J Med Genet 45:479-480 doi:10.1136/jmg.2008.058503
  • Correspondence
    • PostScript

Lack of C20orf133 and FLRT3 mutations in 43 patients with Kabuki syndrome in Japan

  1. H Kuniba1,2,13,
  2. M Tsuda1,13,
  3. M Nakashima1,13,
  4. S Miura1,13,
  5. N Miyake2,13,
  6. T Kondoh2,
  7. T Matsumoto2,
  8. H Moriuchi2,
  9. H Ohashi3,13,
  10. K Kurosawa4,
  11. H Tonoki5,
  12. T Nagai6,13,
  13. N Okamoto7,
  14. M Kato8,
  15. Y Fukushima9,13,
  16. K Naritomi10,13,
  17. N Matsumoto11,13,
  18. A Kinoshita1,13,
  19. K-i Yoshiura1,13,
  20. N Niikawa1,12,13
  1. 1
    Departments of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
  2. 2
    Departments of Pediatrics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
  3. 3
    Division of Medical Genetics, Saitama Children’s Medical Center, Iwatsuki, Japan
  4. 4
    Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan
  5. 5
    Department of Pediatrics, Tenshi Hospital, Sapporo, Japan
  6. 6
    Department of Pediatrics, Dokkyo University School of Medicine Koshigaya Hospital, Koshigaya, Japan
  7. 7
    Department of Planning and Research, Osaka Medical Center and Research Institute for Maternal and Child health, Osaka, Japan
  8. 8
    Department of Pediatrics, Yamagata University School of Medicine, Yamagata, Japan
  9. 9
    Department of Medical Genetics, Shinshu University School of Medicine, Matsumoto, Japan
  10. 10
    Department of Medical Genetics, University of the Ryukyus, Nishihara, Japan
  11. 11
    Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  12. 12
    Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, Tobetsu, Japan
  13. 13
    Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Tokyo, Japan
  1. Dr K-i Yoshiura, Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki 852-8523, Japan; kyoshi{at}
  • Received 23 February 2008
  • Revised 5 March 2008
  • Accepted 6 March 2008

Kabuki syndrome (KS, OMIM 147920), also known as Niikawa–Kuroki syndrome, is a multiple congenital anomalies/mental retardation (MCA/MR) syndrome characterised by a peculiar facial appearance, skeletal abnormalities, joint hypermobility, dermatoglyphic abnormalities, postnatal growth retardation, recurrent otitis media and occasional visceral anomalies. Although some studies have ruled out several loci from the candidacy for KS, any putative disease gene loci or candidate genes remain unidentified.

In a recent issue of the journal, Maas et al1 reported that exon 5 of the C20orf133 gene at 20p12.1 was disrupted by a 250 kb de novo microdeletion in a patient with KS; they also screened for mutations in C20orf133 and FLRT3 (a nested gene located within intron 3 of C20orf133) in 19 additional patients with KS, but failed to detect such mutations or deletions in any of them. It remains unclear whether the two genes are responsible for the pathogenesis of KS, and if so, how frequently the deletion at the locus is found in KS patients. Herein we describe the results of a deletion assay for the exon 5 in C20orf133 and a mutation analysis of C20orf133 and FLRT3 among 43 patients with KS in Japan. In addition, we also show the results of a copy number analysis at 20p12.1 by Human Mapping 250K Nsp Array among 18 patients with KS in Japan.

Ethics approval for this study was obtained from the Committee for the Ethical Issues on Human Genome and Gene Analysis in Nagasaki University. The subjects studied consisted of 43 patients (20 girls and 23 boys) with KS from Japan for mutation analysis and a deletion assay, and of 18 patients (nine girls and nine boys) with KS for copy number analysis. Genomic DNA was isolated by the standard method. Genomic sequences were retrieved from the UCSC genome browser. GenBank accession numbers of NCBI of C20orf133 and FLRT3 are NM_080676 and NM_198391, respectively.

C20orf133 and FLRT3 were directly sequenced. Polymerase chain reaction (PCR) primers used for the two genes were those described by Maas et al,1 but as some primers did not work in our laboratory, we newly designed the primes. Direct sequencing of C20orf133 and FLRT3 did not show any pathogenic nucleotide changes in the 43 patients. Observed five nucleotide changes in the patients were all found in normal Japanese individuals as well—that is, a first substitution, c.173C>T (p.T58I) in C20orf133, already registered in the database of single nucleotide polymorphisms (SNPs) as rs2990505, was identified in 14 patients (12 heterozygous and two homozygous status). A second substitution, c.1069T>C (p.S357P) in C20orf133, not registered in the database, was observed in 10 patients (heterozygous status) and in 35 of 159 normal Japanese individuals (29 heterozygous and six homozygous status). A third substitution, g.14257801T>C in exon 2 (5′UTR exon) of FLRT3, found in nine patients (heterozygous status), was registered as rs761998. A fourth substitution, c.765A>G (p.Q255Q) in FLRT3, not registered in the database, was found in one patient (heterozygous status) and in three of 137 normal Japanese individuals (heterozygous status). The last nucleotide change, heterozygous deletions of three nucleotides, g.14257934_14257936delCAG in exon 2 (5′UTR exon) of FLRT3, not registered in dbSNP, was found in nine patients, and in four of 81 normal Japanese individuals.

Deletion assay involving exon 5 of C20orf133 was performed by quantitative real-time PCR on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, California, USA). ALB gene was chosen as a reference gene,2 which had no copy number polymorphism (CNP) in the Database of Genomic Variants (DGV, Primers and fluorogenic probes were designed with the assistance of Primer Express v1.5 (Applied Biosystems). Primer sequences are available on request. The quantitative PCR indicated that none of the 43 patients had any copy number changes involving C20orf133 exon 5. The average quotient (SD) of the target/reference genes in patients was 1.090 (0.124) with a range (SD) of 0.934–1.291 (0.025–0.426), and the control persons as well (data not shown).

According to the DGV (last updated 29 Nov 29 2007), a ∼368 kb deletion (chromosome 20, nucleotide numbers (nt) 14,606,364–14,974,100 bp), involving exon 5 of C20orf133 (nt 14,613,489–14,613,605), has been reported in one of 506 unrelated healthy Northern German and 270 HapMap individuals (registered as Variation_9315, a normal loss).3 Thus, it is possible that the 250 kb deletion at 20p12.1 in a KS patient reported by Maas et al1 was a rare copy number variation. However, some CNPs may possibly play more important roles in human phenotypic variations than SNPs.4 5 For example, Balikova et al reported a unique novel syndrome caused by the amplification of large genomic regions, ∼750-kb at cytoband 4p16, known to be copy number variation.6 Therefore, we must be careful to check copy number changes for an MCA/MR syndrome.

To search particular microdeletion/duplication at cytoband 20p12.1, we performed copy number analysis among 18 patients with KS by DNA oligomicroarray hybridisation using the GeneChip Human Mapping 250 K Nsp Array (Affymetrix, Santa Clara, California, USA). Data at the target region were analysed using GTYPE (GeneChip Genotyping Analysis Software), CNAT (GeneChip Chromosome Copy Number Analysis Tool) and Partek Genomic Suite v6.3 (Partek Inc, St Louis, Missouri, USA). Two copy number changes were found among 18 patients. Neither of them was registered in the DGV, but they were less likely to be pathogenic because both of them were within intronic sequences—that is, a ∼100 kb deletion within intron 5 of C20orf133 was detected in one patient (patient 3 in fig 1). Its physical positions and log2 ratios were: chromosome 20, nt 15,000,514 bp (logR −0.4136), 15,014,439 (−0.4586), 15,034,442 (−0.4831), 15,066,513 (−0.4519), and 15,102,706 (−0.4330). In another patient (patient 14 in fig 1), a ∼30 kb region was suggested as duplication and the region was located within intron 4 of the C20orf133. The physical positions and log2 ratios were: chromosome 20, nt 14,527,943 bp (LogR 0.4113), 14,553,038 (0.4443), 14,557,957 (0.4204) and 14,563,924 (0.3878). The other 16 patients with KS did not show significant copy number changes at the region. Thus, particular copy number changes at the region were not detected in these patients with KS.

Figure 1 Copy number analysis at 20p12.1 among 18 patients with Kabuki syndrome. Each patient in log2 ratio plot is shown by a different colour. A bar of Heat Map representing gain for red and loss for green is divided in each patient. Deletion at intron 5 of C20orf 133 is indicated in patient 3. Duplication at intron 4 of C20orf133 is suggested in patient 14 (arrows in logR plot; deep green or deep red in HeatMap).

In summary, we performed a mutation analysis for C20orf133 and FLRT3, a deletion assay for exon 5 of C20orf133 in 43 patients with KS and a copy number analysis by DNA oligomicroarray among the 18 patients with KS in Japan. These studies did not reveal pathogenic alterations in the patients. Therefore, our findings unfortunately could not support the working hypothesis that the C20orf133 and/or FLRT3 were the causative gene in most Japanese KS patients.


We are very grateful to the patients and their parents for their participation in this research. We also thank Ms Yasuko Noguchi, Ms Ayano Goto, and Ms Miho Ohga for their technical assistance. NN was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan, and was supported by SORST from Japan Science and Technology Agency (JST) (Nos. 17019055 and 19390095, respectively). KY was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare.


  • Competing interests: None declared.

  • Ethics approval: Ethics approval for this study was obtained from the Committee for the Ethical Issues on Human Genome and Gene Analysis in Nagasaki University.