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Papers - MIYA Fuyuki

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Biallelic loss-of-function variants in the centriolar protein CCP110 leads to a ciliopathy-like phenotype.

Suzuki H, Muramatsu Y, Miya F, Asada H, Yamada M, Nishimura G, Kosaki K, Takenouchi T

European journal of medical genetics 70 104955 2024.08

ISSN 1769-7212

CCP110 (centriolar coiled coil protein 110, also known as CP110) is one of the essential proteins localized in the centrosome that plays critical roles in the regulation of the cell cycle and also in the initiation of ciliogenesis. So far, no human congenital disorders have been identified to be associated with pathogenic variants of CCP110. Mice with biallelic loss-of-function variants of Ccp110 (Ccp110−/−) are known to manifest multiple organ defects, including a small body size, polydactyly, omphalocele, congenital heart defects, cleft palate, short ribs, and a small thoracic cage, a pattern of abnormalities closely resembling that in “ciliopathies” in humans. Herein, we report a 7-month-old male infant who presented with growth failure and skeletal abnormalities, including a narrow thorax and severe brachydactyly. Trio exome analysis of the genomic DNA of the patient and his parents showed that the patient was a compound heterozygote for truncating variants of CCP110, including a frameshift variant NM_001323572.2:c.856_857del, p.(Val286Leufs*5) inherited from the father, and a nonsense variant NM_001323572.2:c.1129C>T, p.(Arg377*) inherited from the mother. The strikingly similar pattern of malformations between Ccp110−/− mice and the 7-month-old male infant reported herein carrying unequivocal truncating CCP110 variants strongly supports the contention that CCP110 is a novel disease-causative gene.

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Truncating variants of the sterol recognition region of SHH cause hypertelorism phenotype rather than hypotelorism-holoprosencephaly.

Yamada M, Mizuno S, Inaba M, Uehara T, Inagaki H, Suzuki H, Miya F, Takenouchi T, Kurahashi H, Kosaki K

American journal of medical genetics. Part A 194 ( 8 ) e63614 2024.08

ISSN 1552-4825

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism.

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SALL4 deletion and kidney and cardiac defects associated with VACTERL association.

Watanabe D, Nakato D, Yamada M, Suzuki H, Takenouchi T, Miya F, Kosaki K

Pediatric nephrology (Berlin, Germany) 39 ( 8 ) 2347 - 2349 2024.08

ISSN 0931-041X

Congenital anomalies of the kidney and urinary tract (CAKUT) can be a part of the VACTERL association, which represents the non-random combination of the following congenital anomalies: vertebral anomalies, anal anomalies, cardiac anomalies, tracheal-esophageal anomalies, kidney anomalies, and limb anomalies. VACTERL association is generally considered to be a non-genetic condition. Exceptions include a patient with a heterozygous nonsense SALL4 variant and anal stenosis, tetralogy of Fallot, sacro-vertebral fusion, and radial and thumb anomalies. SALL4 encodes a transcription factor that plays a critical role in kidney morphogenesis. Here, we report a patient with VACTERL association and a heterozygous 128-kb deletion spanning SALL4 who presented with renal hypoplasia, radial and atrio-septal defects, and patent ductus arteriosus. The present report of SALL4 deletion, in addition to a previously reported patient with VACTERL association phenotype and SALL4 nonsense mutation, further supports the notion that SALL4 haploinsufficiency can lead to VACTERL association.

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SCN8A Encephalopathy with a Significant Long-Term Response to Lacosamide

Yokoyama A., Nakamura T., Ichinose F., Miya F., Kato M., Matsuo M.

Journal of Pediatric Neurology 22 ( 6 ) 470 - 474 2024.06

ISSN 13042580

Developmental and epileptic encephalopathy associated with SCN8A variants (i.e., SCN8A encephalopathy) causes early-onset epilepsy, involuntary movements, hypotonia, and developmental delay. Sodium channel blockers are effective for treating SCN8A encephalopathy; however, the long-term effects are unknown. Herein, we report the long-term efficacy of lacosamide (LCM) treatment in a patient with SCN8A encephalopathy. Our patient, a 7-year-old girl, presented with a hyperekplexia-like excessive startle response, drug-resistant epilepsy with sinus arrest, and prolonged respiratory failure during the neonatal period. The patient was diagnosed with SCN8A encephalopathy caused by a de novo pathogenic variant of SCN8A: c.3979A > G; p.Ile1327Val. The patient experienced tonic clustered seizures daily, with dramatic responses to high doses of LCM, lasting approximately 3 years.

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West症候群を発症したPTEN病的バリアントの女児例

門田茉莉, 占部良介, 中井まりえ, 星野英紀, 加藤光広, 宮冬樹, 三牧正和

日本小児科学会雑誌 ((公社)日本小児科学会) 128 ( 6 ) 828 - 832 2024.06

ISSN 0001-6543

West症候群(WS)では近年多くの原因遺伝子が知られるようになった。今回、WSの原因遺伝子としては一般的ではないPTEN(Phosphatase and Tensin Homolog)にバリアントをもつWSの女児例を経験した。周産期歴には特に異常なく、生後7ヵ月時にシリーズ形成する癲癇性スパズムを発症した。初診時に筋緊張低下と運動発達遅延および頭囲拡大を認め、発作間欠期脳波検査でヒプサリズミアがみられWSと診断した。頭部MRIでは脳回の構造異常や過誤腫はみられず、ACTH療法とその後の抗発作薬内服治療によって発作は消失し、脳波異常も改善したが、中等度～重度の精神運動発達遅滞がみられた。遺伝子解析でPTENに既知の病的バリアント(NM 000314:exon6:c.517C>T:p.(R173C))が同定された。一般に生殖細胞系列のPTEN病的バリアントを原因とする疾患の表現型としてCowden症候群やBannayan-Riley-Ruvalcaba症候群、Proteus症候群などが知られており、これらはいずれも過誤腫を伴う疾患で、PTEN Hamartoma Tumor Syndromeと総称されている。既報ではPTENバリアントによって脳形成障害をきたした場合でも癲癇を発症する割合は低く、本例のように脳回の構造異常を伴わずにWSをきたした症例の報告はこれまでになかった。

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration.

Yamada M, Maeta K, Suzuki H, Kurosawa R, Takenouchi T, Awaya T, Ajiro M, Takeuchi A, Nishio H, Hagiwara M, Miya F, Matsuo M, Kosaki K

Scientific reports 14 ( 1 ) 6506 2024.03

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon–intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

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A case of infantile epileptic spasms syndrome and autism spectrum disorder with an RFX3 mutation.

Torio M, Maeda K, Akamine S, Kawakami S, Matsubara Y, Miya F, Kato M, Kira R

Seizure 112 11 - 14 2023.11

ISSN 1059-1311

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Neurochemistry evaluated by magnetic resonance spectroscopy in a patient with FBXO28-related developmental and epileptic encephalopathy.

Sano K, Miya F, Kato M, Omata T, Takanashi JI

Brain & development 45 ( 10 ) 583 - 587 2023.11

ISSN 0387-7604

Background: Mutations in the FBXO28 gene, which encodes FBXO28, one of the F-box protein family, may cause developmental and epileptic encephalopathy (DEE). FBXO28-related DEE is radiologically characterized by cerebral atrophy, delayed/abnormal myelination, and brain malformation; however, no neurochemical analyses have been reported. Case report: A female Japanese infant presented with severe psychomotor delay, epileptic spasms, and visual impairment. Whole-exome sequencing revealed a de novo variant of the FBXO28 gene, leading to the diagnosis of FBXO28-related DEE. Magnetic resonance (MR) spectroscopy at 6, 12, and 32 months revealed decreased N-acetylaspartate and choline-containing compounds and increased levels of myoinositol. Conclusion: MR spectroscopy revealed neurochemical derangement in FBXO28-related DEE, that is, disturbed myelination secondary to neuronal damage with astrogliosis.

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Time-dependent cell-state selection identifies transiently expressed genes regulating ILC2 activation.

Tanaka Y, Yamagishi M, Motomura Y, Kamatani T, Oguchi Y, Suzuki N, Kiniwa T, Kabata H, Irie M, Tsunoda T, Miya F, Goda K, Ohara O, Funatsu T, Fukunaga K, Moro K, Uemura S, Shirasaki Y

Communications biology 6 ( 1 ) 915 2023.09

The decision of whether cells are activated or not is controlled through dynamic intracellular molecular networks. However, the low population of cells during the transition state of activation renders the analysis of the transcriptome of this state technically challenging. To address this issue, we have developed the Time-Dependent Cell-State Selection (TDCSS) technique, which employs live-cell imaging of secretion activity to detect an index of the transition state, followed by the simultaneous recovery of indexed cells for subsequent transcriptome analysis. In this study, we used the TDCSS technique to investigate the transition state of group 2 innate lymphoid cells (ILC2s) activation, which is indexed by the onset of interleukin (IL)-13 secretion. The TDCSS approach allowed us to identify time-dependent genes, including transiently induced genes (TIGs). Our findings of IL4 and MIR155HG as TIGs have shown a regulatory function in ILC2s activation.

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Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences.

Yamada M, Nitta Y, Uehara T, Suzuki H, Miya F, Takenouchi T, Tamura M, Ayabe S, Yoshiki A, Maeno A, Saga Y, Furuse T, Yamada I, Okamoto N, Kosaki K, Sugie A

European journal of medical genetics 66 ( 8 ) 104804 2023.08

ISSN 1769-7212

DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.

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Precise definition of the breakpoints of an apparently balanced translocation between chromosome 3q26 and chromosome 7q36: Role of KMT2C disruption.

Yamada M, Suzuki H, Miya F, Kosugiyama K, Ujiie T, Tonoki H, Kosaki K

Congenital anomalies 63 ( 4 ) 121 - 124 2023.07

ISSN 0914-3505

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Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling.

Rashid MI, Ito T, Miya F, Shimojo D, Arimoto K, Onodera K, Okada R, Nagashima T, Yamamoto K, Khatun Z, Shimul RI, Niwa JI, Katsuno M, Sobue G, Okano H, Sakurai H, Shimizu K, Doyu M, Okada Y

Scientific reports 13 ( 1 ) 8146 2023.05

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.

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Topologically associating domain underlies tissue specific expression of long intergenic non-coding RNAs.

Hamba Y, Kamatani T, Miya F, Boroevich KA, Tsunoda T

iScience 26 ( 5 ) 106640 2023.05

Accumulating evidence indicates that long intergenic non-coding RNAs (lincRNAs) show more tissue-specific expression patterns than protein-coding genes (PCGs). However, although lincRNAs are subject to canonical transcriptional regulation like PCGs, the molecular basis for the specificity of their expression patterns remains unclear. Here, using expression data and coordinates of topologically associating domains (TADs) in human tissues, we show that lincRNA loci are significantly enriched in the more internal region of TADs compared to PCGs and that lincRNAs within TADs have higher tissue specificity than those outside TADs. Based on these, we propose an analytical framework to interpret transcriptional status using lincRNA as an indicator. We applied it to hypertrophic cardiomyopathy data and found disease-specific transcriptional regulation: ectopic expression of keratin at the TAD level and derepression of myocyte differentiation-related genes by E2F1 with down-regulation of LINC00881. Our results provide understanding of the function and regulation of lincRNAs according to genomic structure.

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Molecular basis of carotid body tumor and associated clinical features in Japan identified by genomic, immunohistochemical, and clinical analyses.

Yoshihama K, Mutai H, Sekimizu M, Ito F, Saito S, Nakamura S, Mikoshiba T, Nagai R, Takebayashi A, Miya F, Kosaki K, Ozawa H, Matsunaga T

Clinical genetics 103 ( 4 ) 466 - 471 2023.04

ISSN 0009-9163

Carotid body tumor (CBT) is classified as a paraganglioma (PGL). Here, we report the genetic background, protein expression pattern, and clinical findings of 30 Japanese CBT cases. Germline pathogenic or likely pathogenic (P/LP) variants of genes encoding succinate dehydrogenase subunits (SDHs) were detected in 15 of 30 cases (50%). The SDHB variants were the most frequently detected, followed by SDHA and SDHD variants. One case with SDHAF2 variant was bilateral CBT, and other two multiple PGL cases were not detected P/LP variants. The three cases with germline variants that could be tested did not have somatic P/LP variants of the same genes. Immunohistochemical analysis showed negative SDHB signals in CBT tissues in five cases with germline P/LP variants of SDHB, SDHD, or SDHA. In addition, SDHB signals in CBT tissues were negative in four of nine cases without germline P/LP variants of SDHs. These findings suggest the involvement of unidentified molecular mechanisms affecting SDHs.

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Diagnosis of Prader-Willi syndrome and Angelman syndrome by targeted nanopore long-read sequencing.

Yamada M, Okuno H, Okamoto N, Suzuki H, Miya F, Takenouchi T, Kosaki K

European journal of medical genetics 66 ( 2 ) 104690 2023.02

ISSN 1769-7212

The CpG island flanking the promoter region of SNRPN on chromosome 15q11.2 contains CpG sites that are completely methylated in the maternally derived allele and unmethylated in the paternally derived allele. Both unmethylated and methylated alleles are observed in normal individuals. Only the methylated allele is observed in patients with Prader-Willi syndrome, whereas only the unmethylated allele is observed in those with Angelman syndrome. Hence, detection of aberrant methylation at the differentially methylated region is fundamental to the molecular diagnosis of Prader-Willi syndrome and Angelman syndromes. Traditionally, bisulfite treatment and methylation-sensitive restriction enzyme treatment or methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) have been used. We here developed a long-read sequencing assay that can distinguish methylated and unmethylated CpG sites at 15q11.2 by the difference in current intensity generated from nanopore reads. We successfully diagnosed 4 Prader-Willi syndrome patients and 3 Angelman syndrome patients by targeting differentially methylated regions. Concurrent copy number analysis, homozygosity analysis, and structural variant analysis also allowed us to precisely delineate the underlying pathogenic mechanisms, including gross deletion, uniparental heterodisomy, uniparental isodisomy, or imprinting defect. Furthermore, we showed allele-specific methylation in imprinting-related differentially methylated regions on chromosomes 6, 7, 11, 14, and 20 in a normal individual together with 4 Prader-Willi patients and 3 Angelman syndrome patients. Hence, presently reported method is likely to be applicable to the diagnosis of imprinting disorders other than Prader-Willi syndrome and Angelman syndrome as well.

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Splicing QTL analysis focusing on coding sequences reveals mechanisms for disease susceptibility loci.

Yamaguchi K, Ishigaki K, Suzuki A, Tsuchida Y, Tsuchiya H, Sumitomo S, Nagafuchi Y, Miya F, Tsunoda T, Shoda H, Fujio K, Yamamoto K, Kochi Y

Nature communications (Nature Communications) 13 ( 1 ) 4659 2022.08

Splicing quantitative trait loci (sQTLs) are one of the major causal mechanisms in genome-wide association study (GWAS) loci, but their role in disease pathogenesis is poorly understood. One reason is the complexity of alternative splicing events producing many unknown isoforms. Here, we propose two approaches, namely integration and selection, for this complexity by focusing on protein-structure of isoforms. First, we integrate isoforms with the same coding sequence (CDS) and identify 369-601 integrated-isoform ratio QTLs (i2-rQTLs), which altered protein-structure, in six immune subsets. Second, we select CDS incomplete isoforms annotated in GENCODE and identify 175-337 isoform-ratio QTL (i-rQTL). By comprehensive long-read capture RNA-sequencing among these incomplete isoforms, we reveal 29 full-length isoforms with unannotated CDSs associated with GWAS traits. Furthermore, we show that disease-causal sQTL genes can be identified by evaluating their trans-eQTL effects. Our approaches highlight the understudied role of protein-altering sQTLs and are broadly applicable to other tissues and diseases.

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Utility of tissue-specific gene expression scores for gene prioritization in Mendelian diseases.

Kato D, Mitsuhashi S, Miya F, Saitoh S, Okamoto N, Tsunoda T, Kochi Y

Journal of human genetics (Journal of Human Genetics) 67 ( 12 ) 739 - 742 2022.08

ISSN 1434-5161

In genetic testing of Mendelian diseases, it is a bioinformatics challenge to effectively prioritize disease-causing candidate genes listed from massively parallel sequencing. Tissue specificity of the gene expression levels may give a clue because it may reflect tissue-specific disease manifestation. However, considering poor correlations between mRNA and protein expression in some genes, it is not clear whether transcriptomics- or proteomics-based tissue specificity should be used to prioritize candidate genes. Therefore, we compared the efficiency of tissue-specific scores (TS scores) obtained from transcriptome and proteome data in prioritizing candidate genes for whole exome sequencing (WES) analysis of Mendelian disease patients. We show that both Protein and RNA TS scores are useful in prioritizing candidate genes in WES analysis, although diseases like coagulopathies get more benefit from Protein TS score. This study may provide useful evidence in developing new methods to effectively identify novel disease-causing genes.

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First phase 1 clinical study of olaparib in pediatric patients with refractory solid tumors.

Takagi M, Ogawa C, Iehara T, Aoki-Nogami Y, Ishibashi E, Imai M, Kimura T, Nagata M, Yasuhara M, Masutani M, Yoshimura K, Tomizawa D, Ogawa A, Yonemori K, Morishita A, Miyamoto S, Takita J, Kihara T, Nobori K, Hasebe K, Miya F, Ikeda S, Shioda Y, Matsumoto K, Fujimura J, Mizutani S, Morio T, Hosoi H, Koike R

Cancer (Cancer) 128 ( 15 ) 2949 - 2957 2022.08

ISSN 0008-543X

Background: The survival of patients with high-risk, refractory, relapsed, or metastatic solid tumors remains dismal. A poly(ADP-ribose) polymerase (PARP) inhibitor could be effective for the treatment of pediatric solid tumors with defective homologous recombination. Methods: This open-label, multicenter phase 1 clinical trial evaluated the safety, tolerability, and efficacy of olaparib, a PARP inhibitor, in pediatric patients with refractory solid tumors to recommend a dose for Phase 2 trials. Olaparib (62.5, 125, and 187.5 mg/m2 twice daily) was administered orally every day (1 cycle = 28 days) using a standard 3 + 3 dose-escalation design. Patients aged 3–18 years with recurrent pediatric solid tumors were eligible. Pharmacokinetic and pharmacodynamic analyses were performed. Results: Fifteen patients were enrolled and received olaparib monotherapy, which was well tolerated. The recommended phase 2 dose for daily administration was 187.5 mg/m2 twice daily. Pharmacokinetics were dose proportional. The area under the concentration-time curve from 0 to 12 h and the peak plasma concentration for 187.5 mg/m2 twice daily in children were comparable to previous data obtained in a 200-mg, twice-daily cohort and lower than those in the 300-mg twice-daily cohort in adults. Pharmacodynamic studies demonstrated substantial inhibition of PARP activity. Two partial responses were observed in patients with Wilms tumor and neuroblastoma. Conclusions: This report is the first clinical trial to describe the use of a PARP inhibitor as monotherapy in children. Olaparib was well tolerated, with preliminary antitumor responses observed in DNA damage response-defective pediatric tumors. Lay summary: This Phase 1 trial evaluated the efficacy and safety of olaparib in patients with refractory childhood solid tumors. Olaparib was well tolerated, achieving objective response in 2/15 patients. The DNA damage response was attenuated in nearly one-half of advanced neuroblastoma patients, demonstrating the utility of the PARP inhibitor. The results support further investigation of olaparib as a new treatment for DNA damage-response or repair-defective pediatric cancers.

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Deciphering complex rearrangements at the breakpoint of an apparently balanced reciprocal translocation t(4:18)(q31;q11.2)dn and at a cryptic deletion: Further evidence of TLL1 as a causative gene for atrial septal defect.

Yamada M, Suzuki H, Miya F, Takenouchi T, Kosaki K

American journal of medical genetics. Part A (American Journal of Medical Genetics, Part A) 188 ( 8 ) 2472 - 2478 2022.08

ISSN 1552-4825

When a de novo balanced reciprocal translocation is identified in patients with multiple congenital abnormalities, attempts are often made to infer the relationship between the phenotype of the patient and genes in the proximity of the breakpoint. Here, we report a patient with intellectual disability, atrial septal defect, syndactyly, and cleft lip and palate who had an “apparently balanced” de novo reciprocal translocation t(4:18)(q31;q11.2) as well as a 7-Mb cryptic deletion spanning the HOXD cluster on chromosome 2q31 that was unrelated to the reciprocal translocation. Further analysis using a nanopore long-read sequencer showed complex rearrangements on both derivative chromosomes 4 and 18 and the deleted chromosome 2. First, the TLL1 locus, which is associated with atrial septal defect, was disrupted by the rearrangement involving chromosome 4. Second, the deleted interval at 2q31 included the entire HOXD cluster, the deletion of which is known to cause toe syndactyly, and the DLX1 and DLX2 loci, which are responsible for cleft lip and palate. Among the haplo-sensitive genes within the deleted interval on 2q31, only the RAPGEF4 gene is known to be associated with an autistic phenotype. Hence, most of the clinical features of the patient could be ascribed to specific genomic rearrangements. We have shown the effectiveness of long-read sequencing in defining, in detail, the likely effects of an apparently balanced translocation and cryptic deletion. The results of the present analysis suggest the possibility of phenotypic prediction through a detailed analysis of structural abnormalities, including balanced translocations and deletions.

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De novo non-synonymous DPYSL2 (CRMP2) variants in two patients with intellectual disabilities and documentation of functional relevance through zebrafish rescue and cellular transfection experiments.

Suzuki H, Li S, Tokutomi T, Takeuchi C, Takahashi M, Yamada M, Okuno H, Miya F, Takenouchi T, Numabe H, Kosaki K, Ohshima T

Human molecular genetics 31 ( 24 ) 4173 - 4182 2022.07

ISSN 0964-6906

Collapsin response mediator protein 2 (Crmp2) is an evolutionarily well-conserved tubulin-binding cytosolic protein that plays critical roles in the formation of neural circuitry in model organisms including zebrafish and rodents. No clinical evidence that CRMP2 variants are responsible for monogenic neurogenic disorders in humans presently exists. Here, we describe two patients with de novo non-synonymous variants (S14R and R565C) of CRMP2 and intellectual disability associated with hypoplasia of the corpus callosum. We further performed various functional assays of CRMP2 variants using zebrafish and zebrafish Crmp2 (abbreviated as z-CRMP2 hereafter) and an antisense morpholino oligonucleotide [AMO]-based experimental system in which crmp2-morphant zebrafish exhibit the ectopic positioning of caudal primary (CaP) motor neurons. Whereas the co-injection of wild-type z-CRMP2 mRNA suppressed the ectopic positioning of CaP motor neurons in Crmp2-morphant zebrafish, the co-injection of R566C or S15R, z-CRMP2, which corresponds to R565C and S14R of human CRMP2, failed to rescue the ectopic positioning. Transfection experiments of zebrafish or rat Crmp2 using plasmid vectors in HeLa cells, with or without a proteasome inhibitor, demonstrated that the expression levels of mutant Crmp2 protein encoded by R565C and S14R CRMP2 variants were decreased, presumably because of increased degradation by proteasomes. When we compared CRMP2-tubulin interactions using co-immunoprecipitation and cellular localization studies, the R565C and S14R mutations weakened the interactions. These results collectively suggest that the CRMP2 variants detected in the present study consistently led to the loss-of-function of CRMP2 protein and support the notion that pathogenic variants in CRMP2 can cause intellectual disabilities in humans.

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