Long-range PCR in next generation sequencing: A low cost approach for large & complex genes

The article by Vanaja et al1 published in this issue of the Indian J Med Res addresses an upcoming molecular detection strategy for lysosomal storage disorders (LSDs). Though individually rare, collectively LSDs affect approximately 1 in every 5000-8000 births worldwide2. A specific diagnosis of LSDs has always been a challenge for clinicians due to the clinical variability, and lack of a specific phenotype3. There are numerous pathways which are involved in lysosomal biogenesis, and at various stages, changes can lead to adverse metabolic effects and manifestation of different subtypes of the same disorder.

The diagnosis depends primarily on enzyme assays and preferably on molecular characterization of pathogenic variations. Different molecular techniques are available, starting from simple amplification-refractory mutation system PCR, PCR-RFLP (restriction fragment length polymorphism), Sanger sequencing, next generation sequencing (NGS) based tests and multiplex ligation-dependent probe amplification. All these except NGS are meant for targeted testing either designed for specific variations or specific genes. These methods are useful for testing hotspot mutations or if the targeted genes are small. In addition, all these techniques have many variable efficiencies in amplifying the target regions, and require multiple optimization processes, especially those having GC-rich areas. Currently, mutations in over 50 genes are known which cause various LSDs. Direct sequencing is considered the method of choice for mutation detection in several laboratories till now; however, this method again requires multiple steps of PCR, has limitation pertaining to the size of amplicons primarily to coding regions and is time-consuming and finally turns out to be expensive4.

Nowadays, NGS is primarily utilized as an instrumental technology for identifying single gene defects with a comprehensive approach in undiagnosed patients with early-onset symptoms and atypical cases. It can be used either for ‘targeted’ sequencing of selected gene panels or for ‘untargeted’ approaches based on exome or genome analysis. Hence, NGS based detection of LSDs has become a valuable alternative to other well-established biochemical assays having an advantage of monitoring a broader spectrum of diseases in a single test. Targeted gene panel assesses multiple genes of interest in parallel, saving time and cost of running multiple separate assays. Alexander Gheldof et al5, in 2018, developed a LSD panel comprising of 50 genes specific for LSD, which increased the diagnostic yield. Currently, there are many LSD gene panels available commercially.

In 1992, Barnes6 developed new PCR conditions that allowed amplification of upto 5 kb fragmant6. Long range (LR)-PCRs can amplify amplicons sizes between 3-5 kb to over 30 kb by modifying the polymerases which facilitated genomic mapping and sequencing. When LR-PCR was combined with sequencing, it achieved a higher sensitivity in a faster and more cost-effective way for detecting genetic variations over normal PCR methods. Over the years, LR-PCR has proved to be a cost-effective option for sequencing candidate genomic regions, particularly combination with NGS platforms. One of the major benefits of LR-PCR is, having the option of designing primers taking into account the intronic regions as far as 200-300 nucleotides away from the intron-exon junctions which is not possible in direct sequencing method and if larger genomic rearrangements involving more than one exon are involved it often get missed.

Literature review shows that researchers have used LR-PCR for NGS based studies for large/multiple genes like for autosomal dominant polycystic kidney disease, inherited retinal disease and for genotyping of autoinflammatory disorders and all these studies have opined that it is a flexible, versatile and a cost-effective technique7-10. Limitations include inability to detect deletions spanning primer-binding regions which in turn cause allele dropout and the assay would not detect any copy number variation (CNV) and the affected regions would show only homozygous-appearing variants.

In a nutshell, the authors have used a comprehensive method which can generate more information than traditional sequencing and offers an lucrative choice for testing the entire group of genes implicated in LSDs at one go. Such a strategy would be helpful for screening several large complex genes such as tuberous sclerosis, cystic fibrosis and neurofibromatosis I.