SOX12 and NRSN2 Are Candidate Genes for 20P13 Subtelomeric Deletions Associated with Developmental Delay

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SOX12 and NRSN2 Are Candidate Genes for 20p13 Subtelomeric Deletions Associated with Developmental Delay

ARTICLE in AMERICAN JOURNAL OF MEDICAL GENETICS PART B NEUROPSYCHIATRIC GENETICS · DECEMBER 2013 Impact Factor: 3.42 · DOI: 10.1002/ajmg.b.32187 · Source: PubMed

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Gerald Cox Ankita Patel Sanofi Aventis Group Baylor College of Medicine

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Pawel Stankiewicz Yiping Shen Baylor College of Medicine Boston Children's Hospital

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Available from: Yiping Shen Retrieved on: 25 September 2015 RESEARCH ARTICLE

Neuropsychiatric Genetics SOX12 and NRSN2 Are Candidate Genes for 20p13 Subtelomeric Deletions Associated with Developmental Delay Yu An,1,2 Sami S. Amr,3,4,5 Alcy Torres,3,6 Laura Weissman,3,7 Peter Raffalli,3,6 Gerald Cox,3,8 Xiaoming Sheng,2 Va Lip,2 Weimin Bi,9 Ankita Patel,9 Pawel Stankiewicz,9 Bai-Lin Wu,1,2,3 and Yiping Shen2,3,10* 1Institutes of Biomedical Sciences, Children’s Hospital and MOE Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai, China 2Department of Laboratory Medicine, Boston Children’s Hospital, Boston, Massachusetts 3Harvard Medical School, Boston, Massachusetts 4Brigham and Woman’s Hospital, Boston, Massachusetts 5Partners Healthcare Center for Personalized Genetic Medicine, Cambridge, Massachusetts 6Department of Neurology, Boston Children’s Hospital, Boston, Massachusetts 7Developmental Medicine Center, Boston Children’s Hospital, Massachusetts 8Genetics Program, Boston Children’s Hospital, Boston, Massachusetts 9Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 10Shanghai Children’s Medical Center, Jiaotong University School of Medicine, Shanghai, China

Manuscript Received: 29 March 2013; Manuscript Accepted: 26 June 2013

20p13 telomeric/subtelomeric deletions are clinically significant but are currently under-investigated. So far only five molecularly How to Cite this Article: delineated cases have been reported in literature and no candi- An Y, Amr SS, Torres A, Weissman L, date genes have been sufficiently implicated. Here, we present six Raffalli P, Cox G, Sheng X, Lip V, Bi W, new deletion cases identified by chromosomal microarray anal- Patel A, Stankiewicz P, Wu B-L, Shen Y. ysis (CMA). We also review 32 cases combined from literature 2013. SOX12 and NRSN2 Are Candidate and databases. We found that most 20p13 deletion patients Genes for 20p13 Subtelomeric Deletions exhibit significant developmental delay. Dysmorphic features Associated With Developmental Delay. are common but a consistent pattern was not recognized. Re- duced cognitive ability was frequent. Based on pathogenic dele- Am J Med Genet Part B 162B:832–840. tions delineated in this study, we mapped the smallest overlapping region and identified two nervous system expressing genes (SOX12 and NRSN2) as candidate genes that may be involved in the developmental defects in 20p13 microdeletion. Ó 2013 Wiley Periodicals, Inc.

Key words: 20p13; microdeletion; developmental delay; Yu An and Sami S. Amr contributed equally to this study. SOX12; NRSN2 No conflict of interest for all authors. Grant sponsor: Wellcome Trust; Grant sponsor: Shanghai “Eastern Scholar” program; Grant sponsor: 973 National Basic Research Program of China; Grant number: 2010CB529601. INTRODUCTION Correspondence to: Yiping Shen, Ph.D., FACMG, Shanghai Children’s Medical Center, 1678 Subtelomeric deletions of 20p have been reported as pathogenic Dong Fang Road Shanghai, China 200127. imbalances in patients with developmental delay or intellectual E-mail: [email protected] disability [Ravnan et al., 2006]. Compared to the well reported Article first published online in Wiley Online Library interstitial and terminal 20p deletions involving the JAG1 gene, (wileyonlinelibrary.com): 6 September 2013 which is responsible for Alagille syndrome [Kalousek, 1976; Byrne DOI 10.1002/ajmg.b.32187

Ó 2013 Wiley Periodicals, Inc. 832 AN ET AL. 833 et al., 1986; Sauter et al., 2003], small terminal deletions of the short disorder. On physical examination, he had narrow palpebral arm of chromosome 20 (20pter) that do not span the JAG1 gene, fissures, hypoplastic ear helices, high arched palate, hypoplastic and only affect the 20p13 region, have not been well characterized fifth toenail, high arched feet, and color blindness. He had two and are currently under-reported. To date, a total of 17 patients EEGs which revealed some paroxysmal activities and very promi- have been reported in literature with subtelomeric deletions of 20p, nent hyper-synchronous bursts in sleep that lasted up to about 4 sec 12 of which were detected by fluorescence in situ hybridization but was not enough to support a clinical diagnosis of a seizure (FISH) and the range and size of these deletions were not described disorder. [Baker et al., 2002; Ravnan et al., 2006]. The remaining five 20p deletions were known to be localized to 20p13, however the clinical information of the patients carrying the 20p13 deletions was Patient 2 limited. Sebat et al. [2007] reported two patients diagnosed with Patient 2 is an 11-year-6-month-old Caucasian male who was autism and Asperger’s syndrome with de novo 20p13 deletions that diagnosed with Landau–Kleffner syndrome, autism, and global were 216 kb and 1.1 Mb in size respectively. McGill et al. [2010] developmental delay. He had delay in speech and language, social, described two patients with development delay and other features, gross and fine motor skills, and he began to walk at an age of who carried de novo 20p13 deletions that were 1.2 and 1.7 Mb in 19 months. He has a history of bouts of asthma, diarrhea, and size. More recently, Moutton et al. [2012] reported a de novo hypertonia and also has delayed closure of the anterior fontanelle, 2.08 Mb deletion in a patient with psychomotor retardation, amblyopia of the left eye, astigmatism, and late eruption of first epilepsy and low-set ears with overfolded helices. In addition, there tooth. At 2 years of age, his EEG was noted to have abnormal are a total of 27 cases currently collected in chromosomal micro- readings. At 9 years and 8 months, he presented with a short and array databases: Database of Chromosomal Imbalance and Pheno- stocky body habitus, external genitalia Tanner stage 1, and macro- type in Humans using Ensembl Resources (DECIPHER) [Firth cephaly. He had a normal neurological examination. His overall et al., 2009] and The International Standards for Cytogenomic cognitive abilities by the Woodcock–Johnson Test of Cognitive Arrays Consortium (ISCA). Here, we report six new 20p13 deletion Abilities, 3rd edition were in the low range with a standard score of cases and provide a review of clinical phenotypes observed in 20p13 85 (16th percentile). The other standard scores included a verbal deletion patients. Based on all pathogenic deletions, we mapped the comprehension at 92, concept formation at 86, and visual matching smallest overlapping region (SOR) to a 47 kb interval that contains at 86. On physical examination, distinctive features were noted only two genes. including deep-set eyes, broad nasal alae, prominent forehead with bossing, normal ears, round and flat face, short philtrum and MATERIALS AND METHODS inverted upper lip, left single palmar crease, prominent finger pads, and partial 2–3 syndactyly of both feet. Patients 1–4 were identified among 8,065 consecutive cases with clinical indications of developmental delay, intellectual disability, and/or multiple congenital anomalies referred for clinical chromo- Patient 3 somal microarray analysis (CMA) between November 2006 and Patient 3 is a 3-years-4-months-old Caucasian female diagnosed June 2012. Medical chart review was approved by the Institutional with communication disorder, not otherwise specified and a Review Board of the Boston Children’s Hospital. CMA was per- developmental coordination disorder. She had a history of both formed using Agilent 244 K comparative genomic hybridization delayed attainment of motor milestones including walking at (CGH) array as previously described [Shen et al., 2007]. Parental approximately 19 months, as well as expressive language skills. FISH was performed for Patients 1 and 2. Two cases (Patients 5 and Her most recent developmental evaluation at 2 years and 6) ascertained from Baylor College of Medical were identified using 6 months of age included administration of the Bayley Scales V8.1 array [Boone et al., 2010]. Additionally, a total of 27 cases of Infant and Toddler Development, on which she received a presented in DECIPHER (http://decipher.sanger.ac.uk) and ISCA cognitive score of 95 and a language score of 79 which are average (https://www.iscaconsortium.org/) databases were accessed on to below average compared to children of her age. She had good January 11, 2013. All genomic coordinates are in hg19. social relatedness and strong play skills and did not exhibit behaviors consistent with an autism spectrum diagnosis during administration of the Autism Diagnostic Observation Schedule. Patient 1 Her profile continued to be notable for significant expressive Patient 1 is a 16-year-9-month-old Caucasian male diagnosed with language delay including vulnerabilities in vocabulary, higher global developmental delay, pervasive developmental delay-not level language, basic conversation and articulation and also for otherwise specified (PDD-NOS) with hypernasal and dysarthric delays in motor development as well as poor motor planning and speech. Developmentally, he was late in speaking sentences, coordination. Physical examination of the head revealed some toilet training, and acquisition of fine motor skills. He walked frontal bossing, downslanting palpebral fissures, slightly deep set without assistance at 16 months. At 8 years of age, his intellectual eyes, a short nasal root with broad tip and anteverted nares. Ears functioning is in the average range compared to his peers (Verbal I. are normal in position, slightly protuberant. Neurological exam Q. 93, Performance I.Q. 90, and Full Scale I.Q 90 by Wechsler revealed cranial nerves II-XII grossly intact. Reflexes were 2þ in Intelligence Scale for Children [WISC]). The Achenbach structured the lower extremities and difficult to be elicited in the upper questionnaires revealed significant features of an attention deficit extremities. No seizures were reported. 834 AMERICAN JOURNAL OF MEDICAL GENETICS PART B

Patient 4 All patients had delays in motor, social, speech and language. Patient 4 is a 16-year-old Caucasian boy diagnosed with global Cognition was often reduced but not always abnormal. Features developmental delay, attention deficit hyperactivity disorder of autism and ADHD were also present in several patients. Dys- (ADHD), mixed expressive-receptive language disorder and prob- morphic features were present in most of the patients but a lems secondary to abuse and neglect. He was born with an unde- consistent pattern was not detected. scended testicle and exhibited facial tics and arm twitches. His The registered position and range of deletions identified at 20p13 neuropsychiatric and behavioral issues are complicated, with a in each of the six patients are shown in Figure 1. Custom tracks in volatile mood, aggressive behavior, self-injurious behavior, but UCSC genome browser in Figure 2 show all 20p13 deletion cases in does not have any obsessive- compulsive-like behaviors. The score literature and databases (13 cases from DECIPHER, 14 cases from of Behavior Assessment System for Children (BASC) exam was in ISCA, 5 from literature, and 6 from this study). In our cohort, the “At Risk” range for externalizing behaviors of hyperactivity, parental testing in three patients confirmed that the deletion aggression, and conduct problems and also in the “At Risk” range occured de novo, while the remaining three were not tested for for depression anxiety and attention problems with regard to both parents. Based on all reported pathogenic deletions and de internalizing behaviors. His fine motor output was awkward and novo deletions (Fig. 2), the smallest overlapping region (SOR) effortful. The diagnosis of autistic spectrum disorder was raised but deleted across patients with 20pter deletions was determined to be a remained questionable. He used minimal language and his formu- 47 kb interval spanning chr20:297,444–344,879 (hg19). Remark- SOX12 lation was noted with articulation errors, retrieval problems, syn- ably, this region encompasses only two genes, (MIM tactic difficulties, and considerable pronoun and preposition 601947) and NRSN2 (MIM 610666), neither of which has been SOX12 confusion. The level of well below expectation in motor skills, previouslylinkedto disease. No common CNVs that include NRSN2 social and communication, personal living skills, and community and have been reported in the Database of Genomic living skills resulted in his significant delays in his adaptive behav- Variants (DGV, http://projects.tcag.ca/variation, Fig. 2). iors. Physical examination revealed little dysmorphism and no reported seizure activity. DISCUSSION Earlier FISH studies identified 20p subtelomeric deletion as one of Patients 5 and 6 the clinically significant imbalances detected in patient populations Both patients presented with moderate developmental delay [Ravnan et al., 2006], but so far only five 20p13 deletions, four of and dysmorphic features but limited clinical information was which overlap, have been documented with defined size and available. location. More telomeric and subtelomeric 20p13 deletion cases have recently been detected by CMA and reported in cytogenomic RESULTS disorder databases such as DECIPHER and ISCA (Table II). As observed in our patients that received parental testing, the majority Array comparative genomic hybridization (aCGH) analysis was of reported 20p13 deletions are de novo events which strongly performed on Patients 1–6. Patient 1 carried a telomeric 1.4 Mb suggest a pathogenic nature of the deletions. Two deletions deletion at 20p13 (chr20:266,000–1,676,018) and a 3.0 Mb dupli- reported in DECIPHER database (255061 and 257240) were inher- cation at 9q33.1 (chr9:119,860,019–122,853,348). FISH analysis ited from similarly affected parents further supporting the patho- indicated that his two chromosome abnormalities were unrelated genicity of 20p13 deletions. There is only one deletion case (250840) and were not due to a parental balanced translocation. Maternal in the DECIPHER database which was reported to be inherited testing revealed the duplication but not the deletion. Patient 2 from a normal parent, suggesting the possibility of incomplete carried a 1.1 Mb subtelomeric deletion at 20p13 (chr20:121,521– penetrance. Patient 4 in this study was adopted and the possibility of 1,269,566). FISH testing of both parents did not detect this deletion, its deletion was inherited from a normal parent cannot be excluded. indicating this is a de novo deletion. In addition, a 197 kb duplica- All Patients shared a common deleted 47 kb SOR that includes tion was also identified at 14q13.2 (chr14:35,512,239–35,709,374). SOX12 and NRSN2 both of which have not been previously Parental testing was not done for this duplication. Patient 3 carried a associated with disease. 799 kb deletion at 20p13 (chr20:60,747–860,076) and a 55 kb SOX12 is a group C member of Sry-related high-mobility group duplication at 22q12.1 (chr22:29,066,032–29,121,120). Parental (HMG) box (Sox) transcription factors. It was formally called as testing revealed that the 20p13 deletion occurred de novo and SOX22 and was found to be abundantly expressed in the central the 22q12.1 duplication was identified in the patient’s phenotypi- nervous system in human embryos [Jay et al., 1997]. It has long been cally normal mother. Patient 4 carried a 274 kb deletion at 20p13 postulated that SOX12 could be a candidate gene for an unknown (chr20:297,444–571,151) and a 302 kb duplication at 8q12.1 intellectual disability disorder associated with multiple malforma- (chr8:57,747,635–58,049,848). This patient was adopted and family tion based on its significant expression levels in neuronal and history as well as parental samples was unavailable. A single deletion mesenchymal tissues [Jay et al., 1997; Dy et al., 2008]. However, at 20p13 (chr20:102422–1227535) was detected in Patient 5, how- there is no evidence to date supporting the direct involvement of ever parental testing was not carried out. Patient 6 carried a single de SOX12 in human developmental disorders. The other two members novo deletion at 20p13 (chr20:102422–508612). of the SoxC group are SOX4 and SOX11, which have been shown to The clinical features of our patients, as well as other reported play key roles in important developmental processes [Schilham patients with sufficient clinical details, are summarized in Table I. et al., 1996; Sock et al., 2004], but currently the physiological NE AL. ET AN

TABLE I. Comparison of Clinical Features Found in Our Patients and Other Reported Patients With 20pter Deletions

This study This study This study This study McGill et al., McGill et al., Moutton et al., Patient 1 Patient 2 Patient 3 Patient 4 Patient 1 Patient 2 Patient 1 Size of deletion 1.4 Mb 1.1 Mb 799 kb 274 kb 1.7 Mb 1.2 Mb 2.08 Mb (20qter) Inheritance Not in mother, father De novo De novo Unknown De novo De novo De novo not tested Additional CNVs 3 Mb del at 9q33.1 197 kb dup at 55 kb dup at 22q12.1 302 kb dup at /// 14q13.2 8q12.1 Gender Male Male Female Male Female Female Female Age 16 years 9 months 11 years 6 months 3 years 4 months 16 years 11 months 15 years 19 years Physical abnormalities Head Normal Macrocephaly Relative Normal Normal Microcephaly Relative macrocephaly microcephaly Facies Normal Round and flat NA Normal with pointed Broad NA Long-pointed chin chin Forehead Normal Frontal bossing Frontal bossing Normal Normal NA Normal Eyes Narrow palpebral Deep set Downslanting Slightly Microcorneas, NA Normal fissures palpebral fissures, hyperteloric marked edema of slightly deep set the lids, reversed eyes epicanthal, synophrys, telecanthus Nose Normal Broad nasal ala A short nasal root with Normal Underdeveloped Long thin nose, Bulbous broad tip and nasal bridge, hypoplastic nasal anteverted nares up-turned nose tip Ears Hypoplastic helix Normal Normal Normal Thicked, Low-set and Low-set, posteriorly rotated posteriorly dysplastic, thick rotated ears overfolded helices with thickened overfolded helices Lips Normal Everted philtrum and Normal Bowed upper lip Normal NA Thin vermilion inverted upper lip Mouth/teeth High arched palate Delayed eruption of Normal Normal Normal High arched palate, Delayed first tooth dental crowding, permanent teeth persistence of eruption primary teeth Skin Normal Skin tag Normal Normal Two ear pits on the NA NA left lobe Digits Normal Syndactyly NA Normal with long NA Short, broad thumbs Brachydactyly toes Hypoplastic nails Positive Negative NA Negative NA Positive Broad fingertips (5th toe nail) Neonatal Jaundice None Gastroesophageal NA Respiratory stress Mild jaundice Jaundice complications reflux Delayed closure of Negative Positive NA NA Positive Positive Negative fontenalle (Continued) 835 836 TABLE I. (Continued) This study This study This study This study McGill et al., McGill et al., Moutton et al., Patient 1 Patient 2 Patient 3 Patient 4 Patient 1 Patient 2 Patient 1 Growth for age Height 60th percentile 11th percentile 10th percentile 3rd–10th 5th percentile <3rd percentile Minus 1.5 SD (165.9 cm at 14 (129.4 cm at 9 (86.5 cm at 2 percentile (66 cm at 6 (145.6 cm at15 (154 cm at 19 years 9 months) years 8 months) years 4 months) (121.2 cm at 8 months) years 4 months) years) years 8 months)

Weight 75th percentile 79th percentile 10th percentile 10th–25th 25th percentile 3rd percentile Minus 0.8 (63 kg at 14 (36.2 kg at 9 (11.8 kg at 2 years 4 percentile (8.26 kg at 6 (40.6 kg at 15 SD (51 kg at 19 years 9 months) years 8 months) months) (22.3 kg at 8 months) years 4 months) years) years 8 months) Head 75th percentile 98th Percentile 75th percentile (49 cm) 50th percentile 50th percentile 75th percentile Minus 1.5 SD circumference (55.5 cm) (55 cm) (51 cm) (45 cm) (54.5 cm) (53 cm) Age of 14 years 9 months 9 years 8 months 2 years 4 months 8 years 8 months 6 months 15 years 4 months 19 years evaluation Genitalia NA Tanner stage 1 NA NA NA NA NA Neurological abnormalities Hearing NA NA Normal Normal NA NA NA Vision NA NA NA Normal NA Poor visual acuity Peripheral retinopathy Cranial nerves/ Normal Normal Normal Normal Normal Normal Normal muscle tone EEG Positive Positive NA Negative NA Positive Normal at rest; abnormalities abnormal during hyperpnoea, and light stimulation Seizure Negative Negative NA Negative Neonatal seizure Generalized Absence-epilepsy tonic-clonic seizure Global developmental delay Walk Delayed Delayed Delayed Delayed Delayed Delayed Delayed Speech and Delayed Delayed Delayed Delayed NA Delayed Delayed MRCNJUNLO EIA GENETICS MEDICAL OF JOURNAL AMERICAN language Social Delayed Delayed Normal Delayed NA NA Delayed Motor Delayed Delayed Delayed Delayed Delayed Delayed Delayed Cognitive Normal (IQ 90) Low-average range Normal NA NA Mild ID (IQ 57) Moderate ID (IQ 48) abilities Autism PDD-NOS Positive Negative Negative NA NA NA diagnosis Autistic features Positive Positive NA NA NA NA Decreased social interaction, stereotypical movement of hands Other ADHD Landau–Kleffner NA ADHD, behavior issue NA NA NA neuropsychiatric syndrome disorders

NA, not ascertained. ATB PART AN ET AL. 837

FIG. 1. Array CGH scatter plots of the six patients with deletions at 20p13 (Patients 1–6 from top to bottom). The two ends of each plot were registered at 20pter on left and the border of 20p13 and 20p12.3 on right. Each dot represents a probe. Red dots represent log2 ratios of Cy5/Cy3 greater than 0.25 and green dots represent log2 ratios less than 0.25. Black dots represent log2 ratios between 0.25 and 0.25. The pink shaded region for each patient represents the 20p13pter deletion in that individual.

function of SOX12 remains largely unknown [Dy et al., 2008; the function of Sox12 in mice is believed to be compensated by the Bhattaram et al., 2010; Penzo-Mendez, 2010; Mu et al., 2012]. other two members in a nonreciprocal manner [Hoser et al., 2008] The three SoxC proteins exhibited overlapping expression patterns as evidenced by the lack of phenotypic consequence of Sox12 and functional redundancy [Dy et al., 2008; Hoser et al., 2008]. knockout mice. However, inactivating Sox12 worsened the disease Being the weakest transcription factor in group C [Dy et al., 2008], phenotype of Sox4þ/11þ/ mice suggesting that Sox12 has a 838 AMERICAN JOURNAL OF MEDICAL GENETICS PART B

FIG. 2. Top panel shows a genome view of all deletion cases (red colored custom tracks) relative to the genomic coordinates and RefSeq genes at 20p13 region, extracted from Human Genome Build 37 (hg19). Bottom panel shows the zoomed-in smallest overlapping region (SOR) encompassing two genes (SOX12 and NRSN2) at a 47 kb interval (chr20: 297, 444–344, 879).

minor contribution to mouse development and organogenesis either gene is not tolerated and may affect the human health and [Hoser et al., 2008]. It is possible that human SOX12 has an normal development. Our study, which identifies NRSN2 and expression pattern and function that is different from its orthologs SOX12 as candidate genes for developmental delay seen in patients in other species limiting the utility of knockout mouse models in with 20p13 deletions, further supports a role for these genes in understanding the contribution of SOX12 to the clinical phenotype disease pathogenesis. seen in 20pter deletion patients. The second candidate gene It should be noted that in addition to the chromosome 20p13 identified in the SOR interval, NRSN2, is a homolog of Neuro- deletions, additional CNVs were identified in four patients. Patient p24/Neurensin-1, which encodes a small neuronal membrane 1 carries a large 3 Mb duplication on chromosome 9q33.1, which protein. Its expression is limited in brain tissues including cerebral was also identified in the phenotypically normal mother. Despite cortex, thalamus/hypothalamus and hippocampus. Intracellularly, the large size of this duplication, it only encompasses three genes, neurensin-2 is localized in small vesicles in neural cells [Nakanishi only one of which, TLR4 (MIM 603030), was listed in OMIM as et al., 2006]. The role of NRSN2 in neurodevelopment is currently disease causing, specifically resulting in a slightly increased predis- unknown. position to colorectal cancer due to a missense variant. Therefore, it Although the function of NRSN2 and SOX12 in human devel- is unlikely that the 3 Mb duplication observed in Patient 1 is related opment is largely unknown, no polymorphic CNVs overlap either to his phenotype. Patient 2 carries a 197 kb duplication of unknown gene in healthy individuals listed in the Database of Genetic clinical significance at 14q13.2. There is no evidence to indicate that Variation (http://projects.tcag.ca/variation). In addition, sequenc- this duplication is clinically relevant. Patient 3 carries a 55 kb ing data from over 10,000 individuals from a broad population by duplication on chromosome 22q12.1, which was also observed the NHLBI GO Exome Sequencing Project (ESP) reveals a lack of in her phenotypically normal mother. A similar duplication has loss-of-function variants in NRSN2 and SOX12, even at low fre- been detected in a normal individual; therefore this duplication is quencies (http://evs.gs.washington.edu/EVS/; accessed on 01/13). unlikely to be associated with patient’s phenotype. Patient 4 carries These observations in the general population suggest that the loss of a 302 kb duplication at 8q12.1 involving only one gene IMPAD1 AN ET AL. 839

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