Clinical and molecular cytogenetic analyses of four families with 1q21.1 microdeletion or microduplication

Abstract Background Little information is available regarding the penetrance of 1q21.1 copy number variants (CNVs). In the present study, we explored the clinical significance of 1q21.1 microdeletion or microduplication. Methods In four families, chromosome karyotype was analyzed using G‐banding karyotype analysis technology. CNVs were detected using array‐comparative genomic hybridization (aCGH) and then a quantitative polymerase chain reaction (qPCR) was used to validate candidate CNVs. Sequence signature in the breakpoint region was analyzed using University of California Santa Cruz (UCSC) databases. Results Except for karyotype 45, XX, der (13, 14) (q10, q10) in the mother (I2) of family 2, the karyotype was normal in all other members of the four families. In the mother (I2) and fetus (II2) of family 1, in newborn (II1) of family 2 and in fetus (II1) of family 3, there was 1.22‐Mb heterozygous microdeletion in the chromosome 1q21.1q21.2 region. The child (II1) of family 4 had a 1.46‐Mb heterozygous microduplication in the chromosome 1q21.1q21.2 region. The results of the qPCR were consistent with that of aCGH. There was large number of low copy repeats (LCRs) in the breakpoint region found by analysis of the UCSC database, and multiple LCRs were matched with sequences in the chromosome 1 short‐arm region. Conclusions 1q21.1 microdeletion and microduplication exhibit a variety of clinical manifestations and the specificity of their clinical features is not high. The penetrance of the distal 1q21.1 microdeletion may be affected by other factors in the present study. In summary, we report the discovery of a new distal 1q21.1 microduplication, which enriches the CNV spectrum in the 1q21.1 region and is conducive to prenatal genetic counseling.

mildly dysmorphic facies and developmental delay. However, there is no clinically recognizable syndrome, and some individuals with this microdeletion do not present obvious clinical findings. [3][4][5] The 1q21.1 recurrent microdeletion is inherited in an autosomal teddominant manner, with 18-50% of deletions occurring de novo and 50-82% being inherited from their parents. No genotype-phenotype correlations are observed in those with the 1q21.1 recurrent microdeletion. Little information is available regarding penetrance of the 1q21.1 recurrent microdeletion. Similar to several other recurrent microdeletions (e.g. 16p11.2, 15q13.3), the 1q21.1 recurrent microdeletion can be inherited from the parents with minimally abnormal or completely normal clinical findings. In addition, several relatives of probands with the same 1q21.1 microdeletion have a normal phenotype or only mild manifestations. 6,7 Because the number of individuals published to date is limited, the exact phenotypic consequences remain unclear. In the present study, we describe the clinical phenotype and molecular cytogenetics of three families with 1q21.1 microdeletions.
1q21.1 duplication syndrome is a rare aberration of chromosome 1 with multiple congenital malformations, including developmental delay, autism spectrum disorder, dysmorphic features and congenital heart anomalies. Congenital heart malformations occur in approximately 18% and 29% of patients with proximal and distal 1q21.1 microduplications, respectively. These comprise a broad spectrum of abnormalities, including left-sided, right-sided, conotruncal and septal defects. 8 In the present study, we describe the clinical phenotype and molecular cytogenetics of one family with 1q21.1 microduplication.
The distal 1q21.1 microduplication was discovered for the first time in the present study, enriching the CNV spectrum in the 1q21.1 region and providing a basis for clinic and prenatal genetic counseling.

| MATERIALS AND METHODS
All study methods were approved by the Ethics Committee of Henan Provincial Peoples Hospital. Written informed consent was provided by all subjects who enrolled in the study, as well as their parents.

| Subjects
Four participants including two fetuses with malformations, a child with Tetralogy of Fallot and a normal newborn, were from four Chinese families without a family history of congenital malformation. Parents of these patients went to the Medical Genetics Institute for genetic counseling. A routine clinical examination was performed. Detailed birth, medical data and clinical manifestations were collected. The pedigree of four families is shown in Figure 1.

| G-banding karyotype analysis
Amniotic fluid (10 ml) and venous blood (3 ml) were collected from two fetuses and other members of the four families, respectively. Amniotic fluid (1 ml) or venous blood (1 ml) was inoculated into RPMI1640 at 37°C for 72 h. Colchicine was added 1 h before collecting samples.
The chromosomes were prepared using the routine method and then underwent Giemsa staining followed by analysis of 30 mitotic phases under a microscope using a fully automatic karyotype analysis system (Leica Microsystems, Wetzlar, Germany). The karyotype was described based on the International Naming System of Human Cell Genetics (ISCN2013).   (Table S1). GADPH served as a reference gene.

| Sequence analysis of the breakpoint region
The sequence signature in the breakpoint region was analyzed using UCSC databases, as well as National Center for Biotechnology Information (NCBI). FIGURE 2 Fetal encephalomeningocele. Behind the fetal head, there is a 56 × 52 mm fluid sonolucent area that is multilocular as a result of the existence of septations with a 15 × 9 mm high-level echo FIGURE 3 The fetus has complete atrioventricular septal defect. The four-chamber view of the fetus shows the disappearance of cross section with a 6.75-mm defect FIGURE 4 Tetralogy of Fallot combined with acleistocardia. Left ventricular long-axis view shows the ventricular septal defect and aortic overriding (left). Large arterial short-axis view shows the main pulmonary artery, left pulmonary artery, right pulmonary artery stenosis and the increased thickness of the right ventricular anterior wall (right)

| RESULTS
In family 1, prenatal diagnosis and assay of genomic CNVs were required because the type-B ultrasonic instrument showed encephalomeningocele ( Figure 2).in the fetus (II2). In family 2, karyotype analysis and assay of genomic CNVs were required because the mother (I2) had karyotypic abnormality. In family 3, prenatal diagnosis and assay of genomic CNVs were required because the type-B ultrasonic instrument showed complete atrioventricular septal defect ( Figure 3).in the fetus (II1). In family 4, karyotype analysis and assay of genomic CNVs were required because there were the symptoms of dyspnea and cyanosis, systolic ejection murmurs were available beside the left sternal border and between the second and fourth ribs, and the type-B ultrasonic instrument showed tetralogy of Fallot combined with acleistocardia ( Figure 4) in the child (II1). All members of the four families were followed up and their clinical data were collected (Table 1).
Except for karyotype 45, XX, der (13, 14) (q10, q10) in the mother   As proposed by Girirajan et al., 26 several other possibilities, such as FIGURE 9 The UCSC database indicates a large number of repeated similar fragments in the 1q21.1q21.2 region with CNVs epigenetic phenomena, expression or regulatory variation among genes in the vicinity of the unbalanced region, the unmasking of recessive alleles and the possibility of a 'two-hit' model, may account for the phenotypic variability of some genomic diseases.  27 Helicase protein encoded by the GHD1L gene is involved in repair by transformation of ATP into poly ADP-ribose, and is closely related to DNA repair after chromatin unwinding. 29 Adenylate activating protease β2 subunit encoded by the PRKAB2 gene is found to have high expression in the right ventricular outflow tract. 30 Because there are very few related studies, the effects of other genes in this region on the development and differentiation of tissues and organs remain to be further explored.
In the present study, we investigated clinical data and genetics in the four families aiming to explore the relationship between if the CNVs inherited from parents are found, their pathogenicity is not completely denied because the pathogenic penetrance of these CNVs may be affected by other factors, and so it is also necessary to evaluate fetal development using other prenatal diagnosis technology. In addition, the present study discovered a new distal 1q21.1 microduplication that enriches the CNV spectrum in the 1q21.1 region.
In summary, the present study provides molecular genetic data and is conducive to genetic counseling with respect to CNV-related diseases.