Inherited deletion of 9p22.3‐p24.3 and duplication of 18p11.31‐p11.32 associated with neurodevelopmental delay: Phenotypic matching of involved genes

Abstract We describe a 3.5‐year‐old Iranian female child and her affected 10‐month‐old brother with a maternally inherited derivative chromosome 9 [der(9)]. The postnatally detected rearrangement was finely characterized by aCGH analysis, which revealed a 15.056 Mb deletion of 9p22.3‐p24.3p22.3 encompassing 14 OMIM morbid genes such as DOCK8, KANK1, DMRT1 and SMARCA2, and a gain of 3.309 Mb on 18p11.31‐p11.32 encompassing USP14, THOC1, COLEC12, SMCHD1 and LPIN2. We aligned the genes affected by detected CNVs to clinical and functional phenotypic features using PhenogramViz. In this regard, the patient's phenotype and CNVs data were entered into PhenogramViz. For the 9p deletion CNV, 53 affected genes were identified and 17 of them were matched to 24 HPO terms describing the patient's phenotypes. Also, for CNV of 18p duplication, 22 affected genes were identified and six of them were matched to 13 phenotypes. Moreover, we used DECIPHER for in‐depth characterization of involved genes in detected CNVs and also comparison of patient phenotypes with 9p and 18p genomic imbalances. Based on our filtration strategy, in the 9p22.3‐p24.3 region, approximately 80 pathogenic/likely pathogenic/uncertain overlapping CNVs were in DECIPHER. The size of these CNVs ranged from 12.01 kb to 18.45 Mb and 52 CNVs were smaller than 1 Mb in size affecting 10 OMIM morbid genes. The 18p11.31‐p11.32 region overlapped 19 CNVs in the DECIPHER database with the size ranging from 23.42 kb to 1.82 Mb. These CNVs affect eight haploinsufficient genes.


| INTRODUC TI ON
Neurodevelopmental disorders (NDDs) are a heterogeneous class of conditions that impact brain development with deficits in several domains of cognitive function that affect daily life. It has been estimated that at least 30% of NDDs are caused by genetic factors. 1 NDDs include intellectual disability (ID), autism spectrum disorder, and attention-deficit/hyperactivity disorder. 2 New high-throughput technologies, such as whole exome and whole genome sequencing, have revolutionized the diagnostic approach for NDDs, but there is still an ongoing debate on the choice of the optimal first-tier diagnostic test. 3 However, for the detection of copy-number variants (CNVs), array comparative genomic hybridization (aCGH), with an average diagnostic yield between 15% and 20%, continues to be widely used as a first-tier diagnostic test. 4 CNVs are not only a prevalent source of genomic variation in the general population, but are also a major cause of NDDs and related disorders. 5 Here, we present a 3.5-year-old Iranian female child and her affected 10-month-old brother affected with derivative chromosome 9, originating from a balanced translocation t(9;18) (p22;p11.31) present in the mother. Characterization of the postnatally detected rearrangement by aCGH showed partial 9p monosomy and partial 18p trisomy. In line with the objectives and scope of this study, we matched patient's phenotypes to the genes within the detected CNVs using PhenogramViz. Facilitating a phenotypeguided interpretation of CNVs, this software is available as a plugin for CytoScape (V.3.8.2) and integrates data from different sources (including OMIM, MGI, ZFIN and Orphanet) with the selected HPO terms and then visualizes gene-to-phenotype associations as a 2D network of nodes and edges, which is called a phenogram. 6,7 Furthermore, we used DECIPHER for in-depth characterization of genes that overlap the detected CNVs along with other relevant information such as gene/disease association information and predictive scores (e.g., pLI score) and also comparison of patient phenotypes with 9p and 18p genomic imbalances. For finding specific gene-phenotype associations, small CNVs affecting only one gene was our focus. 8 Since the first report of partial monosomy 9p in 1973, 9 over 100 cases have been documented. 10 Unlike full or mosaic trisomy 18 or partial trisomy 18q, trisomy 18p has been rarely reported with only about 32 cases of trisomy 18p having been published to date. 11 Reported cases involving a translocation between chromosomes 9 and 18 resulting in partial monosomy 9p and partial trisomy 18p are even rarer. Our review of published reports found only one such report. 12 2 | MATERIAL S AND ME THODS

| Blood sampling and karyotyping
Blood samples were collected from all members of the studied family. Informed consents were obtained. The study was approved by the regional ethics committee at the Mashhad University of Medical Sciences, Mashhad, Iran. Chromosomal culture and karyotyping of both children and the parents were performed on routinely cultured peripheral blood lymphocytes. A total of 20 metaphase spreads were analysed in each case according to the GTG banding technique.
The maximum banding resolution achieved was 500-600 bands.

| Copy number variation analysis using aCGH
Whole genome oligo aCGH was performed using SurePrint G3 ISCA V2 8X60K whole genome oligo array version 2 and was analysed using Agilent Cytogenomic software v4. The array consists of 60,000 spots with overall median probe spacing of 60 kb and higher near to 500 targeted disease regions. The sample was hybridized against the male sample used as reference.

| Bioinformatic analysis
For aligning the genes affected by detected CNVs to clinical and functional phenotypic features, we used PhenogramViz software.

| Clinical assessment
A 3.5-year-old Iranian female child (Patient 1) and his 10-month-old brother (Patient 2) both with dysmorphic features and congenital malformations were referred for chromosomal analysis and genetic counselling. She was the first child born to healthy, nonconsanguineous parents after a full-term pregnancy and normal delivery. The mother was aged 27 years and the father 31 years at the time of the baby's birth. Obstetric history revealed a history of one spontaneous abortion occurring at 10 weeks of gestational age.
It seemed that patient 2 has similar complaints, which was determined upon clinical investigation. The remaining family history was unremarkable.
At clinical examination, patient 1's speech, gross motor and social milestones were prominently delayed. She had a trigonocephalic head, low posterior hairline, highly arched eyebrows, wide intermammillary distance, wide nasal bridge, long philtrum, thin upper lip vermilion, open mouth with downturned corners, mild micrognathia, hand flapping and repetitive movements, tapering fingers, and pes planus ( Figure S1). She also had a small ventricular septal defect (VSD) as demonstrated by echocardiography. Her transferrin and iron-binding capacity (TIBC) was high (500 μg/dl). Additional laboratory investigations, including brain and renal/urinary tract ultrasonography, blood urea, blood sugar, thyroid profile, creatinine, calcium, alkaline phosphatase, phosphorus, and serum electrolytes were all normal. Her brother had also relatively similar clinical manifestations along with cryptorchidism. Furthermore, his brain MRI showed a hypoplastic (thin) corpus callosum, cavum septum pellucidum, enlarged sylvian cistern, delayed myelination, and trigonocephaly ( Figure 1).

| Phenotypic matching using PhenogramViz
For phenotypic matching, the patient's phenotype and CNV data were provided and entered to PhenogramViz. For the 9p deletion CNV, 53 affected genes were identified and 17 of them were matched to 24 HPO terms describing patient's phenotypes ( Figure S4). Additionally, for the 18p duplication, 22 affected genes were identified and six of them were matched to 13 phenotypes ( Figure S5). Note: pLI, The probability that a gene is intolerant to loss-of-function (LOF) mutations. Values range from 0 to 1 and genes with larger values (closer to one) are more intolerant of mutations. %HI, haploinsufficiency score, according to DECIPHER high ranks (e.g., 0%-10%) indicate that a gene is more likely to exhibit haploinsufficiency, low ranks (e.g., 90%-100%) indicate a gene is more likely to not exhibit haploinsufficiency. G2P, Gene2Phenotype is an online system designed to facilitate the development, validation, curation and distribution of large-scale, evidence-based datasets for use in diagnostic variant filtering (https://www.decip herge nomics.org/). and GLDC were matched to neurodevelopmental phenotypes, but no matching was seen for genes located in 18p11.31-p11.32 (Figure 3).
In support of the role of the KANK1 gene, in a study by Lerer et al., 17 neuroimaging for nine children belonging to a four-generation family with deletion of the KANK1 gene showed brain atrophy and ventriculomegaly. Hypothetically, ventriculomegaly may be act as a common ancestor phenotype between KANK1 and enlarged sylvian cistern. Accumulation of CSF in the ventricles due to mutations in the MPDZ gene may be also cause ventriculomegaly. 18 The other potential disease-associated gene for the patient 2's observed neuroimaging phenotypes is TGIF1 located at 18p11.3. As a dosagesensitive gene, TGIF1 was matched to four neuroimaging phenotypes including hypoplasia of corpus callosum, cavum septum pellucidum, enlarged sylvian cistern, and trigonocephaly.
Trigonocephaly as a notable craniofacial feature in our patients (Figures 1 and S1) was matched also to FREM1 gene, which resides in the 300 kb interval on 9p24.3 region. This segment for the first time was defined by Swinkels et al. 19 as a candidate for metopic

| Genital phenotypes
Genital abnormalities are estimated to be present in up to 70% of patients with partial 9p monosomy. 21 The DMRT genes (DMRT1,

ACK N O WLE D G E M ENTS
We would like to express our deep gratitude to the all members of the studied family and the staff at Pardis Pathobiology and Genetics Laboratory for their helpful collaboration.

FU N D I N G I N FO R M ATI O N
This study was supported by NIH NHGRI U24HG011449 and Medical Genetics Research Center (MGRC), Mashhad University of Medical Sciences, Mashhad, Iran.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest relevant to the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.