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BACKGROUND: The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. METHOD: We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. RESULTS: ECNano achieved deep on-target depth of coverage (DoC) at average > 100× and > 98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30× DoC. ECNano obtained an average read length of 1000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30× DoC. Clair-ensemble achieved > 99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days. CONCLUSION: We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.

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BACKGROUND: Combined oxidative phosphorylation deficiency 26 (COXPD26) is an autosomal recessive disorder characterized by early onset, developmental delay, gastrointestinal dysfunction, shortness of breath, exercise intolerance, hypotonia and muscle weakness, neuropathy, and spastic diplegia. This disease is considered to be caused by compound heterozygous mutations in the TRMT5 gene. CASE PRESENTATION: In this study, we report a female child with COXPD26 manifesting as shortness of breath, gastrointestinal dysmotility, severe developmental delay, muscle hypotonia and weakness, exercise intolerance, renal and hepatic defects, and recurrent seizures with spastic diplegia. Interestingly, the hepatic feature was first observed in a COXPD26 patient. Medical exome sequencing with high coverage depth was employed to identify potential genetic variants in the patient. Novel compound heterozygous mutations of the TRMT5 gene were detected, which were c.881A>C (p.E294A) from her mother and c.1218G>C (p.Q406H) and c.1481C>T (p.T494M) from her father. CONCLUSION: The newly emerged clinical features and mutations of this patient provide useful information for further exploration of genotype-phenotype correlations in COXPD26.

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BACKGROUND: The DYNC1H1 gene encodes a part of the dynamic protein, and the protein mutations may further affect the growth and development of neurons, resulting in degeneration of anterior horn cells of the spinal cord, and a variety of clinical phenotypes finally resulting in axonal Charcot-Marie-Tooth disease type 20 (CMT20), mental retardation 13 (MRD13) and spinal muscular atrophy with lower extremity predominant 1 (SMA-LED). The incidence of the disease is low, and it is difficult to diagnose, especially in children. Here, we report a case of DYNC1H1 gene mutation and review the related literature to improve the pediatrician's understanding of DYNC1H1 gene-related disease to make an early correct diagnosis and provide better services for children. CASE SUMMARY: A 4-mo-old Chinese female child with adducted thumbs, high arch feet, and epileptic seizure presented slow response, delayed development, and low limb muscle strength. Electroencephalogram showed abnormal waves, a large number of multifocal sharp waves, sharp slow waves, and multiple spasms with a series of attacks. High-throughput sequencing and Sanger sequencing identified a heterozygous mutation, c.5885G>A (p.R1962H), in the DYNC1H1 gene (NM_001376) of the proband, which was not identified in her parents. Combined with the clinical manifestations and pedigree of this family, this mutation is likely pathogenic based on the American Academy of Medical Genetics and Genomics guidelines. The child was followed when she was 1 year and 2 mo old. The magnetic resonance imaging result was consistent with the findings of white matter myelinated dysplasia and congenital giant gyrus. The extensive neurogenic damage to the extremities was considered, as the results of electromyography showed that the motor conduction velocity and sensory conduction of the nerves of the extremities were not abnormal, and the degree of fit of the children with severe contraction was poor. At present, the child is 80 cm in length and 9 kg in weight, with slender limbs and low muscle strength, and still does not raise her head. She cannot sit or speak. Speech, motor, and mental development was significantly delayed. There is still no effective treatment for this disease. CONCLUSION: We herein report a de novo variant of DYNC1H1 gene, c.5885G>A (p.R1962H), leading to overlapping phenotypes (seizure, general growth retardation, and muscle weakness) of CMT20, MRD13, and SMA-LED, but there is no effective treatment for such condition. Our case enriches the DYNC1H1 gene mutation spectrum and provides an important basis for clinical diagnosis and treatment and genetic counseling.

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BACKGROUND: Fetal central nervous system abnormalities often associated with infant death or severe disability. The etiology in fetuses with CNS abnormalities who have normal karyotypes and copy number variants (CNVs) remains unclear, which increases the difficulty in following management and the assessment of prognosis. METHOD: 11 unrelated fetuses with CNS abnormalities and their parents were enrolled. Genomic DNA was obtained and then trios-medical exome sequencing (trios-MES) including 4000 genes (fetuses and their parents) was performed after both karyotyping and chromosome microarray showed negative results. RESULTS: Pathogenic and likely pathogenic variants were identified in five of 11 cases (5/11, 45.5%), including five novel mutations and two recurrent mutations in ISPD, L1CAM, and GRIN2B genes. Most cases (4/5, 80%) carried one or two recessive mutations, indicating a high recurrent risk. CONCLUSION: Exome sequencing should be considered for fetuses with CNS abnormalities following negative results of karyotyping and chromosome array. Trios-MES as one of exome sequencing is a potential method for the diagnosis of these fetuses.

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OBJECTIVE: To evaluate the clinical application of medical exome sequencing (MES) for prenatal diagnosis of genetic diseases related to fetal structural anomalies detected by prenatal ultrasound examination. STUDY DESIGN: A total of 105 fetuses with structural anomalies were negative results in both Quantitative fluorescent polymerase chain reaction (QF-PCR) and chromosomal microarray analysis (CMA). Then trio-based MES was further used for identifying the potential monogenic diseases in these fetuses. Coding regions and known pathogenic non-coding regions of over 4000 disease-related genes were interrogated, and variants were classified following the guidelines of American College of Medical Genetics (ACMG). RESULTS: The 105 fetuses with structural anomalies were categorized into 12 phenotypic groups. A definitive diagnosis was achieved in 19% (20/105) of the cases, with the identification of 21 pathogenic or likely pathogenic variants in 14 genes. The proportion of patients with diagnostic genetic variants varied between the phenotypic groups, with the highest diagnostic yield in the cardiovascular abnormalities (44%), followed by the skeletal and limb abnormalities (38%) and brain structural abnormalities (25%). In addition, 12 fetuses were detected variants of unknown significance (VOUS), while the relevance of phenotypes and variants would further evaluated. CONCLUSION: MES can identify the underlying genetic cause in fetal structural anomalies. It can further assist the management of pregnancy and genetic counseling. It was demonstrated the importance of translating prenatal MES into clinical practice.

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BACKGROUND: Intellectual disability (ID) is a heterogeneous neurodevelopmental disorder with a complex genetic underpinning in its etiology. Chromosome microarray (CMA) is recommended as the first-tier diagnostic test for ID due to high detection rate of copy number variation (CNV). METHODS: To identify an appropriate clinical detection scheme for ID in Han Chinese patients, whole genome low-coverage sequencing was performed as the first-tier diagnostic test, and medical exome sequencing (MES) as the second-tier diagnostic test for patients with negative results of CNVs. RESULTS: A total of 19 pathogenic CNVs in 16/95(16.84%) ID patients and 10 pathogenic single-nucleotide variations (SNVs), including 6 novel mutations in 8/95(8.42%) ID patients were identified on whom no pathogenic CNVs were discovered. The detection rate of CNVs in ID with multiple congenital anomalies (MCA) subgroup was significantly higher than ID with autism spectrum disorders and other IDs subgroups. And the single-nucleotide variations showed a higher occurrence rate in the other IDs subgroup. CONCLUSIONS: There were differences in the diagnostic yields of different variation types among the three ID subgroups. Our findings provided a new perspective on appropriate clinical detection scheme in different ID subgroups based on statistically significant differences among the three ID subgroups. The application of whole genome low-coverage sequencing as the first-tier diagnostic test for ID with MCA subgroup and MES as the first-tier diagnostic test for other ID subgroup was considered as an efficient clinical detection scheme.

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OBJECTIVE: The L1 cell adhesion molecule (L1CAM) gene, encodes the L1 cell adhesion molecule, is involved in the central nervous system development. Its mutations result in L1 syndrome which is associated with brain malformation and nervous developmental delay. CASE REPORT: We presented three fetuses with hydrocephalus and agenesis of the corpus callosum detected by ultrasound, followed by medical exome sequencing (MES) test with L1CAM mutations: two known missense mutation c.551G > A (p. R184Q) and c.1354G > A (p. G452R), and a novel frameshift mutation c.1322delG which causes the early termination of translation (p. G441Afs∗72). By utilizing multiple computational analysis, all the variants were scored to be likely pathogenic. CONCLUSION: Combined use of ultrasound and MES to identify the molecular etiology of fetal anomalies may contribute to expanding our knowledge of the clinical phenotype of L1 syndrome observed in the south Chinese population.

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BACKGROUND: Centronuclear myopathy (CNM), a subtype of congenital myopathy (CM), is a group of clinical and genetically heterogeneous muscle disorders. Centronuclear myopathy is a kind of disease difficult to diagnose due to its genetic diversity. Since the discovery of the SPEG gene and disease-causing variants, only a few additional patients have been reported. METHODS: A radiograph test, ultrasonic test, and biochemical tests were applied to clinical diagnosis of CNM. We performed trio medical exome sequencing of the family and conservation analysis to identify variants. RESULTS: We report a pair of severe CNM twins with the same novel homozygous SPEG variant c. 8710A>G (p.Thr2904Ala) identified by clinical trio medical exome sequencing of the family and conservation analysis. The twins showed clinical symptoms of facial weakness, hypotonia, arthrogryposis, strephenopodia, patent ductus arteriosus, and pulmonary arterial hypertension. CONCLUSIONS: Our report expands the clinical and molecular repertoire of CNM and enriches the variant spectrum of the SPEG gene in the Chinese population and helps us further understand the pathogenesis of CNM.

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INTRODUCTION: A range of cerebrocortical development malformations (MCD) ranging from simplified gyral patterns to the complete loss of gyri and sulci is associated with mutations in a cluster of highly homolog ß-tublin genes, such as TUBB2A and TUBB2B. CASE REPORT: The fetus had pachygyria, asymmetrical perisylvian polymicrogyria, dysplasia of the lateral sulcus and insula, agenesis of the splenium and partial agenesis of the body corpus callosum, cerebellar superior vermian hypoplasia with agenesis of the inferior vermis. Karyotype and microarray were normal. Trio Medical Exome Sequencing detected a de novo novel heterozygous mutation c.862G > A (p.E288K) in the tubulinpathy genes. Long-range PCR and Sanger sequencing specific for TUBB2A and TUBB2B gene detected a heterozygous variant c.862G > A specific to TUBB2B. CONCLUSION: The combination of LR-PCR amplification and medical exome sequencing allows mutational assessment in tubulinopathy genes. Our study expands the spectrum of malformations associated with mutations in the ß-tubulin gene TUBB2B.

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