Chromosomal Abnormality in Fetuses with Isolated Antenatal Hydronephrosis: Update for Prenatal Diagnosis and Genetic Counseling


 Background: The prenatal finding of fetuses with antenatal hydronephrosis (ANH) gives a significant dilemma for the clinicians. Which patients require invasive prenatal diagnosis? Though previous literatures have recommended the use of chromosomal microarray analysis (CMA)for fetuses with CAKUT, the cutoff value for CMA have no current consensus on fetuses with ANH. In this article, we aimed to detect chromosomal abnormalities in fetuses with isolated severe ANH (anterior-posterior renal pelvic diameter (APRPD) ≥ 10mm) by CMA, summarized the literatures and proposed recommendations for the prenatal genetic diagnosis according to APRPD.Methods: Fetuses (n=84) with isolated severe ANH (APRPD ≥ 10mm) were evaluated by CMA. According to APRPD measurements at second trimester, we classified the cases into two groups: (1) Group A: cases with APRPD of 10–15 mm(N=57); (2) Group B: cases with APRPD ≥ 15 mm(N=27).The prenatal and postnatal outcomes were assessed by ultrasonic examination and telephone follow-up.Results: Overall, one case with 18 trisomy was identified. Clinically significant copy number variants (pathogenic or likely pathogenic CNVs) were identified in 11.9% (10/84) cases, including 3.5% (3/84) of pathogenic CNVs. The detection rates were 5.2% (3/57), 25.9% (7/27) for group A and group B, respectively. There was statistically significant differences in the frequency of clinic significant CNVs in the two groups (p＜0.05). Conclusion: CMA is valuable in prenatal genetic diagnosis of fetuses with severe ANH（APRPD ≥ 10mm）, regardless of whether other ultrasonic abnormalities were observed. This cohort should be followed up during the pregnancy.


Introduction
Antenatal hydronephrosis (ANH) or pyelectasis refers to dilation of the renal pelvis and calices. It is one of the most common abnormalities among the prenatal ultrasound screening affecting approximately 1-5% of all pregnancies [1] .According to APRPD measurements during the second trimesters, the degrees of ANH were classi ed to mild(APRPD 4 mm-7 mm), moderate(APRPD7mm-10 mm), and severe AHN( ≧ 10 mm) [2] .In more than half of the cases, these ndings resolve spontaneously by the end of gestation or during the rst year of life. However, There was a signi cant increased risk of postnatal pathology per degree, showing pathologic outcomes including ureteral re ux(VUR), obstructive uropathy ( OU: including ureteropelvic junction obstruction, ureterovesical junction obstruction and congenital megaureter) and so on [3] , Pathologic ANH has been associated with an increased risk of genetic disorders. For instance, Miguel Verbitsky performed a largest study on the copy number variants (CNVs) landscape of congenital anomalies of the kidney and urinary tract (CAKUT). Among the subcategories, cases with VUR were affected by a high CNV burden 37.2%(245/659)of cases with CNVs(≥ 100 kb), 38.9%(199/512)of cases with OU were re ected by CNV burden(≥ 100 kb) [4] ; Faure found thirteen CNVs identi ed in 12 boys with posterior urethral valves (29% of the cohort), including two pathogenic CNVs and 11 of unknown signi cance [5] .
In the prenatal domain, the isolated fetal pyelectasis during second-trimester targeted ultrasound was reported to be soft marker associated with trisomy 21 [6] . Meanwhile, several studies have described CNVs detected in fetuses with ANH [7] [8] [9] [10] . However, most of these cases were usually associated with additional ultrasound abnormalities. Moreover, these literatures have not comprehensively investigated the risk of CNVs with varying degrees of APRPD or those aspects of ANH that predict chromosome abnormality. Our study focus on the isolated ANH, we performed chromosomal microarray analysis (CMA) to identify chromosomal abnormalities in the fetuses with severe ANH(APRPD ≧ 10 mm) to determine whether or not the degree of ANH is associated with the risk of CNVs and the recommended cutoff value for CMA.

Diagnostic yield of CMA testing for fetuses with ANH
We analyzed a total of 84 prenatal cases of ANH by CMA. These cases had no other sonographic ndings except ANH. The overall diagnostic yield of CMA testing for fetuses with ANH was 25% (21/84), including 1 trisomy 18 and 20 CNVs. Among the detected CNVs, clinically signi cant CNVs (pathogenic and likely pathogenic) were identi ed in 11.9% (10/84) cases, including 3.5% (3/84) of pathogenic CNVs. The incidence rates of clinically signi cant CNVs were 5.2% (3/57) and 25.9% (7/27) in Group A and Group B, respectively. There were statistically signi cant differences in the incidence rates of all CNVs and clinically signi cant CNVs between the two groups (p 0.05; Table 2) the maternal age, gestational age at amniocentesis and parity did not differ between the two groups according to t tests and Chi-squared tests. Additional details are presented in Table 1.  In group A, CNVs were detected in 10.5% (6/57) cases, included three clinically signi cant CNVs, one benign CNV, and two VOUS. We compared the frequency of clinically signi cant CNVs with that in the control cohort in Chinese population [14] . The detected rate of clinically signi cant CNVs in group A was signi cantly higher than that in the control cohort (3/57 vs. 9/3748, P = 0.0). In case 2 (table3), the deleted region on chromosome15q11. characterized by microtia, patellar applasia/hypoplasia, and a proportionate short stature [15] .In case 10 (table 3), a de novo deletion fragment in 17q12 region covered 99.97% region of RCAD (renal cysts and diabetes) syndrome.
The genetic variants were not inherited from their parents in case 4, 5, 7 ,8 and case 9 (table 3). Moreover, all overlapped with deletion/duplation syndromes or containing OMIM -morbid gene partly, but no de nite pathogenic evidence had been reported. Hence, these disorders were classi ed as likely pathological. In case 4, a de novo deletion of 0.55 Mb was detected on 11p15.5. This repetitive fragment overlapped with 1.79% region of Homozygous 11p15-p14 Deletion syndrome [OMIM:606528], known as congenital hyperinsulinism (CHI) and profound hearing loss [16] .

Follow-up information
Eighteen cases were excluded for the following reasons: loss of follow-up (n = 7) and elective termination (n = 11) including 3 with chromosomal abnormalities, 6 with severe bilateral hydronephrosis, 1 with right ureteral atresia and 1 with congenital renal agenesis. Finally, 66 cases had a complete set of follow-ups during the prenatal and postnatal period. The total rate of normalization was 81.8% (54/66) in uterus or after birth. During postnatal follow-up period, 11 cases (16.6%, 11/66) were treated with surgery due to pelvic uretero junction obstruction (PUJO) (10/11) and ureter stricture (1/11). For all these children, the treatment of operation was effective, the postoperative renal morphology recovered signi cantly, and the renal function could be preserved, except one child was suspected to have a nephroblastoma at 8 months of age. None of the children had mental and growth retardation during the follow -up period. Discussion: Recommendations for fetuses with prenatally diagnosed ANH remain controversial, especially regarding identi cation of the most accurate and detailed genetic causes. Previous studies have concluded that isolated fetal pyelectasis is related to an increased risk of aneuploidy, more than doubles the odds of trisomy 21 from maternal serum screening test results [6] . However, the diagnostic yield of CNVs in fetuses with isolated ANH remains uncertain. Some studies have reported the overall detection rates of pathogenic CNVs varied from 3.2-9% in fetuses with ANH and 4.5% of fetuses with isolated ANH [7][8] [9] [10] (Table 4). In our study, CMA revealed the clinically signi cant CNVs (pathogenic and likely pathogenic) of 11.9% (10/84) in fetuses with isolated severe ANH, including pathogenic CNVs of 3.5% (3/84), similar to that in previous reports. Its results demonstrated the increased risk for clinically signi cant CNVs in isolated ANH, compared to the general population. These ndings strongly supported the practice of routine CMA analysis in pregnancies with isolated several ANH. Meanwhile, the relative risk for clinically signi cant CNVs in cases with APRPD 10-15 mm was signi cantly higher than the control population. Although, there is a discrepancy between CNV and maternal age, it has been reported that advanced maternal age only re ected in chromosome aneuploidy, chromosome structural abnormalities did not increase with the mother's age [17] . Moreover, we incorporated fetuses with APRPD of 10 mm and progressed to determine the cutoff value for application of CMA. Because the value of 10 mm and less measured at 2nd Trimester was also the cutoff value for fetal mild bilateral pyelectasis, which was a benign condition especially when the renal pelvis regressed [18] [19] . Based on these ndings, we suggest that cases with APRPD of 10 mm and further progressed could be offered for invasive procedures.
In cases with APRPD of greater than or equal to 15 mm (group B), seven cases of CNVs with clinical signi cance were detected, providing a 25.9% (7/27) incremental yield of detecting CNVs. the frequency of clinically signi cant CNVs in group B were obviously higher than that in group A. This result is in accordance with conclusions of previous studies showing that a larger anterio rposterior pelvic diameter (APRPD) is associated with increased likelihood of surgery due to the correspondent higher grade of obstruction and consequently to higher risk of renal function [20] . Therefore, we speculated that the detected rate of clinical signi cance CNVs increased with their APRPD.
During the follow-up period, no obvious mental and growth retardation were found in all the children.
Previous studies have assessed the clinical signi cance of CNVs in children with CAKUT. Fature et al [21] carried out a follow up study and found that patients with CNVs had a worse prognosis, compared with the control group. Miguel Verbitsky [22] found that patients with pathogenic CNVs had a nominally reduced estimated glomerular ltration rate, elevated cystatin C levels, and increased proteinuria at study enrollment. These studies showed that genomic imbalances could have pleiotropic effects on renal function. In conclusion, compared to fetuses without CNVs, fetuses with CNVs have a relatively poor prognosis in renal function, and should be recommended for close monitoring of renal function after birth.

Conclusions
The limitation of our study is the small sample size. This was a small study from a single center. Thus, further studies are required to perform multicenter surveys with larger sample sizes in order to con rm our ndings. In conclusion, our data suggest an association between CNVs and ANH. The abnormality rate of CNVs increased with their APRPD. CMA should be recommended to fetuses with APRPD ≧10 mm at 2nd trimester. Meanwhile, the cases had no other ultrasound anomalies, including structural anomalies and soft markers. According to APRPD described by Lee et al [11] , we classi ed the ANH into 3 groups at 2nd Trimester: 1) mild ANH (APRPD 4 to < 7 mm), 2) medium ANH (APRPD 7 to 10 mm), and 3) severe ANH (APRPD ≧ 10 mm). All the fetuses were checked with at least twice time, which had to be repeated 2-3 weeks. Only the cases that APRPD was unchanged or became worse entered the study. The cases with severe ANH (APRPD ≧ 10 mm) underwent invasive aminio puncture. According to APRPD values, the cohort was divided into two groups: those with APRPD of 10-15 mm (Group A) and those with APRPD of 15 mm or more (Group B). Data for maternal age, gestational age at screening, value of APRPD, parity, karyotype and CMA results were showed in table1 and Fig. 1.

Subjects
In order to estimate the effect on incidence, we compared the frequency of the clinically signi cant CNVs (pathogenic and likely pathogenic) with that in the control cohort of Chinese populations, based on a retrospective study by Ye Shi et al [12] . The author analyzed 4,224 women (over 35 years old at delivery) who accepted prenatal diagnosis. Among them, 3748 cases with normal ultrasound were referred to invasive testing due to advanced maternal age. In this group, 9 (0.2%) CNVs with associated clinical signi cance were reported.
This work was approved by the Medical Ethics Committee of Nanjing Maternity and Child Health Care Hospital. Prenatal genetic counseling was carried out by trained clinical geneticists regarding the advantages and potential risks of CMA, including possible ndings of uncertain signi cance, nonpaternity, consanguinity, and adult-onset diseases.

Chromosomal microarray analysis
Genomic DNA was extracted from 10 ml amniotic uid samples using a QIA amp DNA Mini Kit (Qiagen, Germany). Human cyto12 SNP array (Illumina, San Diego, CA) comprising approximately 300,000 SNP probes with average marker spacing of roughly one probe every 10 kb was applied for the whole-genome scan. SNP array experiments were performed as previously described in our laboratory [13] and molecular variants of uncertain signi cance (VOUS) and benign CNVs. CNVs were de ned as pathogenic if 1) the CNVs was documented as clinically signi cant in multiple peer-reviewed publications, regardless of its penetrance and expressivity, or 2) the CNVs overlapped a smaller interval with clearly established clinical signi cance. CNVs coinciding with known polymorphic CNVs or have been reported in multiple peerreviewed publications or databases as benign variants were considered benign. CNVs that did not t any of the above criteria were considered as VOUS. All above experimental and analytical methods were performed based on the previous report as reference in our laboratory [13] . In this study, we reported only pathogenic CNVs and VOUS.

Follow-up study
Follow-up information was obtained through telephone interview and pregnant women medical records.
The results of follow-up included prenatal and postnatal outcome. Prenatal variables included gestational age at spontaneous resolution, whether combined with other structural anomalies, and elective termination. Postnatal outcome included non-favourable outcome and normalization. Normalization was de ned as absence of pelvic or ureteral dilatation (APRPD of renal pelvis not detectable or < 5 mm) and normal con guration of both kidneys and bladder according to postnatal ultrasound. Non-favourable postnatal outcome was de ned as persistent anomalies and need of surgery. The average follow-up period was 3 years.

Statistical analysis
Statistical analyses were performed on maternal clinical characteristics and experimental results using SPSS software (IBM, SPSS statistics). Categorical data were presented as means and standard deviations, the signi cance of differences were determined using independent exponent t tests and Chisquared tests. Results with p values (p < 0.05) were considered statistically signi cant.

Declarations
Ethics approval and consent to participate This study was approved by the Medical Ethics Committee of Nanjing Maternity and Child Health Care Hospital. Informed consent was obtained from parents.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests