There are over 750 million people worldwide affected with chronic kidney disease (CKD), a disease burden that is much higher than those living with diabetes, cancer or even AIDS/HIV ¹ . Inherited kidney diseases and renal ciliopathy syndromes are one of the major contributors to CKD burden, where up to 10% of adults and over 70% of children reaching end stage kidney disease (ESKD) are expected to harbour genetic causes ² . Renal ciliopathy syndromes typically lead to cystic kidney disease and include autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease and nephronophthisis, with a growing number of genetic causes implicated. The most common genetic causes of autosomal recessive renal ciliopathies would include PKHD1, NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290 and TMEM67 ³ . However, studying such rare diseases has considerable challenges mainly due to the small size of patient cohorts negatively affecting progress of treatments and commercial feasibility. Collaborative research and progress of new technologies and methodologies are strategic to overcoming these challenges.

WES is becoming part of routine clinical and diagnostic practice ² . Focusing only on protein-coding regions through WES decreases the sequencing costs and produces manageable genetic data for interpretation, which enhances its extensive usage in diagnosis leading to the discovery of previously unrecognized renal disease genes and disorders ^{2, 4} . In the case of heterogeneous renal ciliopathies, WES has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants ^{5, 6} . In this study, WES was used to determine the genetic causes of cystic kidney disease and renal ciliopathy syndromes in a group of 8 unrelated Omani children from consanguineous families, carefully selected with regard to clinical phenotype and in whom no genetic testing had previously been performed. As this study shows, the focus of nephrogenetics in Oman is primarily to establish an accurate genetic diagnosis to explain clinical phenotypes using the significantly improved diagnostic power of genomic technologies.

In paediatric populations, CKD is a major contributor to health-care burden leading to severe morbidity and mortality. At least 17% of those with ESKD are considered as CKD with unknown aetiology, where the primary kidney disease is not clear ¹⁶ . In addition, the primary clinical diagnosis of CKD patients is often inaccurate ¹⁶ . Thus, in the developing era of precision medicine, WES is used as an essential tool that provides novel diagnostic perspectives for the detection of the causes of CKD. Knowledge of genetic causes has valuable clinical implications in therapeutic intervention, improving prognosis, guide family counselling or managing settings of kidney transplantation ¹⁷ . Despite being rare, inherited kidney diseases represent one of the most common causes of CKD and ESKD, accounting for up to 10% of adults and almost all children commencing renal replacement therapy ¹⁸ . The possibility of monogenic causes in those with unknown aetiology of CKD or with atypical clinical presentation is assumed to be high ¹⁶ . At least 500 different genetic causes have been associated with childhood CKD ¹⁹ .

In this pilot study, we examined the utility of WES in the diagnosis of 8 different Omani children with childhood onset CKD related to cystic kidney disease and a suspected inherited renal ciliopathy. A conclusive genetic diagnosis was achieved in half of the cases. Positive WES findings allow a precise molecular diagnosis and targeted clinical management as well as informing family planning and facilitating proper genetic counselling. In four of the children (M46, M48, P18 and N36) the molecular genetic findings confirmed the suspected clinical diagnosis. The identification of a molecular genetic diagnosis in all these families can provide accurate genetic advice about the parent’s reproductive choices and the possibility of preimplantation genetic diagnosis (PGD) or early genetic testing of a foetus in future pregnancies.

A wide range of genetic studies have been performed in childhood CKD populations and different diagnostic yields were achieved due to differences in the inclusion criteria or patients and the study design. In a study of families with inherited kidney disease, Mallett , et al. ²⁰ reported a diagnostic yield of 46%, reflecting the significant ability of WES in underlying the potential genetic causes of most renal phenotypes. In another recent study ² , Groopman et al. reported higher diagnostic yield in patients with congenital and cystic kidney disease (23.9%). Furthermore, regardless of the primary kidney diagnosis, higher diagnostic yield was associated with a positive family history of CKD, history of parental consanguinity and presentations of extra-renal features ^{2, 6} . Thus using a combination of homozygosity mapping along with WES genotype data is always recommended as a powerful approach for consanguineous families to identify rare genetic causes ²¹ .

Although WES provides massive amounts of genetic data, 4 patients remained unsolved in this study. Despite an analysis of both homozygous alleles, compound heterozygous alleles and heterozygous de novo alleles, families M43, M44, M47 and P3 remained unsolved. A very rare homozygous allele in NPHP3 was noted in family M44. Mutations in NPHP3 have been associated with very early and severe ciliopathy syndromes including Meckel syndrome ²² , which matches the phenotype of this family. Proving the pathogenicity of this synonymous change now requires RNA analysis as well as searching for this allele in patients with a similar phenotype. Transcript aware annotation of genomic variants will be the next major step in utilising the data from WES effectively ^{23, 24} . The identification of a novel loss of function allele in IFT140 in family P3 that failed to be confirmed following Sanger sequencing acts as a cautionary reminder to validate all WES findings before genetic reporting. The WES read depth of this allele was <10 and therefore sequencing coverage remains an important consideration when choosing diagnostic sequencing modalities.

Interpretation of many novel and extremely rare variants is still limited by the incomplete knowledge of the total human protein-coding genes as well as the incorrect annotation of variants pathogenicity and incorrect association of genes with the disease in the literature. At present, up to 70% of protein-coding genes have no recognized human disease phenotype ²⁵ . False gene-disease associations are present in the literature ^{26, 27} and clinically valuable databases of variants pathogenicity, such as Human Gene Mutation Database (HGMD ^®), comprise various errors causing benign variants being falsely selected out of the data and allocated as plausible diagnosis ²⁸ . This situation is predicted to improve as further genomes are sequenced, including large data collections containing populations of both healthy individuals and patients with rare diseases. In addition, studying more families with similar clinical phenotypes from the same population may facilitate linking novel undiscovered genes to the disease phenotype in those unsolved patients.

In this study, WES confirmed the clinical diagnosis in 4 children. In a similar study of large consanguineous or familial cohort ( n = 79) of children clinically diagnosed with NPHP, genetic diagnostic yield of 63% was reported, of which the clinical diagnosis was confirmed in 64% and changed to different molecular diagnosis in the remaining 36% ¹¹ .

This study has some limitations, including small sample size that does not give a generalized image of broader childhood renal ciliopathy in the population from Oman. However, an enhanced assessment of the utility of WES in the clinical diagnostic practice of these disorders may be given through systematic WES analysis of a larger, unselected cohort. Moreover, the diagnostic gap in this study may be caused by the common technical limitation of WES, including the missed detection of structural variant breakpoints, sequencing difficulties for regions with repetitive elements or guanine-cytosine (GC)-rich regions, and limited discrimination between highly homologous genomic regions with pseudogenes. These limitations are attributed to the short-read lengths that are utilized to generate high genomic coverage and depth ²⁹ . These limitations are assumed to be resolved through using long-read sequencing platforms that compromise these technical challenges and improve the detection of genetic variants ²⁹ . Thus, the emerging future of long-read sequencing based whole genome sequencing (WGS) could enhance the diagnostic yield of patients with inherited renal ciliopathies and provide more conclusive primary kidney disease diagnosis. This can be supported by recent reports of WGS obtaining higher molecular diagnostic yield compared with WES, where 20–40% of those unsolved by WES were genetically conclusive by WGS ³⁰ . In particular, WGS has recently been used to successfully identify a deep intronic allele in NPHP3 leading to nephronophthisis ³¹ and with such approaches, defining deleterious intronic alleles will allow an increase in the diagnostic yield of WGS.

Recent advancements in medical genetics through the use of massively parallel sequencing have not only advanced the discovery of novel causative variants, genes and phenotypes, but also contributed to the re-classification of diseases and phenotypes into novel gene-based ontologies ³² . However, all types of next generation sequencing (NGS)-based testing (Target panel, WES and WGS) have some shared limitations, including the inability to obtain enough coverage of genomic regions with highly repetitive GC-content sequence, such as that in MUC1 gene. In his study of six unrelated families with medullary cystic kidney disease type 1 (MCKD1) ³³ , Kirby et al. highlighted the challenges of these technologies in detecting the causative monogenic causes of some Mendelian disorders, such as MCKD1, where only long-range polymerase chain reaction and molecular cloning successfully performed the task. Moreover, in many patients with acquired diseases, NGS testing is of limited importance and transformation of genetic results into clinical setup may be challenging ³² . In the field of kidney disease, the majority of genetic testing studies are narrowed to a research setting, thus until now the knowledge of its diagnostic efficacy in clinical practice is still limited ¹⁶ . In addition, managing the medical ethics raised by these technologies, including uncertain variants and incidental findings, and balancing the social concerns is still challenging ³⁴ .

WES of patients with different inherited cystic kidney diseases and renal ciliopathies shows promise as a diagnostic tool, especially in well selected patients with a high coefficient of inbreeding and/or with a syndromic presentation. It has the potential to resolve those cases with clear suspicion of renal ciliopathies, as well as those with uncertain aetiology causing CKD. The fact that ~50% of patients remain without genetic diagnosis after WES highlights the need for improved sequencing techniques and interpretation tools, driven by constantly evolving knowledge regarding the genetic architecture of diseases. The clinical impacts of positive WES results on therapeutic choice, genetic counselling and guidance of kidney transplant are critical. Indeed, professional genetic counselling on the prospective effects of a positive test result is crucial, bearing also in mind the possibility of incidental findings. Although further studies from the Omani population are required, we predict an expanding impact of NGS-based diagnosis, both gene panels and WES in clinical practice in the very near future.