Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed nextgeneration sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gainof-function alleles in Ras-like without CAAX 1 (RIT1) and mitogenactivated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that nextgeneration sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.human genetics | developmental diseases | whole exome sequencing | PTPN11 | RAS

The identification of mosaicism is important in establishing a disease diagnosis, assessing recurrence risk, and genetic counseling. Next-generation sequencing (NGS) with deep sequence coverage enhances sensitivity and allows for accurate quantification of the level of mosaicism. NGS identifies low-level mosaicism that would be undetectable by conventional Sanger sequencing. A customized DNA probe library was used for capturing targeted genes, followed by deep NGS analysis. The mean coverage depth per base was approximately 800×. The NGS sequence data were analyzed for single-nucleotide variants and copy number variations. Mosaic mutations in 10 cases/families were detected and confirmed by NGS analysis. Mosaicism was identified for autosomal dominant (JAG1, COL3A1), autosomal recessive (PYGM), and X-linked (PHKA2, PDHA1, OTC, and SLC6A8) disorders. The mosaicism was identified either in one or more tissues from the probands or in a parent of an affected child. When analyzing data from patients with unusual testing results or inheritance patterns, it is important to further evaluate the possibility of mosaicism. Deep NGS analysis not only provides insights into the spectrum of mosaic mutations but also underlines the importance of the detection of mosaicism as an integral part of clinical molecular diagnosis and genetic counseling.

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Summary Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of β-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.

Purpose: Next-generation sequencing (NGS) has been widely applied to clinical diagnosis. Target-gene capture followed by deep sequencing provides unbiased enrichment of the target sequences, which not only accurately detects single-nucleotide variations (SNVs) and small insertion/deletions (indels) but also provides the opportunity for the identification of exonic copy-number variants (CNVs) and large genomic rearrangements. Method:Capture NGS has the ability to easily detect SNVs and small indels. However, genomic changes involving exonic deletions/ duplications and chromosomal rearrangements require more careful analysis of captured NGS data. Misaligned raw sequence reads may be more than just bad data. Some mutations that are difficult to detect are filtered by the preset analytical parameters. "Loose" filtering and alignment conditions were used for thorough analysis of the misaligned NGS reads. Additionally, using an in-house algorithm, NGS coverage depth was thoroughly analyzed to detect CNVs. Results:Using real examples, this report underscores the importance of the accessibility to raw sequence data and manual review of suspicious sequence regions to avoid false-negative results in the clinical application of NGS. Assessment of the NGS raw data generated by the use of loose filtering parameters identified several sequence aberrations, including large indels and genomic rearrangements. Furthermore, NGS coverage depth analysis identified homozygous and heterozygous deletions involving single or multiple exons. Conclusion:Our results demonstrate the power of deep NGS in the simultaneous detection of point mutations and intragenic exonic deletion in one comprehensive step.

Balamuthia amoebic encephalitis has a subacute-to-chronic course and is almost invariably fatal owing to delayed diagnosis and a lack of effective therapy. Here, we report a 13-year-old girl with cutaneous lesions and multifocal granulomatous encephalitis. The patient underwent a series of tests and was suspected as having tuberculosis. She was treated with various empiric therapies without improvement. She was finally correctly diagnosed via nextgeneration sequencing of the cerebrospinal fluid. The patient deteriorated rapidly and died 2 months after being diagnosed with Balamuthia mandrillaris encephalitis. This study highlights the important clinical significance of nextgeneration sequencing, which provides better diagnostic testing for unexplained paediatric encephalitis, especially that caused by rare or emerging pathogens.

Triple-negative breast cancer (TNBC) is an operational term for breast cancers lacking targetable estrogen receptor expression and HER2 amplifications. TNBC is, therefore, inherently heterogeneous, and is associated with worse prognosis, greater rates of metastasis, and earlier onset. TNBC displays mutational and transcriptional diversity, and distinct mRNA transcriptional subtypes exhibiting unique biology. High-throughput sequencing has extended cancer research far beyond protein coding regions that include non-coding small RNAs, such as miRNA, isomiR, tRNA, snoRNAs, snRNA, yRNA, 7SL, and 7SK. In this study, we performed small RNA profiling of 26 TNBC cell lines, and compared the abundance of non-coding RNAs among the transcriptional subtypes of triple negative breast cancer. We also examined their co-expression pattern with corresponding mRNAs. This study provides a detailed description of small RNA expression in triple-negative breast cancer cell lines that can aid in the development of future biomarker and novel targeted therapies.

Background: Paxlovid is recognized as an effective medication in preventing the progression of coronavirus disease of 2019 (COVID-19) to severe form in adults; however, its efficacy has remained unknown in pediatric cases. This study aimed to analyze the feasibility, safety, and efficacy of Paxlovid treatment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children aged 6-14 years. Methods: We conducted a cohort study based on prospectively collected clinical data. We recruited 5 pediatric cases with underlying diseases treated with Paxlovid from 7 April 2022 to 26 May 2022 and 30 age-matched patients with underlying diseases who were not treated with Paxlovid as controls. The safety and efficacy of Paxlovid were primarily assessed by inter-group comparisons. Results: Of the 5 Paxlovid-treated cases, including 1 male and 4 females, 3 and 2 cases were mildly and moderately ill, respectively. The underlying diseases included congenital heart defects, cerebral palsy, Down syndrome, and leukemia. Only 1 patient had received 1 dose of an inactivated SARS-CoV-2 vaccine. Paxlovid was initiated within 5 days after the onset of symptoms in all cases. Comedications were used in 2 cases. In the safety analyses, after Paxlovid initiation, 1 patient had transient diarrhea, and 1 patient had transiently elevated liver enzymes [alanine transaminase (ALT), 125 U/L; aspartate transaminase (AST), 83 U/L; normal range, <40 U/L]. In the efficacy analyses, all 5 Paxlovid-treated cases recovered, with the respective viral shedding times of 11, 4, 10, 9, and 9 days. Compared with age-matched controls, the viral shedding times were not significantly different between groups.Conclusions: Based on the current small sample size study, Paxlovid is a feasible option for treating SARS-CoV-2-infected children aged 6-14 years with underlying diseases. However, the safety and efficacy of Paxlovid warrant further large-scale studies.