In patients with lysosomal storage disorders (LSDs) such as lysosomal acid lipase deficiency (LAL-D), using long-range polymerase chain reaction (LR-PCR) amplification for assessment purposes has been shown to be cost-effective, reliable, and easily customizable, according to findings from a molecular study published in the Indian Journal of Medical Research.

LSDs comprise a group of genetic metabolic disorders that are caused by a deficiency in lysosomal enzymes or defects in other lysosomal components. When lysosomal enzyme assays are not available or not practical to perform, molecular genetic testing is necessary for diagnostic validation. In fact, an accurate diagnosis in such situations is known to be “essential for the proband, as well as for the family, for appropriate management, prognostication, surveillance, genetic counseling, and prenatal/pre-implantation genetic testing.”

The researchers of the current study sought to design and validate a cost-effective, scalable, readily customizable molecular genetic testing approach for LSDs, including LAL-D. Their strategy involved the use of LR-PCR, which was based on targeted enrichment, followed by next-generation sequencing (NGS) for molecular genetic assessment of LSDs.

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The study was conducted between November 2014 and November 2017 at the Diagnostics Division of the Centre for DNA Fingerprinting and Diagnostics, located in Hyderabad, Telangana, India. Across all age-groups, participants were diagnosed with an LSD based on clinical features and imaging studies. In individuals with suspected LSDs, their specific diagnosis was verified by molecular genetic testing, an enzyme assay, or findings from their unaffected carrier parents.

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In all participants, peripheral blood samples were obtained. The study samples were divided into 2 groups:

  • Group A: affected individuals and/or parents who had previously undergone molecular genetic testing, via Sanger sequencing, in whom the disease responsible for the genetic variants had already been identified in 7 genes
  • Group B: patients with a biopsy-proven or an enzymatically confirmed diagnosis of a specific LSD and/or their unaffected carrier parents, who had not yet undergone molecular genetic testing

In Group A, 28 DNA samples of affected probands and/or carrier parents were used for the first phase of the study, which comprised validation and standardization of the study method. Thirty DNA samples from Group B were used in the second phase of the study. Samples of probands and carrier parents were obtained in an effort to confirm the utility of the technique for detecting homozygous and heterozygous variants.

The study demonstrated the ability to apply and validate a strategy that was based on the in-house development of discriminatory amplicons via LR-PCR amplification for the targeted capture of LSD genes of interest, which was followed by NGS of pooled samples. The 2 key advantages of using this technique of amplicon libraries are its significant reduction in costs and its ability to easily customize the genes of interest compared with the use of commercially available targeted enrichment kits.

“Overall, LR PCR-based targeted gene enrichment combined with NGS appears to be a reliable, clinically practical, easily customizable, scalable and cost-effective approach for mutation analysis of LSDs such as LAL-D and can be used even in resource-poor settings,”  the researchers concluded.


Vanaja MC, Jain JMN, Dalal A, Ranganath P. Long-range PCR amplification-based targeted enrichment & next generation sequencing: a cost-effective testing strategy for lysosomal storage disorders. Indian J Med Res. Published online July 7, 2023. doi:10.4103/ijmr.IJMR_2707_20