A feasible molecular diagnostic strategy for rare genetic disorders within resource-constrained environments

Abstract

Timely and accurate diagnosis of rare genetic disorders is critical, as it enables improved patient management and prognosis. In a resource-constrained environment such as the South African State healthcare system, the challenge is to design appropriate and cost-effective assays that will enable accurate genetic diagnostic services in patients of African ancestry across a broad disease spectrum. Next-generation sequencing (NGS) has transformed testing approaches for many Mendelian disorders, but this technology is still relatively new in our setting and requires cost-effective ways to implement. As a proof of concept, we describe a feasible diagnostic strategy for genetic disorders frequently seen in our genetics clinics (RASopathies, Cornelia de Lange syndrome, Treacher Collins syndrome, and CHARGE syndrome). The custom-designed targeted NGS gene panel enabled concurrent variant screening for these disorders. Samples were batched during sequencing and analyzed selectively based on the clinical phenotype. The strategy employed in the current study was cost-effective, with sequencing and analysis done at USD849.68 per sample and achieving an overall detection rate of 54.5%. The strategy employed is cost-effective as it allows batching of samples from patients with different diseases in a single run, an approach that can be utilized with rare and less frequently ordered molecular diagnostic tests. The subsequent selective analysis pipeline allowed for timeous reporting back of patients results. This is feasible with a reasonable yield and can be employed for the molecular diagnosis of a wide range of rare monogenic disorders in a resource-constrained environment.

Mutation analysis and clinical profile of South African patients with Neurofibromatosis type 1 (NF1) phenotype

“…The negative cohort did not have SpliceAI delta scores suggesting high confidence of pathogenicity. No clinically significant variants were identified in other RASopathy genes ( CBL, LZTR1, KRAS, NRAS, PTPN11, RAF1, SOS1, RIT1, SOS2, RASA1, BRAF, MAP2K1, MAP2K2, SPRED1, NF1, NF2, HRAS, SHOC2, RRAS, RASA2, A2ML1 ) that were included in the targeted panel ( Mudau et al, 2023 ). In the negative cohort, 3/5 (60%) patients, for whom the information was available, had a documented first degree relative with clinically-diagnosed NF1.…”

“…Agilent SureSelect QXT and IonTorrent Ion Ampliseq capture assays to target the exons and exon/intron boundaries of the NF1 (NM_001042492.3) , SPRED1 ( NM_152594.3 ). Other RASopathy genes were also sequenced ( Mudau et al, 2023 ), these included; A2ML1 (NM_144670.6), BRAF (NM_004333.4), CBL (NM_005188.4), HRAS (NM_005343.4), KRAS (NM_033360.4), LZTR1 (NM_006767.3), MAP2K1 (NM_002755.3), MAP2K2 (NM_030662.3), NF2 (NM_000268.4), NRAS (NM_002524.3), PTPN11 (NM_0002834.3), RAF1 (NM_002880.3), RASA1 (NM_002890.3), RASA2 (NM_006506.5), RIT1 (NM_006912.6), RRAS (NM_006270.5), SHOC2 (NM_007373.4), SOS1 (NM_005633.4), SOS2 (NM_006939.4). The two targeted gene panels utilised in the current study were custom designed using Agilent SureDesign Software (Agilent Technologies,United States) and ThermoFisher Ion AmpliSeq Designer (ThermoFisher Scientific, United States).…”

“…There is currently no information available in the literature about the molecular aetiology in South African patients of African ancestry with NF1. A study performed in our laboratory demonstrated that NGS targeted gene panels are a cost-effective strategy that can be employed for the molecular diagnosis of a wide range of monogenic disorders in a low resource setting ( Mudau et al, 2023 ). The current study aims to develop a molecular testing strategy that would identify the majority of NF1 variants and would be feasible for molecular diagnostic laboratories to implement, in low resource settings.…”