This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PVS1,PS1,PS3,PM2.

The splice site c.2694-2_2694-1del variant has been reported to segregate with Meckel syndrome and embryonic lethality in families (Bergmann et. al., 2008). An experimental study has shown that this variant results in altered splicing, likely by usage of a cryptic splice site, resulting in a prematurely truncated protein (Fiskerstrand et. al., 2010). The c.2694-2_2694-1del variant is novel (not in any individuals) in 1000 Genomes and has allele frequency of 0.02759% in gnomAD database. This variant has been reported to the ClinVar database as Pathogenic/Likely Pathogenic. Donor and acceptor splice site variants typically lead to a loss of protein function (Baralle D, Baralle M., 2005), and loss-of-function variants in NPHP3 are known to be pathogenic (Halbritter et. al., 2013). For these reasons, this variant has been classified as Pathogenic. In the absence of another reportable variant, the molecular diagnosis is not confirmed. The same variant (c.2694-2_2694-1del) has been observed in her father.

PVS1,PM2_p

As part of Carrier Screening testing performed at First Genomix, this variant was identified in a heterozygous state in a patient who is not affected with this condition.

NPHP3: PVS1, PS3:Supporting, PS4:Supporting

The variant is observed at an extremely low frequency in the gnomAD v4.1.0 dataset (total allele frequency: 0.023%). Predicted Consequence/Location: Canonical splice site: predicted to alter splicing and result in a loss or disruption of normal protein function. Multiple pathogenic loss-of-function variants are reported downstream of the variant. In silico tools predict the variant to alter splicing and produce an abnormal transcript [SpliceAI: 1.00 (spliceogenicity >=0.2, non-spliceogenicity <0.1)]. The variant has been reported at least twice as pathogenic with clinical assertions and evidence for the classification (ClinVar ID: VCV000220868 /PMID: 18371931 /3billion dataset). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

Canonical splice site variant in a gene for which loss-of-function is a known mechanism of disease; Published functional studies demonstrate this variant leads to loss of splice acceptor resulting in skipping of exon 20 (Molinari et al., 2018); This variant is associated with the following publications: (PMID: 27894351, 28973083, 30002499, 34426522, 32055034, 31980526, 32552793, 32901917, 20007846, 18371931, 33726816, 27535533)

The heterozygous c.2694-2_2694-1del variant in NPHP3 was identified by our study in one individual with renal-hepatic-pancreatic dysplasia 1. Trio exome analysis showed this variant to be in trans with a likely pathogenic variant (ClinVar Variation ID: 635041). The c.2694-2_2694-1del variant in NPHP3 has been previously reported in 22 unrelated individuals with NPHP3-related disease (PMID: 33532864, PMID: 28973083, PMID: 32055034, PMID: 32552793, PMID: 32901917, PMID: 27894351, PMID: 33726816, PMID: 23559409, PMID: 26673778, PMID: 30002499, PMID: 33323469), but has been identified in 0.04% (14/30612) of South Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs963574014). Although this variant has been seen in the general population in a heterozygous state, its frequency is not high enough to rule out a pathogenic role. Of these 22 previously reported unrelated individuals (PMID: 33532864, PMID: 28973083, PMID: 32055034, PMID: 32552793, PMID: 32901917, PMID: 27894351, PMID: 33726816, PMID: 23559409, PMID: 26673778, PMID: 30002499, PMID: 33323469), 12 were homozygotes (PMID: 32055034, PMID: 32552793, PMID: 27894351, PMID: 18371931, PMID: 20007846) and 4 were compound heterozygotes who carried pathogenic or likely pathogenic variants in trans (PMID: 33532864, ClinVar Variation ID: 659899; PMID: 28973083, ClinVar Variation ID: 988261; PMID: 23559409, ClinVar Variation ID: 96511, ClinVar Variation ID: 693989 ; PMID: 30002499, ClinVar Variation ID: 262696; PMID: 33323469, ClinVar Variation ID: 1454640), which increases the likelihood that the c.2694-2_2694-1del variant is pathogenic. This variant has also been reported in ClinVar (Variation ID: 220868) and has been interpreted as pathogenic by multiple submitters. RT-PCR analysis performed on affected tissue shows evidence of altered splicing of exon 20 (PMID: 32552793, PMID: 30002499, PMID: 20007846). A different nucleotide change that also results in a splice acceptor variant at the same site, c.2694-2A>T (ClinVar Variation ID: 1524627), has been previously reported likely pathogenic, and the variant being assessed here, c.2694-2_2694-1del, is predicted by SpliceAI to have a similar effect on splicing. This variant is located in the 3' splice region. Computational tools do suggest an impact to splicing. However, this information is not predictive enough to determine pathogenicity. There is an in-frame cryptic splice site 21 bases from the intron-exon boundary, providing evidence that this variant may delete 7 amino acids instead of causing loss of function. However, this information is not predictive enough to determine pathogenicity. Loss of function of the NPHP3 gene is an established disease mechanism in autosomal recessive renal-hepatic-pancreatic dysplasia 1. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive renal-hepatic-pancreatic dysplasia 1. ACMG/AMP Criteria applied: PVS1_Moderate, PS1_Supporting, PS3_Moderate, PM3_VeryStrong (Richards 2015).

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with Meckel syndrome 7 (MIM# 267010), nephronophthisis 3 (MIM# 604387) and renal-hepatic-pancreatic dysplasia 1 (MIM# 208540). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0210 - Splice site variant proven to affect splicing of the transcript with a known effect on protein sequence. RT-PCR done on whole blood RNA of an affected individual demonstrated exon 20 skipping, which is expected to result in a frameshift (PMID: 30002499). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD <0.01 for a recessive condition (v2: 78 heterozygotes, 0 homozygotes). (SP) 0311 - An alternative nucleotide change at the same canonical splice site is present in gnomAD (v3: 1 heterozygote, 0 homozygotes). (SB) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It has been reported in at least ten individuals described to have nephronophthisis, end stage renal disease or ciliopathy syndrome and consistently classified as pathogenic by diagnostic laboratories in ClinVar (PMID: 28921755, 30002499, 32055034). (SP) 1206 - This variant has been shown to be paternally inherited (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

PVS1, PS3, PS4, PM2, PM3, PP1

Absent from controls (or at extremely low frequency if recessive) in Genome Aggregation Database, Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium.;For recessive disorders, detected in trans with a pathogenic variant.;Patient's phenotype or family history is highly specific for a disease with a single genetic etiology.;Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.;Null variant in a gene where loss of function (LOF) is a known mechanism of disease.

Variant summary: NPHP3 c.2694-2_2694-1delAG is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing and loss of NPHP3 function. Several computational tools predict a significant impact on normal splicing: Two predict the variant abolishes a 3' acceptor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 0.00027 in 251300 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in NPHP3 causing Joubert Syndrome And Related Disorders (0.00027 vs 0.0004), allowing no conclusion about variant significance. c.2694-2_2694-1delAG has been observed in multiple homozygous individuals affected with Joubert Syndrome And Related Disorders (e.g. Al-Hamed_2022). These data indicate that the variant is very likely to be associated with disease. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publication has been ascertained in the context of this evaluation (PMID: 34853893). ClinVar contains an entry for this variant (Variation ID: 220868). Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change affects a splice site in intron 19 of the NPHP3 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or altered protein product. This variant is present in population databases (rs751527253, gnomAD 0.05%). Disruption of this splice site has been observed in individual(s) with Meckel-Gruber-like syndrome and/or nephronophthisis-related ciliopathy (PMID: 18371931, 20007846, 23559409, 26673778). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 220868). Studies have shown that disruption of this splice site results in activation of a cryptic splice site, and produces a non-functional protein and/or introduces a premature termination codon (PMID: 20007846). For these reasons, this variant has been classified as Pathogenic.

The NPHP3 c.2694-2_2694-1delAG variant is predicted to result in a deletion affecting a canonical splice site. This variant has been reported in the homozygous and compound heterozygous states in multiple individuals with NPHP3 related disorders (Bergmann et al. 2008. PubMed ID: 18371931; Meng et al 2017. PubMed ID: 28973083; Shaheen et al 2016. PubMed ID: 27894351; Shamseldin et al. 2021. PubMed ID: 34645488). Expression assays reveal that this variant leads to skipping of exon 20 (Maddirevula et al. 2020. PubMed ID: 32552793). This variant is reported in 0.046% of alleles in individuals of South Asian descent in gnomAD. This variant is interpreted as pathogenic.

The heterozygous c.2694-2_2694-1delAG variant was identified by our study in the compound heterozygous state, with a pathogenic variant, in one individual with nephronophthisis. This variant is pathogenic based off of multiple reports in ClinVar and the literature.