Targeted Next-Generation Sequencing

Library Preparation and Sequencing

• The library was quantified using the Qubit dsDNA HS analysis kit on a Qubit 2.0 fluorometer.
• Emulsion PCR was performed on the OneTouch2 system using the Ion PGM HI-Q OT2 kit.
• Template enrichment was done using Ion OneTouch ES, and Ion 318 Chip v2 was prepared for loading.

Targeted Next-Generation Sequencing

• Libraries were prepared using Ion AmpliSeq Library Kit v2.0 for the patient and two other patients affected by hereditary diseases.
• The panel contains three primer pools, and amplification was performed using the Ion Ampliseq HiFi Mix.

RNA Extraction and RT-PCR

• Total RNA was isolated from leukocytes using the RNeasy Mini Kit.
• cDNA was performed using Superscript III Reverse transcriptase.
• cDNA amplification of the CTNS gene was performed to confirm the deletion using specific primers.

Sanger Sequencing

• The reaction products were analyzed by 1% agarose gel electrophoresis.
• Sanger sequencing was done using dye terminator chemistry and run on an automated sequencer.

Data Availability and Ethics Approval

• The datasets used and/or analyzed during the study are available from the corresponding author upon request.
• Written informed consent for molecular genetic testing was obtained from the patient and the future couple.
• The study was approved by The Ethics Committee of the National Institute of Health in Rabat, Morocco.

Cystinosis

• Cystinosis is a rare autosomal recessive lysosomal storage disorder described in 1903 as a familial cystine accumulation.
• It's a systemic disease, and the kidney involvement is the most serious clinical event.
• In more than 90% of patients, it drives them to end-stage renal disease before the age of 20.

Genetic Counseling

• Genetic counseling and targeted parental carrier testing, prenatal, and preimplantation genetic diagnosis in subsequent pregnancies, and targeted premarital genetic testing in at-risk couples are recommended.
• This is because the couple has a family history of a severe form of cystinosis.

CTNS Gene

• The CTNS gene is associated with cystinosis.
• The Ion AmpliSeq Inherited Disease Panel can provide a high degree of multiplexed target selection of all exons and some gene introns associated with more than 700 unique genetic diseases.
• The sequence data analysis detected a potential deletion spanning exons 4 and 5 in the CTNS gene.

Case Study

• The patient, a 22-year-old girl, was diagnosed with cystinosis in early childhood.
• She has a severe form of cystinosis, which progressed towards renal failure at the age of 6 years old.
• Her sister's husband and the couple (V-1 and V-2; Fig. 1) came to the center for genetic counseling.

Molecular Studies

• The authors used Invitrogen kit to extract genomic DNA from peripheral blood lymphocytes.
• The Qubit dsDNA HS Assay Kit was used to quantify DNA with Qubit Fluorometer.
• The sequence data analysis detected a potential deletion spanning exons 4 and 5 in the CTNS gene.
• The candidate variants were obtained when variant frequency at a given position is ≥20% and variant coverage is ≥20×.