Prenatal Diagnosis and Molecular Cytogenetic Characterization of Copy Number Variations on 4p15.2p16.3, Xp22.31, and 12p11.1q11 in a Fetus with Ultrasound Anomalies: A Case Report and Literature Review
Chromosomal rearrangements, such as duplications/deletions, can lead to a variety of genetic disorders. Herein, we reported a prenatal case with right aortic arch and aberrant left subclavian artery, consisting of a complex chromosomal copy number variations. Routine cytogenetic analysis described the chromosomal karyotype as 46,XY, add (2)(q37) for the fetus. However, the chromosomal microarray analysis (CMA) identified a 22.4 Mb duplication in chromosome 4p16.3p15.2, a 3.96 Mb microduplication in 12p11.1q11, and a 1.68 Mb microdeletion in Xp22.31. Fluorescence in situ hybridization (FISH) using a chromosome 4 painting probe was found to hybridize to the terminal of chromosome 2q on the fetus, thus confirming that the extra genetic materials of chromosome 2 was actually trisomy 4p detected through CMA. Meanwhile, the parental karyotypes were normal, which proved that the add (2) was de novo for fetus. The duplication of Wolf-Hirschhorn syndrome critical region (WHSCR) and X-linked recessive ichthyosis associated with Xp22.31 deletion separately were considered potentially pathogenic causes although other abnormalities involving these syndromes were not observed. For prenatal cases, the combined utilization of ultrasonography, traditional cytogenetic, and molecular diagnosis technology will enhance better diagnostic benefits, offer more detailed genetic counselling, and assess the prognosis of the fetuses.
Trisomy 4p syndrome, partial or almost duplication of the short arm in chromosome 4, is a rare chromosomal anomaly in clinic . Since it was initially described as a distinct clinical entity in the 1970s, more than 80 cases of trisomy 4p had been reported [1, 2]. Most trisomy 4p cases involve at least the distal half of 4p (i.e., 4p14-4pter) or more . Trisomy 4p has been associated with various clinical symptoms, such as growth retardation, psychomotor delay, mental retardation, and dysmorphic features such as facial dysmorphism, microcephaly, skeletal abnormalities, and heart defects [2, 4]. The formation mechanism of trisomy 4p usually results from an unbalanced segregation of a parental balanced translocation or a de novo duplication . Currently, it is difficult to establish clear genotype-phenotype correlations for trisomy 4p cases that usually exhibit different sizes of duplicated region accompanied by another chromosomal deletion .
Xp22.3 microdeletion is associated with several genetic syndromes in males, which includes short stature, X-linked recessive chondrodysplasia punctata, X-linked mental retardation, X-linked ichthyosis, and Kallmann syndrome [5, 6].
Trisomy 12 is a rare chromosomal disorder resulting in spontaneous abortion easily. Partial duplications are associated with diverse clinic phenotypes, ranging from normal phenotypes to severe physical defects in different organ systems .
Here, we report a prenatal case with abnormal sonography findings, consisting of a de novo 4p15.2p16.3 duplication translocated to the terminal of the long arm in chromosome 2, a Xp22.31 microdeletion and a 12p11.1q11 duplication. Meanwhile, we also compare the clinical features of the cases involving similar 4p duplication as described in the literature and databases.
2. Materials and Methods
A 23-year-old, gravida 1, para 0, woman underwent amniocentesis for cytogenetic and chromosomal microarray analysis (CMA) at 25 weeks of gestation due to the right aortic arch and aberrant left subclavian artery in prenatal sonography findings. The pregnant woman and her husband were nonconsanguineous and healthy. They both showed no family history of diabetes mellitus or congenital malformations. The wife denied any exposure to alcohol, teratogenic agents, irradiation, or infectious diseases during this pregnancy. Our study protocol was approved by the Ethics Committee of the First Hospital of Jilin University (No. 2019-261), and written informed consent was obtained from the couple.
2.2. Cytogenetic Analysis
Cytogenetic studies were carried out on G-band metaphases collected from cultured amniotic fluid cells and peripheral blood cells of the couple. Chromosome preparations were obtained according to G-banding technique with a resolution between 300 and 400 bands. We analyzed twenty metaphases for the fetus and the couple. And the chromosomal karyotypes were described according to the ISCN 2016 nomenclature .
2.3. Chromosomal Microarray Analysis (CMA)
Following written consent from the pregnant woman, 10 mL of uncultured amniotic fluid cells was collected through amniocentesis. Genomic DNA was isolated using Qiagen micro kit with the manufacturer’s protocol. Then, the procedures are conducted through CytoScan 750K array (Affymetrix, Santa Clara, CA, USA), in accordance with the manufacturer’s protocol and our previous study . The procedure included genomic DNA extraction, digestion and ligation, PCR amplification, PCR product purification, quantification and fragmentation, labeling, array hybridization, washing, and scanning. Thresholds for genome-wide screening were set at ≥200 kb for gains and ≥100 kb for losses. The detected copy number variations were comprehensively estimated by comparing them with published literature and the public databases: (1) Database of Genomic Variants (DGV) (http://dgv.tcag.ca/dgv/app/home), (2) DECIPHER (http://decipher.sanger.ac.uk/), (3) ISCA https://www.iscaconsortium.org/), (4) ECARUCA (http://www.ecaruca.net), and (5) OMIM (http://www.ncbi.nlm.nih.gov/omim).
2.4. Fluorescence In Situ Hybridization (FISH)
Based upon the results of karyotype analysis and CMA results, fluorescence in situ hybridization (FISH) using a whole chromosome painting probe specific for chromosome 4 (Cytocell Technologies, Cambridge, UK) was performed on metaphase slides for the fetus to confirm the existence of trisomy 4p, according to the manufacturer’s protocol.
The routine cytogenetic analysis described the chromosomal karyotype as 46,XY, add (2)(q37) (Figure 1). However, the CMA detection identified a complex chromosomal copy number variations (CNVs): arr [GRCh37] 4p16.3p15.2(68345-22489538)×3; arr [GRCh37] 12p11.1q11 (33909111-37869107)×3; and arr [GRCh37] Xp22.31 (6455151-8135568)×0 (Figure 2). It was inferred that the extra genetic materials of chromosome 2q might be the partial 4p duplication. Based upon the speculation, we informed the couple that peripheral chromosome studies will be performed to confirm that add (2) was parentally inherited or de novo. Meanwhile, the whole chromosome painting probe specific for chromosome 4 was applied to confirm whether the segment of trisomy 4p was located on chromosome 2. The G-banding analysis demonstrated that the parental karyotypes were normal, which illustrated that the add (2) in the fetus was de novo. FISH using chromosome 4 painting probe was found to hybridize to the terminal of chromosome 2q on the fetus, thus confirming that the existence of add (2) was actually the 4p duplication detected through CMA (Figure 3). The couple finally chose to terminate the pregnancy according to genetic counselling based upon abnormal cytogenetic and molecular genetic analysis.
In our study, we report a prenatal case with right aortic arch and aberrant left subclavian artery in ultrasonography. The fetus carried a 4p16.3p15.2 duplication attached to the terminal of chromosome 2q, a Xp22.31 microdeletion, and a 12p11.1q11 microduplication, which were identified by the combined application of G-banding technique, CMA, and FISH analysis. To the best of our knowledge, these chromosomal complex rearrangements have not been reported previously.
Trisomy 4p, as a rare chromosomal disorder, is a distinct syndrome due to various degrees of duplicated regions in the short arm of chromosome 4 . Trisomy 4p cases usually presented growth retardation and psychomotor retardation with or without seizures, as well as various major and minor anomalies, including microcephaly, prominent glabella, bulbous nose, retrognathia, pointed chin, short neck, enlarged ears, rocker-bottom feet, arachnodactyly, and camptodactyly [2, 11].
To better interpret the phenotype-karyotype correlations, we summarized the clinical manifestations of cases with similar/overlapping 4p15.2p16.3 duplication according to literature review and databases, as shown in Table 1 [12–17]. The age of the patients ranged from 2 months to 27 years. Among these duplications, 4/10 were parentally inherited, 3/10 patients were de novo, and 3/10 patients were not available. The high frequencies of different clinical manifestations were as follows: ear anomaly (7/10), growth retardation (6/10), limb anomaly (6/10), intellectual disability (5/10), psychomotor retardation (5/10), short neck (4/10), widely spaced nipples (3/10), and microcephaly (3/10). It was worth noting that varying degrees of abnormal craniofacial features could be observed in all cases. To our knowledge, right aortic arch and aberrant left subclavian artery were not reported in prenatal cases with 4p15.2p16.3 duplication before. Unbalanced segregation of a parental balanced translocation or inversion involving chromosome 4 would usually lead to partial trisomy 4p, accompanied by another chromosome or 4q monosomy , and the karyotypes of 6/10 cases in our review were consistent with this description. The couple accepted chromosomal karyotypic analysis to testify whether the imbalanced genomic aberrations of the fetus resulted from parentally balanced chromosomal rearrangement or not. The normal karyotypes of the couple illustrated that the add (2) in the fetus was de novo. Afterwards, molecular genetic analysis proved the existence of 4p duplication, which was translocated to the terminal of chromosome 2q. Generally speaking, more research should be gathered to describe a distinct phenotype-karyotype correlation on pure 4p duplication.
Chromosomal rearrangements, such as duplications/deletions, can result in a series of genetic diseases . Dosage-sensitive genes, due to the chromosomal duplication or deletion, could result in a wide range of clinic phenotypes, including heart disease, cancers, and neuropsychiatric disorders . Based upon the DECIPHER database, the morbid genes located in the three regions and their associated diseases are summarized in Table 2. According to the literature review and databases, we delineated the potentially pathogenic genes probably exhibiting a “dose effect” and related duplication/deletion syndrome, which could predict the possible phenotypes in clinic and poor prognosis for the fetus.
In our study, the CMA detected a duplication of 4p15.2p16.3 containing the critical 4p16.3 region, the deletion of which was associated with Wolf-Hirschhorn syndrome (WHS; OMIM: 194190), first described in the 1960s . Patients with this deletion usually presented growth impairment, intellectual disability, congenital malformations, distinctive craniofacial appearance, and seizures [22, 23]. Two critical regions are involved in the Wolf-Hirschhorn syndrome critical region (WHSCR): WHSCR1 (WHSC1 and WHSC2 genes included) and WHSCR2 (WHSC1 and LETM1 genes included). The gene WHSC1 (OMIM: 602952) is primarily associated with the clinic features of Wolf-Hirschhorn syndrome, including developmental retardation and abnormal facial appearance. And the gene LETM1 (OMIM: 604407) is proposed to be responsible for seizures in WHS patients . However, more investigations revealed that trisomy 4p patients with WHSCR also presented development/psychomotor retardation and craniofacial and skeletal malformations [2, 18]. Patients with half 4p duplication were characterized by multiple congenital anomalies, mental retardation, broad nasal bridge, and ear anomalies , and 4p16 duplication has been correlated with overgrowth and mental retardation . According to the ClinGen database, there is some evidence for the triplosensitivity in association with the WHSCR duplication. Although the fetus in our report only showed right aortic arch and aberrant left subclavian artery at present, it could be speculated that other abnormal symptoms might gradually appear in the late pregnancy or after birth.
In our study, the CMA detected a 1.68 Mb deletion in Xp22.31, a most genomic instable region in the short arm of chromosome X. The genes in this deleted region included STS, HDHD1, VCX, and PNPLA4. STS (OMIM: 300747), located in Xp22.31, encodes steroid sulfatase as metabolic precursors for estrogens, androgens, and cholesterol. The deletion of this causative gene is closely associated with X-linked recessive ichthyosis (XLI; OMIM: 308100), which is a typically inherited disorder characterized by scaly skin on the scalp, trunk, neck, and extremities after birth. The incident rate is about 1/1500 in males, and approximately 90% XLI patients are involved in STS gene deletion . During the process, the deficiency of the enzyme steroid sulfatase (STS) is critically responsible for abnormal phenotypes . According to the ClinGen database, there is sufficient pathogenic evidence for haploinsufficiency associated with STS. Based upon the DECIPHER database and published literature, intellectual disability and learning difficulties can also be observed in patients with XLI. Xp22.3 deletion in XLI males can be associated with mental retardation, which should be taken seriously . HDHD1 (OMIM: 306480), often missing in XLI, encodes a pseudouridine-5-phosphatase. It is specifically involved in dephosphorylation of a modified nucleotide present in RNA . PNPLA4 (OMIM: 300102) might play a role in regulating epidermal homeostasis . The VCX (OMIM: 300229) gene might not be associated with intellectual development, while potential correlation between the VCX gene and cognitive development and/or communication still requires further research . Based upon the above, the male fetus was recognized as a potential XLI patient.
In addition, a duplication in 12p11.1q11 was detected. ALG10 (OMIM: 618355), encoding a membrane-associated protein, might be associated with nonsyndromic hearing impairment in mice. Currently, there is no pathogenic evidence on ALG10 duplication .
For visible chromosomal anomalies of unknown origin, traditional cytogenetic analysis can hardly identify the aberrations due to the low banding resolution, but the application of CMA could detect the extra or missing genetic materials quickly and assess the clinic significance through genome browsers and public databases, which offers a more precise genetic counselling for these patients [34, 35]. In our study, 4p15.216.3 duplication involving WHSCR and Xp22.31 deletion associated with XLI were detected simultaneously in the fetus, which might lead to unavoidable poor prognosis, so the couple finally chose termination of pregnancy. Considering normal karyotypes of the couple, CMA should also be carried out for the couple before they intend to conceive again. Considering the possibility of the mother being a Xp22.32 deletion carrier, preimplantation genetic testing could be considered. Equally, prenatal diagnosis is essential after the wife gets pregnant.
In our study, we delineate a prenatal case with ultrasound findings, consisting of a de novo partial 4p15.2p16.3 duplication, a Xp22.31 microdeletion, and a 12p11.1q11 microduplication. Among the three CNVs, 4p15.2p16.3 was translocated to the terminal of the long arm in chromosome 2, which was testified by FISH. Although no other abnormalities were observed, WHSCR duplication and X-linked recessive ichthyosis involved in the critical duplicated/deleted regions were considered pathogenic factors. For prenatal cases, the combined utilization of prenatal ultrasound screening and traditional cytogenetic and molecular genetic analysis will exert better diagnostic benefits and offer more significant cytogenetic information, so more precise genetic counselling could be given in clinic.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
We express our sincere gratitude to all staff of the Center for Reproductive Medicine and Center for Prenatal Diagnosis, First Hospital, Jilin University for their excellent work. This work was supported by the Finance Department Health Special Project of Jilin Province, China (grant number: JLSCZD2019-022).
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