Nobuyoshi Shimizu

Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism

Parkinsons disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP), maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253 (ref. 4); the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, we have now isolated a complementary DNA clone of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3–7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product ‘Parkin’.

DMY is a Y-specific DM-domain gene required for male development in the medaka fish

Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. Here, we used recombinant breakpoint analysis to restrict the sex-determining region in medaka fish (Oryzias latipes) to a 530-kilobase (kb) stretch of the Y chromosome. Deletion analysis of the Y chromosome of a congenic XY female further shortened the region to 250u2009kb. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only the DM-related PG17 was Y specific; we thus named it DMY. Two naturally occurring mutations establish DMYs critical role in male development. The first heritable mutant—a single insertion in exon 3 and the subsequent truncation of DMY—resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. During normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for testicular development and is a prime candidate for the medaka sex-determining gene.

Role of TBX1 in human del22q11.2 syndrome.

BACKGROUNDnDel22q11.2 syndrome is the most frequent known chromosomal microdeletion syndrome, with an incidence of 1 in 4000-5000 livebirths. It is characterised by a 3-Mb deletion on chromosome 22q11.2, cardiac abnormalities, T-cell deficits, cleft palate facial anomalies, and hypocalcaemia. At least 30 genes have been mapped to the deleted region. However, the association of these genes with the cause of this syndrome is not clearly understood.nnnMETHODSnTo test for the chromosomal deletion at 22q11.2, we did fluorescence in-situ hybridisation analysis with ten probes on 22q11.2 in 235 unrelated patients with clinically diagnosed del22q11.2 syndrome. To investigate mutations in the coding sequence of TBX1, we also did genetic analysis in 13 patients from ten families who have the 22q11.2 syndrome phenotype but no detectable deletion of 22q11.2.nnnFINDINGSn96% (225 of 235) of patients had a defined 1.5-3-Mb deletion at 22q11.2. We identified three mutations of TBX1 in two unrelated patients without the 22q11.2 deletion-one with sporadic conotruncal anomaly face syndrome/velocardiofacial syndrome and one with sporadic DiGeorges syndrome-and in three patients from a family with conotruncal anomaly face syndrome/velocardiofacial syndrome. We did not record these three mutations in 555 healthy controls (1110 chromosomes; p<0.0001).nnnINTERPRETATIONnOur results suggest that the TBX1 mutation is responsible for five major phenotypes in del22q11.2 syndrome. Therefore, we conclude that TBX1 is a major genetic determinant of the del22q11.2 syndrome.

A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene.

Identification of eight members of the Argonaute family in the human genome.

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.

Insertion of β-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (∼68-bp) β-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of β-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

Absence of the Candidate Male Sex-Determining Gene dmrt1b(Y) of Medaka from Other Fish Species

Although the sex-determining genes are known in mammals, Drosophila, and C. elegans, little is known in other animals. Fishes are an attractive group of organisms for studying the evolution of sex determination because they show an amazing variety of mechanisms, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems and modifications thereof. In the fish medaka, dmrt1b(Y) has recently been found to be the candidate male sex-determining gene. It is a duplicate of the autosomal dmrt1a gene, a gene acting in the sex determination/differentiation cascade of flies, worms, and mammals. Because in birds dmrt1 is located on the Z-chromosome, both findings led to the suggestion that dmrt1b(Y) is a non-mammalian Sry with an even more widespread distribution. However, although Sry was found to be the male sex-determining gene in the mouse and some other mammalian species, in some it is absent and has obviously been replaced by other genes that now fulfil the same function. We have asked if the same might be true of the dmrt1b(Y) gene. We find that the gene duplication generating dmrt1b(Y) occurred recently during the evolution of the genus Oryzias. The gene is absent from all other fish species studied. Therefore, it may not be the male-sex determining gene in all fishes.

Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region

We have identified and cloned a novel gene (DGCR8) from the human chromosome 22q11.2. This gene is located in the DiGeorge syndrome chromosomal region (DGCR). It consists of 14 exons spanning over 35kb and produces transcripts with ORF of 2322bp, encoding a protein of 773 amino acids. We also isolated a mouse ortholog Dgcr8 and found it has 95.3% identity with human DGCR8 at the amino acid sequence level. Northern blot analysis of human and mouse tissues from adult and fetus showed rather ubiquitous expression. However, the in situ hybridization of mouse embryos revealed that mouse Dgcr8 transcripts are localized in neuroepithelium of primary brain, limb bud, vessels, thymus, and around the palate during the developmental stages of embryos. The expression profile of Dgcr8 in developing mouse embryos is consistent with the clinical phenotypes including congenital heart defects and palate clefts associated with DiGeorge syndrome (DGS)/conotruncal anomaly face syndrome (CAFS)/velocardiofacial syndrome (VCFS), which are caused by monoallelic microdeletion of chromosome 22q11.2.

The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor

The bodies of most teleost fish species are covered with specialized subepithelial structures known as scales. The scale is an epithelial appendage that differentiates from the dermal mesenchyme. Mammals, on the other hand, have no scales, but instead their bodies are covered with hair. Although their appearances are quite different, scales and hair can be considered structurally similar in that both of them are epithelial appendages distributed over the body surface in an orderly pattern. This analogy suggests that they may have the same evolutionary origin. But, to date, no molecular evidence has been presented that links scales and hair. A mutation at the rs-3 locus of medaka (Oryzias latipes) leads to almost complete loss of scales. We demonstrated that the rs-3 locus encodes ectodysplasin-A receptor (EDAR), which is required for the initiation of hair development in mammals. We identified a novel transposon inserted in the first intron of EDAR, which causes aberrant splicing. This work shows that EDAR is required for scale development in fish and suggests that it is an evolutionarily conserved molecule that is required for the development of epithelial appendages in vertebrates.