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