Introduction

Kabuki syndrome (KS, OMIM 147920), also known as Niikawa–Kuroki syndrome, is a multiple congenital anomaly/mental retardation (MCA/MR) syndrome characterized by a distinct facial appearance, skeletal abnormalities, joint hypermobility, dermatoglyphic abnormalities, postnatal growth retardation, recurrent otitis media and occasional visceral anomalies.1, 2 The prevalence was estimated to be 1/32 000 in Japan3 and 1/86 000 in Australia and New Zealand.4 Although most cases were sporadic, at least 14 familial cases have been reported. It is assumed that KS is an autosomal dominant disorder, considering the equal male-to-female ratio of patients and parent–child transmission pattern in some familial cases.5

The cause of KS remains unknown, even though at least 400 patients have been diagnosed in a variety of ethnic groups since 1981.3, 4, 5, 6, 7 Some works have ruled out several loci; for example, 1q32–q41, 8p22–p23.1 and 22q11, as candidates for KS.8, 9, 10, 11, 12, 13 A study of array-based comparative genomic hybridization (CGH) showed a disruption of the C20orf133(MACROD2) gene by 250 kb deletion in a patient with KS,14 but the following mutation screening for the gene failed to find a pathogenic base change within exons in 19 other patients with KS14 and in 43 Japanese patients.15 Another study of array CGH with 0.5–1.2 Mb resolution reported that 2q37 deletions were detected in two patients with Kabuki-like features, but their facial features were not typical for KS.16 To date, no concordant specific lesion has been found by whole-genome scan with array CGH in a bacterial artificial chromosome (BAC) clone with 0.5–1.5 Mb resolution.16, 17, 18

Chromosomal aberration analysis by high-resolution oligonucleotide array technologies in recent years, called molecular karyotyping, enables us to detect submicroscopic pathogenic copy number alterations, which were undetectable even by BAC array CGH.19, 20 As not a few MCA/MR syndromes are because of chromosomal copy number aberration, we hypothesize that some sort of microdeletion/microduplication causes KS. Herein, we report the results of molecular karyotyping in 17 patients using GeneChip 250K array and those of mutation screening of candidate genes in 41 patients with KS in Japan.

Materials and methods

Subjects

The subjects for molecular karyotyping consisted of 18 patients (nine girls and nine boys) at entry. The subjects for mutation screening consisted of 41 patients (20 girls and 21 boys), including the aforementioned 18 patients. The diagnoses of KS were confirmed by experts of clinical genetics, although written permission for the use of facial photographs in publications was not obtained. These Japanese patients showed a normal karyotype at a 400-band level, and were earlier reported with no pathogenic genome copy number change by 1.5 Mb-resolution BAC array CGH.18 Genomic DNA was isolated by the standard method from their peripheral blood leukocytes or in part from their lymphoblastoid cell lines. Experimental procedures were approved by the Committee for the Ethical Issues on Human Genome and Gene Analysis at Nagasaki University.

Molecular karyotyping

DNA oligomicroarray hybridization, using the GeneChip Human Mapping 250K Nsp Array (Affymetrix, Santa Clara, CA, USA), was carried out for 18 patients with KS, following the provided protocol (Affymetrix). Data were analyzed using GTYPE (GeneChip Genotyping Analysis Software) to detect copy number aberration and visualized using CNAG (Copy Number Analyser for GeneChip) version 3.21 References for non-paired analysis of CNAG were chosen from eight unrelated individuals of HapMap samples from the Affymetrix website (http://www.affymetrix.com/support/). The resolution of this procedure was estimated as 30–100 kb. CNAG version 3 was linked with the University of California Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu/) assembly May 2004, and then its physical position was referred to the data assembly on March 2006 in the UCSC genome browser after adjustment.

Validation of deletion

Quantitative PCR (qPCR) analysis to validate deletions was run on a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany) using an intercalating dye, SYTO9 (Molecular probes, OR, USA), which is an alternative to SYBR green I.22 Absolute quantification was carried out using a second derivative max method. A standard curve of amplification efficiency for each set of primers was generated with a serial dilution of genomic DNA. A corrected gene dosage was given as the ratio of a target gene divided by an internal control gene. The copy number was obtained from a calibration under the assumption that the control genome was diploid.

Target genes of copy number aberration were as follows: SUMF1 (for patient K9); MAMDC2 (for patient K16); and CETN1 (for patient K34). The primer sequences of these genes are available in the online supplementary file. Internal control diploid genes were OAZ2 and USP21. Primer sets of the control genes for genomic DNA were selected from the Real Time PCR Primer Sets website (http://www.realtimeprimers.org/). The control genes were confirmed to have no copy number variants on the Database of Genomic Variants (DGV) updated on 26 June 2008 (http://projects.tcag.ca/variation/). BLAST searches confirmed all primer sequences specific for the gene.

Samples were analyzed in triplicate in a 384-well format in a 10 μl final volume containing about 2 ng genomic DNA, 0.5 μM forward primer, 0.5 μM reverse primer, 0.1 Units TaKaRa ExTaq HS version (TaKaRa, Kyoto, Japan), 1 × PCR buffer, 200 μM dNTP and 0.5 μM SYTO9. The amplification conditions consisted of an initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 10 s and extension at 72 °C for 15 s The data were analyzed using LightCycler 480 Basic Software (Roche Diagnostics) and the melting curve was checked to eliminate non-specific products from the reaction.

Mutation screening of candidate genes

Candidate genes, identified within a detected deletion, consisted of four genes: TRPM3 (NM_001007471 and NM_206946), KLF9 (NM_001206), SMC5 (NM_015110) and MAMDC2 (NM_153267) located at 9q21.12–q21. 11. The entire coding region and splice junctions of the genes were sequenced on an automated sequencer 3130xl (Applied Biosystems, Foster City, CA, USA) using BigDye version 3.1 (Applied Biosystems). Genomic sequences were retrieved from the UCSC genome browser (assembly: March 2006). PCR primers were designed with the assistance of Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). The primer sequences are available in the online supplementary file. Resultant electropherograms were aligned using ATGC version 3.0 (Software Development, Tokyo, Japan) and inspected visually to find DNA alterations.

In silico analysis

Relations among deleted genes were assessed using online software, PANTHER (Protein Analysis Through Evolutionary Relationships, http://www.pantherdb.org), to determine whether the genes involve some developmental pathway or biological process.23 The novel synonymous base substitutions found in the mutation screening were examined for their potential activation of the cryptic splice site by comparison between wild-type allele and mutated allele using the GeneSplicer program (http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml).

Results

Molecular karyotyping and validation of deletion

The entries of molecular karyotyping were 18 patients with KS (K1, K3, K5, K6, K7, K8, K9, K11, K12, K13, K16, K18, K20, K21, K22, K23, K34 and K38). We eliminated the data of patient K3 from copy number analysis, because it showed low quality data; that is, a single-nucleotide polymorphism (SNP) call rate of 82.51% and a quality control performance detection rate of 74.09%, probably because of DNA degradation during long-term storage. The other patients showed high call rates, enough for copy number analysis (SNP call rate of 90.07–97.72% and detection rate of 91.52–99.77%). We identified nine deleted regions, the lengths of which were between 35 kb and 1.27 Mb, and nine duplicated regions, of lengths between 72 and 495 kb, in the 17 patients analyzed (Table 1). As for the nine duplications detected, five of them were concordant to several observed gains in DGV, and four of them in each patient did not contain any known genes.

Table 1 Detected genomic copy number aberrations in 17 patients with Kabuki syndrome

It is interesting that the deleted region of 9q21.11–q21.12 (1.27 Mb in patient K16), which had not been registered in DGV, harbored four known genes: transient receptor potential cation channel, subfamily M, member 3 (TRPM3), Kruppel-like factor 9 (KLF9), structural maintenance of chromosomes protein 5 (SMC5) and MAM domain containing 2 (MAMDC2) (Figure 1d). The deletion of 3p26.2 (205 kb in patient K9, Figure 1a) had involved a non-coding exon of the SUMF1 gene. The deletion of 18p11.32 (35 kb in patient K34, Figure 1b) containing the CETN1 gene had one registration in DGV as Variation_5044, which described only one observed loss and 14 observed gains in 95 individuals. The deletion of 4q13.2 (1.26 Mb in patient K23, Figure 1c) and 20p12.1 (152 kb in patient K6) did not carry any coding exon of any gene. The regions of 14q11.2 (116 kb in patient K5) and 15q11.2 (972 kb in patient K1 and K23) were non-pathological deletions with as many registrations as observed losses in DGV.

Figure 1
figure 1

Chromosome view of Copy Number Analyser for GeneChip (CNAG) analysis. Each dots represent fluorescent intensity on each single-nucleotide polymorphisms (SNP) probe of GeneChip 250K NspI array (Affymetrix). Solid lines indicate copy number analyzed with CNAG. Arrows show detected deletions. (a) Chromosome (Ch) 3 of patient K9, 205 kb deletion in 3p26.2 involving an exon of SUMF1gene. (b) Chromosome 18 of patient K34, 35 kb deletion in 18p11.32, containing the CETN1 gene. (c) Chromosome 4 of patient K23, 1.26 Mb deletion in 4q13.2, not involving any known gene. (d) Chromosome 9 of patient K16, 1.27 Mb deletion in 9q21.11–q21.12, harboring four genes: TRPM3, KLF9, SMC5 and MAMDC2. The University of California Santa Cruz genome browser denotes the cytobands, genes and probe setting of Affymetrix 250K NspI array within the region. No copy number variation was registered here in the Database of Genomic Variants updated 26 June 2008. FISH, fluorescent in situ hybridization.

To validate the deletion of the detected region, we confirmed the loss of heterozygosities of the SNP probes present there using GTYPE (data not shown) and carried out qPCR. The regions of SUMF1 on 3p26.2 (for patient K9) and of MAMDC2 on 9q21.11–q21.12 (for patient K16) had one copy in each patient compared with those in unaffected individuals (Figure 2). The deletion of CETN1 on 18p11.32 (for patient K34) was inherited from his unaffected mother. As samples from the parents of patient K16 were unavailable, it was not possible to examine whether the deletion of 9q21 was de novo. But the deletion was not found in 95 normal Japanese individuals using qPCR (data not shown).

Figure 2
figure 2

Validation of deletion with quantitative PCR (qPCR). qPCR confirmed a loss of one copy in each patient: SUMF1 at 3p26.2 for patient K9; MAMDC2 at 9q21.1 for patient K16; CETN1 at 18p11.32 for patient K34. The deletion of patient K34 was inherited from his unaffected mother. UA, unaffected individual. Error bars, s.d.

As a consequence of this copy number analysis, we considered the next four genes as candidate genes for KS: TRPM3, KLF9, SMC5 and MAMDC2.

Mutation screening and in silico analysis

Table 2 shows the results from mutation screening of the four candidate genes in 41 patients with KS. Ten base substitutions were found in the 41 patients, consisting of six registered SNPs, one unregistered SNP and three silent mutations. In addition, SUMF1 (NM_182760) and CETN1 (NM_004066) were also screened, but no mutations were detected (data not shown).

Table 2 Mutation screening of candidate genes in 41 patients with Kabuki syndrome

We checked the three silent mutations for splice site alteration using the GeneSplicer program, but no activation of the cryptic splice site was predicted. Although PANTHER classification of the four candidate genes did not show significant correlation for biological processes or pathway because of its small scale in number, some genes associated with developmental biology; that is, DNA repair (SMC5) and mRNA transcription regulation (KLF9).

Discussion

We used high-resolution oligonucleotide array of GeneChip 250K NspI with a resolution of 30–100 kb and tried to find causative deletions or mutated genes for KS. Our molecular analysis did not strongly identify the causative gene for KS, but we identified a locus that possibly contributed to KS.

The deletion in patient K16, with a length of 1.27 Mb at 9q21.11–q21.12, harbored four known genes: TRPM3, KLF9, SMC5 and MAMDC2 (Figure 1d). Unfortunately, her parents' DNAs were unavailable, but the region is unlikely to be a copy number variant (CNV) because it has not been known as CNV in DGV; moreover, the deletion was not found in 95 normal Japanese individuals using qPCR.

As mutation screening in the 41 patients with KS showed no pathogenic base substitution in these genes, we cannot state that these genes are major genetic factors for KS. However, it is presumable that the genes have some etiological roles for KS because of its genetic heterogeneity. Ontology of the PANTHER classification suggested that the three genes were associated with developmental biology, such as mRNA transcription regulation. Moreover, the 1.27 Mb region of 9q21 was included in an earlier reported candidate locus of cleft lip/palate by meta-analysis of linkage analysis.24 Patient K16 actually had velopharyngeal insufficiency because of a submucous cleft palate. Therefore, it is reasonable to consider that the deleted genes cooperated with the development of a cleft palate, which is often accompanied by KS.

Although the 152 kb deletion within intron 5 of C20orf133 (MACROD2) in patient K6 did not involve any coding exon and her parents' DNAs were unavailable, the deletion was neither registered as CNV in DGV nor was it found in 95 normal Japanese individuals by qPCR (data not shown). Maas et al.14 reported de novo 250 kb deletion, including exon 5 of C20orf133 (MACROD2), in a patient with KS. Direct sequencing for the gene in 62 other patients with KS did not detect mutations,14, 15 but the gene may be one of the causative genes for KS in consideration of its genetic heterogeneity.

We focused this study on KS on deletion/duplication detected using oligonucleotide array and mutation screening of the coding genes within the region. One limitation of this study is its resolution. As a matter of course, a higher resolution array can detect smaller genomic rearrangements, which were undetectable in the same patient, as we showed here compared with an earlier study of BAC array CGH.18 Although SNP probes are useful to examine loss of heterozygosity as a collateral evidence in deletions, unevenly distributed probes of the SNP array have a disadvantage for CNV detection. As various platforms on oligonucleotide array have developed, higher resolution platforms will have to be applied to search tiny genomic rearrangements in patients with KS. Another limitation is that we assumed that a single copy number change caused KS. It remains to be elucidated whether CNV association25 contributes towards manifestations of KS. If further investigation with refined array technologies cannot find the etiology of KS, the direction of study for KS will have to be changed to find de novo sequence alteration or methylation aberration, including in the non-coding genomic regions.

In summary, we applied molecular karyotyping with GeneChip 250K array to detect copy number aberrations in 17 patients with KS and screened four candidate genes in 41 patients with KS. We could not identify causative DNA alteration for KS, but the locus, 9q21.11-q21.12, including TRPM3, KLF9, SMC5 and MAMDC2, may contribute to the cleft palate of KS. Further investigations will be needed as various array platforms have the potential to specify genomic alterations for KS.