平成23.24年度「独創性のある生命科学研究」個別研究課題 1）先天性甲状腺機能低下症の分子遺伝学的基盤の解明

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High prevalence of DUOX2 mutations in Japanese patients with thyroid dyshormonogenesis and transient hypothyroidism

We used next generation sequencing (NGS) to analyse the 16S rRNA bacterial gene amplicons based on the V3-V4 region and the ITS2 regions from fungi and plants derived from honeybee samples. Amplicon libraries, were prepared using the 16S Metagenomic Sequencing Library Preparation, Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System (Illumina®) protocol. NGS raw data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA686953. The presented data can be used to compare the metagenomics samples from different honeybee population all over the world. Higher load of fungi, and bacteria groups as: Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and unassigned other bacteria was observed for Nosema cearana and neogregarines infected honeybees. Healthy honeybees had higher load of plant pollens, and bacteria groups as: γ-proteobacteria, Orbales, Gilliamella, Snodgrassella, γ-proteobacteria, Enterobacteriaceae. More details you can find in research article [1] Ptaszyńska et al. 2021. Datasets: Excel 1. In the excel file are the row original information form NGS of composition of bacteria from 16S_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September). Excel 2. In the excel file are the row original information form NGS of composition of fungi and plant pollen from ITS_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September). Excel 3. In the excel file are the row original information form NGS of composition of bacteria from 16S_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples. Excel 4. In the excel file are the row original information form NGS of composition of bacteria from ITS_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples. Dataset 5. Table 1A. One-way ANOVA report from the correlation between Polish honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. Dataset 6. Table 1B. One-way ANOVA report from the correlation between Polish honeybees’ health status and fungi and plant pollens detected on the basis of ITS NGS analysis. Dataset 7. Table 2A. One-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. Dataset 8. Table 2B. One-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis

成長異常,発育遅延,てんかん,奇異な顔貌を伴った永続型新生児糖尿病（著者の要望によるタイトル） 成長異常,発育遅延,てんかん,奇異な顔貌を伴った恒久的新生児糖尿病（医中誌のタイトル）

雑誌掲載版We report the case of 15-mo-old girl who manifested neonatal diabetes mellitus associated with IUGR, developmental delay, epilepsy dysmorphic features and growth disturbance. She was born at 38 wk of gestation after normal pregnancy. Her birth weight was 2262 g (<10%tile) and height 45.0 cm (<10%tile). Since birth she gradually presented failure to thrive and presented hyperglycemia (glucose 692 mg/dl), ketoacidosis and elevated HbA1c level (7.7%) at 3 mo of age. The anti-GAD antibody was negative and the serum CPR level was 0.5 ng/ml. The girl was treated with intensive insulin therapy from 3 mo of age, however, her weight did not improve and was only 3305 g (-4.6 SD) at 5 mo of age. When admitted to our hospital, she was recognized as having dysmorphic features such as prominent forehead, downturned mouth, bilateral ptosis, arthrogryposis, hypertonia and umbilical hernia. Because she presented frequent hypoglycemic episodes under the intensive insulin injection regimen, treatment was changed to a twice a day injection schedule. Subsequently, the patient did not show any hypoglycemic episodes and she gradually gained a weight. However, at 7 mo of age, seizures developed and her EEG was hypsarrhythmic. She is now 15 mo of age but she cannot sit without support. She is still short and her height is 64.8 cm (-3.9 SD)

Schematic diagramas showing an overview of mutation detection methods.

<p>(<b>A</b>) In patients with McCune-Albright syndrome, the proportion of mutation-carrying cells (colored red) is low in peripheral blood leukocytes (PBL). (<b>B</b>) In the present study, PCR amplification was conducted in the absence (<i>left panel</i>) or presence (<i>right panel</i>) of the peptide nucleic acid (PNA) probe. The PNA probe preferentially hybridizes to wildtype PCR fragments (colored black) and inhibits their amplification. This results in enrichment of mutant PCR fragments (colored red). We used chimeric PCR primers, containing both locus-specific and adapter sequences, to generate amplicons that are sequenced on the Illumina platform. (<b>C</b>) PCR without the PNA probe produces PCR amplicons, of which relative proportion between wildtype (colored black) and mutant (colored red) is similar to PBL (<i>left panel</i>). In contrast, PNA treatment enriches mutant amplicons (<i>right panel</i>). (<b>D</b>)<b>,</b> PCR amplicons were analyzed by both Sanger sequencing and next generation sequencing (‘NGS’). Due to low mutation abundance, mutations cannot be detected in amplicons generated without the PNA probe (<i>left panel</i>), while they can be detected in PNA-treated amplicons (<i>right panel,</i> an arrow indicates the mutation). In the MiSeq platform, clonal clusters, each derived from a single DNA molecule, are generated on a flow cell, and are sequenced base-by-base simultaneously and independently. The diagrams under the schematic flow cells show imaginative optically scanned data of the cycle corresponding to the mutated nucleotide. In a sample without PNA treatment, the mutant amplicons can be recognized on the flow cell (<i>left panel</i>). Mutant-enriched samples are also analyzable by NGS (<i>right panel</i>).</p

Characteristics of the study subjects.

<p>Abbreviations: FD, osseous fibrous dysplasia; MAS, McCune-Albright syndrome; N.D., not detected; NGS, next generation sequencing; PNA, the peptide nucleic acid method; PNA-NGS, combinatory use of PNA and NGS; PP, precocious puberty</p>*<p>Hyperprolactinemia and GH-producing adenoma</p>**<p>GH-producing adenoma</p

Results of the serial dilution study.

<p>(<b>A</b>) Cloned mutant DNA (R201H mutation) was diluted into cloned wildtype DNA to 1/10 (10%), 1/100 (1%), 1/333 (0.3%), 1/1,000 (0.1%), 1/3,333 (0.03%) and 1/10,000 (0.01%). Serially diluted DNA samples were PCR-amplified with or without the peptide nucleic acid (PNA) probe (‘PNA’ and ‘Conventional’. Each PCR product was analyzed by Sanger sequencing and next generation sequencing (NGS). Partial chromatograms encompassing the GNAS codon 201 (indicated by CGT) are shown. Relative abundance of the c.602 nucleotide (G, A, T and C) measured by NGS is aligned with each chromatogram. The G allele is wildtype, while the A allele is the R201H mutant. Values with a positive test result (defined by z-score of measured mutant abundance; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060525#s2" target="_blank">Materials and Methods</a> for details) are colored red. Experiment-specific reference ranges are also shown. In the 12 chromatograms, the mutant signal could be detected in two PNA-treated samples and one non-treated sample (indicated by red arrows). Contrastingly, the mutation could be detected down to 0.03% by NGS alone, and down to 0.01% by combinatory use of PNA and NGS. (<b>B</b>) A serial dilution plot showing a linear correlation between true mutation abundance and measured mutation abundance. Note that both axes are logarithmic. Comparison of mutation abundance values of PNA-treated and untreated samples revealed that the fold enrichment by PNA is about 7, and it was independent of initial abundance.</p