Heterozygous variants of uncertain significance in NPHS1 and CRB2 in a newborn with congenital nephrotic syndrome of the Finnish type and multiple fetal anomalies: a case report

Introduction

Background

Congenital nephrotic syndrome (CNS) is a rare condition that occurs within the first three months of life. The most frequently encountered form of CNS is the Finnish type (CNF), which is inherited as a recessive allele of NPHS1 (1-3). Variants of uncertain significance (VUS) have been reported in CNS (4), severe short stature (5), Fabry disease (6), Lynch syndrome (7), cancer (8), and others (9).

Rationale and knowledge gap

The current knowledge does not provide a definite answer regarding whether the VUS types are associated with a health condition, making the uncertainty surrounding the variant’s involvement in disease development challenging to determine (10). This highlights the need for further research to understand the pathogenicity of VUS in CNF. There are likely undiscovered VUS mutations associated with the dysfunction of the regulatory mechanism of NPHS1 transcription and the malfunction of its product, the nephrin protein. The VUS can disrupt the function of the exonic splicing enhancer (ESE) and CTCF (CCCTC-binding factor), impair normal gene expression, and contribute to diseases (11,12). The ESE motifs are discrete sequences that serve as binding sites for specific serine/arginine-rich (SR) proteins, a family of structurally related and highly conserved splicing factors (13). The CTCF and its binding motifs play a critical role in regulating exon splicing by controlling the accessibility of spliceosome machinery to pre-messenger RNA (mRNA) (11). Another issue is related to the uncovered relationship of VUS with enhancers. Enhancers are typically non-coding regulatory sequences defined by the presence of CTCF motifs and other regulatory elements (14-16). CTCF plays a critical role in the functions of ESEs, 5' untranslated region (5'UTR), and enhancers by establishing the necessary three-dimensional (3D) genome and chromatin environment (11,17). Also, it is possible that the CNF occurrence due to NPHS1 of the VUS type results from an unreported or unexplored amino acid change, which leads to a malfunctioning nephrin protein.

Objective

There is still little consensus on the causes of VUS-related disease, and no widely accepted explanation exists. This study presents information about previously unreported VUS in NPHS1 and CRB2 found in a newborn girl diagnosed with CNF and several fetal anomalies. The NPHS1-VUS and CRB2-VUS impact the CTCF and ESE, respectively, and were identified in the exons of the corresponding genes. The findings support a link between these variants and the clinical symptoms associated with nephrin dysfunction, which is encoded by NPHS1 (1-3), and component 2 of the crumbs cell polarity complex, encoded by CRB2 (18,19). We present this case according to the CARE reporting checklist (available at https://acr.amegroups.com/article/view/10.21037/acr-24-246/rc).

Case presentation

A female newborn was delivered via cesarean section (CS) at 32.5 weeks of gestational age, based on an 18-week maternal ultrasound (US) that showed multiple fetal anomalies, including bilateral enlarged and multicystic kidneys, a split cerebellum, and an invisible stomach, necessitating the CS. The baby’s birth weight was 1.670 kg, in the 10th percentile, and considered a small size for gestational age. She was admitted to the Neonatal Intensive Care Unit (NICU) for prematurity, respiratory distress, hypoglycemia, and further investigation of abnormalities on the fetal sonogram. On the day of life (DOL) 2, chest and abdominal X-rays showed a stomach in the left upper quadrant (LUQ) (Figure 1A). At three months of age, X-rays showed an enlarged heart (cardiomegaly), a central venous catheter to give intravenous fluids and drugs, and a nasogastric (NG) tube for draining liquid or air from the stomach and delivering medicines (Figure 1B). The abdominal US on the DOL 2 showed both kidneys at the upper level of normal size (Figure 2A). At three months of age, the abdominal US showed bilateral kidneys at the upper limit of normal without obstruction or cysts (Figure 2B). The patient was also found to have hypoalbuminemia with an albumin level of 0.5 g/dL, total protein of 1.6 g/dL (low), and normal creatinine (Cr) of <0.2 mg/dL (Table S1). Pediatric nephrology was consulted, and urine analysis (UA), urinary protein, and creatinine ratio (Ur Pr/Cr) were ordered as per nephrology recommendation, which was in the nephrotic range (Table S1). Hypoalbuminemia with normal Cr was attributed to poor maternal nutrition due to inadequate care and prematurity (Gestational age <37 weeks). The patient was started on high-protein total parenteral nutrition (TPN) due to a nephrotic range of proteinuria, persistent hypoalbuminemia, and worsening generalized edema. Albumin infusion 0.5 g/kg Q8h and Lasix 0.5 mg/kg was initiated at DOL 3. At DOL 5, the patient was diagnosed with CNS with Ur Pr/Cr of 164 mg/dL, urinary protein was 2,144 mg/L, and urinary Cr was <13 mg/dL. Immunoglobulin G (IgG) level was also low, <140 mg/dL. At DOL 7 and 8, we started 0.01 mg/kg of Enalapril twice daily and 0.5 mg/kg/day of Indomethacin to reduce proteinuria. Given the patient’s low immunoglobulin level and high risk of infection, we also began intravenous immunoglobulin 500 mg/kg every 3–4 weeks. The patient developed hypertension (HTN) during the NICU stay, and the enalapril dosage was adjusted to control it accordingly. We checked the thyroid panel every 2 weeks according to the endocrinologist’s recommendation. Synthroid 25 mcg was started due to hypothyroidism (thyroid-stimulating hormone 9.11, free thyroxine 40.81) on DOL 54 (Table S1). Labs were repeated every week on Monday, and medication doses were adjusted according to laboratory results and the patient’s weight. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration and its subsequent amendments. Written informed consent for publication of this case report and accompanying images was not obtained from the patient’s guardians or the relatives after all possible attempts were made.

Figure 1 Chest and abdominal X-rays of a female newborn. (A) At 2 days old, the chest X-rays showed the stomach in the left upper quadrant, and (B) at 3 months old, the X-rays revealed cardiomegaly, a central venous catheter, and a NG tube. DOL, day of life; NG, nasogastric.

Figure 2 Ultrasound images of the infant’s kidneys. (A) The ultrasound taken on day 2 of life showed both kidneys at the upper level of normal size. (B) The ultrasound at 3 months of age showed a bilaterally enlarged kidney. Lt, left; Rt, right.

Genetic analysis

The genetic analysis was performed on the whole blood sample collected from the infant on DOL 7 and analyzed by next generation sequencing (NGS) at Mayo Clinic Laboratories/Mayo Clinic. The analysis evaluated 54 genes associated with focal segmental glomerulosclerosis and nephrotic syndrome (Table S2), revealing five heterozygous VUS across four genes: two in NPHS1 and one each in NUP160, ALG1, and CRB2 (Table S3). The NPHS1 VUS: c.2150A>G; p.Tyr717Cys, and c.3024A>G; p.Arg1008=, mapped at exons 16 and 22, respectively, both submitted to ClinVar-NCBI (National Center for Biotechnology Information) (Figure 3A). Figure 3B and Figure S1 show the c.2150A>G, an unreported missense variant that leads to p.Tyr717Cys, likely affecting the function of the Ig strand F, which is the fibronectin type 3 (FN3) domain of the nephrin protein encoded by NPHS1, whereas c.3024A>G; p.Arg1008= is of synonymous type mapped at the FN3 domain of NP_004637.1. The latest version of NPHS1-mRNA (NM_004646.4) gives 725 nt for the exon-1, compared to 214 nt for exon-1 in the earlier version, NM_004646.3 (NCBI-Nucleotide). The NPHS1 exon-1, as shown in NM_004646.4 mRNA, is largely a regulatory sequence composed of 667 nt and hosts 19 CTCF motifs. The recent annotation release (RS_2024_08) from NCBI-Gene shows that the human NPHS1 locus, Gene ID: 8564, occupies a region of 27,133 nt on the reverse strand, with the forward strand enhancers and silencers overlapping several NPHS1 exons (Figure S2). The NPHS1-NM_004646.4 mRNA shows that 92% of the exon 1 sequence is a regulatory untranslated sequence that overlaps the enhancer sequence LOC127891331, whereas the LOC127891330 enhancer overlaps the NPHS1 exons 2 and 3. The two silencers, LOC129664432 and LOC129664433, overlap NPHS1 exons 14 and 10, respectively (Figure S2). Our analysis revealed CTCF motifs in the NPHS1 exons 16 and 22 (Figure 4A), as well as in enhancers and silencers (Table S4), and 19 CTCF motifs were identified in NPHS1 exon-1. The CTCF binding motifs are critical for shaping the 3D genome (20-23), and changes in any CTCF motifs affect the outcomes of alternative splicing across the NPHS1 setting. Figure 4A,4B also show ESE motifs identified in exons NPHS1 16 and 22; only VUS in the NPHS1-e16 aligns with the CTCF motif and is likely to disrupt the CTCF functions, thereby impacting pre-mRNA splicing. A detailed analysis of other 3 genes shown in Figure 3. Several ESE motifs are identified in NUP160 exon-27, which hosts the c.3271C>T VUS, but do not align with the ESE motifs. The NUP160 exon-27 lacks a CTCF motif (Figure S3A). The ALG1 c.739C>T VUS is in exon-6 and does not align with identified CTCF and ESE motifs (Figure S3B). Based on our data analysis, we decided to exclude the NUP160 and ALG1 variants from further research because they probably won’t significantly impact infant conditions. The CRB2 c.1654G>T VUS is in exon-7 and aligned with one of the ESE sites; consequently, it may disrupt exonic splicing (Figure S3C). This observation may clarify the role of the CRB2 c.1654G>T VUS in the infant’s multiple conditions beyond CNF and the severity of CNF. CRB2 is associated with cystic kidney disease (MIM 219730) and kidney defects (OMIM 609720). It is essential for podocyte foot process arborization, slit diaphragm formation, and proper trafficking of nephrin functioning (OMIM 609720).

Figure 3 The two identified NPHS1 VUS. (A) The ClinVar-NCBI lists two NPHS1 VUS variants, c.2150A>G and c.3024A>G, submitted by Mayo Clinic Laboratories/Mayo Clinic. (B) The positions of the two variants along reference Nephrin precursor: NP_004637.1. VUS, variants of uncertain significance.

Figure 4 The identified regulatory elements in NPHS1 exons 16 (e16) and 22 (e22). (A) The CTCF motifs were identified by the JASPAR database (/jaspar.elixir.no). The ESE motifs were identified by ESEfinder 3 (esefinder.ahc.umn.edu). (B) CTCF motifs are highlighted in red, ESE motifs are underlined, and the normal base of the mutated nucleotide is highlighted in blue. ESE, exonic splicing enhancer.

Discussion

In the presented case study, an infant was diagnosed with CNF along with multiple fetal anomalies. NGS testing identified genetic VUS that have unclear health effects, posing a significant challenge to clinical diagnosis. The identified variants in the infant are previously unreported heterozygous variants in four genes: NPHS1 c.2150A>G; p.Tyr717Cys, CRB2 c.1654G>T; p.Ala552Ser, NUP160 c.3271C>T; p.Arg1091Cys and ALG1 c.739C>T; p.Arg247Cys (Table S3). Our hypothesis is based on the premise that the identified variants may impact the regulatory sequences of affected genes. We examined ESEs, 5'UTR, and/or CTCF (CCCTC-binding factor), which are essential for splicing and gene expression (11,12,17). The NPHS1 c.2150A>G VUS emerged as the most compelling candidate when our analysis indicated that it impacts the CTCF motif in exon-16 (Figure 4). A detailed analysis of the other three genes indicated that, in addition to NPHS1-VUS, the CRB2 c.1654G>T variant affects the ESE motif in exon-7 (Figure S3), making it another promising candidate for explaining the observed clinical symptoms. These data provide valuable insights into how VUS may lead to dysfunctional NPHS1 and CRB2, resulting in impaired nephrin production, and how component 2 of the crumbs cell polarity complex proteins may be nonfunctional.

The strength of the study lay in the connection between NGS analysis and clinical observations obtained from X-rays, US imaging, and biochemical tests, which provided essential information indicating that the infant suffered from CNF (OMIM 256300) caused by NPHS1-VUS. Conversely, the CRB2-VUS is involved in some of the observed fetal anomalies (MIM 219730, OMIM 609720). The limitation of the study is that, although VUS is a recognized genetic mutation, its effects on health still require further understanding. Our analysis may clarify the connection between NPHS1-VUS, CRB2-VUS, and the clinical symptoms of the infant, but it must be viewed within certain limitations that go beyond the scope of this study. These limitations include the need for experimental evidence regarding the impact of VUS on the CTCF in NPHS1 c.2150A>G exon-16 and ESE in CRB2 c.1654G>T. Also, there is concern about potential autosomal recessive polycystic kidney disease (ARPKD); the presence of ARPKD or renal dysplasia could not be excluded from the US exam. However, this concern may lessen when considering the impact of the CRB2 VUS on the splicing of exon-7, which could potentially account for the observed fetal anomalies.

On the other hand, ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/429811/) provides an example of a similar VUS identified in an infant with a CNS condition carrying the NPHS1 homozygous variant, c.3024A>G (AGA>AGG), leading to p.Arg1008= of a synonymous type. The CNS condition was likely caused by the malfunctioning DNA binding sites of the transcription factors FOXL1 and FOXC1 (4).

We provided answers from this study that correlate CNF with fetal anomalies involving VUS. The replacement of tyrosine (Tyr) by cysteine (Cys) at the Ig strand F of nephrin protein can impact its structure and function, particularly for proteins within the immunoglobulin (Ig) superfamily, such as nephrin (24). Previous studies have shown that most mutations of the NPHS1 gene occur in the nephrin protein Ig-like domains, which are crucial for forming the molecular filter and filtering blood (25-27). The CTCF motifs are identified in NPHS1 exons 16 (VUS), 22, and one, along with those in the enhancers overlapping exons 1 and 3, and play a critical role in topologically associating domain (TAD) formation and splicing (20-23). The potential interaction of the impact of NPHS1 c.2150A>G on the CTCF site and the consequence of CRB2 c.1654G>T on the ESE motif may be clarified by considering the functions of CTCF and its binding sites, which are critical in TAD and 3D genome formation as well as exon splicing (11,20-23). CTCF disruption in NPHS1-exon16 likely impacts the functions of other CTCF motifs in enhancers associated with TAD (Table S4), NPHS1-5'UTR (15,16,28). CTCF–CTCF interactions are well recognized for primarily initiating chromatin loops and significantly influencing the regulation of exon alternative splicing by linking distant DNA regions (29,30). About one-third of all diseases are believed to stem from the effects of genetic variants on gene splicing (31).

The implications of genetic diagnosis are significant for CNF as it aids in managing and treating patients. Before active treatment, not all infants with CNF and multiple fetal anomalies may thrive (1-3,32). This study could enhance our understanding of the pathogenicity of VUS, particularly the relationship between NPHS1-VUS and CRB2-VUS. Action is necessary to offer valuable guidance for future research and clinical practice in this area field.

Conclusions

The infant’s CNF condition, along with several fetal anomalies, was detected through clinical observation. NGS analysis revealed four genes with unreported VUS: NPHS1, NUP160, ALG1, and CRB2. Of these, NPHS1 and CRB2 are likely contributing to the infant’s illness. The NPHS1-VUS variant on exon 16 may result in nephrin dysfunction, an essential protein for the development and function of the slit diaphragm in the kidney filtration barrier. The CRB2-VUS is located on exon-7 and may cause focal segmental glomerulosclerosis type 9 (FSGS9) and cystic kidney disease. Both exons contain ESE and CTCF motifs involved in alternative splicing. Changes in these regulatory sequences due to the VUS may contribute to the observed infant’s CNF condition, along with several fetal anomalies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://acr.amegroups.com/article/view/10.21037/acr-24-246/rc

Peer Review File: Available at https://acr.amegroups.com/article/view/10.21037/acr-24-246/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://acr.amegroups.com/article/view/10.21037/acr-24-246/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions relatedto the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration and its subsequent amendments. Written informed consent for publication of this case report and accompanying images was not obtained from the patient’s guardians or the relatives after all possible attemptswere made.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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