16p13.11 microduplication is a rare genetic disorder with incomplete penetrance and variable expressivity, associated with a range of neurodevelopmental and congenital disorders such as developmental delay, intellectual disability, language deficits, behavioral abnormalities, dysmorphic facial features, attention deficit hyperactivity disorder (ADHD), autism, and cardiovascular anomalies (Hamad et al., 2023). The 16p13.11 microduplication is a susceptibility factor for various neurodevelopmental disorders, though its molecular mechanisms, and candidate genes remain unclear (Watson et al., 2014). Chromosome 16 is rich in segmental duplications (SDs), also known as low copy repeats (LCRs), which account for more than 10% of its euchromatic content (Loftus et al., 1999; Martin et al., 2004; Ramalingam et al., 2011). These chromosome rearrangements are an important cause leading to neurodevelopmental disorders (Ingason et al., 2011).

The pathogenicity of 16p13.11 microdeletion has been well-established, yet the clinical significance of 16p13.11 microduplication remains controversial (Allach El Khattabi et al., 2020). Ullmann (Ullmann et al., 2007) firstly reported the pathogenicity of 16p13.1 microduplication, primarily linking it to intellectual disability and autism spectrum disorders. While Hannes (Hannes et al., 2009) identified recurrent reciprocal duplication of 16p13.1 was a common population variant (5/1682, 0.29%) suggesting that it may be a rare benign variation. In recent years, numerous copy number variations (CNVs) have been detected resulting from the widespread clinical application of chromosome microarray analysis (CMA). It is now broadly accepted that 16p13.1 microduplication may be associated with cognitive impairment, developmental delay, behavioral abnormalities, delayed language development, schizophrenia, and a variety of congenital anomalies, including cardiovascular abnormalities, skeletal abnormalities, aortic disease, renal and urinary system malformations (Kirov et al., 2009; Gumuslu et al., 2015; Erhart et al., 2018; Calderoni et al., 2020). However, individuals with normal phenotypes and unaffected relatives have also been identified, that complicating the interpretation of clinical significance of 16p13.11 microduplications in the context of prenatal genetic counseling, particularly.

Notably, most previous literatures on 16p13.11 microduplications have focused on adults and children, but limited data available on prenatal fetal phenotypes. In this study, 15 fetuses with recurrent 16p13.11 microduplication were identified using single-nucleotide polymorphism microarray (SNP array) technology. We retrospectively analyzed the prenatal diagnosis indications, parental origin, pregnancy outcome and follow-up results of fetus with 16p13.11 microduplication syndrome to further explore the genotype-phenotype correlations and elucidate the underlying genetic mechanisms of 16p13.11 microduplication syndrome.

The short arm of chromosome 16 (16p) is a hotspot region for chromosomal copy number variations (CNVs), particularly the 16p13.11 region (Ciaccio et al., 2017; Redaelli et al., 2019; Atli et al., 2022). This region consists of three single-copy sequence intervals (I, II, III), flanked by low copy repeats (LCRs) (Ingason et al., 2011), which predispose to chromosomal rearrangements through non-allelic homologous recombination (NAHR). Intervals I and II are critical for copy number variation (Nagamani et al., 2011). In this study, SNP array analysis of amniotic fluid of 3,451 pregnant women revealing 15 fetuses with varying sizes of microduplications in the 16p13.11 region, ranging from 0.79 to 2.82 Mb. The breakpoints of 15 cases with 16p13.11 microduplications covered different regions across the three single-copy sequence intervals (I, II, III). Notably, There were 9 cases whose breakpoints encompassed intervals I and II, and all cases involved interval II. The concentration of breakpoints in intervals I and II suggests that these two regions may harbor key genes associated with clinical phenotype, consistent with previous findings (Nagamani et al., 2011). Interval I located between 15.12 and 15.15 Mb, contains 3 OMIM genes, including NTAN1, which encodes an asparagine-specific N-terminal amidase, which is an enzyme involved in regulating the half-life of proteins in vivo. Inactivation of the Ntan1 in mice is associated with abnormal neural activities, such as changes in social behavior, impaired memory, impairment of spatial and non-spatial learning abilities (Kwon et al., 2000; Balogh et al., 2001). Interval II located between 15.51 and 16.29 Mb, contains 7 OMIM genes, including NDE1, which is highly expressed in the brain and plays a pivotal role in the growth and development of cerebral cortex (Luttik et al., 1998; Nagamani et al., 2011). Knockout of Nde1 in mice leads to abnormalities in the cerebral cortex and microcephaly (Feng and Walsh, 2004). Moreover, NDE1 gene is regarded as a strong candidate gene associated with phenotypes of neural development (Soto-Perez et al., 2020). Interval II identified as a dosage-sensitive region, represents the core pathogenic locus of 16p13.11 (Tropeano et al., 2013), which consistents with our findings that all cases encompassed interval II.

The 16p13.11 microduplication is recognized as a neurodevelopmental susceptibility locus with incomplete penetrance and variable expression, which is an important impact on developmental delay, learning difficulties and behavioral abnormalities (Coe et al., 2014; Nuttle et al., 2016). The phenotype in adults and children has been well described in the previous literatures, such as cognitive impairment, behavioral abnormalities, cardiac and aortic malformations, skeletal abnormalities and other organ abnormalities (Balogh et al., 2001; Hamad et al., 2023).

However, there are few reports on the prenatal phenotype associated with this microduplication. Redaelli (Redaelli et al., 2019) detected 366 prenatal specimens using comparative genomic hybridization (aCGH) and found that one fetus with prenatal indications of non-immune edema carried 16p13.11 microduplications. Dabkowska (Dabkowska et al., 2020) identified a 16p13.11 microduplication in a fetus with encephalocele. Cai (Cai et al., 2022) found 15 fetuses with 16p13.11 microduplication by SNP array detected from prenatal diagnosis of 9,000 pregnant women, which indicated the most relevant prenatal indicators were structural or soft marker anomalies on ultrasound. Similarly, in this study, ultrasound abnormalities were the most common indication for prenatal diagnosis observed in 6 out of 15 cases, including 3 with structural abnormalities and 3 with soft marker anomalies. Hamad (Hamad et al., 2023) noted congenital cardiac anomalies in 16 of 206 patients with 16p13.11 microduplication, with the most common feature of which was ventricular septal defect. Houcinat (Houcinat et al., 2015) described a family with congenital renal and urinary system anomalies associated with 16p13.11 microduplication, in which the father had unilateral kidney dysplasia and the son suffered from chronic kidney disease and pelvic ureteral obstruction. Shi (Shi et al., 2021) reported that the rate of chromosomal abnormalities was as high as 29.6% on fetal omphalocele. These findings highlight the association between ultrasound abnormalities or soft marker anomalies and chromosomal rearrangements involving 16p13.11 region (Lan et al., 2020; Su et al., 2021). In our cohort, NIPT suggested chromosomal abnormalities in 5 out of 15 cases, 3 of which involved microduplication of chromosome 16. With the widespread clinical application of NIPT, the detection rate of CNVs involving 16p13.11 microduplications has been greatly improved (Du et al., 2018). Advanced maternal age, a known high-risk factor for chromosomal abnormalities (Wang et al., 2019), was the prenatal diagnosis indication in 5 out of 15 cases carrying 16p13.11 microduplications, possibly contributing to the higher detection rate in our study. The specific relationship between advanced age factors and the occurrence of 16p13.11 microduplications is still unclear.

Initially speculated a “benign variant” due to its presence in phenotypically normal parents, however, the pathogenicity of 16p13.11 microduplication is gradually recognized as more patients detected. (Hannes et al., 2009; Nagamani et al., 2011). The penetrance is estimated at around 7%–10.6% (Kendall et al., 2019; Kirov et al., 2014), and the duplication may be inherited from asymptomatic parents or occur de novo (Redaelli et al., 2019). It has been reported that approximately 93.6% of CNVs inherited from unaffected parents (Nagamani et al., 2011; Hamad et al., 2023). In our study, 7 cases underwent pedigree verification, and one case was de novo (1/7), while the remaining 6 were inherited (6/7), accounting for about 85.7% inherited from asymptomatic parents, consistent with previous literature. Notably, 5 cases were inherited from the mother (5/7), while one case had paternal inheritance (1/7), indicating a higher maternal inheritance rate. Tropeano (Tropeano et al., 2013) found a male bias in the 16p13.11 region, but there was no gender bias in our samples (7 males and 8 females), likely due to the small sample size.

Our follow-up revealed a 15.4% penetrance rate via telephone follow-up, which is higher than the levels reported in the literatures (Kendall et al., 2019; Kirov et al., 2014; Martin et al., 2020). There are two children exhibiting clinical phenotypes: one with ventricular septal defect and the other with omphalocele. This discrepancy may be affected by the limited sample size and the nature of telephone follow-up of our study, which may influence parental reports Additionally, genetic factors beyond 16p13.11 CNVs, such as other undetected mutations, could contribute to the phenotypes, but further testing was declined by their parents. Children with 16p13.11 microduplications might lack of specific phenotypes and the abnormalities may progressively emerge during growth or relatively mild. Although no abnormalities are evident in the neonatal period, affected individuals may show varying degrees of cognitive impairments and behavioral abnormalities with increasing age (Delicado et al., 2014). Therefore, ongoing monitoring of the growth and development of individuals with 16p13.11 microduplications is crucial.

In conclusion, when CNVs in the 16p13.11 region are detected during fetal development, clinical decisions should be based on a comprehensive analysis of fetal ultrasound findings, familial history and detailed genetic counseling instead of terminating the pregnancy solely. The potential for varying phenotypic expression within the same family underscores the importance of considering the origin in the parental generation and recurrence risks in comprehensive genetic counseling.

This study has several limitations. First, the sample size was small, with only 15 cases of fetal 16p13.11 microduplications detected. Second, the point mutations of genes were not detected by SNP array used in this study, but the next-generation sequencing was not employed. Third, the longest follow-up case in our study was tracked up to 5 years and clinical phenotypes associated with 16p13.11 microduplications are mainly observed in adults and children. So it is essential to conduct long-term follow-ups of these children in future studies to observe their growth and development. In future, research with larger sample size and longer follow-up period are necessary to better understand the genotype-phenotype correlation and provide more comprehensive genetic counseling.

The use of SNP array is an effective tool for diagnose variations in the 16p13.11 region. Our findings indicate that microduplication of 16p13.11 is strongly associated structural and soft marker anomalies detected via prenatal ultrasound. The application of NIPT (non-invasive prenatal testing) has enhanced the detection rate of prenatal 16p13.1 microduplications. Simultaneously, comprehensive prenatal phenotypic analyses, follow-ups on assessments of pregnancy outcomes and postnatal follow-ups for fetuses with 16p13.11 microduplications were conducted in this study. The majority of these fetuses were found to be in good health after birth, offering valuable insights for clinical genetic counseling.