A novel chromosome 2q24.3‐q32.1 microdeletion in a fetus with multiple malformations

Abstract Background Terminal or interstitial deletion of chromosome 2q is rarely reported but clinically significant, which can result in developmental malformations and psychomotor retardation in humans. In the present study, we analyzed this deletion to comprehensively clarify the relationship between phenotype and microdeletion region. Methods We collected clinical records of the fetus and summarized patient symptoms. Subsequently, genomic DNA was extracted from fetal tissue or peripheral blood collected from parents. In addition, whole‐exome sequencing (WES) and copy number variation sequencing (CNV‐seq) were performed. Results The fetus presented a previously unreported interstitial deletion of 2q24.3‐q32.1. WES and CNV‐seq revealed a de novo 18.46 Mb deletion at 2q24.3‐q32.1, a region involving 94 protein‐coding genes, including HOXD13, MAP3K20, DLX1, DLX2, SCN2A, and SCN1A. The fetus had upper and lower limb malformations, including camptodactyly and syndactyly, along with congenital cardiac defects. Conclusion Herein, we report a fetus with a novel microdeletion of chromosome 2q24.3‐q32.1, likely a heterozygous pathogenic variant. Haploinsufficiency of HOXD13 might be related to limb deformity in the fetus.

short stature, moderate-to-severe developmental delay, microcephaly, hypotonia, specific craniofacial dysmorphisms, and upper/lower limb deformities associated with HOXD genes. 5 Previously, chromosome deletions were discovered by Giemsa banding. 6 Chromosome deletions spanning over 5 Mb are microscopically visualized on chromosome-banded karyotypes. Given the development of nextgeneration sequencing technology, CNV sequencing (CNV-seq) has been widely employed in recent years. Compared with conventional methodology, CNV-seq has advantages such as high throughput, high resolution, and relatively low cost. 7 Moreover, CNV-seq can detect deletions above 100 Kb.
Herein, we describe a novel interstitial heterozygous deletion that encompasses the 2q24.3-q32.1 chromosomal region, as determined using CNV-seq and whole exosome sequencing (WES). The deletion was found to affect 94 genes, of which 33 are associated with diseases, including HOXD13, MAP3K20, DLX1, DLX2, SCN2A, and SCN1A. We analyzed the clinical features and genes on the deletion region to further interpret the relationship between the deletion region and phenotype. 8 2 | ME THODS

| Participants
The proband and parents were enrolled at The First Affiliated Hospital of Wenzhou Medical University. Written consent was obtained from the parents of the fetus prior to commencing the study. All study protocols were reviewed and approved by the ethics committees of The First Affiliated Hospital of Wenzhou Medical University. Relevant clinical records (symptoms, appearance and duration of symptoms, physical and ultrasound examination) were collected and examined.

| DNA extraction
According to the manufacturer's standard instructions, genomic DNA was extracted from the fetal muscle and his parents' peripheral blood samples conserved in EDTA using the Tissue Genome DNA Extraction Kit DP341 and Blood Genome DNA Extraction Kit DP329 (TianGen). DNA purity and concentration were determined using the Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Genomic DNA was stored at −20°C until use.

| WES
Briefly, ultrasound was used to break genomic DNA into 250-300 bp fragments. DNA libraries were constructed by end filling, adapter ligation, and polymerase chain reaction amplification. 9 Then, the DNA libraries underwent hybridization capture and were enriched by the xGen Exome Research Panel v2.0 (IDT). High throughput sequencing was performed on the DNBSEQ-T7 platform (Beijing Genomics Institute). After filtration and quality control, clean reads were aligned to the University of California Santa Cruz (UCSC) human reference genome (hg19) using the Burrows-Wheeler mapping algorithm. 10 Combined with OMIM, HGMD, SwissVar, Clinvar, and dbSNP, the genetic variation was analyzed, classified, and annotated with the American College of Medical Genetics (ACMG).

| CNV-seq
The DNA libraries were single-ended sequenced on the DNBSEQ-T7 platform (Beijing Genomics Institute), with a sequencing depth of 0.2x. Raw sequencing reads were processed according to the quality control standards and subsequently compared with the hg19 of the UCSC using Burrows-Wheeler Alignment. 10 Using read counts, Z-scores, and log2Ratio, the in-house bioinformatics pipeline evaluated CNVs. 7 The candidate CNVs were filtered with the Accurate Diagnosis of Genetic Diseases Cloud Platform (Quanpu).

| Clinical data
The male fetus (the proband) was the second child of young and nonconsanguineous parents. The maternal pregnancy was uncomplicated.
Family history included a spontaneous abortion (embryo arrest) at 8 weeks of gestation. No consanguinity was reported. Prenatal care showed no history of exposure to radiation and toxic agents. At the 23rd week of gestation, the fetus exhibited increased anterior nasal skin and nuchal fold ( Figure 1A  As shown in the ultrasonic image, the distance between the thumb, index finger, and middle finger was increased, with splaying between the index and middle fingers ( Figure 1J). His feet were symmetrical with complete cutaneous syndactyly of the second and third digits ( Figure 1K). The necropsy was refused.

| DISCUSS ION
Herein, the proband presented camptodactyly, syndactyly, proximally placed fourth finger, ventricular septal defect, and aortic dysplasia. We identified a novel heterozygous interstitial deletion at chromosome 2q24.3-32.  heterozygous deletion of Map3k20 did not induce abnormal morphological changes.
Severe limb deformities, including split hand and monodactyly, have also been reported, and DLX1 and DLX2 are speculated to be novel candidate genes. 5,14,28 However, upper and lower limb malformations in the examined fetus did not confirm this possibility.
Theisen et al. 29 38 The heterozygous deletion of SCN2A mainly induces autism spectrum disorders and intellectual disability. 39 However, given the death of our proband, several potential symptoms could not develop, and no neurological examinations, such as cerebral magnetic resonance and electroencephalogram, could be performed. Deletion of SCN2A and SCN1A genes did induce notable clinical effects in our proband.
In summary, we report a de novo interstitial deletion of 2q24.3-q32.1. This genomic segment involves 94 protein-coding genes, and 33 of these are related to recognizable clinical phenotypes. This case study further supports the role of HOXD13 haploinsufficiency in limb defects. Furthermore, we identified possible causative genes by analyzing gene function and phenotype. Certain defects may be due to the cumulative effect of genes in deleted fragments.

ACK N OWLED G M ENTS
We thank the patient and his family for their understanding and cooperation. We also acknowledge the technical support provided by Chigene (Beijing) Translational Medical Research Center Co., Ltd.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.