Review Article, J Genet Disor Genet Rep Vol: 12 Issue: 2

Cilia Disorders in the Genomics Era: A Narrative Review on the Impact of Next Generation Sequencing on Ciliopathy Diagnostics

Sani Eskinazi^*

Department of Health Sciences, McGill University, Montreal, Canada

*Corresponding Author: Sani Eskinazi, 
Department of Health Sciences, McGill University, Montreal, Canada
E-mail: eskinazi@mail.mcgill.ca

Received date: 16 November, 2022, Manuscript No. JGDGR-22-80078;

Editor assigned date: 18 November, 2022, PreQC No. JGDGR-22-80078 (PQ);

Reviewed date: 02 December, 2022, QC No. JGDGR-22-80078;

Revised date: 17 February, 2023, Manuscript No. JGDGR-22-80078 (R);

Published date: 24 February, 2023, DOI: 10.4172/2327-5790.1000136

Citation: Eskinazi S (2023) Cilia Disorders in the Genomics Era: A Narrative Review on the Impact of Next Generation Sequencing on Ciliopathy Diagnostics. J Genet Disor Genet Rep 12:2.

Abstract

Keywords: Ciliopathies; Clinical genetics; Next-generation sequencing; Heterogeneity; Cilia

Introduction

Cilia are present on almost every cell type in the human body [1]. They are microtubule based cellular projections that extend from the plasma membrane. Despite their prevalence, for many years, these hair like projections, particularly non motile cilia, were thought to be vestigial structures with no important function [2]. However, after years of scientific disregard, “the cilium has emerged as a key organelle in numerous physiological and developmental processes”.

Cilia are assembled on basal bodies, which are derived from centrioles, and have a microtubule based axoneme [3]. Cilia can be either motile, as seen in ependymal cells that line brain vesicles, or immotile/sensory, as seen in the photoreceptor cells of the retina or the collecting ducts of the kidney [4,5]. Motile cilia enable cell movement and the movement of fluids across the surface of cells. Sensory (primary) cilia are specialized for signal transduction and act as ‘antennae’, sensing extracellular signals like growth factors, odorants, and developmental morphogens.

Research has revealed that defective primary and motile cilia can result in a number of human diseases, including retinal degeneration, polycystic kidney disease, and primary ciliary dyskinesia [6]. Since cilia are ubiquitous, mutations in cilia related genes can cause multi organ disorders, which are called ciliopathies. Ciliopathies can be either autosomal dominant or recessive and diagnosing these disorders can be very challenging as clinical presentations are often very heterogeneous [7]. This creates a clinical need to optimize genetics based diagnostic tools for ciliopathies. Moreover, there is a knowledge gap in which genetic variations are causative for ciliopathies. Here, I review the implications of using next generation sequencing tools to diagnose ciliopathies and to identify novel genetic variants that are associated with cilia defects. I will highlight the transition from landmark linkage analysis to high throughput screens, while also discussing the strengths of different approaches, which include targeted next generation, whole exome, and whole genome sequencing. Based on this synthesis, I argue that whole exome sequencing is a sufficient first pass in clinical settings, while whole genome sequencing is more informative for basic science research.

Literature Review

Early linkage mapping studies associated some disorders with variants in cilia genes

A landmark study in the field of cilia genetics was the identification of a candidate gene that associated with monogenic Polycystic Kidney Disease (PKD) [8]. PKD is a common and often lethal multi organ disease with around 12.5 million people affected worldwide [9]. The authors of this influential paper identified four families that had PKD like symptoms and relied on traditional genome mapping techniques to identify the location of a potential causative gene. They used genetic markers and looked for linkages with a potential disease allele. Ultimately, linkage analysis and lod score calculations allowed researchers to map the PKD-1 gene to the short arm of chromosome 16. However, at the time, researchers did not know that this gene was related to the primary cilium. The link was established about a decade later when another research group found a functional homologue of PKD-1 in a model organism, C. elegans [10]. These authors generated PKD-1 knock out organisms and saw that PKD-1 is part of the signaling cascade that is necessary for cilia formation. This was one of the first instances where a cilia related gene was associated with a human disorder and undoubtedly generated a lot of interest in finding genes that encode for structural or functional cilia proteins.

With an increase in genome mapping experiments, another group of researchers looked for genetic associations for a disease that is characterized by retinal dystrophy, obesity, renal malformations, and learning disabilities [11]. This disease is now coined Bardet-Biedl Syndrome (BBS) and for many years its molecular basis remained elusive. This was mostly due to the fact that different researchers were mapping it to different loci, with at least six loci being associated with phenotypic BBS [12]. Different research groups focused on different genetic loci and translated them to model organism experiments to see where the encoded protein localized. The commonality was that these proteins localized to the base of the primary cilium, the basal body [13,14]. We now know that the basal body is necessary for cilia formation and, in most cases cilia maintenance, in ciliated cells [15,16]. The fact that defects in the primary cilium can cause multi organ disorders further fueled interest in dissecting the molecular mechanisms of ciliopathies.

Sanger sequencing to diagnose known ciliopathies

As scientists started to learn more about the genetic causes of ciliopathies, Sanger sequencing of exons in known cilia genes became the standard for clinical diagnostics. Researchers built on the aforementioned work on BBS, which in 2011 was associated with 15 cilia genes. In one study, researchers did Sanger Sequencing on 55 families with BBS [17]. Sequencing 142 exons showed that 84% of the patients had potentially pathogenic variations in one of the 12 cilia genes. The mutational analysis also revealed 21 novel mutations in these known genes, including 10 novel missense variations and 2 frameshift mutations. These results show the high genetic heterogeneity that can lead to BBS and ciliopathies at large. In addition, this heterogeneity can lead to the following two challenges. Firstly, it can make predictive genotyping challenging, meaning that it is difficult to know whether or not someone who does not show a ciliopathy phenotype, but has a variant in a ciliary gene, will develop clinical symptoms. Secondly, Sanger sequencing relies on previous knowledge of ciliopathy genes, but it is possible that known genes or exons of genes are not adequate to get a whole genetic picture of a ciliopathy patient. Given the large genetic heterogeneity, it is possible that exons that were not sequenced might have pathogenic variants or there might be mutations in other unidentified ciliary genes.

Targeted next generation sequencing

These challenges associated with Sanger sequencing coincided with a period where next generation sequencing tools were becoming more and more prevalent in research and clinical settings. After linking many renal disorders too cilia perturbations, scientists aimed to identify the mutation profile of ciliary genes in autosomal recessive PKD patients. Thus, targeted next generation sequencing emerged as a cost effective and rapid way to assess variants in cilia genes. An important study assessed allelic variants in 191 structural and functional genes of the primary cilium in tissue samples from 7 PKD patients. This was a significantly more high throughput experiment compared to early studies that found one or two genes associated with familial PKD. Illumina sequencing revealed pathogenic variants in 39 genes encoding various structural components of the primary cilium. The most frequently mutated genes were those that encode centriolar and centrosomal proteins; other studies have also shown mutations in centriolar genes as the most common causes of ciliopathies. Since centrioles are essential for cilia formation it is likely that these patients do not form cilia and as such have severe disease phenotypes.

Interestingly, none of the samples showed mutations in Intra Flagellar Transport (IFT) proteins, which are a group of proteins that carry essential protein cargo to the tip of the cilium. This highlights the genetic heterogeneity of PKD since the same phenotype can be traced back to an autosomal recessive mutation in an IFT protein in another study. However, I believe that the lack of IFT proteins could also be due to the small sample size of this study (n=7 patients). Moreover, this also highlights a weakness of targeted sequencing. It is possible that IFT genes were not sequenced comprehensively, preventing the researchers from identifying causal variants in these highly redundant genes. Given a large number of studies that show IFT proteins as causal factors in renal ciliopathies, I believe that IFT proteins are important for proper cilia function and thus a genetic cause for ciliopathies. The authors of this study did not conduct extensive sequencing of all known IFT proteins. Overall, targeted next generation sequencing has been key in highlighting genetic variations that lead to cilia defects, but can miss some pathogenic variants that are implicated in ciliopathies.

Whole exome sequencing is an effective diagnostic tool and integral for identifying novel ciliopathy genes

As the cost of per nucleotide next generation sequencing continued to decrease, researchers started to complement targeted studies with exome wide approaches. Whole Exome Sequencing (WES) yields a more comprehensive analysis of genetic variations that lead to ciliopathies. Moreover, WES can reveal novel genes that are important for cilia function, while still generating rapid and high depth results. WES was used on six families with members that had ambiguous ciliopathy like symptoms. It is important to highlight that these patients had not received an official ciliopathy diagnosis. Despite this ambiguous clinical presentation, all the patients had mutations in BBS genes, which are a group of genes that encode for basal body proteins. This group of BBS proteins, sometimes referred to as the ‘BBSome’ transport essential signal receptor proteins to the cilium and thus defects in the ‘BBSome’ impair the cilium’s ability to detect external signals. BBSome defects result in many ciliopathy phenotypes, as mentioned previously. Moreover, WES revealed mutations in three other genes that were proposed to be “novel candidate cilia genes.” The functions of these genes are unknown, but they are all differentially expressed in dividing cells, suggesting that they might play a role in centriole function, which is essential for ciliogenesis. Examples like this show that WES is a useful strategy to diagnose patients with unclear phenotypes, while also revealing novel genes that might be related to cilia biology.

WES can also allow clinicians to understand the underlying genetics of chronic kidney disorders, a common ciliopathy phenotype. Knowing that kidney failure is due to primary cilia defects can guide optimal treatment. In this pursuit, a research group did WES on around 100 patients that had chronic kidney disease before age 25 and found that 7 of them had mutations in cilia genes. Interestingly, one patient with a ciliopathy variant did not have any known family history of renal disease, so the mode of transmission remains an open question. Regardless, WES is an informative tool for ciliopathy patients that are in need of kidney transplantation management.

Moving onto disorders associated with defects in motile cilia, we can see that a large number of studies have also relied on WES. An important study analyzed Primary Ciliary Dyskinesia (PCD), a defect of motile cilia that leads to chronic respiratory symptoms. This disease has an autosomal recessive mode of inheritance and is a known ciliopathy. Researchers utilized whole genome sequencing on one family and WES on another family and saw that all PCD patients had a homozygous loss of function in a ciliary axoneme gene. However, this was not the only gene associated with PCD. Another group identified nine related subjects with PCD from geographically dispersed Amish communities and performed WES of two affected individuals and their unaffected parents. WES confirmed the previously known autosomal recessive mode of inheritance and identified a missense mutation in the HEATR2 gene as the causal variant in these families. This gene is essential for the stability of the inner microtubule structure that allows for the movement of motile cilia. This study shows that WES can be an effective diagnostic tool for PCD and also reveals that mutations in various ciliary genes can be sufficient for PCD. This begs the question of do all mutations that are linked to PCD generate the same phenotype? Next generation sequencing has been key in linking changes in different ciliary genes to phenotype severity. As of 2020, more than 40 different genes have been reported that cause PCD. There have been attempts at linking certain genes to milder phenotypes, but I think it is important to exercise caution as these links are made based on small numbers of patients with variations in any one gene. An example is DNAH9 mutations, which a protein that is essential for axoneme stability, being associated with a mild respiratory phenotype. Overall, WES is shown to be effective at diagnosing ciliopathies associated with defects in motile and/or primary cilia.

Is whole genome sequencing more robust at diagnosing ciliopathies?

A growing body of literature shows that WES is an effective way of diagnosing ciliopathies, and yet exome sequencing does not take into account a very large portion of the DNA of patients. This begs the question of would whole genome sequencing be more robust. While there is not literature that directly addresses this question, an important point to note is that almost all known causative variants that lead to ciliopathies are in the coding regions of the genome. Several studies have used WGS in families that have ciliopathies and only identified variants in coding regions.

However, WGS can be more effective at diagnosing patients that have atypical symptoms. Many studies show that WGS is impactful in identifying novel developmental disorders, and ciliopathies should not be an exception. While WES is more prevalent, WGS is a valuable tool when diagnosing patients that have a diverse spectrum of disease characteristics, particularly since it can reveal changes in non-coding regions of other genes that can be contributing to the ciliopathy like phenotype. In one recent study, scientists did WGS on 4 children with suspected ciliopathies. The authors identified that all 4 children had mutations in cilia genes, but two patients had additional pathogenic variants for non-coding regions that are associated with liver disease.

In addition, WGS is important at identifying structural changes and copy number defects. One study that conducted WGS in 11 different families found a tandem duplication of 3 exons of an IFT gene in 8 families, which all had uncharacterized ciliopathies. Importantly, this IFT protein, which is involved with carrying the building blocks of cilia to the tip of the axoneme, had been missed by whole exome sequencing. This study shows that ciliopathies are not just caused by nucleotide level variants or deletions and that WGS is instrumental in detecting structural rearrangements, including copy number changes. Moreover, others studies have suggested that whole genome sequencing is more powerful than whole exome sequencing at detecting exome variants. Lastly, it is known that transcription level regulation is an important part of ciliogenesis. There is some evidence that mutations in ciliogenesis regulators would lead to mild to severe ciliopathies. For instance, proteins that regulate the expression levels of two cilia genes were shown to be associated with joubert syndrome. Mutations in this regulatory gene was shown to give rise to an ambiguous ciliopathy like phenotype. Such genetic mechanisms will be revealed as WGS on ciliopathy patients get more prevalent.

Overall, WES has been very effective at identifying known and find novel genetic variants that lead to ciliopathies. It is likely that it is sufficient as a first pass in clinical settings. However, I do not think that a targeted gene panel would be robust enough to rule out a cilia defect for a patient that has ciliopathy like symptoms. WES would be an important follow-up for patients that had a ‘normal’ gene panel sequencing. In addition, WGS can play an important role in research by tackling a knowledge gap in cilia biology: Can ciliopathies be associated with variations in non-coding regions of the genome?

Challenges of using whole exome and whole genome sequencing as diagnostics

We have demonstrated that whole exome and/or whole genome sequencing are important venues for ciliopathy detection and the identification of novel genes that are implicated in such diseases. Yet there are challenges associated with using WES and/or WGS in clinic for diagnostic purposes. Firstly, cilia genes tend to be highly variable and lack mutation hot spots. Many variants generate wild type cilia and thus it can make diagnosing clinically ambiguous patients challenging. Moreover, different families tend to have different variations in the same gene, making developing precise diagnostic tools a complex task. Secondly, some cilia genes have duplicated regions in other parts of the chromosome. Take the PKD1 gene, for example. Several studies have shown that the high sequence similarity between the pseudogenes and their parent genes can obscure the detection of the pathogenic mutation. An added challenge is the fact that pseudogenes for original ciliopathy associated genes are located proximal to the original gene.

Moreover, mutations in the same ciliary gene can give rise to different symptoms, making it challenging for physicians to initiate genetic sequencing or interpret the results. For instance, mutations in the CEP290, a centrosomal protein, can lead to retina phenotypes and/or kidney problems. It is possible that it can cause other symptoms as well and if physicians are not aware of them it might be difficult for them to associate genetic variants with the phenotype. Since the boundaries that separate ciliopathy symptoms are very fluid wide scale implementation can be a challenge.

Lastly, there are some medical ethics challenges associated with using WES in clinic. WES may identify mutations in genes unrelated to the studied disease, which has major implications for patients and their relatives. This would not be the case with targeted sequencing and yet we have talked about the limitations of targeted sequencing in identifying novel ciliopathies. In addition, we do not know what the implications of a novel variant will be. As mentioned previously, many variants of unknown significance tend to be unproblematic for patients. Do these patients benefit from knowing that they have this variant? It is worth noting that these problems are seen in the context of other genetic disorders and need to be tackled via a multidisciplinary collaboration that includes genetic counselors, physicians, and ethics specialists.

Discussion of future directions for ciliopathy genomics

The increase in genomics data continues to reveal new cilia genes that are causative for ciliopathies. However, we are still far from a comprehensive list of ciliopathy genes. Firstly, I we are far from generating a complete genetic map of a wild type cilium. Analysis of single cell RNA sequencing data and WGS will continue to reveal new genes that regulate cilia function. Secondly, scientists will continue to discover novel genetic variants in known cilia genes that contribute to ciliopathies. Thirdly, while I highlighted some molecular mechanisms that lead to ciliopathies, the exact process through which many of these genes impact the cilium is unknown, making translating our findings to therapeutics an open question in the field.

Another open question in the field of ciliopathies is whether or not we can link mutations in specific genes to a phenotype. As we have seen through this review, similar variants can lead to drastically different phenotypes and this makes precision treatment challenging. This can be tackled with a meta-analysis and bioinformatics approach. If we can combine next generation sequencing and phenotype data for ciliopathy patients, we can generate a ‘ciliopathy’ databank that associates each gene and variant to a phenotype. As this potentially open source databank grows, scientists can notice trends and make statistically significant genotype to phenotype interpretations.

Discussion

Furthermore, most research has focused on ciliopathies as monogenic (autosomal dominant or recessive) disorders. It would be interesting to see if some patients have multiple mutations in different cilia genes. Due to the basal body’s close relationship to centrioles, it is likely that multiple mutations lead to embryonic lethality, however this might not be the case with all individuals. There are model organisms that are viable and show severe defects after introducing mutations in multiple cilia genes. So, it would be clinically important to see if an increase in the number of cilia mutations correlate with more severe ciliopathy phenotypes.

Lastly, there has been an increasing interest in studying links between cilia defects and neurodevelopmental disorders. In a recent preprint, a large research group associated autism spectrum disorder, intellectual disabilities, and attention deficit disorders with de novo or inherited variants in ciliogenesis regulators. The study established that, in their cohort of 36 people, deleterious variants in transcription factors that regulate ciliogenesis are important causes for the aforementioned disorders. So far, most research on ciliopathies have been conducted on inherited mutations, but this study paves the way for research that looks at the impact of de novo mutations in cilia biology.

Conclusion

Here, I reviewed the impact of next generation sequencing techniques on our knowledge of cilia defects and ciliopathy diagnostics. Over the last two decades the field has evolved drastically: From using chromosome mapping to utilizing next generation genome wide sequencing, which has allowed scientists to identify novel genetic variants that cause ciliopathies. These high throughput screens have been critical in showing the genetic heterogeneity of ciliopathies, alongside highlighting that cilia defects can cause multi organ level phenotypes. This synthesis revealed that mutations in genes that encode basal body (BBSome, CEP290, PKD-1), IFT, and microtubule based axoneme proteins (HEATR2, DNAH9) can be causative factors for ciliopathies, with no clear correlation with clinical symptoms. Importantly, I attempted to associate mutations in specific genes to clinical phenotypes, and similar to other reviews saw that mutations which disrupt similar cellular processes can lead to varying clinical phenotypes. Thus, I believe that the real clinical power of this data will come when we integrate results from different studies that looked at the genetics of ciliopathies. As we start generating large biobanks for ciliopathies, we will be able to make more accurate diagnostics based on whole exome or whole genome sequencing data. Perhaps with a robust understanding of the impact of specific genetic variants, we can start creating evidence based treatments for unique ciliopathies. Tailoring treatment towards each patient’s genetics and clinical phenotype will pave the way towards an era of precision medicine for ciliopathies.

Acknowledgments

I would like to thank Jonathan Pritchard and Alyssa Lyn Fortier for valuable discussions about the manuscript.

References

1. Ishikawa H, Marshall WF (2011) Ciliogenesis: Building the cell’s antenna. Nat Rev Mol Cell Biol 12:222-234.

   [Crossref] [Google Scholar] [PubMed]

2. Horani A, Ferkol TW, Dutcher SK, Brody SL (2016) Genetics and biology of primary ciliary dyskinesia. Paediatr Respir Rev 18:18-24.

   [Crossref] [Google Scholar] [PubMed]

3. Reiter JF, Leroux MR (2017) Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol 18:533-547.

   [Crossref] [Google Scholar] [PubMed]

4. Vladar EK, Stearns T (2007) Molecular characterization of centriole assembly in ciliated epithelial cells. J Cell Biol 178:31-42.

   [Crossref] [Google Scholar] [PubMed]

5. Reeders ST, Breuning MH, Davies KE, Nicholls RD, Jarman AP, et al. (1985) A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature 317:542-544.

   [Crossref] [Google Scholar] [PubMed]

6. Chebib FT, Torres VE (2016) Autosomal dominant polycystic kidney disease: Core curriculum 2016. Am J Kidney Dis 67:792-810.

   [Crossref] [Google Scholar] [PubMed]

7. Barr MM, Sternberg PW (1999) A polycystic kidney disease gene homologue required for male mating behaviour in C. elegans. Nature 401:386-389.

   [Crossref] [Google Scholar] [PubMed]

8. Forsythe E, Beales PL (2013) Bardet biedl syndrome. Eur J Hum Genet 21:8-13.

   [Crossref] [Google Scholar] [PubMed]

9. Leppert M, Baird L, Anderson KL, Otterud B, Lupski JR, et al. (1994) Bardet biedl syndrome is linked to DNA markers on chromosome 11 q and is genetically heterogeneous. Nat Genet 7:108-112.

   [Crossref] [Google Scholar] [PubMed]

10. Katsanis N, Beales PL, Woods MO, Lewis RA, Green JS, et al. (2000) Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with bardet-biedl syndrome. Nat Genet 26:67-70.

    [Crossref] [Google Scholar] [PubMed]

11. Mykytyn K, Nishimura DY, Searby CC, Shastri M, Yen H jan, et al. (2002) Identification of the gene (BBS1) most commonly involved in bardet-biedl syndrome, a complex human obesity syndrome. Nat Genet 31:435-438.

    [Crossref] [Google Scholar] [PubMed]

12. Magescas J, Eskinazi S, Tran MV, Feldman JL (2021) Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia. Curr Biol 31:2410-2417.

    [Crossref] [Google Scholar] [PubMed]

13. Breslow DK, Holland AJ (2019) Mechanism and regulation of centriole and cilium biogenesis. Annu Rev Biochem 88:691-724.

    [Crossref] [Google Scholar] [PubMed]

14. Chen J, Smaoui N, Hammer MBH, Jiao X, Riazuddin SA, et al. (2011) Molecular analysis of bardet-biedl syndrome families: Report of 21 novel mutations in 10 genes. Invest Ophthalmol Vis Sci 52:5317-5324.

    [Crossref] [Google Scholar] [PubMed]

15. Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis E (2013) The next generation sequencing revolution and its impact on genomics. Cell 155:27-38.

    [Crossref] [Google Scholar] [PubMed]

16. Skalicka K, Hrckova G, Vaska A, Baranyaiova A, Kovacs L (2018) Genetic defects in ciliary genes in autosomal dominant polycystic kidney disease. World J Nephrol 7:65-70.

    [Crossref] [Google Scholar] [PubMed]

17. Qin H, Rosenbaum JL, Barr MM (2001) An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol 11:457-461.

    [Crossref] [Google Scholar] [PubMed]