Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss of function mutations in either TSC1 or TSC2. Genotype and phenotype analyses are conducted worldwide, but there have been few large-scale studies on Japanese patients, and there are still many unclear points. This study analyzed 283 Japanese patients with TSC (225 definite, 53 possible, and 5 genetic diagnoses). A total of 200 mutations (64 TSC1, 136 TSC2) were identified, of which 17 were mosaic mutations, 11 were large intragenic deletions, and four were splicing abnormalities due to deep intronic mutations. Several lesions and symptoms differed in prevalence and severity between TSC1 and TSC2 patients and were generally more severe in TSC2 patients. Moreover, TSC2 missense and in-frame mutations may attenuate skin and renal symptoms compared to other TSC2 mutations. Genetic testing revealed that approximately 20% of parents of a proband had mild TSC, which could have been missed. The patient demographics presented in this study revealed a high frequency of TSC1 patients and a low prevalence of epilepsy compared to global statistics. More patients with mild neuropsychiatric phenotypes were diagnosed in Japan, seemingly due to a higher utilization of brain imaging, and suggesting the possibility that a significant amount of mild TSC patients may not be correctly diagnosed worldwide.

Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.

Alternative splicing is a regulated process by which eukaryotic genes may produce diverse biological products. Defects in the process typically affect cellular function and can lead to disease. Next-generation sequencing (NGS) technologies have been developed to detect alternative splicing events; however, the alternative splicing events detected by standard RNA-Seq may or may not be derived from full-length RNA. The SMARTer method provides full-length double-strand cDNA synthesis, and the resulting gene expression patterns correlate strongly with standard RNA-Seq. However, it also yields non-specific genomic DNA amplification. We improved the SMARTer method by employing a target-capture full-length double-strand cDNA sequencing method. High-fidelity, full-length cDNA is generated by the SMARTer method, followed by target-specific capture with exon probes. The expression pattern observed with this SMARTer Capture method was highly correlated with the results of the original SMARTer method. The number and accuracy of the detected splicing events were increased by eliminating non-specific genomic DNA amplification by the SMARTer Capture. Compared to the original SMARTer method, the SMARTer Capture provided 4-fold greater detection of alternative splicing events at the same read number, and it took less than 1/100 of read number to detect the same number of splicing events. The percent splicing in index (PSI) of the SMARTer Capture is highly correlated with the PSI of the SMARTer. These results indicate that the SMARTer Capture represents an improvement of the SMARTer method to accurately characterize alternative splicing repertories in targeted genes without biases.

Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.