Methods

Patient Recruitment

Patients recruited in this study were part of a national project on “Iran MucoPolysaccharidosis REdiagnosis Study (IMPRESsion)”, which has been carried out through a collaboration between “Fetal Health Hope Generation Research Center” (FHHGRC) and “National Scientific Committee on Inborn Errors of Metabolism” (NSCIEM) from September 2018 to February 2020. Patients with the clinical and/or biochemical diagnosis of MPS who were registered by NSCIEM participated in this study. Patients were recalled for re-examination and blood sampling through 6 designated provincial centers located in the cities of Tehran, Tabriz, Mashhad, Ahvaz, Isfahan, and Shiraz. Families were assigned to the most accessible provincial center based on their place of residence.
Patients who were assigned to Tehran provincial centers were directly referred to FHHGRC. For these patients, pre-test and post-test genetic counseling was provided and medical photo and video documentation were carried out. Also, peripheral blood samples were drawn from the probands, their parents, affected/unaffected siblings, and other affected relatives, if available. For patients referred from centers other than Tehran, collected peripheral blood samples from the probands and their parents/siblings/other affected relatives coupled with their clinical data and signed consent forms were sent to the FHHGRC by mail. In these patients, history taking, and pedigree drawing was performed through phone contacts with families by genetic counselors.
The ethnicity and geographical origin of the probands and their parents (including the country, province, city, and village, if applicable) were recorded in detail. Probands or parents born in large cities were inquired about the geographical origin of their previous generations (e.g. grandfather, grandmother) to avoid misrepresentation of the data with regard to the fact that the proband’s birthplace could have been affected by immigration of the previous generations to the large cities. Written informed consents were obtained from the patients or their parents/guardians. The study was approved by the research center review board and all procedures were performed in accordance with the ethical standards of the local committee and the revised version of the Helsinki Declaration in 2013. Details of the pedigrees, clinical data and the results of biochemical tests or previous genetic tests were completely reviewed, summarized and tabulated by a genetic counselor. There were two groups of families based on their genetic testing status:
Families who had positive findings in their previous genetic tests (Group A): in these patients, the previously reported variants were re-analyzed according to the American College of Medical Genetics and Genomic (ACMG) guideline. Sanger verification and segregation analysis were then carried out if indicated.
Families with no previous genetic tests or no finding in their tests (Group B): these patients went through our comprehensive genetic approach to MPSs, starting from targeted NGS, followed by complementary Sanger sequencing of poorly covered regions, Sanger verification and/or segregation analysis. Details of the applied approach are discussed below.

DNA Preparation and Next-Generation Sequencing

Venous blood sample was used for DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Germany). Targeted capture NGS was performed using Ion Ampliseq Inborn Errors of Metabolism Research Panel V2 (Life Technologies). The capture panel consisted of 9681 amplicons (1.05 Mbp) covering coding regions and flanking exon/intron boundaries of 594 genes associated with inborn errors of metabolism with an average gene coverage of 99.17%. Amplicon length and average insert size were 254 bp and 207 bp respectively.
The library preparation for targeted capture was carried out according to the manufacturer’s protocol. Briefly, 100 ng of DNA quantified by Qubit DNA HS assay (Life Technologies) was used. The amplicons were then ligated to the barcode-labelled X adapter and the Ion Sphere Particle (ISP) compatible P adapter. The library was purified with AMPure XP Beads (Beckman Coulter, Pasadena, CA) and the final library concentration was measured using the Ion Library TaqMan® Quantitation Kit (Life Technologies). Following pooling equimolar libraries, automated template preparation and chip loading were carried out using Ion Chef System (Life Technologies). The Ion Proton Sequencing 200 kit (Life Technologies) was used for sequencing on a PI chip V2 following the manufacturer’s protocol.

Variant Identification