A 17q21.31 microdeletion encompassing the MAPT gene in a mentally impaired patient

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1 Human Cytogenetics Case Report Cytogenet Genome Res 114:89 92 (2006) DOI: / A 17q21.31 microdeletion encompassing the MAPT gene in a mentally impaired patient a b c d M.C. Varela A.C.V. Krepischi-Santos J.A. Paz J. Knijnenburg d b a K. Szuhai C. Rosenberg C.P. Koiffmann a Human Genome Study Center, and b Laboratory of Human Genetics, Department of Genetics and Evolutionary Biology, University of São Paulo, c Neurology Unit, Children Institute, University of São Paulo, School of Medicine, São Paulo (Brazil); d Laboratory of Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden (The Netherlands) Manuscript received 14 September 2005; accepted in revised form for publication by M. Schmid, 20 October Abstract. About 15% of patients with a clinical phenotype of Angelman syndrome (AS) have an unknown etiology. We report a patient with features reminiscent of AS, including a pattern of characteristic facial anomalies as well as speech impairment, developmental delay and frequent laughter. In addition, the patient had features not commonly associated with AS such as heart malformations and scoliosis. She was negative in SNURF-SNRPN exon 1 methylation studies and the G-banded karyotype was normal. Array-based comparative genomic hybridization disclosed a deletion of maximally 1 Mb at 17q The deleted region contains the MAPT gene, implicated in late onset neurodegenerative disorders, and the STH and NP_ genes. Another gene, such as CRHR1, might also be included based on maximum possible size of the deletion. We suggest that microdeletions within the 17q21.31 segment should be considered as a possible cause of phenotypes resembling AS, particularly when easily controlled seizures and/or cardiac abnormalities are also present. Copyright 2006 S. Karger AG, Basel Angelman syndrome (AS) (Angelman, 1965; Varela et al., 2004) is a neurobehavioral disease that comprises developmental delay, severe mental retardation, absent speech, seizures, ataxia, outbursts of laughter, microcephaly, brachycephaly, macrostomia and prognathism. Gait is described as wide-based, with arms held flexed and upheld at the elbows. Classical AS is caused by the loss of expression of maternally Supported by grants from the State of São Paulo Foundation for Support of Research (FAPESP) and the Brazilian National Foundation for Research (CNPq). Request reprints from Carla Rosenberg Departamento de Genética e Biologia Evolutiva, IB USP Rua do Matão, andar sala São Paulo, SP (Brazil) telephone: ; fax: Carlarosenberg45@aol.com M.C.V. and A.C.V.K.-S. contributed equally to this work. imprinted gene(s) mapped to the chromosome region 15q11]q13. Four different mechanisms are known to cause the AS phenotype: 70% have a maternal deletion within 15q11]q13; 2 3% exhibit paternal uniparental disomy of chromosome 15 (UPD15); 2% have a mutation in the imprinting center and 8% have mutations in the UBE3A gene. Additionally, 15% of AS cases have an, as yet, undetected genetic mechanism. We report a patient with features reminiscent of AS, and a microdeletion detected by array-based comparative genomic hybridization (array-cgh). Materials and methods Patient description Here we report a female patient first seen at 2 years of age who was referred for genetic testing for AS. Informed written consent was obtained from the parents. She was the first child born to non-consanguineous, healthy Caucasian parents at 38 weeks of gestation by Cesarean section. Birth weight was 2,560 g (2.5! P! 10), length was 44.5 cm Fax karger@karger.ch S. Karger AG, Basel /06/ $23.50/0 Accessible online at:

2 Results and discussion Fig. 1. Facial appearance of the propositus at the age of 2 (left) and 3 (right) years. ( P! 2.5) and OFC 34 cm (p25). Apgar scores were 9 at 1 min and 10 at 5 min. She presented poor sucking, neonatal hypotonia and mild developmental delay: she could raise her head at 6 months, sat without support at 8 months, walked at 19 months, and started to speak a few words around 2 years of age. At physical examination, she had brachycephaly, occipital groove, upslanting palpebral fissures, hypertelorism, anteverted nares, protruding tongue, micrognathia, prognathia, wide mouth, short philtrum, wide-spaced teeth, frequent drooling, strabismus, hypopigmented skin, light hair and eye color compared to family members ( Fig. 1 ). Her weight was 11.8 kg (25! P! 50 th centile), height 90 cm (75! P! 90 th centile), and head circumference 47.5 cm (50! P! 75 th centile). She also presented swallowing disorders, heart malformations including atrial septal and ventricular septal defects, and persistent ductus arteriosus, congenital left hip dislocation, scoliosis and impaired vision (hypermetropia). A single clonic seizure episode occurred at age of 13 months but a brain MRI was normal. She exhibited speech impairment with minimal use of words, non-verbal communication, frequent laughter and smiling with an apparently happy demeanor, excessive chewing/mouthing behavior, bruxism and attraction to/fascination with water. Array-CGH The array-cgh procedures were performed as previously described (Knijnenburg et al., 2005; Rosenberg et al., 2006). Briefly, slides containing triplicates of 3,500 large insert clones spaced at 1.0 Mb density over the full genome were produced in the Leiden University Medical Center. The large insert clone set used to produce these arrays was provided by the Wellcome Trust Sanger Institute (UK), and information regarding the full set is available at the Wellcome Trust Sanger Institute mapping database site, Ensembl ( DNA amplification, spotting on the slides and hybridization procedures were based on published protocols (Carter et al., 2002; Fiegler et al., 2003). Target imbalances were determined based on log2 ratios of the average of their replicates, and sequences were considered as amplified or deleted when outside the range. Fluorescence in situ hybridization (FISH) FISH experiments were performed on metaphase spreads from the patient to validate the presence of the imbalance identified by array- CGH analyses, and from her parents to determine if the deletion had been inherited. Clones shown to be deleted by array-cgh ( Fig. 2 ) were hybridized following standard procedures (Rosenberg et al., 1994). Analyses were performed under a Zeiss Axiophot epifluorescence microscope (Carl Zeiss, Jena, Germany). Metaphases were captured with a CCD camera and processed using ISIS software (MetaSystems, Altlussheim, Germany). The patient s karyotype after GTG-banding at the 550 band level resolution was 46,XX. The analysis of SNURF- SNRPN exon 1 methylation patterns amplified by PCR (Zeschnigk et al., 1997) showed one paternal and one maternal band, and excluded microdeletion of 15q11]q13, UPD and maternal mutation in the imprinting center as causes for the AS phenotype. Array-CGH was performed on DNA from peripheral blood from the patient as previously described (Rosenberg et al., 2006). Array clones found to be altered in normal controls or with a large standard deviation were excluded, as described in Rosenberg et al. (2005). The array-cgh disclosed a unique alteration, a deletion of Mb ( Fig. 2 A) at 17q21.31, which was confirmed by fluorescence in situ hybridization using the clone located in the deleted segment, namely RP5-843B9 (FISH Fig. 2 B). FISH on metaphases from the parents showed that the deletion of the patient was de novo. Mickelson et al. (1997) have reported a patient with a phenotype reminiscent of Angelman syndrome and a deletion mapped cytogenetically to the interval 17q23.1]q23.3. They used polymorphic markers to corroborate the supposed location of the cytogenetic deletion but surprisingly failed to show any loss of alleles. Recent information obtained from the Ensembl genome browser confirms that many of the markers used must have been within the cytogenetically determined deletion area. Those markers are physically located within the 17q22]q25 and cover over 22 Mb. Importantly, none of the informative markers would have overlapped the location of the deletion described by us, raising doubts on the accuracy of the estimated cytogenetic location of their deletion; this leaves open the possibility that their deletion could overlap ours. Further, Mickelson et al. (1997) compared their case to four other patients in the literature carrying 17q21]q24 deletions, none of whom exhibited resemblance to the Angelman phenotype. The deletion in our patient is much smaller and more precisely mapped than those described previously and reviewed by Mickelson et al. (1997). Amongst the features most frequently described in their review of patients with 17q21 deletions, our patient exhibited developmental delay, dislocated hips, impaired vision and congenital heart disease, but did not present short stature, microcephaly, symphalangism and proximal thumbs. Although our patient exhibits some features of AS, her developmental delay is milder and her seizures easier to control than generally observed in AS patients with 15q deletions, and is similar to some AS patients with UPD15. In addition, she does not present ataxic gait, characteristic of AS, and she has features not commonly found in AS such as congenital heart abnormalities and scoliosis. One other deletion patient detected by the same array- CGH probe (RP5-843B9) was recently reported by Shaw- Smith et al. (2004) in a screening for submicroscopic chromosome alterations in 50 patients with learning disabilities. It is noteworthy that the phenotype of their patient did not resemble AS, and includes short stature, mild contractures and 90 Cytogenet Genome Res 114:89 92 (2006)

3 Fig. 2. De novo deletion on chromosome 17 in the patient with Angelman syndrome phenotype. ( A ) Chromosome-17 array-cgh profile of the patient shows a deletion of a single clone at 17q The black line indicates the position of the deletion on the 17-ideogram. Underneath, an enlargement of the 17q21 band shows a map of the deleted and non-deleted flanking clones. ( B ) FISH of the clone RP5-843B9 to metaphase chromosomes of the patient s lymphocytes. The white arrows indicate the normal (left) and deleted (right) chromosomes 17. patchy skin pigmentation. The only feature in common with our patient was mental impairment. A critical comparison between the breakpoints of the deletions would provide a better understanding of the phenotypic effect of genes in the deleted areas. The segment on chromosome 17 encompassing the deletion in our patient harbors several low-copy repeats (LCR). These LCRs have been recently associated with a 900-kb inversion which is polymorphic with a high frequency (up to 20%) in European populations (Stefansson et al., 2005). This Cytogenet Genome Res 114:89 92 (2006) 91

4 region contains several genes, including the microtubule associated protein tau gene (MAPT), associated with late onset neurodegenerative disorders such as Alzheimer and the Parkinson-Dementia syndrome (MIM ). Other genes within the deleted clone are saitohin (STH), mainly expressed in placenta, muscle, and fetal and adult brains, and NP_ , with unknown function (Swiss-Prot/TrEMBL; The total possibly deleted area ( Fig. 2 A) contains other genes with known function, namely CRHR1 (expressed in cerebellum, cerebral cortex, pituitary and olfactory lobe), and NSF, expressed in the central nervous system and diminished in schizophrenic patients (Mirnics et al., 2000). Which genes in this area are involved in the phenotype of our patient remains to be determined. We suggest that deletion or alteration of one or more genes within this 1-Mb region should be considered as a possible genetic cause for patients resembling the AS phenotype and for whom the well known chromosome 15 mutational mechanisms for AS do not apply, particularly when mild neurodevelopment, easily controlled seizures and/or cardiac abnormalities are present. Acknowledgement We thank Peter L. Pearson for critical reading of the manuscript. References Angelman H: Puppet children A report on 3 cases. Develop Med Child Neurol 7: (1965). Carter NP, Fiegler H, Piper J: Comparative analysis of comparative genomic hybridization microarray technologies: report of a workshop sponsored by the Wellcome Trust. Cytometry 49: (2002). Ensembl: Fiegler H, Carr P, Douglas EJ, Burford DC, Hunt S, Smith J, et al: DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36: (2003). Knijnenburg J, Szuhai K, Giltay J, Molenaar L, Sloos W, Poot M, Tanke HJ, Rosenberg C: Insights from genomic microarrays into structural chromosome rearrangements. Am J Med Genet A 132: (2005). Mickelson EC, Robinson WP, Hrynchak MA, Lewis ME: Novel case of del(17)(q23.1q23.3) further highlights a recognizable phenotype involving deletions of chromosome (17)(q21q24). Am J Med Genet 71: (1997). Mirnics K, Middleton FA, Marquez A, Lewis DA, Levitt P: Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron 28: (2000). Rosenberg C, Janson M, Nordeskjold M, Borresen AL, Vianna-Morgante AM: Intragenic reorganization of RB1 in a complex (4; 13) rearrangement demonstrated by FISH. Cytogenet Cell Genet 65: (1994). Rosenberg C, Knijnenburg J, Bakker E, Vianna-Morgante A, Sloos WC, Otto PA, et al: Array-CGH detection of micro rearrangements in mentally retarded individuals: Clinical significance of imbalances present both in affected children and normal parents. J Med Genet 43: (2006). Shaw-Smith C, Redon R, Rickman L, Rio M, Willatt L, Fiegler H, et al: Microarray based comparative genomic hybridisation (array-cgh) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 41: (2004). Stefansson H, Helgason A, Thorleifsson G, Steinthorsdottir V, Masson G, Barnard J, et al: A common inversion under selection in Europeans. Nat Genet 37: (2005). Varela MC, Kok F, Otto PA, Koiffmann CP: Phenotypic variability in Angelman syndrome: comparison among different deletion classes and between deletion and UPD subjects. Eur J Hum Genet 12: (2004). Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B: A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet 5: (1997). 92 Cytogenet Genome Res 114:89 92 (2006)

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