Nephronophthisis (NPHP) is an autosomal recessive inherited kidney disease that constitutes the most prevalent monogenic causes of kidney failure in the first 3 decades of life and is responsible for 2.4–15% of pediatric patients with kidney failure (Hildebrandt et al., 2009; Luo and Tao 2018). Urine analysis of NPHP patients generally does not illustrate any characteristic urinary abnormalities, as proteinuria and hematuria are often not found until late stages of chronic kidney disease (CKD) (Luo and Tao 2018). A clue to the underlying diagnosis may be finding a loss of urinary concentration ability (Simms et al., 2009) and kidney ultrasound scan findings of corticomedullary cysts and a loss of corticomedullary differentiation (Srivastava et al., 2017); however, these findings are non-specific.

Therefore, it is difficult to detect NPHP using simple laboratory testing and often the diagnosis is delayed until the advanced stages of CKD. A kidney biopsy may show features which include tubular atrophy, tubular basement defects, and interstitial fibrosis but often do not lead to a precise diagnosis, especially if the patient is approaching kidney failure (Simms et al., 2009). Extra-renal manifestations occur in 15% of cases of NPHP, consistent with it being a ciliopathy syndrome and may include retinal defects, liver fibrosis, skeletal defects, and brain developmental disorders (Srivastava et al., 2017).

Recent advances in genetic analysis show excessive phenotypic and genetic heterogeneity of this disorder (Chaki et al., 2011; Tang et al., 2020) with at least 25 different genes having been linked (Luo and Tao 2018; McConnachie et al., 2021). However, pathogenic variants in known NPHP genes can explain only up to one-third of cases of clinically diagnosed nephronophthisis, suggesting further genes may underlie this phenotype (König et al., 2017; Luo and Tao 2018). Here, we report a case of NPHP associated with early kidney failure caused by a homozygous in-frame deletion of GLIS2 diagnosed using WES. Pathogenic variants in GLIS2 remain very rare with only two families previously reported (Attanasio et al., 2007; Halbritter et al., 2013). This case is unique, as it is the first in-frame deletion within GLIS2 to be reported in association with NPHP and the predicted loss of a motif within the zinc finger domain suggests strongly its pathogenicity.

Whole exome sequencing of the proband’s genomic DNA was performed with 96.4% of the exome being covered at least 20-fold. Variants were filtered and analyzed as described, followed by Sanger sequencing validation. Within a large region of homozygosity on chromosome 16, a homozygous novel in-frame deletion variant (NM_032,575.3:c.560_574delACCATGTCAACGATT, p.H188_Y192del) in GLIS2 was identified in the patient and was present in its heterozygous form in both parents but absent in the eldest unaffected sibling from whom DNA was available (Figure 1C). We did not find any other potential pathogenic variants associated with a ciliopathy-related phenotype. Variants in GLIS2 have been previously identified in patients with NPHP (OMIM: 611498) who developed kidney failure in early childhood (Attanasio et al., 2007). This novel deletion variant that leads to a shortened protein missing 5 amino acids (H188, V189, N190, D191, Y192) located in exon 4 of the GLIS2 gene was absent in the public database including 1000G and genomAD. Bioinformatics programs including PROVEAN (deleterious), SIFT (deleterious), and MutPredIndel (score: 0.45615) predicted that this variant (NM_032,575.3:c.560_574delACCATGTCAACGATT, p.H188_Y192del) is deleterious and located at evolutionarily conserved site of the GLIS2 protein close to the zinc-finger C2H2-type DNA-binding domain (Figure 1D). According to ACMG guidelines (Richards et al., 2015), this allele is classified as a variant of uncertain significance (VUS). The variant has been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/1299703/).

Gli-similar 2 (GLIS2) is a member of the Krüppel-like family of transcription factors consisting of five contiguous zinc-finger motifs (Figure 1D) (Zhang et al., 2002). Classical zinc-finger domains (C₂H₂) are among the most common eukaryotic protein domains and often have the consensus sequence Cys-X_2–4-Cys-X₁₂-His-X_2–6-His (where X is any amino acid) (Pavletich and Pabo 1993). Metalloproteins, such as GLIS2 have been shown to co-assemble with Zn²⁺ ions during protein folding into the ββα zinc finger domains (Figure 1E). The His188_Tyr192 variant identified in this study removes 5 amino acids from the α helix of GLIS2 ZF1 (Figure 1D, magenta), removing the terminal sequence of the zinc-finger motif, even though His193 replaces His188 as H₁ (Figures 1E–G). The loss of this motif through an in-frame deletion could potentially result in misfolding of GLIS2. However, there is evidence to show that the individual zinc-finger domains fold independently of the bulk protein structure (Cox and McLendon 2000), and therefore an observed loss of function could be due to altered DNA-binding capacity of the GLIS2 mutant. Downstream functional analyses are required to determine the loss-of-function mechanism.

Making a clinical diagnosis of NPHP is difficult given its non-specific features and therefore many patients with NPHP may be misdiagnosed with another nephropathy or diagnosed as CKD of unknown etiology (Srivastava and Sayer 2014; Snoek et al., 2018). Although clinical findings, kidney ultrasound examination, and kidney biopsy histology in our patient were suggestive of NPHP, a definitive diagnosis was only made following molecular genetic testing. In fact, as many causes of kidney failure may mimic the NPHP phenotype, molecular genetic testing is critical to distinguish NPHP from other inherited forms of kidney diseases.

NPHP is a rare disorder (Levy and Feingold 2000; Hildebrandt 2010) that illustrates genetic heterogeneity with at least 25 different recessive genes having been linked with the disease (Luo and Tao 2018). Almost all NPHP genes encode nephrocystins that localize to primary cilia, except for NPHP-like genes such as XPNPEP3 and SLC41A1 and GLIS2 genes, with protein products located to mitochondria and nucleus, respectively (O'Toole et al., 2010; Srivastava and Sayer 2014; Luo and Tao 2018). Pathogenic variants in these genes can explain only up to one-third of cases and around 60% of cases remain genetically unsolved, suggesting additional genetic causes that have yet to be revealed (König et al., 2017; Luo and Tao 2018). The most common genetic cause of NPHP is variants in NPHP1, associated with 20% of cases (Hildebrandt et al., 2009), while variants in each of the remaining genes possibly contribute up to 1% of cases (Srivastava and Sayer 2014; Luo and Tao 2018).

The human GLIS2, encoding a member of the Kruppel-like zinc-finger protein family named GLIS family zinc-finger two or alternatively nephrocystin-7, is located on chromosome 16p13.3, and it contains 6 exons spanning ∼3.7 kilobases. GLIS2 is located in the nucleus and cytoplasm and is mainly expressed in the kidney and weakly in the heart, lung, placenta, and colon (Zhang et al., 2002). Based on the cell context, GLIS2 can function as activator or repressor of gene transcription. For instance, it works as a repressor of the Hedgehog (Hh) signaling pathway by repressing Hh-dependent expression of Wnt4 as well as repressing transcriptional activation mediated by CTNNB1 in the Wnt signaling pathway. In addition, GLIS2 is required to retain the differentiated epithelial phenotype in kidney cells and in neuron differentiation (Zhang et al., 2002).

In 2007, a homozygous splice-site variant in GLIS2 (c.755+1G > T) was identified in three affected Canadian Oji-Cree children with an autosomal recessive pattern of NPHP (Attanasio et al., 2007). Later, in 2013, a homozygous missense GLIS2 variant (c.523T > C, p.C175R) was identified in a Turkish patient with isolated NPHP who reached kidney failure aged 15 years, as part of a worldwide cohort screen of patients with NPHP-RC (Halbritter et al., 2013). Variants in GLIS2 have been classified as NPHP7 (OMIM 611498) and within the variation database of ClinVar, there are 34 pathogenic variants, of which 31 are copy number variants (CNVs) while three are SNVs (two missense and one splicing) and there are 92 variants of uncertain significance (VUS). Here, we identified in GLIS2 an in-frame deletion variant (NM_032,575.3: c.560_574delACCATGTCAACGATT, p.H188_Y192del) in Omani family with NPHP and early onset kidney failure. As far as we know, this variant is novel and expands the variant spectrum of GLIS2 variants associated with NPHP.

The deletion affects the zinc-finger C2H2-type DNA-binding domain of GLIS2, which plays a role in the recognizing and binding to target DNA as well as mRNA and protein targets (Brayer and Segal 2008). This deletion leads to a change in protein length and feature as the position of the stop-codon in mutated protein will be 520 compared to 525 in the wild-type protein. Further functional studies may be performed in the future to examine the consequences of such deletion in GLIS2 expression level.

In summary, by employing WES, we detected a novel in-frame deletion (NM_032,575.3: c.560_574delACCATGTCAACGATT, p.H188_Y192del) in GLIS2 in an Omani family with NPHP and kidney failure. Therefore, this study has not only broadened the spectrum of variants within GLIS2, but also extended the molecular pathogenesis spectrum of kidney disease in Oman (Al Alawi et al., 2021) and allowed the confirmation of the clinical diagnosis of NPHP leading to kidney failure.