Research Note Cryptic de novo deletion at 2q23.3-q24.1 in a patient with intellectual disability

Jamileh Malbin1*, Mohammad-SadeghFallah2-3*, Zohreh Sharif2, Mahsa Shafaei4, Hamideh Bagherian2, Tahereh PourMostafaei2, Ramiz Aliev1 Sirous Zainali2,5**

1. Azerbaijan National Academy of Science (ANAS), Genetic Resources Institute, Baku, Azerbaijan 2. Kawsar Human Genetics Research Center (KHGRC), Tehran, Iran 3. Cellular and Molecular Endocrine Research Center (CMER), Research Institute for Endocrine Sciences (RIES), Shahid Beheshti University of Medical Sciences, Tehran, Iran 4. Department of pathology and Molecular medicine McMaster Immunology Research Center, McMaster University, Hamilton, Ontario, Canada 5. Department of Molecular Medicine, Biotech Research Center, Pasteur Institute of Iran, Tehran, Iran * These authors contributed equally to the work as the 1st author.

**Corresponding author

Introduction: Structural abnormalities of are one of the most common fndings in individuals with mental disability. Conventional and molecular cytogenetic testing has been applied to detect correlation between these abnormalities and genetic diseases, especially intellectual disability. Interstitial and sub-telomeric deletions in long arm of 2 have been reported in correlation with intellectual disability (ID) by some of these methods. Here we describe a 3.7 Mb deletion in the long arm of in a woman with intellectual disability. Array comparative genomic hybridization (array CGH) revealed a deletion in 2q23.3-q24.1 region. Haplo-insufciency in three in this region (GPD2, FMNL2 and NR4A2) can be considered as causative factors for ID in our patient.

Intellectual disability (ID) is a common disorder affecting 1- 3% of general population. Any chromosomal change, affecting copy number or function, could play an important role in the brain development and intellectual ability. Techniques such as MLPA and array CGH have been used to screen chromosomal imbalances in patients with mental disability and normal karyotype. Large chromosomal deletions and duplications usually change brain function and mental ability (Mulatinho et al., 2012). Several deletions and/or duplications have revealed that chromosome 2 contains many genes participating in brain function (El-Bassyouni et al., 2014; Gijsbers et al., 2009). The 2q24.1 and 2q23.1 regions contain the most repetitious regions, including several genes which are considered as candidate genes responsible for ID. Here we report a patient with 3.7 Mb deletions in 2q23.3-q24.1 region.

Patient A family with non-consanguineous marriage and history of two affected cases (a son and a daughter) with ID was referred for molecular investigation. They also have 5 healthy children and three early childhood deaths (two sons and a daughter) with an unknown diagnosis (Figure-1A).The affected son refused to be evaluated and we could just test the affected daughter. She was a 35 years old female with history of developmental delay, seizures, speech difficulty, and self-mutilation behavior (Figure-1B). At the time of examination, her head circumference, weight and height were 58 cm, 83 Kg, and 166 cm respectively. No other dysmorphic feature was seen except having long fngers. She developed secondary sexual characteristic at the age of 15 and she had regular menstrual period.

Methods Conventional chromosomal study was carried out on peripheral blood at 450-band resolution using GTG banding. DNA was extracted from peripheral blood using salting out techniques (Aljanabi & Martinez, 1997). DNA quality was determined by Nano Drop 2000 spectrophotometer (Nano-Drop Thermo Fisher Scientifc, USA). MLPA study was performed using P245-Kit for microdeletion syndromes screening and P036 & P070 kits (MRC- Holland, Amsterdam; The Netherlands) for sub-telomeric rearrangement as described before (Jehee et al., 2011). Amplifcation products were identifed and quantifed by capillary electrophoresis on an ABI 3130 genetic analyzer (Life Technologies, ABI, USA). Raw data were analyzed using Gene Marker Software V1.85 (Soft Genetics, State College, PA, USA).

Microarray based comparative genomic hybridization (array CGH) analysis was carried out using the Gene Chip ISCA 4X44 whole genome oligo array version 1.1 (Blue Gnome UK ). The array consists of 44000 spots with average resolution of 75 Kbs. Sample was hybridized twice against female and male samples used as controls. DNA was processed according to the manufacturer’s instructions. Total purifed and quantifed genomic DNA was digested, amplifed and then labeled with a biotinylated nucleotide analogue [dUTP/oligo] using terminal deoxynucleotidyltransferase and hybridized. The slides were scanned using Innopsys 700 laser scanner (Innopsys, USA). Images were imported and analyzed by BlueFuse Multi software (Bluefuse systems ltd, USA http://support.illumina.com/array/array_software/bluefuse-multi- software.html). Copy number variations were screened against database of genomic variants and reported variations were excluded.

Results Conventional chromosomal study showed normal karyotype in the affected case. Screening for 21 micro-deletion syndromes and sub-telomeric rearrangement was performed using MLPA method. There were no deletions or duplications in the MLPA study but a sub-telomeric duplication at 4q containing the FRG1 gene was observed using the P070 kit (Figure 2).

Array CGH were performed to confrm the MLPA result. Results showed a 3.7 Mb deletion in 2q23.3-q24.1 region from nucleotide 154,247,108 to 158,021,127 (NCBI36/hg18) (Figure 3). Array CGH in her parents did not show any deletion for this region. Discussion Chromosome 2 (mainly 2q chromosomal region) has been cited frequently in patients with ID and there are many reports which have found abnormalities in 2q23.1-q24.1 regions (Magri et al., 2011; Takatsuki et al., 2010). The applications of newer technologies like array CGH and MLPA have enabled researchers to investigate patients with ID leading to the identifcation of deletions/duplications which may be causative in nature. Since the deleted or duplicated region contain many genes, it is not possible to narrow down the actual causative gene. A more thorough investigation is needed, in most cases, to identify the defnite candidate gene for a given disorder.

In our patient, based on NCBI Browser Resources database (NCBI36/hg18) there are six coding genes (GALNT13 (KIAA1918), KCNJ3 (GIRK1), NR4A2, GPD2, GALNT5 and ERMIN) in the deleted 2q23.3-q24.1 region (Table-1). Our fnding is very similar to a previously reported 3.9 Mb deletion in 2q23.3- q24.1 chromosomal region in a mentally retarded patient (Lybæk et al., 2009). Of the seven candidate genes that were reported to be deleted, six of them are also deleted in our case. Among these ERMIN, NR4A2 and GPD2 genes are considered to be the causative genes of our patient’s symptoms (Lybæk et al., 2009).

Deletions of the GALNT13 gene, which is expressed in different parts of brain (Zhang et al., 2003), and the GALNT5 gene (Guzman-Aranguez, Mantelli, & Argueso, 2009) have been reported previously to be copy number variations (http://projects.tcag.ca/variation). The gene KCNJ3 has been reported to be related to neurologic problems like Schizophrenia (Yamada et al., 2012) and Ataxia (Genini et al., 2007) but there are no reports relating their abnormalities to ID. Mutations in NR4A2 (Nuclear receptor subfamily 4 group A member 2 gene), a member of the steroid–thyroid hormone and retinoid receptor super family are associated with disorders related to dopaminergic dysfunction, including Parkinson disease, Schizophrenia, and Manic depression (Baron et al., 2012), and its deletion may be responsible for self- mutilation observed in our patient; however, the hemizygosity of NR4A2 seems not to cause any signifcant clinical manifestations (Barge-Schaapveld et al., 2013). Mutations in the GPD2 (glycerol- 3-phosphate dehydrogenase 2) gene have been reported in a patient with intellectual disability (Daoud et al., 2009), who had a balanced translocation with breakpoints in the GPD2 (2q24.1) region. They suggested that this gene could be responsible for mental ability and thus it is possible that GPD2 haplo-insfficiency could be related to ID. As this gene is also expressed in the brain, it is hypothesized that GPD2 defciency may lead to ID in a similar way by decreasing ATP production (Barge-Schaapveld et al., 2013). NR4A2 and GPD2 gene deletions have also been described in a patient with 298 kb deletion at chromosome 2q24.1 (Barge- Schaapveld et al., 2013). ERM-like gene (ERMIN) which is located at the boundary of the above deletion, codes for a cytoskeletal protein that is expressed exclusively in oligodendrocytes of the human brain. It was introduced as a novel gene with low expression in an epileptic patient (Wang et al., 2011). Seizures in our patient, can be due to the deletions of NR4A2, GPD2, and ERMIN, while the ID can be explained by haplo- insufficiency of the GPD2 gene (Barge-Schaapveld et al., 2013; Motobayashi et al., 2012).

To identify genes to explain variation in the range of normal intelligence, genome wide linkage study (Posthuma et al., 2005) has revealed signifcant evidence for linkage on chromosome 2q (2q24.1-q31.1) for intelligence.

Although deletions of large parts of a chromosome could cause disorders in patients, it needs more patients to study in order to perform exact correlations between genotype and phenotype. In addition, some haplo-insufficient genes could be compensated by other related genes on other regions of the genome; hence it is difficult to consider a deletion in just one patient as the main cause of ID.

The duplication of the FRG1 gene at 4q35.2 identifed by MLPA could not be confrmed by array CGH. In our opinion the MLPA result may either be non-symptomatic or technical artifact.

In conclusion, the usefulness of array-CGH for detecting submicroscopic aberrations provides alternate explanations for the ID phenotypes. Finally, a potential critical region for the proposed 2q23-q24 microdeletion syndrome has further narrowed down, and we proposed that haplo-insufficiency for FMNL2 and/or NR4A2 genes may compromise normal brain development and function, and the Intellectual Disability can be due to the deletion of GPD2 gene.

ACKNOWLEDGEMENTS The authors extend their gratitude to the patients and their willingness to participate in this study. This work was supported by a grant from the Kawsar Human Genetics Research Center.

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975 and its revision in 2000. Informed consent was obtained from all patients for being included in the study. 'Additional informed consent was obtained from all patients for whom identifying information is included in this article'.

The authors declare that they have no conflict of interest.

References:

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Received 28 February 2015, in revised form 15 August 2015; accepted 20 August 2015

Unedited version published online: 21 August 2015 Figure-1: A: Family pedigree, the proband is marked with an arrow B: Affected case at age 30 Figure.2 Copy number variation in MLPA results in chromosome 4q (duplication) probes in the MRC-Holland P070; A: blue peak as patient sample shows duplication in 4q FRG1 gene, B: arrow shows the point of duplication and C: amount of PCR product In MLPA application Figure 3: Array CGH results reveal a 3.7 Mb deletion in 2q23.3-q24.1; a: deletion in whole genome b: deletion of 30 probes of chromosome 2. Table 1 Genes within the 2q23.3 to 2q24.1 deleted region according to OMIM gene Tissue expression OMIM # gene position function symbol description initiate O-linked glycosylation of mucins by the initial transfer of N- Different part of 154728426- GALNT13a acetylgalactosamine (GalNAc) with an alpha-linkage to a serine or body especially brain 608369 155310489 threonine residue The protein encoded by this gene is an integral brain 1555555093- and inward-rectifier type . The encoded protein, is KCNJ3b 601534 155713014 controlled by G- and plays an important role in regulating heartbeat. This gene encodes a member of the steroid-thyroid hormone- Different part of 157180944- NR4A2c retinoid receptor superfamily. The encoded protein may act as a body 601828 157189287 transcription factor. The protein encoded by this gene localizes to the inner Different part of 157291965- GPD2d mitochondrial membrane and catalyzes the conversion of glycerol-3- brain 138430 157442915 phosphate to dihydroxyacetone phosphate, using FAD as a cofactor Immune, Nervous, 158114340- Posttranslationalmodificationof mucin, polypeptide N- GALNT5 Muscle, 615129 158167913 acetylgalactosaminyltransferase activity Reproductive ERMIN 158,175,125- Mostly in brain Function in brain 610072 158,182,416

aUDP-N-ACETYL-ALPHA-D-GALACTOSAMINE:POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE 13 bPOTASSIUM CHANNEL, INWARDLY RECTIFYING, SUBFAMILY J, MEMBER 3 cNUCLEAR RECEPTOR SUBFAMILY 4, GROUP A, MEMBER 2 dGLYCEROL-3-PHOSPHATE DEHYDROGENASE 2