Takuya Naruto

Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinoma.

Comparative genomic hybridization studies have revealed frequent amplification of the 14q12‐q13 region in esophageal squamous cell carcinoma (ESC) cell lines. To identify genes targeted for amplification, we first defined the minimal common region of amplification using fluorescence in situ hybridization in affected ESC cell lines. The amplicon covered about 6 Mb, between markers D14S1034 and L18528. Then we screened 32 ESC cell lines to discern amplifications and expression levels of 26 expressed sequence tags (ESTs) that had been localized to the amplified region. Five known genes (BAZ1A, SRP54, NFKBIA, MBIP, and HNF3A) and two uncharacterized ESTs (GenBank Accession numbers AA991861 and AA167732) within the amplicon showed amplification and consequent overexpression. Two of these transcripts were amplified in three of the primary ESCs we examined. Our findings suggest that these seven genes are candidate targets of the amplification mechanism and therefore may be associated, together or separately, with development and progression of ESC.

A novel PTCH1 mutation in a patient with Gorlin syndrome

Gorlin syndrome is an autosomal dominant disorder characterized by a wide range of developmental abnormalities and a predisposition to various tumors, and it is linked to the alteration of several causative genes, including PTCH1. We performed targeted resequencing using a next-generation sequencer to analyze genes associated with known clinical phenotypes in an 11-year-old male with sporadic jaw keratocysts. A novel duplication mutation (c.426dup) in PTCH1, resulting in a truncated protein, was identified.

Deep intronic GPR143 mutation in a Japanese family with ocular albinism

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

Delineation of the KIAA2022 mutation phenotype: two patients with X-linked intellectual disability and distinctive features.

Next‐generation sequencing has enabled the screening for a causative mutation in X‐linked intellectual disability (XLID). We identified KIAA2022 mutations in two unrelated male patients by targeted sequencing. We selected 13 Japanese male patients with severe intellectual disability (ID), including four sibling patients and nine sporadic patients. Two of thirteen had a KIAA2022 mutation. Patient 1 was a 3‐year‐old boy. He had severe ID with autistic behavior and hypotonia. Patient 2 was a 5‐year‐old boy. He also had severe ID with autistic behavior, hypotonia, central hypothyroidism, and steroid‐dependent nephrotic syndrome. Both patients revealed consistent distinctive features, including upswept hair, narrow forehead, downslanting eyebrows, wide palpebral fissures, long nose, hypoplastic alae nasi, open mouth, and large ears. De novo KIAA2022 mutations (p.Q705X in Patient 1, p.R322X in Patient 2) were detected by targeted sequencing and confirmed by Sanger sequencing. KIAA2022 mutations and alterations have been reported in only four families with nonsyndromic ID and epilepsy. KIAA2022 is highly expressed in the fetal and adult brain and plays a crucial role in neuronal development. These additional patients support the evidence that KIAA2022 is a causative gene for XLID.

A novel missense mutation of COL5A2 in a patient with Ehlers-Danlos syndrome.

Ehlers–Danlos syndrome (EDS) is a group of inherited connective tissue disorders characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. For molecular diagnosis, targeted exome sequencing was performed on a 9-year-old male patient who was clinically suspected to have EDS. The patient presented with progressive kyphoscoliosis, joint hypermobility and hyperextensible skin without scars. Ultimately, classical EDS was diagnosed by identifying a novel, mono-allelic mutation in COL5A2 [NM_000393.3(COL5A2_v001):c.682G>A, p.Gly228Arg].

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.

Microdeletion of 19p13.3 in a girl with Peutz-Jeghers syndrome, intellectual disability, hypotonia, and distinctive features.

Peutz–Jeghers syndrome (PJS) is a rare autosomal dominant disease characterized by gastrointestinal polyposis and mucocutaneous pigmentation. Germline point mutations in the serine/threonine kinase 11 (STK11) have been identified in about 70% of patients with PJS. Only a few large genomic deletions have been identified. We report on a girl with PJS and multiple congenital anomalies. She had intellectual disability, umbilical hernia, bilateral inguinal hernias, scoliosis, and distinct facial appearance including prominent mandible, smooth philtrum, and malformed ears. She developed lip pigmentation at the age of 12 years but had no gastrointestinal polyps. Array comparative genomic hybridization revealed an approximately 610 kb deletion at 19p13.3, encompassing STK11. Together with previous reports, the identification of common clinical features suggests that microdeletion at 19p13.3 encompassing STK11 constitutes a distinctive phenotype.

De novo duplication of 17p13.1–p13.2 in a patient with intellectual disability and obesity

17p13.1 Deletion encompassing TP53 has been described as a syndrome characterized by intellectual disability and dysmorphic features. Only one case with a 17p13.1 duplication encompassing TP53 has been reported in a patient with intellectual disability, seizures, obesity, and diabetes mellitus. Here, we present a patient with a 17p13.1 duplication who exhibited obesity and intellectual disability, similar to the previous report. The 9‐year‐old proposita was referred for the evaluation of intellectual disability and obesity. She also exhibited insulin resistance and liver dysfunction. She had wide palpebral fissures, upturned nostrils, a long mandible, short and slender fingers, and skin hyperpigmentation. Array comparative genomic hybridization (array CGH) detected a 3.2 Mb duplication of 17p13.1–p13.2 encompassing TP53, FXR2, NLGN2, and SLC2A4, which encodes the insulin‐responsive glucose transporter 4 (GLUT4) associated with insulin‐stimulated glucose uptake in adipocytes and muscle. We suggest that 17p13.1 duplication may represent a clinically recognizable condition characterized partially by a characteristic facial phenotype, developmental delay, and obesity.

West Syndrome in a Patient With Schinzel-Giedion Syndrome

Schinzel-Giedion syndrome is a rare recognizable malformation syndrome defined by characteristic facial features, profound developmental delay, severe growth failure, and multiple congenital anomalies. The causative gene of Schinzel-Giedion syndrome, SETBP1, has been identified, but limited cases have been confirmed by molecular analysis. We present a 9-month-old girl affected by West syndrome with Schinzel-Giedion syndrome. Congenital severe hydronephrosis, typical facial features, and multiple anomalies suggested a clinical diagnosis of Schinzel-Giedion syndrome. Hypsarrhythmia occurred at 7 months of age and was temporarily controlled by adrenocorticotropic hormone (ACTH) therapy during 5 weeks. SETBP1 mutational analysis showed the presence of a recurrent mutation, p.Ile871Thr. The implications in management of Schinzel-Giedion syndrome are discussed.

Frequent silencing of the candidate tumor suppressor TRIM58 by promoter methylation in early-stage lung adenocarcinoma

In this study, we aimed to identify novel drivers that would be epigenetically altered through aberrant methylation in early-stage lung adenocarcinoma (LADC), regardless of the presence or absence of tobacco smoking-induced epigenetic field defects. Through genome-wide screening for aberrantly methylated CpG islands (CGIs) in 12 clinically uniform, stage-I LADC cases affecting six non-smokers and six smokers, we identified candidate tumor-suppressor genes (TSGs) inactivated by hypermethylation. Through systematic expression analyses of those candidates in panels of additional tumor samples and cell lines treated or not treated with 5-aza-deoxycitidine followed by validation analyses of cancer-specific silencing by CGI hypermethylation using a public database, we identified TRIM58 as the most prominent candidate for TSG. TRIM58 was robustly silenced by hypermethylation even in early-stage primary LADC, and the restoration of TRIM58 expression in LADC cell lines inhibited cell growth in vitro and in vivo in anchorage-dependent and -independent manners. Our findings suggest that aberrant inactivation of TRIM58 consequent to CGI hypermethylation might stimulate the early carcinogenesis of LADC regardless of smoking status; furthermore, TRIM58 methylation might be a possible early diagnostic and epigenetic therapeutic target in LADC.