5p deletion syndrome, first described by Lejeune et al. in 1963 (1), is a rare genetic disease that defined as variable-sized deletions of the short arm of chromosome 5. 5p deletion syndrome is one of the most common pathogenic copy number variations (CNVs), with a reported incidence of 1 per 15,000–50,000 live births (2). Typical features of this disorder are post-natal cat-like cry and a peculiar timbre of voice. Other notable features include developmental delay, severe psychomotor and intellectual disability, rounded face, hypertelorism, epicanthic folds, and cardiac, cerebral, renal and gastrointestinal malformations (3). Ultrasound is not recommended for in utero diagnosis of 5p deletion syndrome owing to the non-specific ultrasonographic features (4), which underscores the importance of genetic prenatal diagnosis. Southern Blot was first used for the diagnosis of 5p deletion syndrome in 1989 (5). However, due to the limited coverage of probes, Southern Blot is unable to detect all 5p regions and cannot identify the breakpoint. Karyotyping has been the gold-standard in prenatal diagnosis for decades (6). However, detection of 5p microdeletion is beyond the capability of this technique (7). Fluorescence in situ hybridization (FISH) requires a priori knowledge and it is difficult to detect all microdeletions and microduplications due to the limitations of designed probes (8). Chromosomal microarray analysis (CMA) and copy number variation sequencing (CNV-seq) are reliable and accurate prenatal techniques for identifying CNVs. The latter technique has the ability to identify cryptic CNVs located in regions with insufficient probe coverage on CMA platforms, and it also has a higher sensitivity for detecting low-level mosaicism (9). Phenotypic heterogeneity in different cases of 5p deletion syndrome depends on the specific breakpoints on 5p that involve a variety of genes. All the above-mentioned techniques have shortcomings in detecting the specific breakpoints. Whole genome sequencing (WGS), on the contrary, has the potential to define chromosomal breakpoints at nucleotide-pair resolution (10). WGS combined with Sanger sequencing perform better in detecting chromosomal translocations compared with FISH and karyotyping, show higher resolution and the ability to identify the breakpoint, and may replace traditional cytogenetic methods in the diagnosis of balanced translocation (11, 12).

Herein, we reported three families and two children with CNVs of 5p deletion. The objective of the present study was to assess the genotype-phenotype correlation of 5p deletion syndrome, and redefine the relevant region of 5p deletion syndrome. In addition, we underscored the potential use of WGS for identifying chromosomal breakpoints in prenatal diagnosis.

In family 1 (Figure 1 and Table 1), the pregnant woman (I-1) was 19⁺⁵ weeks of gestation and accepted getting genetic counseling for a history of spontaneous miscarriage. Both the woman (I-1) and her husband (I-2) were phenotypically normal. Amniocentesis was performed due to advanced maternal age, and karyotyping and CNV-seq of the fetus and couples were conducted.

In family 2 (Figure 2 and Table 1), the pregnant woman (II-2) was 19⁺² weeks of gestation, obtaining getting genetic counseling for positive results by non-invasive prenatal testing (NIPT). NIPT was performed on Illumina Nextseq CN500 platform, the quantitated DNA library was sequenced using the single–ended 36 bp sequencing protocol. NIPT result presented a fetal fraction of 12.26%, indicated a 3.20 Mb deletion at 5p15.33:100,000–3,299,999 (z score: –10.212), and showed two chromosome X plus a abnormal chromosome Y which only retained a 0.32 Mb fragment at Yq11.23:28,460,000-28,780,000 (Z score: 6.911). All the other individuals were phenotypically normal. Amniocentesis was performed followed by karyotyping and CNV-seq of the fetus and selected family members (I-2, II-1, II-2, III-1, and III-2). In addition, FISH was conducted on the fetus and selected family members (I-2, II-1, II-2, and III-2).

In family 3 (Figure 4 and Table 1), the pregnant woman (IV-2) was 22 weeks of gestation, receiving genetic counseling for hearing loss and mild intellectual disability. IV-3 was a girl with the phenotype of congenital deafness, moderate intellectual disability, and walking disorder. All the other individuals were phenotypically normal. Amniocentesis was performed followed by karyotyping and CNV-seq of the fetus and family members (III-2, IV-2, IV-3, and V-1). In addition, WGS was carried out on III-2.

Child 1 was a boy with the phenotype of cat-like cry and developmental delay. Both his parents were phenotypically normal. CNV-seq of the family members was conducted.

Child 2 was a girl with the phenotype of hypotonia, dysplasia of corpus callosum, and developmental delay. Both her parents were phenotypically normal. CNV-seq of the family members was conducted.

This study was approved by the Medical Ethics Committee of the Chinese PLA General Hospital (approval number S2016-120-02) and written informed consent was obtained from the family members.

The protocol for diagnostic work up for 5p deletion syndrome was shown in Supplementary Figure 1.

As common CNVs, 5p partial deletions were reported to be usually de novo and caused by paternal translocation in 80–90% of cases, possibly arising from chromosome breakage during gamete formation of males (2). Approximately 10–15% of 5p partial deletions were attributed to balanced parental translocation (14). Here, in family 2, the karyotyping and FISH results demonstrated that the translocation between Yq11.23 and 5p15.33 was the causal lesion. In family 3, both CNV-seq and WGS results showed that the translocation between 5p15.33-p15.31 and 6p25.3-p24.3 was the reason for the CNVs of 5p deletion combined 6p duplication. No translocation was detected in family 1, and the variation was supposed to arise from chromosomal breakage during gamete formation.

The observed variability of phenotypes among individuals with 5p deletion is attributed to the differences of the breakpoints. Studies have demonstrated the correlation between deletion size and clinical features. For example, 5pter-p15.32 is associated with speech delay, the proximal part of 5p15.3 and 5p15.2 may be responsible for the cat-like cry, and deletion of the proximal part of 5p15.2 underlies the typical facial dysmorphism (15, 16). In our study, we report three prenatal diagnosis pedigrees and two children. In these families, 5p deletions with variable sizes were detected in related individuals. However, the pregnant woman in family 1 who carried the 5pter-p15.31 variation was apparently normal, and the fetus (II-1) showed no symptoms after birth. Despite a few reports describing its relation with dyskeratosis congenita, TERT gene is currently only confirmed to be associated with shortened telomeres, and cannot explain all characteristics of the 5p deletion syndrome (17, 18). Hence, according to the ClinGen database and our report, we inferred that the 5pter-p15.31 region (1–7,700,000) may not involve any dosage sensitive genes, and the monosomy of inner 5p15.33 (1–3,220,000) in family 2 and 5pter-p15.31 (1–7,080,000) in family 3 were not pathogenic. Similar cases have been reported previously wherein individuals who carried a 5p terminal (1–4,500,000) deletion and an interstitial deletion at 4,200,304–7,081,712 showed no phenotypic effects (16, 19). Zhang et al. reported a 5p (17,630–4,805,799) deletion pedigree in which the proband showed poor feeding, bradycardia, hoarse cry due to transient bilateral vocal cord dysfunction, motor and language retardation, and specific facial features. The father and sister of the proband were carriers of the same 5p deletion and displayed learning difficulties and behavioral issues, but with no dysmorphic features. However, another sister and brother of the proband displayed behavioral disorder with a normal chromosome 5 (20); therefore the pathogenic cause of affected family members should not be the 5p deletion. Hence, we have always stressed the importance of parental origin verification in the clinical interpretation of fetal CNVs. Shi et al. verified the parental origin of 141 fetal CNVs and identified that 102 CNVs (72.3%) were derived from the parents, with 74 CNVs of likely benign clinical significance (21). In contrast to the benign 5p (1–7,700,000) deletion described by us in family 1, the child 1 carries a 1.04 Mb longer deletion of 5p15.33-p15.31 (1–8,740,000), and showed a high pitched, monotonous cry. This indicates that the 1.04 Mb (chr5:7,700,000–8,740,000) region may be responsible for the cat-like cry, which is more precise than the previously reported cat-like cry critical region. By searching the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) and ClinGen database, we identified only four protein-coding genes involved in this region with no reported dosage sensitivity. These include ADCY2, which partially overlaps the deletion, and the complete sequences of C5orf49, FASTKD3, and MTRR. ADCY2 gene encodes a member of the family of adenylate cyclases, which are membrane-associated enzymes that catalyze the formation of the secondary messenger cyclic adenosine monophosphate (cAMP) in response to G-protein signaling (22). The C5orf49 gene function is not well characterized. Monika et al. reported that C5orf49 gene encodes a protein localized to cilia and has ciliary functions (23). FASTKD3 gene encodes a member of a small family of Fas-activated serine/threonine kinase domain (FASTKD) containing proteins, which is required for normal mitochondrial respiration by enhancing MT-CO1 mRNA translation and mitochondrial complex IV assembly and activity (24, 25). MTRR gene encodes a member of the ferredoxin-NADP (+) reductase (FNR) family of electron transferases, which is responsible for the reactivation of methionine synthase (MTR/MS) activity by catalyzing the reductive methylation of MTR-bound cob (II) alamin (26). MTRR gene has been shown to be associated with Homocystinuria-Megaloblastic Anemia, Cble Complementation Type and Neural Tube Defects, Folate-Sensitive. These findings will be helpful for genotype-phenotype correlation analysis of 5p deletion syndrome.

6p duplication in most patients results from the unbalanced inheritance of a parental translocation, along with deletion of another chromosome (27). The clinical symptoms of these patients are caused by 6p duplication combined with the effect of another chromosome abnormality (28). Since the breakpoints of 6p vary from 6p11 to 6p25, 6p duplication also presents variable clinical characteristics of pre- or post-natal growth restriction with a short final stature, microcephaly, distinct facial features, developmental delay, congenital heart defects, sensorineural hearing impairment, renal complications, speech delay, and intellectual disability (27, 29). A search of the DECIPHER database revealed four patients carrying 6p duplications (patient numbers 284585, 393057, 392292, and 392965) with no other chromosome abnormalities, and they displayed variable phenotypic features including global developmental delay, cryptorchidism, unilateral renal agenesis, abnormality of the thyroid gland, short stature, and microcephaly. All patients showed multisystem anomalies. Additionally, 6p25 duplication may be associated with ocular developmental abnormalities and glaucoma (30). There is also phenotypic heterogeneity among family members with the same 6p25 duplication. For instance, Fontana et al. reported a family with four members carrying a 6p25.3-p25.2 duplication, and all four family members displayed brachydactyly with sporadic cardiac abnormality, hypothyroidism, and special facial features (31). Consistently, the two affected individuals of family 3 in this report also displayed different phenotypes. The pregnant woman (IV-2) showed symptoms of hearing loss and mild intellectual disability, while her younger female cousin (IV-3) had congenital deafness, moderate intellectual disability, and walking problems. The 6p25.3-p24.3 duplication region in family 3 includes 44 protein-coding genes. Of these, 13 genes are included in the Online Mendelian Inheritance in Man database as disease-causing genes (shown in Table 2). The pathogenicity of altered gene dosage remains to be determined. Five out of those 13 genes were reported to be associated with hearing loss or intellectual disability, including forkhead box C1 (FOXC1), serpin family B member 6 (SERPINB6), tubulin beta 2A class IIa (TUBB2A), tubulin beta 2B class IIb (TUBB2B) and anscription factor AP-2 alpha (TFAP2A). FOXC1 was reported as a haploinsufficiency sensitive gene. FOXC1 mutations can lead to Axenfeld-Rieger syndrome which is characterized by developmental defects of the anterior chamber of the eye, maxillary hypoplasia, hypodontia, microdontia, umbilical abnormalities, and sensorineural deafness (32). SERPINB6 gene is associated with deafness, featured as progressive, age-related sensorineural hearing loss (33). Defects of TUBB2A and TUBB2B genes are linked to cortical dysplasia in combination with other brain malformations type 5 and type 7, respectively. The clinical manifestations include cortical dysplasia, hypoplasia of the corpus callosum, global developmental delay, and seizures (34). Davies et al. suggested the potential involvement of TFAP2A in the development of the anterior eye chamber (35). Tekin et al. identified a heterozygous deletion/insertion mutation in the TFAP2A gene in a 4-year-old Turkish girl with Branchiooculofacial syndrome which is characterized by branchial cleft sinus defects, ocular anomalies such as microphthalmia and lacrimal duct obstruction, and a dysmorphic facial appearance (including cleft or pseudocleft lip/palate). In addition, the girl showed the phenotype of sensorineural hearing loss (36). Here in family 3, we performed WGS and Sanger sequencing assays, and identified a breakpoint of chromosome 6 (chr6:10,418,611-10,418,614), which is located in the intron 1 of TFAP2A (NM_001042425). Further study of the involved genes in 6p25.3-p24.3 is essential for elucidating the pathogenesis of 6p25.3-p24.3 duplication.

In summary, CNV-seq is valuable for prenatal diagnosis and provides helpful genetic guidance for families who are at a high-risk of 5p deletion syndrome. In the present study, we redefined relevant region in 5p deletion syndrome, and proposed that the [GRCh37] 5p15.31:7,700,000_8,740,000 region was responsible for the cat-like cry. The importance of parental validation for variant pathogenicity assessment was also demonstrated. Furthermore, we successfully identified the breakpoints in family 3 by performing WGS, confirming the potential of WGS in detecting chromosomal translocations (10, 37). However, more cases are required to confirm the application of WGS in prenatal diagnosis, and further studies should be implemented on gene function to elucidate the genotype-phenotype correlation. In addition, parallel testing with multiple approaches, albeit at a higher cost, would be an appropriate option for prenatal diagnosis in order to shorten the detection cycle and allow for timely follow-up interventions. Our study may pave the way for further research on the prevention, diagnosis, and treatment of related diseases. Interactive confirmation of more case series would be beneficial for genetic counseling, choice of diagnostic methods, as well as pathogenic analysis of variants.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The datasets of CNV-seq generated and analysed during the current study are available in the Sequence Read Archive repository (https://dataview.ncbi.nlm.nih.gov/object/PRJNA779115?review er=vjtb6iiga3mi0209bocdcqdadt).

The studies involving human participants were reviewed and approved by the Chinese PLA General Hospital Medical Ethics Committee (S2016-120-02). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

RH designed the verification experiments and conducted data analysis. GC and PL wrote the manuscript. JW and GZ performed the sample collection. YZ revised the article. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.