Hydrocephalus and Growth Retardation: A Fetal RNU4ATAC-opathy Missed by Whole-Exome Sequencing

Abstract

Introduction

Whole-exome sequencing (WES) is becoming widely available in prenatal diagnosis. However, as with most scientific methods, WES also has its limitations. The aim of the study was to report a fetal case of RNU4ATAC-opathy which was missed by prenatal WES.

Case Presentation

A 28-year-old healthy primigravida was revealed by ultrasound at 20 + 3 weeks of gestation to have a fetus with ventriculomegaly (left 15.1 mm/right 11.9 mm), hypoplastic vermis, and mild growth retardation. Chromosomal microarray analysis and trio WES failed to detect a pathogenic copy number variation and sequence variant. A repeat ultrasound at 23 + 3 weeks showed worsened growth delay and hydrocephalus (left 20.3 mm/right 11.0 mm) with vermis hypoplasia and agenesis of corpus callosum. Further study with whole-genome sequencing (WGS) detected 2 missense mutations of the noncoding RNU4ATAC (NR_023343.1) gene, n.51G>A (rs188343279) and n.16G>A (rs750325275), in the fetus, which were inherited from the father and mother, respectively.

Discussion

Our study highlights the limitation of WES. WGS might be a clinical option for patients who have a structurally abnormal fetus tested negative by WES.

Established Facts

• Whole-exome sequencing (WES), which sequences the protein-coding regions of the human genome, is becoming widely available in prenatal diagnosis.

• There is a growing body of literature indicating that variants in the noncoding genome are associated with congenital anomalies.

Novel Insights

• We first report a RNU4ATAC-opathy fetus with hydrocephalus and growth retardation, extending the current knowledge of phenotypes associated with this disease.

• Our study highlights the limitation of WES. Whole-genome sequencing might be a clinical option for patients who have a structurally abnormal fetus tested negative by WES.

Introduction

Prenatal genetic testing provides information about the genetic constitution of a fetus that will determine perinatal decision-making and management. Next-generation sequencing (NGS) has revolutionized the paradigm of clinical genetic testing. The American College of Medical Genetics and Genomics (ACMG) recommends that genomic sequencing may be considered when a prenatal diagnosis fails to be achieved via routine prenatal methods (karyotyping/microarray) in a pregnancy with fetal structural anomalies [Monaghan et al., 2020]. The emerging data support the clinical utility of prenatal whole-exome sequencing (WES) based on its additional diagnostic yield [Kilby, 2021; Pauta et al., 2022]. However, there are functional variants in noncoding regions that regulate gene expression, such as enhancers and long noncoding RNAs. These noncoding variants, even if genetically identifiable, are not covered by WES and thus would not be detected. We here report such a fetal case with a RNU4ATAC-opathy which was missed by prenatal WES.

Case Report

A 28-year-old G1P0 woman and her husband were healthy, and both reported an unremarkable past health and family history. Nuchal translucency of 1.0 mm and a crown-rump length of 54 mm at 12 weeks were noted. Her first-trimester serum screening reported a negative result. However, an 18 + 3-week ultrasound revealed bilateral ventriculomegaly (left 14.6 mm/right 14.7 mm) with fetal biometry: head circumference (HC) 142 mm (−1.4 SD), abdominal circumference (AC) 103 mm (−4.1 SD), femur length (FL) 23 mm (−1.7 SD). The amniocentesis work-up included cytomegalovirus DNA amplification and single nucleotide polymorphism-based genomic array analysis; both were negative. The ultrasound at 20 + 3 weeks showed ventriculomegaly (left 15.1 mm/right 11.9 mm), hypoplastic vermis, and mild growth retardation (HC 174 mm, −0.2 SD; AC 128 mm, −3.4 SD; FL 29 mm, −1.3 SD). Further genetic testing with trio WES using the remaining fetal DNA sample failed again to detect a monogenic etiology related to the fetal phenotype. A repeat ultrasound at 23 + 3 weeks showed worsened growth delay (HC 198 mm, −1.6 SD; AC 146 mm, −4.9 SD; FL 33 mm, −3.1 SD) and hydrocephalus (left 20.0 mm/right 11.0 mm) with vermis hypoplasia and agenesis of corpus callosum (Fig. 1a). The couple elected to terminate the pregnancy but declined an autopsy. The fetus showed a relatively large head, hypertelorism, midface hypoplasia, micrognathia, and mildly short limbs (Fig. 1b). Reanalysis of WES data using the new postnatal phenotype still reported no disease-causing findings.

Fig. 1.

Two RNU4ATAC variants identified in a fetus with hydrocephalus and growth delay. a Prenatal sonographic findings with hydrocephalus (left) and hypoplastic vermis (right) at 23 weeks. b The aborted fetus. c Chromatograms of the RNU4ATAC variants.

After genetic counseling, whole-genome sequencing (WGS) was used to detect possible variants that might be undetectable by WES. Two missense variants in the RNU4ATAC (NR_023343.1) gene, n.51G>A (rs188343279) and n.16G>A (rs750325275) (Fig. 1c), were identified in the fetus, inherited from the father and mother, respectively. Again genomic variants of protein-coding genes were not demonstrated by WGS. Therefore, the fetus was diagnosed as a RNU4ATAC-opathy.

Discussion and Conclusion

RNU4ATAC, a noncoding gene, codes for a small nuclear RNA, part of the U12-dependent minor spliceosome complex involved in the removal of introns during RNA processing [Beauchamp et al., 2020]. Defects in RNU4ATAC are responsible for 3 rare recessive developmental diseases, namely microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), Roifman syndrome, and Lowry-Wood syndrome, collectively called RNU4ATAC-opathies. Although each disorder has its main distinct phenotype, all have overlapping features, i.e., developmental delay, skeletal dysplasia, and intellectual disability. The n.51G>A identified in our case is a common variant in patients with MOPD1, Roifman syndrome, and Lowry-Wood syndrome, while the n.16G>A variant has been reported only in Roifman syndrome [Merico et al., 2015; Benoit-Pilven et al., 2020].

Most MOPD1 causal variants cluster in the 5′ stem-loop region of the RNA (i.e., position 51), while almost all Roifman syndrome causal variants identified are compound heterozygous, with one variant overlapping the MOPD1-implicated 5′ stem-loop critical region and the other variant occurring at highly conserved positions in the stem II (i.e., position 16). However, a homozygous variant in stem II can be sufficient to cause the full spectrum of features associated with typical Roifman syndrome [Dinur Schejter et al., 2017]. Compared to the 5′ stem-loop variants, which have a stronger effect on minor spliceosome function, variants occurring in the stem II are unique to Roifman syndrome [Abdel-Salam et al., 2012; Shelihan et al., 2018]. This might be the molecular basis for the phenotypic differences between the 2 syndromes. Until now, cases of RNU4ATAC-opathies have seldom been identified prenatally. Our case is the first one of Roifman syndrome identified prenatally based on the molecular diagnosis. Fetal hydrocephalus and vermis hypoplasia have never been reported to be associated with RNU4ATAC-opathies. In another study with 2 affected fetuses in one family, microcephaly and severe growth retardation were the 2 remarkable features associated with compound heterozygous MOPD1-causing variants [Wang et al., 2018]. Interestingly, the 2 RNU4ATAC variants, n.51G>A and n.29T>A, were also initially missed by WES and detected by targeted sequencing of the RNU4­ATAC gene. Although RNU4ATAC-opathies have recognizable phenotypes, an atypical presentation is sometimes observed. Our case thus expands the clinical spectrum in utero and also presents an example that phenotypic heterogeneity can be caused by different variants of the same gene.

By sequencing the protein-coding regions of the human genome, more genetic information can be available by WES than by a sequencing panel. WES can also simultaneously achieve better time and cost efficiency compared to WGS. Thus, WES is becoming widely available for prenatal diagnosis which has a tight timeframe. However, as with most scientific methods, WES has its limitations [Burdick et al., 2020]. As evidenced by our study, RNU4ATAC is not consistently targeted in standard exome capture methodologies that only target protein-coding exons. It can be expected that more such cases will be left undetected. This might also be the case for other noncoding minor spliceosome small nuclear RNA genes (RNU6ATAC, RNU11, and RNU12). The noncoding genes are mostly composed of regulatory elements that control gene expression. There is a growing body of literature indicating that variants in the noncoding genome are associated with congenital anomalies [Scacheri and Scacheri, 2015; Zhang and Lupski, 2015; Carrion-Castillo et al., 2021].

From a technical perspective with unlimited resources and time, WGS is undoubtedly the best NGS-based diagnostic option as it interrogates single nucleotide variations, indels, structural variants, and copy number variations in both the approximately 5% portion of the genome that encodes protein sequences and the 95% of remaining noncoding sequences [Lappalainen et al., 2019]. However, WGS requires more sequencing yield and reagents and produces massive amounts of data that have to be scrutinized and interpreted by sophisticated bioinformatics expertise, increasing both the cost and time required for analysis. A recent study performed trio WGS in parallel with chromosomal microarray plus WES in 111 fetuses with structural or growth anomalies. As expected, WGS detected all pathogenic genetic variants in 22 diagnosed cases identified by chromosomal microarray plus WES, yielding a diagnostic rate of 19.8% (22/110) [Zhou et al., 2021]. The authors acknowledged that the main obstacles of WGS used as a routine NGS approach include informatics and interpretation challenges, huge numbers of variants of unknown significance, and comparatively high cost. Therefore, only when costs and data analysis workload are further reduced to an acceptable level, WGS has the potential to entirely replace WES and other techniques that involve selective capturing of target sequences. Even in this situation, a diagnostic laboratory will still try to look up phenotype-specific gene lists first, in order to reduce interpretation complexities.

In conclusion, we present a prenatal case with a genetic defect caused by variants of the RNU4ATAC gene which is beyond the detecting ability of WES. Our report highlights the limitation of WES. It extends the current knowledge of phenotypes associated with RNU4ATAC-opathies. Indeed, the future prenatal NGS yield will ultimately depend on our knowledge on the specific phenotype-genotype correlations during fetal development. For example, as the phenotype may continue to expand, targeted RNU4ATAC sequencing should be considered in undiagnosed prenatal cases by WES, especially in those with brain malformations and skeletal dysplasia. At present, WGS should be a clinical option for patients who have a structurally abnormal fetus tested negative by WES. Aside from cost, however, the clinical application of WGS will require a host of resources to appropriately interpret and classify variants. Considering that the exome contains approximately 85% of disease-causing variants in human, WES is still the main genetic analysis technique for structurally anomalous fetuses.

Statement of Ethics

This study protocol was reviewed and approved by the Ethical Committee of Guangzhou Women and Children's Medical Center (2021-468B00). Written informed consent was obtained from the patients for publication of the details of their medical case and any accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author Contributions

Conceptualization: Yong-Shan Chen, Dong-Zhi Li. Writing and original draft preparation: Yong-Shan Chen. Clinical data collection: Jie-Fu He, Tao Quan. Molecular genetic data: Shu-Bin Li. Critical review: Yong-Shan Chen, Dong-Zhi Li. All authors analyzed and interpreted the data and approved the manuscript in its final form.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Funding Statement

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.