Analysis of IKBKG/NEMO gene in five Japanese cases of incontinentia pigmenti with retinopathy: fine genomic assay of a rare male case with mosaicism.

Incontinentia pigmenti (IP) is an X-linked dominant genodermatosis that is usually lethal in utero in males, though exceptionally they survive very rarely either with Klinefelter syndrome or a somatic mosaicism. We performed genomic analysis of five Japanese IP patients including a rare boy case, all of whom were definite cases with retinopathy. Four patients including the boy revealed the recurrent exon 4-10 deletion in the sole known causative gene IKBKG/NEMO, which was confirmed by various specific PCR techniques. The boy's saliva DNA showed a mosaicism consisting of the deletion and intact alleles, but his blood DNA did not. Relative quantification analysis of the real-time PCR data by ∆∆CT method estimated the mosaicism ratio of the boy's saliva as 45:55 (deletion:intact). A genomic analysis for the recurrent deletion at the nucleotide sequence level has been performed directly using patient's DNA and it has been clarified that the breakpoints are within two MER67B repeats in the intron 3 and downstream of exon 10. This is the first report of the assay for the mosaicism ratio of a male IP case with a recurrent exon 4-10 deletion of IKBKG/NEMO and the sequencing analysis of the breakpoints of the recurrent deletion directly using patient's sample.

Identification of susceptibility loci for light-induced visual impairment in rats.

Bright light exposure in animals results in the selective degeneration of the outer retina, known as "retinal photic injury" (RPI). The susceptibility to RPI differs among rat strains. WKY rats display susceptibility to RPI with extensive retinal degeneration observed in the sagittal eye specimen, whereas LEW strain rats are resistant to it, showing only slight or no degeneration. In the present study, we first established an ethological screening method using the Morris water maze to discern differential susceptibility among the living rats. WKY and LEW were crossed to produce the first filial generation (F1) offspring. Maze-trained individuals were exposed to bright, white light. The screening test results demonstrated that the susceptibility to light-induced visual impairment in rats is a dominant Mendelian susceptibility trait, as F1 rats were susceptible to visual impairment like WKY rats. Therefore, F1 rats were backcrossed with recessive LEW to produce the first backcross offspring (BC1). Subsequent recurrent backcrossing while selecting for the susceptibility, indicated a segregation ratio of ca. 24% in BC1 and BC2 generations, indicating the involvement of two or more genes in the susceptibility. Further, microsatellite analysis of BC1-to-BC4 individuals using microsatellite markers mapped two susceptibility loci on chromosome segments 5q36 and 19q11-q12, named RPI susceptibility (Rpi)1 and Rpi2, respectively. This study provides an insight into mechanisms underlying differential susceptibility, which could help decipher the mechanism underlying the onset/progression of human age-related macular degeneration.

Recommendations of the 2006 Human Variome Project meeting.

Lists of variations in genomic DNA and their effects have been kept for some time and have been used in diagnostics and research. Although these lists have been carefully gathered and curated, there has been little standardization and coordination, complicating their use. Given the myriad possible variations in the estimated 24,000 genes in the human genome, it would be useful to have standard criteria for databases of variation. Incomplete collection and ascertainment of variants demonstrates a need for a universally accessible system. These and other problems led to the World Heath Organization-cosponsored meeting on June 20-23, 2006 in Melbourne, Australia, which launched the Human Variome Project. This meeting addressed all areas of human genetics relevant to collection of information on variation and its effects. Members of each of eight sessions (the clinic and phenotype, the diagnostic laboratory, the research laboratory, curation and collection, informatics, relevance to the emerging world, integration and federation and funding and sustainability) developed a number of recommendations that were then organized into a total of 96 recommendations to act as a foundation for future work worldwide. Here we summarize the background of the project, the meeting and its recommendations.

The KM-parkin-DB: A Sub-set MutationView Database Specialized for PARK2 (PARKIN) Variants.

We previously isolated PARKIN (PARK2) as a gene responsible for a unique sort of Parkinson disease, namely Autosomal Recessive Juvenile Parkinsonism (ARJP). In this study, we surveyed all the available literature describing PARK2 gene/Parkin protein mutations found in Parkinson disease patients. Only carefully evaluated data were deposited in the graphical database MutationView (http://mutview.dmb.med.keio.ac.jp) to construct KM-parkin-DB, an independent sub-set database. Forty-four articles were selected for data curation regarding clinical information such as ethnic origins, manifested symptoms, onset age, and hereditary patterns as well as mutation details including base changes and zygosity. A total of 366 cases were collected from 39 ethnic origins and 96 pathogenic mutations were found. PARK2 gene mutations were found also in some general Parkinson disease patients. The majority (63%) of mutations in PARK2 were restricted to two particular domains (UBL and RING1) of the Parkin protein. In these domains, two major mutations, a large deletion (DelEx3) and a point mutation (p.Arg275Trp), were located.

The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74-90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

Interaction between optineurin and the bZIP transcription factor NRL.

Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN-interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co-expression of HA-tagged NRL and FLAG-tagged OPTN in HeLaS3 cells followed by immunoprecipitation and Western blotting with anti-tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN-binding protein to show eye-specific expression. A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.

A prioritization analysis of disease association by data-mining of functional annotation of human genes.

Complex diseases result from contributions of multiple genes that act in concert through pathways. Here we present a method to prioritize novel candidates of disease-susceptibility genes depending on the biological similarities to the known disease-related genes. The extent of disease-susceptibility of a gene is prioritized by analyzing seven features of human genes captured in H-InvDB. Taking rheumatoid arthritis (RA) and prostate cancer (PC) as two examples, we evaluated the efficiency of our method. Highly scored genes obtained included TNFSF12 and OSM as candidate disease genes for RA and PC, respectively. Subsequent characterization of these genes based upon an extensive literature survey reinforced the validity of these highly scored genes as possible disease-susceptibility genes. Our approach, Prioritization ANalysis of Disease Association (PANDA), is an efficient and cost-effective method to narrow down a large set of genes into smaller subsets that are most likely to be involved in the disease pathogenesis.

DNA sequence and analysis of human chromosome 8.

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635 kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10.

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

KMeyeDB: a graphical database of mutations in genes that cause eye diseases.

KMeyeDB (http://mutview.dmb.med.keio.ac.jp/) is a database of human gene mutations that cause eye diseases. We have substantially enriched the amount of data in the database, which now contains information about the mutations of 167 human genes causing eye-related diseases including retinitis pigmentosa, cone-rod dystrophy, night blindness, Oguchi disease, Stargardt disease, macular degeneration, Leber congenital amaurosis, corneal dystrophy, cataract, glaucoma, retinoblastoma, Bardet-Biedl syndrome, and Usher syndrome. KMeyeDB is operated using the database software MutationView, which deals with various characters of mutations, gene structure, protein functional domains, and polymerase chain reaction (PCR) primers, as well as clinical data for each case. Users can access the database using an ordinary Internet browser with smooth user-interface, without user registration. The results are displayed on the graphical windows together with statistical calculations. All mutations and associated data have been collected from published articles. Careful data analysis with KMeyeDB revealed many interesting features regarding the mutations in 167 genes that cause 326 different types of eye diseases. Some genes are involved in multiple types of eye diseases, whereas several eye diseases are caused by different mutations in one gene.

Hair roots as an mRNA source for mutation analysis of Usher syndrome-causing genes.

mRNA is an important tool to study the effects of particular mutations on the mode of splicing and transcripts. However, it is often difficult to isolate mRNA because the organ or tissue in which the gene is expressed cannot be sampled. We previously identified two probable splicing mutations (c.6485+5G>A and c.8559-2A>G) during the mutation analysis of USH2A in Japanese Usher syndrome (USH) type 2 patients, but we could not observe their effects on splicing because the gene is expressed in only a few tissues/organs, and is not expressed in peripheral lymphocytes. In this study, we used hair roots as a source of mRNA of USH-causing genes, and successfully detected the expression of seven, except USH1C and CLRN1, of the nine USH-causing genes. We used RNA extracted from the hair roots of a patient who has both c.6485+5G>A and c.8559-2A>G mutations in USH2A in a compound heterozygous state to observe the effects of these mutations on transcripts. Reverse-transcription PCR analysis revealed that c.6485+5G>A and c.8559-2A>G inactivated splice donor and splice acceptor sites, respectively, and caused skipping of exons. Thus, RNA extracted from hair roots is a potential powerful and convenient tool for the mutation analysis of USH-causing genes.

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70-80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

A case of childhood glaucoma with a combined partial monosomy 6p25 and partial trisomy 18p11 due to an unbalanced translocation.

Background: Chromosomal deletion involving the 6p25 region results in a clinically recognizable syndrome characterized by anterior eye chamber anomalies with risk of glaucoma and non-ocular malformations (6p25 deletion syndrome). We report a newborn infant case of childhood glaucoma with a combination of partial monosomy 6p25 and partial trisomy 18p11 due to an unbalanced translocation.Materials and methods: The patient was a 0-year-old girl. Both eyes showed aniridia and left eye Peters anomaly with multiple malformations. To identify the chromosomal aberrations in the patient with clinically suspected 6p25 deletion syndrome, we performed cytogenetic analysis (G-banding and multicolor fluorescent in-situ hybridization) and array-based comparative genomic hybridization (array-CGH) analysis.Results: Cytogenetic analyses revealed a derivative chromosome 6 with its distal short arm replaced by an extra copy of the short arm of chromosome 18. Array-CGH analysis detected a 4.6-Mb deletion at 6pter to 6p25.1 and 8.9-Mb duplication at 18pter to 18p11.22. To determine the breakpoint of the unbalanced rearrangement at the single-base level, we performed a long-range PCR for amplifying the junctional fragment of the translocation breakpoint. By sequencing the junctional fragment, we defined the unbalanced translocation as g.chr6:pter_4594783delinschr18:pter_8911541.Conclusions: A phenotype corresponding to combined monosomy 6p25 and trisomy 18p11 presented as childhood glaucoma associated with non-acquired (congenital) ocular anomalies consist of aniridia and Peters anomaly and other systemic malformations. To the best of our knowledge, this is the first report which demonstrated the breakpoint sequence of an unbalanced translocation in a Japanese infant with childhood glaucoma.

Analysis of eighteen deletion breakpoints in the parkin gene.

Parkin mutations are responsible for the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP). On initial screening of Japanese patients with AR-JP, we had found that approximately half of the parkin mutations are deletions occurring between exons 2 and 5, forming a deletion hot spot. In this study, we investigated the deletion breakpoints of the parkin mutations in 22 families with AR-JP and examined the possible association between these deletion events and meiotic recombinations. We identified 18 deletion breakpoints at the DNA nucleotide sequence level. Almost all these deletions were different, indicating that the deletion hot spot was generated by recurrent but independent events. We found no association between the deletions and specific DNA elements. Recent copy number variation (CNV) data from various ethnic groups showed that the deletion hot spot is overlapped by a highly polymorphic CNV region, indicating that the recurrent deletion mutation or CNV is observable worldwide. By comparing Marshfield and deCODE linkage maps, we found that the parkin deletion hot spot may be associated with a meiotic recombination hot spot, although such association was not found on comparison with recent high-resolution genetic maps generated from the International HapMap project. Here, we discuss the possible mechanisms for deletion hot spot formation and its effects on human genomes.

Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland.

BACKGROUND: Among the salivary gland carcinomas, carcinoma in pleomorphic adenoma has been regarded as a representative carcinoma type which arises secondarily in the background of a pre-existent benign pleomorphic adenoma. It is still poorly understood how and which benign pleomorphic adenoma cells transform into its malignant form, carcinoma ex pleomorphic adenoma. METHODS: We have established five cell systems from a benign pleomorphic adenoma of the parotid gland of a 61-year-old woman. They were characterized by immunofluorescence, classical cytogenetics, p53 gene mutational analysis, fluorescence in-situ hybridization, and histopathological and immunohistochemical examinations of their xenografts, to demonstrate their potency of secondary transformation. RESULTS: We established and characterized five cell systems (designated as SM-AP1 to SM-AP5) from a benign pleomorphic adenoma of the parotid gland. SM-AP1 to SM-AP3 showed polygonal cell shapes while SM-AP4 and SM-AP5 were spindle-shaped. SM-AP1-3 cells were immunopositive for keratin only, indicating their duct-epithelial or squamous cell differentiation, while SM-AP4/5 cells were positive for both keratin and S-100 protein, indicating their myoepithelial cell differentiation. Chromosome analyses showed numeral abnormalities such as 5n ploidies and various kinds of structural abnormalities, such as deletions, translocations, derivatives and isodicentric chromosomes. Among them, der(9)t(9;13)(p13.3;q12.3) was shared by all five of the cell systems. In addition, they all had a common deletion of the last base G of codon 249 (AGG to AG_) of the p53 gene, which resulted in generation of its nonsense gene product. Transplanted cells in nude mice formed subcutaneous tumors, which had histological features of squamous cell carcinoma with apparent keratinizing tendencies. In addition, they had ductal arrangements or plasmacytoid appearances of tumor cells and myxoid or hyaline stromata, indicating some characteristics of pleomorphic adenoma. CONCLUSION: This study demonstrates in vitro that certain cell types from pleomorphic adenoma are able to clone and survive over a long term and develop subcutaneous tumors in nude mice. The histological features of squamous cell carcinoma from the transplanted cell systems in nude mice might suggest a secondary onset of malignancy from a pre-existing benign adenoma.

A Japanese family with cone-rod dystrophy of delayed onset caused by a compound heterozygous combination of novel CDHR1 frameshift and known missense variants.

We analyzed two siblings in a Japanese family with delayed onset cone-rod dystrophy (CRD) using whole-exome sequencing. A novel frameshift c.1106dup (p.H370Afs*17) variant and a known missense c.2027 T > A (p.I676N) variant in CDHR1 were identified. Both patients shared the same variants, although they displayed a significant difference in disease severity. A meta-analysis of the relationship between the severity and the variant type was performed using the reported cases in the literature and did not reveal a definitive correlation.

Clinical characteristics of a Japanese patient with Bardet-Biedl syndrome caused by BBS10 mutations.

PURPOSE: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by retinal dystrophy, renal dysfunction, central obesity, mental impairment, polydactyly, and hypogonadism. Only limited information on BBS is available from Japanese patients. In addition, there are currently no reports of Japanese patients with BBS caused by BBS10 mutations. The purpose of this study was to present the characteristics of a Japanese patient with BBS caused by BBS10 mutations. PATIENT AND METHODS: The patient was a 22-year-old Japanese woman. Comprehensive ophthalmic examinations, including visual acuity measurements, perimetry, electroretinography (ERG), fundus autofluorescence imaging, and optical coherence tomography, were performed. Trio-based whole-exome sequencing was performed to identify potential pathogenic mutations, confirmed by Sanger sequencing. RESULTS: The patient showed neither renal malformation nor dysfunction, and visual impairment seemed to be relatively mild for BBS. The fundus examination revealed diffuse retinal degeneration without pigmentary deposits, and ERG scans showed undetectable responses. She had a history of surgically corrected polydactyly, and displayed symptoms of obesity. There was also a menstrual irregularity that could require progestin administration. Genetic analysis revealed compound heterozygous BBS10 mutations in the patient: a novel missense mutation c.98G>A [p.(G33E)], and a novel nonsense mutation c.2125A>T [p.(R709*)]. CONCLUSION: To our knowledge, this is the first description of a Japanese patient with BBS caused by BBS10 mutations. The clinical characteristics of our patient were mild, as neither renal impairment nor legal blindness was observed. Early diagnosis would play a role in providing counseling, and in some cases, therapeutic interventions for BBS patients.

Visual Outcomes in Japanese Patients with Retinitis Pigmentosa and Usher Syndrome Caused by USH2A Mutations.

PURPOSE: EYS and USH2A are the most common causative genes for retinitis pigmentosa (RP) in Japan. We determined the clinical outcomes for USH2A-related non-syndromic RP or Usher syndrome type II (USH2). METHODS: Two non-syndromic RP and 11 USH2 patients with previously identified USH2A mutations were included. Their complete history and medical records were collected using standard procedures. Visual fields and acuity were compared with those of patients with EYS mutations. Clinical analyses were based on ophthalmic and otolaryngologic examinations. RESULTS: In all patients, the fundus displayed changes typical of RP. Most patients showed relatively well-preserved visual acuity in their thirties or forties, with rapid deterioration in their fifties. Concentric constriction started in the twenties or thirties, and no effective residual visual field was observed after the fifties. CONCLUSIONS: The visual outcome for non-syndromic RP or USH2 patients with USH2A mutations is consistent with that for RP patients with EYS mutations.