ß 2007 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 143A:916–920 (2007) Contiguous Deletion of the NDP, MAOA, MAOB, and EFHC2 Genes in a Patient With Norrie Disease, Severe Psychomotor Retardation and Myoclonic Epilepsy L. Rodriguez-Revenga,1,2 I. Madrigal,1,2 L.S. Alkhalidi,3 L. Armengol,4 E. Gonza´lez,4 C. Badenas,1,2 X. Estivill,1,5 and M. Mila`1,2* 1Biochemistry and Molecular Genetics Department, Hospital Clı´nic, Barcelona, Spain 2IDIBAPS (Institut d’Investigacions Biome`diques August Pi i Sunyer), Barcelona, Spain 3Department of Health and Medical Services, Rashid Hospital, Dubai, United Arab Emirates 4Genes and Disease Programme, Centre for Genomic Regulation (CRG), Barcelona Biomedical Research Park, Barcelona, Spain 5Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Spain Received 20 February 2006; Accepted 7 September 2006 Norrie disease (ND) is an X-linked disorder, inherited as a Clinical features of the proband include bilateral retinal recessive trait that, therefore, mostly affects males. The gene detachment, microcephaly, severe psychomotor retardation responsible for ND, called NDP, maps to the short arm of without verbal language skills acquired, and epilepsy. The chromosome X (Xp11.4-p11.3). We report here an atypical identification and molecular characterization of this case case of ND, consisting of a patient harboring a large reinforces the idea of a new contiguous gene syndrome that submicroscopic deletion affecting not only the NDP gene would explain the complex phenotype shared by atypical but also the MAOA, MAOB, and EFHC2 genes. Microarray ND patients. ß 2007 Wiley-Liss, Inc. comparative genomic hybridization (CGH) analysis showed that 11 consecutive bacterial artificial chromosome (BAC) clones, mapping around the NDP gene, were deleted. These clones span a region of about 1 Mb on Xp11.3. The deletion Key words: microarray comparative genomic hybridiza- was ascertained by fluorescent in situ hybridization (FISH) tion; Norrie disease; NDP gene; MAOA and MAOB genes; analysis with different BAC clones located within the region. EFHC2 gene; contiguous gene syndrome How to cite this article: Rodriguez-Revenga L, Madrigal I, Alkhalidi LS, Armengol L, Gonza´lez E, Badenas C, Estivill X, Mila` M. 2007. Contiguous deletion of the NDP, MAOA, MAOB, and EFHC2 genes in a patient with Norrie disease, severe psychomotor retardation and myoclonic epilepsy. Am J Med Genet Part A 143A:916–920. INTRODUCTION time of clinical examination. ND is inherited as an X- linked recessive trait and therefore it mostly affects Norrie disease (ND, OMIM# 310600) is a rare males. The gene responsible for ND, named NDP inherited neurodevelopmental disorder of congeni- gene, maps to the short arm of chromosome X tal blindness that occurs early during embryogenesis (p11.4-p11.3) [Chen et al., 1992; Sims et al., 1992]. [Norrie, 1927; Warburg, 1971]. It is characterized by The function of the gene product is still unknown, bilateral and severe retinal malformations and although most observations are consistent with a opacity of the lens. In addition to ocular symptoms, primary role of norrin in vascular development and a certain portion of patients present mental dis- turbances, often with psychotic features, and/or sensorineural deafness. The hearing loss is progres- sive and recent evidence shows that its penetrance appears to be complete, although there is some Grant sponsor: ‘‘Fondo de Investigacio´n Sanitaria’’, Spain; Grant variability in the clinical severity and the age of onset numbers: PI050776, PI050159, FS041126, RGPG, (G03/184); Grant [Rehm et al., 2002]. Even though these symptoms are sponsor: Genome Spain. *Correspondence to: M. Mila`, Biochemistry and Molecular Genetics considered as classical in ND, the clinical diagnosis is Department, Hospital Clı´nic, C/Villarroel 170, 08036 Barcelona, Spain. often difficult, mainly because more than half of E-mail: [email protected] patients are neither deaf nor mentally delayed at the DOI 10.1002/ajmg.a.31521 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a NORRIE DISEASE 917 proliferation [Shastry and Trese, 2003; Michaelides NDP gene was PCR amplified in different segments et al., 2004]. containing complete exon, acceptor, and donor Atypical ND patients harboring large DNA dele- splicing sites sequences. PCR conditions for each tions have also been reported. Those fewer patients, exon and the primer sequences used were as in addition to the retinal abnormalities, show other previously described by Meindl et al. [1992]. PCR clinical features, including microcephaly, growth amplification products were further sequenced on delay, severe to profound mental retardation, immu- an automated sequencer (ABI3100; Applied Biosys- nodeficiency, and epileptic seizures [Donnai et al., tems, Foster City, CA). 1988; Zhu et al., 1989; Suarez-Merino et al., 2001]. We report here on the clinical and molecular Microarray Comparative Genomic characterization of a new case of a large deletion of Hybridization (CGH) Xp11.4-p11.3, including the NDP gene. Genotype– phenotype correlation has allowed us to establish The tiling path X chromosome-specific array the cause of epilepsy that this patient shows. The consists >1,600 genomic bacterial artificial chromo- identification of this case reinforces the idea of a new some (BAC) clones derived from the X chromosome, contiguous gene syndrome that could explain the with an average density of one BAC every 100 kb on complex phenotype shared by these patients. the X chromosome, plus internal quality control BACs from autosomal chromosomes. All clones had three replicates on the array. The production of the CLINICAL REPORT X-array, probe preparation, and hybridization on The patient is a 7-year-old boy, who was born as the array were performed at the microarray unit of the second child of healthy unrelated parents. He the Center for Genomic Regulation (CRG). The set of was born at term after an uneventful pregnancy but at clones used to produce this array was purchased at 5 weeks of age he had to be taken into a hospital due the Children Hospital Oakland Research Institute to chest infection. At that time he was found to have (http://bacpac.chori.org/home.htm). DNA amplifi- bilateral yellowish reflex in both eyes. Retinal cation, spotting on the slides, and hybridization dysplasia was suggested as the diagnosis, and procedures had been published elsewhere [Fiegler another molecular laboratory confirmed ND after et al., 2003; Wang et al., 2004]. Test and control DNAs molecular analysis. The family reported a maternally were labeled by random priming using the BioPrime related nephew with similar clinical features. Patient Array CGH Genomic Labeling System (Invitrogen, was not seen again until 5 years when the family Life technologies, Carlsbad, CA). Dye swap was used visited a specialist seeking for genetic counseling to reduce false positives due to dye-related artifacts. since they were planning for a third child. Physical Two hybridization experiments were performed for examination at that age showed that the patient was each sample: in the forward experiment the array below the third centile for both height and weight was hybridized with a mixture of patient’s DNA (<3 SD), and that his head size could be considered labeled with Cy3-dCTP and the reference (control) as microcephalic. He was blind with whitish-yellow DNA labeled with Cy5-dCTP, while in the reverse retrolental masses that contained proliferated blood experiment patient’s DNA was labeled with Cy5- vessels, and he exhibited severe psychomotor dCTP and co-hybridized with reference (control) retardation without verbal language skills acquired. DNA labeled with Cy3-dCTP, to a second array. Neither hearing abnormalities nor other congenital Genomic imbalances were determined based on log2 findings were detected. At this time, brief seizures ratios of the average of their replicates, and were observed and antiepileptic treatment with sequences were considered as amplified or deleted valproic acid was started. Latest examinations when reported in the direct and reverse experiment revealed a normal MRI and a diffuse slow wave outside the Æ0.30 range. dysfunction without any epileptiform activity by EEG. No more seizures episodes had occurred after Fluorescent In Situ Hybridization (FISH) the antiepileptic treatment was initiated. Currently, Analysis the patient is regularly followed by pediatric and neuropediatric specialists due to severe psychomo- BAC/PAC clones located in the region identified as tor retardation, epilepsy, microcephaly, and blind- deleted by array CGH were purchased from the BAC/ ness. PAC Resources of the Children’s Hospital Oakland Research Institute (CHORI, Oakland, CA). The order of the probes according to the current mapping data MATERIAL AND METHODS is: centromere—RP1-95C20–RP1-27K14–RP6- Molecular Analysis 218J18–RP11-334D18—telomere. Clones were grown and DNA was extracted as manufacturer’s Genomic DNA samples were obtained from instructions. Afterwards, approximately 200 ng of peripheral blood of patient and his family. The each DNA sample were labeled with Spectrum American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a 918 RODRIGUEZ-REVENGA ET AL. Orange or Spectrum Green by nick translation (Vysis, Downers Grove, IL). Then 100 ng of each sample were precipitated with 5 mg of human cot-1 DNA (Vysis, Downers Grove, IL) and dissolved in 10 mLof hybridization buffer (Vysis, Downers Grove, IL). FISH using this solution as a probe was performed in a chromosome preparation from the patient and patient’s mother. Briefly, the slide was denatured by immersion in 70, 85, and 100% ethanol. The probe was also denatured at 738C for 5 min. After application of 10 ml of probe, the slide was incubated in a moist chamber for 16 hr at 378C. After hybridization, the slide was washed in 0.4 Â SSC at 728C during 2 min and 2 Â SSC at room temperature during 30 sec; chromosomal DNA was counter- stained with DAPI (Vysis, Downers Grove, IL).