The clinical translation of next-generation sequencing has created a paradigm shift in the diagnostic assessment of individuals with suspected rare genetic diseases. Whole-exome sequencing (WES) simultaneously examines the majority of the coding portion of the genome and is rapidly becoming accepted as an efficient alternative to clinical Sanger sequencing for diagnosing genetically heterogeneous disorders. Among reports of the clinical and diagnostic utility of WES, few studies to date have directly compared its concordance to Sanger sequencing, which is considered the clinical "gold standard". We performed a direct comparison of 391 coding and noncoding polymorphisms and variants of unknown significance identified by clinical Sanger sequencing to the WES results of 26 patients. Of the 150 well-covered coding variants identified by Sanger sequencing, 146 (97.3%) were also reported by WES. Nine genes were excluded from the comparison due to consistently low coverage in WES, which might be attributed to the use of older exome capture kits. We performed confirmatory Sanger sequencing of discordant variants; including five variants with discordant bases and four with discordant zygosity. Confirmatory Sanger sequencing supported the original Sanger report for three of the five discordant bases, one was shown to be a false positive supporting the WES data, and one result differed from both the Sanger and WES data. Two of the discordant zygosity results supported Sanger and the other two supported WES data. We report high concordance for well-covered coding variants, supporting the use of WES as a screening tool for heterogeneous disorders, and recommend the use of supplementary Sanger sequencing for poorly-covered genes when the clinical suspicion is high. Importantly, despite remaining difficulties with achieving complete coverage of the whole exome, 10 (38.5%) of the 26 compared patients were diagnosed through WES.
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http://dx.doi.org/10.1002/mgg3.223 | DOI Listing |
Med Mycol
December 2024
UR 3738 - CICLY - Equipe Inflammation et immunité de l'épithélium respiratoire, Faculté de Médecine Lyon-Sud Charles Mérieux, Université Claude Bernard Lyon 1, Lyon, France.
Cryptococcus neoformans/gattii and Histoplasma capsulatum var. capsulatum may present atypical histopathological features inducing diagnostic errors. We aimed to estimate the frequency of these atypical features on formalin-fixed tissue samples (FT) and to assess the relevance of an integrated histomolecular diagnosis using specific Histoplasma capsulatum PCR and panfungal PCR followed by Sanger sequencing and/or targeted-massive parallel sequencing (MPS).
View Article and Find Full Text PDFZh Nevrol Psikhiatr Im S S Korsakova
December 2024
Bochkov Research Centre for Medical Genetics, Moscow, Russia.
A fifth world case of autosomal recessive Siddiqi syndrome (SIDDIS) related to ene is presented. In a consanguineous Lezgin (a Dagestan ethnicity) family, there were two affected brothers aged 28 yrs (proband, personally examined) and 32 yrs. Whole-exome sequencing followed by familial Sanger sequencing detected a novel missence variant c.
View Article and Find Full Text PDFEur J Neurol
January 2025
School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
Background: The regulatory role of the apolipoprotein E (APOE) ε4 allele in the clinical manifestations of spinocerebellar ataxia type 3 (SCA3) remains unclear. This study aimed to evaluate the impact of the APOE ε4 allele on cognitive and motor functions in SCA3 patients.
Methods: This study included 281 unrelated SCA3 patients and 182 controls.
Eur J Neurol
January 2025
Service de Génétique Médicale, CHU Bordeaux, Bordeaux, France.
Purpose: Heterozygous pathogenic variants in SPAST are known to cause Hereditary Spastic Paraplegia 4 (SPG4), the most common form of HSP, characterized by progressive bilateral lower limbs spasticity with frequent sphincter disorders. However, there are very few descriptions in the literature of patients carrying biallelic variants in SPAST.
Methods: Targeted Sanger sequencing, panel sequencing and exome sequencing were used to identify the genetic causes in 9 patients from 6 unrelated families with symptoms of HSP or infantile neurodegenerative disorder.
Biotechniques
December 2024
Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, Colombia.
In 2006, a PCR method was introduced to subtype by Sanger sequencing of an ≈610 bp amplicon of the 18S rRNA gene. This method, known as barcoding-PCR, has become widespread, although the primer pair used can amplify non- sequences, which can result in false positives. Barcoding-PCR is most effective with DNA extracted from cultures, limiting its sensitivity when used directly with stool samples.
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