Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic die

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing.

BACKGROUND: Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. RESULTS: We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. CONCLUSION: We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing.

BACKGROUND: Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. RESULTS: We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. CONCLUSION: We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Abstract Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than 15 kbp-Spanning Coding Sequence of the SACS Gene

In this study, we developed and analytically validated a fully automated, robust confirmation sensitive capillary electrophoresis (CSCE) method to perform mutation scanning of the large SACS gene. This method facilitates a rapid and cost-effective molecular diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Critical issues addressed during the development of the CSCE system included the position of a DNA variant relative to the primers and the CG-content of the amplicons. The validation was performed in two phases; a retrospective analysis of 32 samples containing 41 different known DNA variants and a prospective analysis of 20 samples of patients clinically suspected of having autosomal recessive spastic ataxia of Charlevoix-Saguenay. These 20 samples appeared to contain 73 DNA variants. In total, in 32 out of the 45 amplicons, a DNA variant was present, which allowed verification of the detection capacity during the validation process. After optimization of the original design, the overall analytical sensitivity of CSCE for the SACS gene was 100%, and the analytical specificity of CSCE was 99.8%. In conclusion, CSCE is a robust technique with a high analytical sensitivity and specificity, and it can readily be used for mutation scanning of the large SACS gene. Furthermore this technique is less time-consuming and less expensive, as compared with standard automated sequencing

Future of Dutch NGS-Based Newborn Screening:Exploring the Technical Possibilities and Assessment of a Variant Classification Strategy

In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation.</p

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

BackgroundExome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques.ResultsWe compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70x there is only minimal loss in sensitivity for SNV and CNV detection.ConclusionWe conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Reanalysis of exome negative patients with rare disease: a pragmatic workflow for diagnostic applications

BACKGROUND: Approximately two third of patients with a rare genetic disease remain undiagnosed after exome sequencing (ES). As part of our post-test counseling procedures, patients without a conclusive diagnosis are advised to recontact their referring clinician to discuss new diagnostic opportunities in due time. We performed a systematic study of genetically undiagnosed patients 5 years after their initial negative ES report to determine the efficiency of diverse reanalysis strategies. METHODS: We revisited a cohort of 150 pediatric neurology patients originally enrolled at Radboud University Medical Center, of whom 103 initially remained genetically undiagnosed. We monitored uptake of physician-initiated routine clinical and/or genetic re-evaluation (ad hoc re-evaluation) and performed systematic reanalysis, including ES-based resequencing, of all genetically undiagnosed patients (systematic re-evaluation). RESULTS: Ad hoc re-evaluation was initiated for 45 of 103 patients and yielded 18 diagnoses (including 1 non-genetic). Subsequent systematic re-evaluation identified another 14 diagnoses, increasing the diagnostic yield in our cohort from 31% (47/150) to 53% (79/150). New genetic diagnoses were established by reclassification of previously identified variants (10%, 3/31), reanalysis with enhanced bioinformatic pipelines (19%, 6/31), improved coverage after resequencing (29%, 9/31), and new disease-gene associations (42%, 13/31). Crucially, our systematic study also showed that 11 of the 14 further conclusive genetic diagnoses were made in patients without a genetic diagnosis that did not recontact their referring clinician. CONCLUSIONS: We find that upon re-evaluation of undiagnosed patients, both reanalysis of existing ES data as well as resequencing strategies are needed to identify additional genetic diagnoses. Importantly, not all patients are routinely re-evaluated in clinical care, prolonging their diagnostic trajectory, unless systematic reanalysis is facilitated. We have translated our observations into considerations for systematic and ad hoc reanalysis in routine genetic care. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13073-022-01069-z