Kabuki Make-Up Syndrome Is Not Caused byMicrodeletion Close to the Van Der WoudeSyndrome Critical Region at 1q32-q41

Yoshio Makita,1* Koki Yamada,2,3 Akie Miyamoto,1 Akimasa Okuno,1 and Norio Niikawa2

1Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan2Department of Human Genetics, Nagasaki University School of Medicine, Nagasaki, Japan3Department of Ophthalmology, Nagasaki University School of Medicine, Nagasaki, Japan

We reported on a 5-year-old Japanese girlwith clinical manifestations of Kabukimake-up syndrome (KMS) and van derWoude syndrome (VWS). Since the concur-rence of the two syndromes is known in fourpatients, including ours, it suggests a com-mon cause. Assuming that the association ofthe two syndromes was caused by a micro-deletion involving the putative KMS/VWSgenes, we carried out fluorescence in situhybridization and microsatellite analysesusing PAC clones and dinucleotide repeatmarkers spanning the VWS1 critical regionat 1q32-q41. No deletion was detected at theVWS1 critical region. Am. J. Med. Genet. 86:285–288, 1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: Kabuki make-up syndrome;van der Woude syndrome;contiguous gene syndrome;microdeletion

INTRODUCTION

Kabuki make-up (Niikawa-Kuroki) syndrome (KMS)is an MCA/MR syndrome characterized by mild to mod-erate mental retardation, postnatal growth retarda-tion, skeletal anomalies, unusual dermatoglyphic pat-terns, and a facial appearance resembling the Kabukiactors’ make-up. KMS patients, often have hypodontiaand cleft palate. Although more than 100 KMS patientsare known [Niikawa et al., 1998; Philip et al., 1992;

Schrander-Stumpel et al., 1994; Ilyina et al., 1996; Wil-son, 1998] since the first descriptions by Niikawa et al.[1981] and by Kuroki et al. [1981], the cause of thesyndrome remains unknown. Most patients are spo-radic, but a few families are known with possible par-ent-child transmission of the disorder [Halal et al.,1989; Kobayashi and Sakuragawa, 1996; Tsukahara etal., 1997]. This suggests autosomal dominant inheri-tance with a chromosomal microdeletion as an under-lying mechanism. This assumption was tested once,based on a similarity of anomalies in KMS with thosein the 22q11.2 deletion (DiGeorge/velocardiofacial)syndrome, but no deletion was observed by fluores-cence in situ hybridization (FISH) in the critical region[Li et al., 1996].

Van der Woude syndrome (VWS, MIM*119300) is anautosomal dominant disorder characterized by lower-lip pits, cleft lip and palate, and hypodontia. VWS1 wasassigned to 1q32-q41 [Bocian and Walker, 1987; Mur-ray et al., 1990] and VWS2 to 2q34-q36 [Frezal andSchinzel, 1991]. The VWS1 critical region (VWCR1)was confined to a segment between markers D1S245and D1S414 [Sander et al., 1995].

Franceschini et al. [1993] reported a KMS patientwhose additional manifestations were consistent withVWS. This led us to hypothesize that the presence ofthe two syndromes in an individual was caused by amicrodeletion spanning two putative, contiguously lo-cated KMS and VWS genes. Here, we report on a5-year-old Japanese girl with KMS and VWS. The re-sults of FISH and microsatellite marker analyses of theVWCR1 will be described.

CLINICAL REPORT

The girl was the first child of a 29-year-old motherand a 33-year-old father, both being healthy and unre-lated. There is no family history of cleft lip or lip pits.The pregnancy and delivery were uneventful. Birthweight was 3,000 g and length 49 cm. The patient pre-sented mild respiratory problems, received oxygensupplementation, and gradually improved. She hadlower lip pits (Fig. 1B) and a cleft soft plate, the latterof which was repaired at age 23 months. The diagnosis

Contact grant sponsor: Ministry of Education, Science, Sportsand Culture of Japan; Contact grant numbers: 08770543 and09770529.

*Correspondence to: Yoshio Makita, M.D., Ph.D., Departmentof Pediatrics, Asahikawa Medical College, Nishikagura 4-5-3-11,Asahikawa, 078-8510, Japan.E-mail: makita5p@asahikawa-med.ac.jp

Received 17 February 1999; Accepted 28 May 1999

American Journal of Medical Genetics 86:285–288 (1999)

© 1999 Wiley-Liss, Inc.

of VWS was made. We first examined her at age 3 yearsbecause of short stature. She measured 89 cm (−2.4 SD)and weighed 11.5 kg. She had arched eyebrows withsparse lateral halves, long eyelashes, long palpebralfissures with mild ectropion at the lateral third of thelower eyelids, prominent ears, and a depressed nasaltip (Fig. 1A) and a deep sacral dimple. Dermatoglyphicanalysis demonstrated increased fingertip ulnar looppatterns (9/10) and hypothenar loop patterns, de-creased total finger ridge counts, and prominent fingertip pads.

At age 5 years, she was admitted for further evalu-ation. The insulin tolerance test and arginine tolerancetest both showed patterns suggestive of partial growthhormone deficiency. Serum levels of other hormoneswere all normal including T3, T4, TSH, LH, and FSH.Radiological examinations showed mild thoracic scolio-sis and spina bifida occulta at the sacral region and adelayed bone age. Orthopantomograph showed the ab-sence of second maxillary premolars. Her IQ was esti-mated at 86 by WIPPSI (modified Wesckler Preschooland Primary Scale of Intelligence). Chromosomes werenormal with a 46,XX karyotype. Her facial appearance,postnatal growth retardation with partial GH defi-ciency, and the unusual dermatoglyphic patterns allsupported the diagnosis of KMS.

MATERIALS AND METHODSPAC Clone Isolation and FISH

A PAC library [Ioannou et al., 1994] was screened bypolymerase chain reaction (PCR) as described previ-ously [Matsumoto et al., 1997]. In short, a PAC clonewas first isolated using a sequence tagged site (STS) atVWCR1 [Sander et al., 1995; Schutte et al., 1996]. Bothends of the PAC-DNA were sequenced by an automatedDNA sequencer (ALFexpress, Pharmacia Biotech, Swe-den) with a primer pair (58–38): T7, AATACGACTCAC-TATAG; SP6, ATTTAGGTGACACTATAG. Based onthe sequence data, new STS primers were designedand used for further PAC-library screening. PCR wascycled 30 times with GeneAmp 9700 system (PerkinElmer Cetus Instruments, Norwalk, CT) at 94°C for 20sec, 55°C for 20 sec, and at 72°C for 30 sec in a reactionmixture containing 10 mM Tris/HCl (pH 8.3), 50 mMKCl, 1.5 mM MgCl2, 0.1% Tween 20, 200 mM dNTPs, 1mM each primer, and 0.5 U Taq polymerase (Perkin-Elmer Cetus).

Epstein-Barr virus-transformed lymphoblastoid celllines were established from the patient and her par-ents, and metaphase chromosomes were prepared fromthe cell lines. FISH was performed using biotin-labeledPAC clones as probes as described previously [Matsu-moto et al., 1997]. FISH signals were detected withfluorescein isothiocyanate conjugated avidin (VectorLaboratories, Burlingame, CA).

Polymorphic Marker Analysis

Parent-child transmissions of alleles at five loci onchromosome 1 were analyzed using five respective di-nucleotide repeat markers that are mapped to VWCR1(Table 1) [Sander et al., 1995; Schutte et al., 1996].Briefly, DNA from the patient and her parents werePCR-amplified with Cy5-labeled primer pairs for thefive markers. PCR was performed in a reaction mixturecontaining 50 ng of genomic DNA, 1 mM each primer,200 mM each dNTP, 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 1.5 mM MgCl2, 0.001% gelatin, and 0.5 U Taqpolymerase under the following conditions: one cycle at94°C for 10 min; further 40 cycles at 94°C for 30 sec fordenaturation, 55°C for 30 sec for annealing, and at72°C for 30 sec for extension; and one cycle of elonga-tion at 72°C for 10 min to ensure complete extension.Ten-fold diluted PCR products in a mixture containing5 mg/ml loading dye (dextran blue 2000 in deionizedformamide) and size markers (Cy5 Sizer, PharmaciaBiotech, Gaithersburg, MD) were denatured at 94°C

Fig. 1. The patient at age 5 years. (A) Lateral view; (B) lower lip pitsindicated by arrows.

TABLE I. Allelotypes at Dinucleotide-Repeat Marker Loci inthe Patient and Her Parents

Marker locus

Allelotype

Father Mother Patient

D1S245 2, 2 1 1, 2D1S471 1, 2 1, 3 1D1S491 1, 2 2 2D1S70 1 1 1D1S205 1, 2 2, 3 1, 2

286 Makita et al.

for 3 min, loaded on 6% acrylamide gel (Ready Mix Gel,Pharmacia Biotech), and electrophoresed in a runningbuffer containing 40 mM Tris-borate (pH 8.0) and 1mM EDTA, using the ALFexpress DNA sequencer at55°C and 38 W/gel. Resulting data were analyzed witha software, Fragment ManagerTM (Pharmacia Biotech,version 1.2) to determine genotypes.

RESULTS AND DISCUSSION

Microsatellite marker analysis revealed that the pa-tient is heterozygous (two copies of alleles) at two

(D1S245 and D1S205) of the five marker loci examined(Table I). Three other markers (D1S471, D1S491, andD1S70) were uninformative in this family. By the PAClibrary screening, eight clones were isolated fromVWCR1 (Fig. 2). They extended from a YAC cloneyAS9L to a clone yAS9R [Schutte et al., 1996], indicat-ing to cover almost all the region. FISH analysis witheach PAC clone as a probe gave twin signals on bothhomologous chromosomes 1 of the patient (Fig. 3).

The girl we describe had clinical manifestations ofboth KMS and VWS. She had all five cardinal mani-festations of KMS and lower lip pits, a manifestation ofVWS. She also had hypodontia and a cleft soft palate,manifestations common to KMS and VWS. To ourknowledge, four such patients, including ours, areknown [Franceschini et al., 1993; Kokitsu-Nakata etal., 1999; G. Oliveira, personal communication]. Cleftlip/palate and hypodontia are main manifestations ofVWS, but they are often present in KMS patients [Nii-kawa et al., 1988; Philip et al., 1992; Schrander-Stumpel et al., 1994; Ilyina et al., 1995; Burke andJones, 1995]. Moreover, a patient with a 1q25-q32.1deletion reported by Hamano et al. [1987] had a facialappearance resembling those of KMS patients. Thesefindings led us to assume that the occurrence of the twosyndromes in each of four individuals are caused by amicrodeletion that surpassed two putative, contiguous-ly located KMS and VWS genes, i.e., a contiguous genesyndrome. This assumption was tested in the presentstudy by FISH and microsatellite marker analyses.However, our patient has no gross deletion at theVWCR1. This suggests that the putative KMS gene isnot located within the region. It remains to be seenwhether the patient has a microdeletion in the VWStype 2 critical region (VWCR2) at 2q34-q36 [Frezal andSchinzel, 1991].

Fig. 2. Integrated physical map of VWCR1, showing VWCR1 (shaded bar, upper row), a PAC contig (middle rows), and reported STS (lower row).Squares depict YAC-derived STSs and polymorphic STSs, and circles indicate new STSs from the PAC clones isolated. The names and order of STSs areaccording to Schutte et al. [1996].

Fig. 3. Fluorescence in situ hybridization on the patient’s chromo-somes, using a PAC clone, 337-A-9. Arrows indicate twin spot signals.

Kabuki Syndrome With Van Der Woude Syndrome 287

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