Kumiko Yanagi

The history of human populations in the Japanese Archipelago inferred from genome-wide SNP data with a special reference to the Ainu and the Ryukyuan populations

The Japanese Archipelago stretches over 4000 km from north to south, and is the homeland of the three human populations; the Ainu, the Mainland Japanese and the Ryukyuan. The archeological evidence of human residence on this Archipelago goes back to >30 000 years, and various migration routes and root populations have been proposed. Here, we determined close to one million single-nucleotide polymorphisms (SNPs) for the Ainu and the Ryukyuan, and compared these with existing data sets. This is the first report of these genome-wide SNP data. Major findings are: (1) Recent admixture with the Mainland Japanese was observed for more than one third of the Ainu individuals from principal component analysis and frappe analyses; (2) The Ainu population seems to have experienced admixture with another population, and a combination of two types of admixtures is the unique characteristics of this population; (3) The Ainu and the Ryukyuan are tightly clustered with 100% bootstrap probability followed by the Mainland Japanese in the phylogenetic trees of East Eurasian populations. These results clearly support the dual structure model on the Japanese Archipelago populations, though the origins of the Jomon and the Yayoi people still remain to be solved.

Mutations in CD96, a Member of the Immunoglobulin Superfamily, Cause a Form of the C (Opitz Trigonocephaly) Syndrome

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

Opitz trigonocephaly C syndrome in a boy with a de novo balanced reciprocal translocation t(3;18)(q13.13;q12.1)

Opitz trigonocephaly C syndrome (OTCS) is a multiple congenital anomaly syndrome characterized by trigonocephaly, mental retardation, a typical facial appearance, redundant skin, joint and limb abnormalities, and visceral anomalies. We describe a patient with the manifestations of OTCS who also had a de novo balanced reciprocal translocation t(3;18)(q13.13q12.1). His phenotype is a mild form with mild developmental delay and no severe visceral anomalies. Our findings suggest the possible existence of a new locus responsible for OTCS either on 3q13.13 or 18q12.1.

Identification of Four Novel Synonymous Substitutions in the X-Linked Genes Neuroligin 3 and Neuroligin 4X in Japanese Patients with Autistic Spectrum Disorder

Mutations in the X-linked genes neuroligin 3 (NLGN3) and neuroligin 4X (NLGN4X) were first implicated in the pathogenesis of X-linked autism in Swedish families. However, reports of mutations in these genes in autism spectrum disorder (ASD) patients from various ethnic backgrounds present conflicting results regarding the etiology of ASD, possibly because of genetic heterogeneity and/or differences in their ethnic background. Additional mutation screening study on another ethnic background could help to clarify the relevance of the genes to ASD. We scanned the entire coding regions of NLGN3 and NLGN4X in 62 Japanese patients with ASD by polymerase chain reaction-high-resolution melting curve and direct sequencing analyses. Four synonymous substitutions, one in NLGN3 and three in NLGN4X, were identified in four of the 62 patients. These substitutions were not present in 278 control X-chromosomes from unrelated Japanese individuals and were not registered in the database of Single Nucleotide Polymorphisms build 132 or in the Japanese Single Nucleotide Polymorphisms database, indicating that they were novel and specific to ASD. Though further analysis is necessary to determine the physiological and clinical importance of such substitutions, the possibility of the relevance of both synonymous and nonsynonymous substitutions with the etiology of ASD should be considered.

Neurobehavioral disorders in patients with Aarskog–Scott syndrome affected by novel FGD1 mutations

Aarskog–Scott syndrome (AS) is an X-linked disorder characterized by short stature, dysmorphic facial appearance, brachydactyly, and shawl scrotum. Although mental retardation is not frequent in patients with the syndrome, this condition has been described in some patients. In particular, attention deficit/hyperactivity disorder has been reported as a common characteristic of AS [Fryns, 1992]. We here describe two unrelated boys with the syndrome, and neurobehavioral, psychiatric disorder, both with a FGD1 mutation (Table I). Patient 1, a 13-year-old boy, had typical clinical manifestations of AS and borderline intelligence (IQ 71). He had difficulty sustaining attention, was easily distracted, and often forgot to bring texts to the school. He also had symptoms of hyperactivity or impulsivity, that is, great difficulty remaining seated in the classroom or in the outpatient department, and acting without thinking. His hyperactivity was pointed out before age 6 years. He thus fulfilled the diagnostic criteria for attention deficit/hyperactivity disorder by the Diagnostic and Statistical Manual for Mental Disorders [DSM, 1994]. His neurobehavioral symptoms are improved by methylphenidate treatment. Patient 2, a 4-year-old boy, had clinical manifestations of suggestive of AS with normal intelligence (Table I). His physical development was normal. He had qualitative impairment in social interaction (i.e., great difficulty in understanding non-verbal signs, intentional actingof people’s unwilling things, giving a lot of ‘wh’ questions), and behavioral oddities (i.e., having clear and precise memory of a way once going, persistence of a position of stealing wheel). His neurobehavioral symptoms meet DSM-IV criteria for Asperger syndrome completely. In order to have molecular evidence of the disease for these two patients, we analyzed them for all exons of the FGD1, NLGN3, and NLGN4 genes after obtaining informed consent from each family. The materials and methods are described previously [Yanagi et al., 2004]. In brief, DNA was isolated from leukocytes of the patients using a standard protocol. Exons and intron/exon boundary regions of FGD1, NLGN3, and NLGN4 were amplified by PCR (primer sequences and PCR conditions are available upon request). PCR product was purified by Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). Then, direct sequencing was performed using a DyeTerminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 310 sequencer (Applied Biosystems). Patient 1 had a missense mutation (1,327G>T) in exon 6 of FGD1, resulting in an arginine-to-leucine change (R433L) within the RhoGEF domain of FGD1 protein. This mutation was not found in 120 control chromosomes analyzed. Patient 2 had a non-sense mutation (2,221G>T) in exon 15 of the gene, which

A commentary on the promise of whole-exome sequencing in medical genetics.

The dawn of next-generation sequencers (NGSs) and innovative sequencing technologies have brought a paradigm shift in medical research and clinical practice. Furthermore, the cost reduction of NGSs enables personalized medicine to come to fruition. However, whole-genome sequencing (WGS) remains expensive when applied to personal genome analysis. WGS generates a large amount of data that requires highperformance computer processing. Targeted whole-exon capture and sequencing [wholeexome sequencing (WES)] is more costeffective when compared with WGS because exons represent only B1–2% of the genome and also higher sequence coverage can be achieved by NGSs. In addition, most Mendelian disorders are caused by exonic mutations or splice-junction mutations, and protein-coding genes harbor B85% of the mutations that have large effects on diseaserelated traits.1 Thus, WES will provide many advantages and lower costs than WGS when analyzing personal genomes. WES was first successfully used in 2010 to discover the gene responsible for Miller syndrome, a Mendelian disorder.2 Since then, WES has been increasingly used as a fast and accurate genomic discovery approach to investigate both rare genetic disorders and common diseases. WES is widely applied across different areas of medicine, because it has the added advantage of reduced cost and requires analysis of a much smaller but essential dataset when compared with WGS. In addition, recent clinical molecular diagnostics have used WES to detect heterogeneous Mendelian diseases.3,4 A recent review of WES approaches in medical genetics describes the usefulness of WES in medicine and medical research and the impact of WES on clinical diagnoses.5 WES approaches have greatly facilitated the discovery of candidate genes or gene variants in Mendelian disorders and rare variants in common diseases and genomic characterization in cancer. Currently, WES is increasingly being applied to disease gene discovery, cancer typing and molecular diagnosis.5 Presently, WES is an essential tool in medical genetics, especially in the research of Mendelian disorders. WES or multigene tests using NGSs are widely applied to heterogeneous disorders including deafness or ciliopathy.5,6 WES is also being increasingly applied to genetic testing for undiagnosed patients.4,5 Yang et al.4 performed WES in undiagnosed patients whose phenotypes were suggestive of potential genetic disorders and achieved a molecular diagnosis for 62 of 250 (25%) patients. Because WES detects individual genetic variation, it can be used to construct a variation database of anthropic and ethnic populations. At the same time, because WES can detect groups of genetic variations that are unrelated to the indication for the first diagnostic purpose but are of medical value for individual patient care, such ‘incidental findings’ pose potential ethical problems that should be strongly considered and discussed in clinical practice.5,7 WES is a widely applied technique in medical genetics that is capable of detecting variations in whole exons. However, in practical use, understanding WES methodology and limitations are important. Current WES techniques are not capable of detecting all of the variations surrounding exons. Detecting variation by WES is limited by the experimental methods, probe coverage and/or platforms used.8–10 Hence, WES may not always detect pathogenic or causative variations in a genetic disease. In addition, because WES is a method to detect genomic sequence variations, when a candidate of causative variation in the disease is detected, it requires verification or support by secondary analyses. In particular, further functional analyses are important to confirm whether the variant is pathogenic or benign. Nevertheless, WES enables the unprecedented low cost and highly efficient analysis of whole exons. WES can be easily used to comprehensively detect individual variations in exons. It is without doubt that WES is a powerful tool in genome analysis, and it greatly progresses medical genetics. Although WESs’ limitations need to be overcome, we anticipate that WES will be used not only in medical research but also in clinical practice for example, molecular diagnosis (whole-gene test) and personal genomics before WGS becomes a common place in medical genetics. Thus, a paradigm shift in medicine by advancement in both WES and WGS is expected to continue.

Novel alternative splicing of human faciogenital dysplasia 1 gene.

ABSTRACT  The human faciogenital dysplasia 1 (FGD1) gene product plays an important role in morphogenesis. Its dysfunction causes Aarskog–Scott syndrome (MIM ♯305400). To characterize the FGD1, we investigated its expression by RT–PCR and Southern blot analysis in normal tissues. We found novel alternative forms of the FGD1. One has a novel exon located in intron 8, named exon 8B (8B FDG1) and the other has an exon in intron 7, exon 7B (7B FGD1). The 8B FDG1 is expressed strongly in the brain, testis, spinal cord, trachea and stomach, and weakly in the thymus and lymphocytes. However, expression of the 7B FGD1 is weak and restricted in the testis and salivary gland. Insertion of each novel exon results in production of a premature termination codon, respectively, and the predicted proteins generated from them have only a proline‐rich domain and an incomplete DH domain which potentially compete with the wild type of FGD1.

Prenatal diagnosis of X‐linked recessive Lenz microphthalmia syndrome

Lenz microphthalmia syndrome comprises microphthalmia–anophthalmia with mental retardation, malformed ears and skeletal anomalies, and is inherited in an X‐linked recessive pattern. In 2004, it was reported that the missense mutation (BCL‐6 co‐repressor gene [BCOR] c.254C>T, p.P85L) in a single family with Lenz microphthalmia syndrome co‐segregated with the disease phenotype. We report a case of prenatal diagnosis for X‐linked recessive Lenz microphthalmia syndrome with the mutation. A 32‐year‐old gravida 5, para 2 Japanese woman was referred to Nagoya City University Hospital at 15 weeks of gestation. After genetic counseling and informed consent, amniocentesis was performed for fetal karyotyping, which was 46,XY. Using the extracted DNA from cultured amniotic cells, fetal search for BCOR c.254C>T mutation was undertaken. The couple requested medical termination of pregnancy, and the postabortion examination confirmed the diagnosis. This is the third report of a BCOR mutation, associated with X‐linked syndromic microphthalmia, and most importantly, it is always the same mutation. The prenatal genetic diagnosis of the Lenz microphthalmia syndrome allowed time for parental counseling and delivery planning.

Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue

The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco’s Modified Eagle’s Medium, RPMI-1640, and William’s E. Of these, William’s E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.