Molecular inversion probes: a low-cost and high throughput sequencing technology for rapid diagnosis of 23 common lysosomal storage diseases in India Ms. Aadhira Nair1, Dr. Jayesh Sheth1, Dr. Madhavi Joshi2, Dr. Chaitanya Joshi3, Dr. Harsh Sheth1 1- FRIGE’s Institute of Human Genetics, Ahmedabad, India. 2- Gujarat State Biotechnology Mission, Gandhinagar, India. 3- Gujarat Biotechnology Research Centre, Gandhinagar, India. ABSTRACT Lysosomal storage disorders (LSDs) constitute the majority of inborn errors of metabolism (IEM) cases in India, owing to its large population and a general practice of consanguineous marriages. With therapeutic options now available for several LSDs, early diagnosis is indispensable. A confirmed genetic diagnosis, in this case, can help in better disease management by providing information to perform prenatal diagnosis and provide genetic counselling for at-risk families. Here, we propose the use of an NGS based method using small molecule molecular inversion probes (smMIPs) to screen suspected patients for 23 common LSDs in India. We have designed 903 MIPs targeting the region of our interest and assessed its capture ability. We obtained the desired product after MIP capture and PCR amplification and also optimized the PCR cycle number using q-PCR to avoid PCR duplicates and data loss. The application of smMIP technology in LSD diagnosis is a first of its kind approach in India and we hope it shall address the LSD burden in India significantly.
INTRODUCTION • LSDs are multisystemic disorders having an underlying genetic cause, comprising over 70 conditions with a collective incidence of 1 in 5000 live births1. Figure 1 depicts the LSD statistics in world and in India.
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Disease
Incidence per 100,000 individuals
Fabry disease
2.5* Per 100,000 males
Gaucher disease
2.0
Metachromatic leukodystrophy
2.5
Pompe disease
2.5
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Figure 1: LSD statistics (A) Incidence of common LSDs in world. (B) Burden of LSDs in India2
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Q-PCR of the MIP captured samples performed in duplicates (Figure 3B). The amplification plot suggests PCR cycle no 21 to be chosen as the optimum cycle number for sufficient amplification of the product PCR at 21 cycles showed desired product ~269bp and primer dimers (Figure 3C).
FUTURE WORK
Figure 2: Schematic representation of molecular inversion probes laboratory procedure. (A) Target capture (B) Gap filling reaction (C) Exonuclease treatment (D) PCR and sequencing library molecules.
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B
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References 1. Platt FM et al, Nat Rev Dis Primers. 2018;4(1):27. 2. Sheth J et al, JIMD Rep. 2014;12:51-63 3. Evan A. et al, Bioinformatics, Volume 30, Issue 18, 2014, 2670– 2672 4. Hiatt JB et al, Genome Res. 2013;23(5):843-854.
Targeted MIP capture carried out for 3 DNA samples. The MIP PCR product analyzed on agarose gel. (Figure 3A) The product of interest corresponding to a size of ~269 bp was observed indicating successful target enrichment by probes. Blank showed presence of self circularized MIPs and no product of interest providing evidence of absence of any contamination.
• Result 2: Run 2- To optimize PCR cycles in order to reduce PCR duplicates and data loss. •
METHODS Literature study shows that there are 23 common LSDs in India2. Molecular inversion probes (MIPs) were designed for all exons and intron-exon boundaries of 23 LSD genes using Mipgen tool 3. All probes were pooled at equimolar concentration to create final pool. Validation cohort: this includes 200 LSD patients with a confirmed enzymatic and molecular diagnosis for a particular LSD. Application cohort: this includes 85 LSD patients with confirmed enzymatic diagnosis for LSD but unknown mutation status. DNA samples subjected to smMIP capture, purification and pooling as per the protocol of Hiatt et al. 20134 (Figure 2). SYBR green based Q-PCR performed for determining the optimum PCR cycle number for sufficient amplification. Sequencing of pooled libraries at an average depth of 200x per amplicon on MiSeq Illumina platform. Data analysis carried out using customized bioinformatics pipeline.
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• Recently, NGS is finding a place in routine diagnosis owing to its ability of assessing several genes simultaneously.
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• Result 1: Run 1- To test the target enrichment ability of probes
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• The general diagnostic route involves iterative rounds of enzyme testing for particular disease, which is costly and time-consuming and can lead to delayed diagnosis of up to 5 years.
• We aim to develop a low cost, high throughput NGS based diagnostic assay for LSDs using small molecule molecular inversion probes (smMIPs).
RESULTS • A total of 903 smMIPs targeting the exons and exon-intron boundaries of 23 LSD genes have been designed using Mipgen tool.
Figure 3: Results of the first target enrichment using smMIPs protocol. (A) Agarose gel electrophoresis image of MIP-PCR product of DNA samples (lane 1-3), blank (lane 4) at 35 cycles (B) Q-PCR amplification plot of the MIP-PCR products. (C) Gel image of MIP-PCR product of DNA samples (lane 1-3), blank (lane 4) at 21 cycles.
• Sequencing of the pooled libraries to assess the distribution of smMIPs. • Evaluation of the accuracy of our assay in variant detection in LSD patients with prior known mutation status. • Assessing the diagnostic yield of our assay in detecting underlying genetic mutations in enzymatically confirmed LSD patients. • Evaluation of the cost effectiveness of our assay in diagnosis of LSDs as compared to the current enzymatic approach used.
CONCLUSION • The assay would serve cost-effective and as a one stop assay to screen suspected patients for the 23 common LSDs. • The timely and correct diagnosis would lead to early therapeutic intervention like ERT, BMT etc. in affected individuals thereby aiding in LSD management. • The assay could be used in future for rapid carrier detection, prenatal and new-born screening in at-risk families.
Acknowledgement The work is being carried out FRIGE’s Institute of Human Genetics and is funded by the Gujarat State Biotechnology Mission (GSBTM/JDR&D/608/2020/459-461). Presenter: Ms. Aadhira Nair FRIGE’s Institute of Human Genetics, Ahmedabad, India. Contact: aadhira.nair@frige.co.in