Deletion at 12q12 increases the risk of developmental delay and intellectual disability

Abstract Single‐nucleotide polymorphism (SNP) arrays have been widely used to identify novel genomic imbalances. Many of these genomic imbalances have been confirmed to interact with developmental delays, intellectual disabilities (IDs), and congenital defects. Here, we identify a Chinese girl with a 3.18‐Mb deletion at 12q12 (human genome build 19: 43,418,911‐46,601,627) who showed postnatal growth delay, low‐set ears, small hands and feet, widely spaced nipples, and blue sclerae. Deletions at 12q12 are extremely rare chromosomal imbalances; only four cases involving a deletion of this type have previously been reported. In these five sporadic cases, all of the patients exhibited developmental issues accompanied by different degrees of ID. A review of DECIPHER patient data revealed an additional six cases involving genomic deletion at 12q12. Many of the patients in these cases exhibited developmental delay and ID. When these patients were included, 91% and 73% of individuals with a deletion in this chromosomal region presented with developmental retardation and ID, respectively. Database searches indicated that this copy number variant (CNV) has not been found in normal humans. Therefore, we suggest that a CNV in this region is a risk factor for developmental retardation and ID.


INTRODUCTION
Growth deficiency occurs in association with many different chromosome abnormalities, including microdeletions and microduplications (Batey et al., 2014;Capalbo, Rienzi, & Ubaldi, 2017;Li et al., 2017). Chromosomal microarray analyses (CMAs) have revealed a large number of copy number variants (CNVs), which refer to a length of DNA larger than 1 kb with a different copy number than that observed in normal humans, on chromosomes. A multitude of CNVs have been found in patients with clinical phenotypes such as autism, developmental delay, and intellectual disability (ID), This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
© 2018 The Authors. Annals of Human Genetics published by University College London (UCL) and John Wiley & Sons Ltd. and many of these CNVs have been confirmed to be associated with diseases (Lowther, Costain, Baribeau, & Bassett, 2017;Takumi, & Tamada, 2018). CNVs that have not been reported in normal humans in database records and do not overlap with genomic regions known to be associated with disorders are temporarily classified as CNVs of unknown significance. Building connections between these novel CNVs and clinical phenotypes remains a considerable challenge. Such investigations are essentially dependent on case accumulation, which requires long-term efforts.
Recently, we encountered a 3-month-old girl with a 3.18-Mb deletion at chromosome 12q12, a CNV of unknown clinical significance. A literature review revealed that 12q12 deletions are extremely rare; only four cases involving such deletions have previously been reported (Carlsen, Frengen, Fannemel, & Misceo, 2015;Failla et al., 2008;Miyake et al., 2004;Tonoki, Saitoh, & Kobayashi, 1998). Although most of these cases were sporadic, the patients in these cases shared certain clinical characteristics. The girl we encountered exhibited growth retardation and ID with the deletion at 12q12. Although 12q12 deletions have previously been observed, the present study is a new demonstration in which an association between this type of deletion and developmental delay has been established. Based on the different rates of occurrence of 12q12 deletion in patients and normal individuals, we suggest that 12q12 deletion is a novel risk factor for developmental delay.

CLINICAL REPORT
The Chinese girl we encountered was born to a 34-yearold mother and a 34-year-old father after 40 weeks of gestation by spontaneous vaginal delivery. She was the second child in the family, and her 12-year-old sister was normal. Both parents were healthy, and consanguinity was denied. The mother had gestational diabetes mellitus and hyperthyroidism. No intrauterine growth retardation was reported. The patient's birth weight was 3.14 kg (50th-75th centile), and her birth length was 46 cm (< 3rd centile). Her Apgar score was unknown. There was second-level meconium staining of the amniotic fluid at her birth. She stayed in the neonatal intensive care unit for 8 days because of aspiration of amniotic fluid. Physical and laboratory examinations revealed congen-ital heart disease, an atrial septal defect, one epulis on the lower half of the oral cavity, hypocalcemia (serum calcium level, 1.52 mmol/L), and high levels of aspartate aminotransferase and uric acid. In addition, the patient was sensitive to egg white and had feeding difficulties.
Other observed anomalies included mildly dysmorphic features (Figure 1 A-F), such as a short neck; upslanting palpebral fissures; large, low-set ears; a broad nasal bridge with anteverted nares; downturned corners of the mouth; widely spaced nipples; fifth finger clinodactyly; small hands (total hand length, 6.5 cm); small feet (total foot length, 7 cm); and blue sclerae.

GENETIC TESTING
This study was approved by ethics committees of our hospital, and consent was obtained from the patient's parents. Gbanding with 300 to 400 bands performed on leukocytes from the patient's peripheral blood revealed a balanced translocation. The patient's karyotype was 46,XX, t (12;14) (q12;q24), which is t (12; 14) (12pter→12q12::14q24→14qter; 14pter→14q24::12q12→12qter). No imbalance at the breakpoint of the translocation was detected by karyotype analysis. CMA was performed on a CytoScan 750K Array (Affymetrix, CA) in accordance with the manufacturer's instructions. Genomic DNA was extracted from peripheral blood and isolated via standard procedures using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden). PCR was performed on a 9700 thermal cycler (AB, Singapore). CMA revealed a 3.18-Mb interstitial deletion at 12q12 (43,418,601,627). No imbalance at the breakpoint of 14q24 was detected. The reciprocal translocation and the deletion at 12q12 were de novo.
Loss-of-function mutations of ARID2 cause an ID syndrome that was reported in 2015 (Shang et al., 2015). SWI/SNF chromatin modifier has been related to neurodevelopmental disorders, including ID and autism. ARID protein is the SWI/SNF subcomplex. Shang et al. reported four patients with mutations of ARID2, who all showed ID and developmental delay, and one of the patients sharing the same phenotype with our patient had atrial septal defect. Deletion of ARID2 possibly caused the congenital heart defect of our patient.
NELL2 protein is neural epidermal growth factor-like-like 2. Previous study has been proved that NELL2 protein plays a part in neuronal proliferation, differentiation, synaptic formation, and plasticity during development and postnatal life (Choi et al., 2014). Jeong et al. investigated NELL2-regulated feeding behavior. A block of NELL2 production led to a decrease in daily food intake followed by a loss in body weight (Jeong et al., 2017). According to the parents' report, our patient showed feeding difficulties in the first 3 months. The girl's appetite was less than 50 mL of milk per day. This might be one of the reasons for the growth deficiency.
Twinfilin 1 encoded by TWF1 is an actin monomer-binding protein. Twinfilin 1 was reported to be involved in ocular coloboma (Rainger et al., 2017). TMEM117 knocked down would alter homeostasis toward cell death and TMEM117 RNAi-facilitated apoptotic cell death (Tamaki et al., 2017). So far no paper published has reported that TWF1 or TMEM117 is connected with developmental delay.
Ten cases involving deletion at 12q12 have previously been reported in the literature (Table 1) and the DECIPHER F I G U R E 2 Genes with high HI scores in the deleted 12q12 regions (modified from the DECIPHER genome browser: https://decipher.sanger.ac.uk) in our patient and 10 other patients. Case 1 is the girl we have reported, case 2 was reported by Carlsen et al. in 2015, case 3 was reported by Failla et al. in 2008, and cases 4 and 5 involved patients 1 and 2 (first reported by Tonoki et al., 1998), respectively, of the patients reviewed by Miyake et al. in 2004. Cases 5 to 11 were obtained from the DECIPHER database [Colour figure can be viewed at wileyonlinelibrary.com] T A B L E 1 Summary of clinical features of five previously reported patients compared with those of the new patient described in this report  (Table 2). Although phenotypes of developmental delay and ID were often observed, associations between 12q12 deletion and such phenotypes have not previously been established, and this microdeletion has not been regarded as pathogenic. Carlson et al. reported a boy with a 1.13-Mb deletion at 12q12 who exhibited growth delay, psychomotor delay, concentration and attention deficits, and an intelligence test score typical of children who are 2 to 3 years younger (case 2, Table 1). In the deleted region, only three protein-coding genes were detected: ANO6, DBX2, and NELL2. Therefore, the authors concluded that these three genes are involved in developmental delay.
Six additional cases (two cases with no provided clinical information were excluded) of 12q12 deletions were found in the DECIPHER database (cases 6-11, Table 2). Three individuals had de novo 12q12 deletions, one individuals had inherited deletions, and inheritance information was not available for the remaining cases. The fact that the parent of one individual (Adam, Mehta, Knight, Hall, & Rossi, 2010) was also affected by a 12q12 deletion suggested that this 12q12 deletion had likely cosegregated with the reported phenotypes in that family.
Developmental delay to varying extents and ID or mental retardation were observed in 91% (10 of 11) and 73% (8 of 11), respectively, of all 11 reported cases involving 12q12 deletion. Three patients were diagnosed with autism spectrum disorder, and two patients had concentration and attention deficits. Craniofacial dysmorphism was noted in our patient and in 5 of the individuals described in the literature and the DECI-PHER database. Thus, at least 55% (6 of 11) of individuals with a reported 12q12 deletion exhibited various craniofacial dysmorphisms.
Certain reported individuals carried additional genomic variations that could have affected their overall phenotypes. Individual 6 had a 5.89-Mb deletion at 12p12.3 with possible clinical significance. There was additional genomic imbalances in case 11 that was likely benign or of uncertain clinical significance.
In the 11 individuals described here, the deleted segments were partially or completely in 12q12. All dosage-sensitive genes in this region are listed in Figure 2.
In two cases (cases 6 and 7), the deleted segments were smaller than 1 Mb and included at least one dosage-sensitive gene; in particular, the affected genes included NELL2 in individual 6 and TWF1 and TMEM117 in individual 7. These findings suggest that NELL2 and either TWF1 or TMEM117 play roles in development. NELL2 mRNA levels were also found to be decreased in patient 2 (Carlsen et al., 2015). Certain genes in 12q12 have the same development-related functions. It appears likely that 12q12 acts as a functional unit in development.
In summary, we presented 11 individuals with 12q12 deletion, including one new subject. Our analysis suggested that a 12q12 microdeletion increases risk for developmental delay and ID. In conclusion, 12q12 deletion should be added to the database of pathophysiological genomic alterations that induce developmental delay, which will be helpful for counselling and management of the patients with developmental delay.