gene: MPS-95 had c.469T>C and c.523_543del in IDUA ; MPS-161 harbored c.46_57del and c.58G>T in IDU ; and MPS-254 had c.1493C>T and c.1496C>T in GALNS. Moreover, MPS-213 was homozygous for c.860C>T inGALNS which was a predominant known variant among our patients and also harbored a homozygous VUS variant in IDUA(c.806C>G). Notably, the clinical manifestations of this patient were in favor of MPS IV, while a single enzyme assay on DBS was suggestive of MPS I. This case was grouped in MPS IV considering the known pathogenic variant in GALNS . There was another patient with dual molecular diagnosis who had a homozygous P/LP variant inGALNS (c.722C>A) as well as a homozygous known nonsense variant in HOGA1 (c.763C>T) which is associated with primary hyperoxaluria, type III.
In terms of pedigree analysis, 32.6% were familial, and 66.3% were sporadic, and 81.8% of the parents had a consanguineous marriage. A breakdown of the consanguineous relations is depicted in Table 2.  Moreover, a majority of the patients were homozygous for the MPS variants (87.2%) (Table 2). The mutational spectrum included 9 (3.5%) CNVs which were then verified by IGV review and confirmed by multiple PCRs targeting the exons within the deleted regions, as well as those encompassing the CNVs. The GALNS gene had the highest number of CNVs (6/9) accounting for 6.7% of all variants identified in this gene (6/89). Details for the detected CNVs are shown in Table 3.
Furthermore, The IMPRESsion study recorded the clinical findings of all the patients with a heatmap of the frequency of symptoms illustrated in Figure 2. The most common symptoms in patients with MPS I, II, and VI recorded in at least 80% of the patients were coarse facies, joint contractures, umbilical hernia, short stature, sleep apnea, and macrocephaly. Among MPS IV patients, the most observed symptoms were umbilical hernia, short stature, corneal clouding, kyphosis, visual impairment, joint hypermobility, and pectus carinatum. MPS III patients displayed symptoms of coarse facies and umbilical hernia, but they also had developmental delay, intellectual disability, developmental regression, and behavioral disorders, which were not recorded in the rest of the MPS patients. (Figure 2)
In terms of geographical origin, 250 patients were Iranian, and 3 were Afghan (five with unknown geographical origin). The most common ethnicities among the Iranians were Fars, Azeri, Lor, and Kurd, with Fars being the most common (117/253, 46.2%), followed by Azeri (43/253, 17%) (Figure 3). We used parental geographical origin to depict the exact geographical origin of the family since the proband’s birthplace could have been affected by immigration of the previous generations to the large cities.
Details of the variants identified in this study are available at Iran Variome MPS Database (Ghaffari and Rafati 2020). The most common variants discovered in MPS VI patients were c.430G>A (15/58, 25.9%), c.962T>C (7/58, 12%), c.281C>A (4/58, 6.8%), c.275C>A (3/58, 5.1%), and c.753C>G (3/58, 5.1%). All 15 patients harboring c.430G>A originated from 3 neighboring provinces located in the southeast of Iran; 7 patients from Yazd, 4 from Sistan-and-Baluchistan, and 4 from Kerman. Furthermore, variant c.962T>C was detected in 6 patients from northwestern provinces including Gilan (3/7), Ardabil (1/7), West Azerbaijan (1/7), and East Azerbaijan (1/7). Likewise, variant c.281C>A was only detected in Khuzestani patients. Overall, the two most prevalent variants in MPS VI account for 37.9% (22/58) of the patients, while the five most prevalent variants account for 55.1% (32/58) of the patients. On the other hand, variant c.430G>A was present in 88.2% (15/17) of all MPS VI patients from Sistan-and-Baluchistan, Kerman, Yazd, Fars, and Hormozgan province, making it a significant variant in southeastern region of Iran. The same feature was observed in c.962T>C and c.281C>A variants, accounting for 85.7% (6/7) of northwestern and 66.7% (4/6) of Khuzestan MPS VI patients respectively.
The most frequent variants among MPS I patients were c.1A>C (11/59, 18.7%) and c.1598C>G (4/59, 6.8%). These two variants alone account for 25.5% of MPS I patients. Variant c.1A>C originated from two distinct regions of Iran, 5 patients from Gilan province, and 6 patients from provinces in the southwestern regions of Iran. Three of the patients carrying the c.1598C>G variant were from the Khorasan region of Iran, while one patient was from Lorestan.
The 4 most common variants in the GALNS gene, c.860C>T, c.319G>A, c.1042A>G, and c.29G>A were responsible for 18.9% (17/90) of the MPS IV patients. The c.319G>A variant was found in 4 patients (4.5%, 4/90) all from the East Azerbaijan province with Azeri ethnicity. This variant was present in 50% (4/8) of all the MPS IV patients from East Azerbaijan. The 4 families harboring the c.1042A>G variant (4.5%, 4/90) originated from Khorasan-Razavi (3/4) and Mazandaran (1/4). The c.29G>A variant originated from two central neighbor provinces in Iran, Chaharmahal and Bakhtiari, and Isfahan.