Introduction

Kabuki syndrome (KS, Niikawa–Kuroki syndrome, MIM: 147920) is a rare multiple congenital anomaly/mental retardation syndrome described simultaneously by Niikawa et al1 and Kuroki et al.2 The estimated frequency of this syndrome is about in 1/32 000 in Japan. Thus far, more than 300 patients have been described in the literature.3 The syndrome is characterized by postnatal growth retardation, distinctive facial features, dermatoglyphic anomalies with persistence of fetal fingertip pads, skeletal dysplasia, and mental retardation. In addition, a broad spectrum of clinical findings are observed in KS patients such as cardiac and kidney malformations, autoimmune thrombopenia and anemia, growth hormone deficiency and premature thelarche (review in Matsumoto and Niikawa4).

The syndrome is usually sporadic. An autosomal dominant mode of inheritance has been proposed in KS because of vertical transmission in several families, a nonincreased rate of consanguinity and a normal sex ratio.5 The multisystemic nature of KS suggests that it might be caused by a microdeletion or microduplication affecting several genes or by a single gene that regulates various target genes.

Recently, Milunsky and Huang6 using comparative genomic hybridization and BAC-FISH studies reported six unrelated patients with KS and an 8p22–8p23.1 duplication. The duplicated region is comprised between BAC clones RP11-112G9 and RP11-92C1. It is approximately 3.5 Mb in size. Furthermore, they reported the presence of a paracentric inversion involving BAC RP11-122N11 in all six patients and the two mothers studied. This inversion is not contiguous to the duplication. In one out of 20 controls a larger inversion was found.

Materials and methods

We collected a series of 24 patients with a clinical diagnosis of KS. They were ascertained through medical genetics units. They met the KS clinical criteria described by Niikawa et al1 and Kuroki et al,2 namely postnatal short stature, mental retardation, skeletal anomalies, dermatoglyphic anomalies, and characteristic facial features (Figure 1). The clinical data are presented in Table 1. All patients were born to unrelated parents and are sporadic cases. Cardiac, renal, ophthalmologic, audiometric, and metabolic investigations including plasma aminoacids, as well as urine organic acids chromatography, and a protein glycosylation test were carried out in all patients. Appropriate ethical approval and written informed consent were obtained. Out of these 24 patients, 10 were published previously (Patients 1, 3, 4, 7, 10, 11, 13, and 14).7, 8, 9

Figure 1
figure 1

Distinctive facial features for cases 1, 2, 8, and 20.

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Table 1 Clinical findings in our 24 KS patients compared to the literature data
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Standard and high-resolution chromosome analyses were obtained from cultured peripheral blood lymphocytes. FISH experiments were performed as described previously.10

All patients were studied with BACs RP11-235F10, RP11-252K12, RP11-31B7 to detect the 8p duplication and BAC RP11-122N11 to study the paracentric inversion as described by Milunsky and Huang.6 In addition, we used in six patients BACs RP11-11P7, RP11-140K14, RP11-112G9, RP11-92C1, RP11-23H1, and RP11-141K9 that are other clones included in the 8p22–8p23.1 duplication. For each patient we counted 10 metaphases and 10 nuclei.

Results

All patients had a normal karyotype at a 500–850 bands resolution level. In all KS patients only one signal on each chromosome was observed using BAC RP11-235F10, RP11-252K12, and RP11-31B7 on metaphases as well as on interphase nuclei (Figure 2a and b). Also no duplication was detected using BACs RP11-11P7, RP11-140K14, RP11-112G9, RP11-92C1, RP11-23H1, and RP11-141K9. Regarding the postulated paracentric inversion, two signals on each homologous chromosome, or two signals on one chromosome and one on the other were observed using BAC RP11-122N11 hybridization on interphase nuclei. Same results were obtained in phenotypically normal controls.

Figure 2
figure 2

FISH results for cases 1 and 8. (a) BACs RP11-252K12 (green) and RP11-31B7 (red) on metaphases (upper) and interphase nuclei (lower). Only two signals were observed for each probe. These two probes are duplicated in patients reported by Milunsky and Huang. (b) BACs RP11-122N11 (green) and RP11-235F10 (red) on metaphase (upper) and interphase nuclei (lower). Only two signals were observed for the BAC RP11-235F10. The BAC RP11-122N11 may generate two signals on each homologous chromosome (right) or on one chromosome (left). This most likely corresponds to a polymorphism (see text).

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Discussion

The aim of the present study of KS patients was to determine the frequency of an 8p22–8p23.1 duplication observed previously by Milunski and Huang.6 Contrary to these authors, we were not able to detect any 8p duplication in our series of 24 Caucasians KS selected according to the clinical Niikawa–Kuroki criteria. Similarly, Miyake et al11 failed to find any duplication in 28 Asiatic patients with typical KS.

Several patients with 8p23 duplication have been reported previously. In most cases this duplication was believed to be a benign variant with no phenotypic effect12 and described as an euchromatic variant (EV).13, 14 This was disputed by Tsai et al15 who reported on 12 patients with an 8p23 duplication and a significant phenotype comprising mainly heart defects. Nevertheless, the clinical features of these patients were not evocative of KS. Some of them presented developmental delay, short stature, cardiac anomalies and hypotonia, but no skeletal and dermatoglyphic anomalies nor facial features characteristic of KS were observed. The phenotypic variability could be related to the size of the duplicated segment as well as to the region involved. In fact, the breakpoints have not been characterized at the molecular level in any of these patients. It is tempting to locate the breakpoints within the olfactory repeat region (ORRs) described by Giglio et al.16, 17 However, it is possible that alternative LCRs can mediate rearrangements with subtle difference in the amount of deleted/duplicated material.18

Based on FISH results obtained with the BAC RP11-122N11, Milunsky and Huang6 suggested that a paracentric inversion may contribute to the occurrence of the 8p duplication. Using the same probe, Miyake et al11 observed duplicated rather than inverted signals. Among 22 KS patients, they found 15 homozygous and seven heterozygous duplications. They observed the same frequency of homozygous and heterozygous duplications in a cohort of 52 normal individuals. Using a combination of multiplex amplifiable probe hybridization and semiquantitative fluorescence in situ hybridization (SQ-FISH), Hollox et al19 observed a variation in DNA copy number across band 8p23.1. They showed that a cluster of at least three antimicrobial β defensin genes is present at 8p23.1. The copy number of this cluster is variable. The most frequent copy numbers are three or four. Chromosomes with seven or eight copies of this unit can be identified at the cytogenetic level in patients carrier of the previously described 8p23.1 EV.19 Interestingly, BAC RP11-122N11 probe maps precisely in the middle of this β-defensin-gene cluster and corresponds to SPAG11 gene. This BAC is estimated to be 10 kb in length (http://genome.ucsc.edu/cgi-bin/hgGateway). Two locations in 8p23 (7.3 and 7.8 Mb) are given in NCBI Clone Registry (http://www.ncbi.nlm.nih.gov/genome/clone/). So, we would like to suggest that the variable number of signals generated by BAC RP11-122N11 results from a polymorphism linked to the genome architecture of this region rather than a paracentric inversion.

In conclusion, using high-resolution chromosomes and BAC-FISH analysis we failed to detect an 8p22–8p23.1 duplication or a paracentric inversion in a series of 24 KS patients. Further studies, for example using micro-array CGH, might be useful to identify a recurrent chromosomal imbalance in KS.