Consanguinity-based analysis of exome sequencing yields likely genetic causes in patients with inherited retinal dystrophy

Inherited retinal dystrophy (IRD) is a group of severe and irreversible vision-threatening disorders, affecting approximately 2 million people worldwide [1]. It is characterized by degeneration and dysfunction of specialized and light-sensitive neurons in the retina, such as cone and/or rod photoreceptor cells [2‐4]. IRD is comprised of a wide spectrum of diseases with enormous genetic heterogeneity [1, 5‐7]. The most common form of IRD is retinitis pigmentosa (RP), which is already known to be attributed to the primary loss of rods, followed by secondary loss of cones. RP initially affects night and peripheral vision, but eventually, patients will experience central vision impairment [8, 9]. Other diseases in this group include cone and rod dystrophy (CORD), Leber congenital amaurosis (LCA), Bietti crystalline dystrophy (BCD) and syndromic diseases such as Usher syndrome and Bardet-Biedl syndrome [10‐13]. Currently, more than 250 disease-causing genes have been identified in patients with IRD. Transmission of these genes has been shown to resemble Mendelian traits, including autosomal recessive (AR), autosomal dominant (AD), and X-linked (XL) modes of inheritance. Although great efforts have been made to dissect the genetic basis of IRD, the genetic causes of IRD in a large proportion of patients remain unknown [14‐16].

Many communities around the world have a long tradition of marrying to relatives. The highest prevalence of inbreeding was observed in North and sub-Saharan Africa, the Middle East, and West, Central, and South Asia [17]. In the rural areas of China, consanguineous marriages such as cousin couples are relatively common, especially among minority ethnic groups. According to data from a population-based survey in the 1990s, the average consanguinity marriage rate among Chinese Han population was 1.4% and it was significantly higher in rural areas than in urban areas, while among Yi population which is one of the major ethnic minority of China, the rate was as high as 14.6% [18]. In the recent decade, nationwide quantitative information on consanguinity is not available, but some population surveys conducted in ethnic minority settlements indicate that the situation of consanguineous marriage has not changed much.

Consanguinity increases the likelihood of pathogenic mutations in a homoallelic state. A higher prevalence of rare hereditary diseases caused by homozygous mutations in AR genes has been observed among members of consanguineous families compared to those of non-consanguineous families [19, 20]. Several studies have also reported genetic causes of IRDs among consanguineous families worldwide, especially in the areas where consanguinity rate is particularly high [21‐24].

However, few studies were performed among patients with IRD in Chinese consanguineous families. Our in-house data show that about 11% of IRD probands have consanguineous parents. Therefore, there is a need for genetic screening and counseling among with IRD patients in Chinese consanguineous families.

To date, next-generation sequencing (NGS) techniques have been widely applied to the research of IRDs, among which whole exome sequencing (WES) has demonstrated the greatest reliability and efficiency in testing genetic mutations [25‐27]. In this study, deep sequencing of all causative genes of IRD was performed on ten probands, and WES data were analyzed with a consanguinity-based analysis strategy. This study not only broadened the genetic mutation spectrum but also enriched knowledge on the genotype–phenotype correlations of IRDs, suggesting that molecular genetic testing using a consanguinity-based approach is of great value for the diagnosis of IRDs.

Consanguineous marriage is prevalent in rural China, which could potentially increase the risk of suffering from hereditary diseases. In consanguineous families, genetic defects that are caused by homozygous mutations inherited respectively from both parents who are carriers of the heterozygous mutation often manifest in the offspring. Incidence of IRDs, for example, could lead to devastating visual disability and negative livelihood impact. However, few studies focused on Chinese consanguineous families with IRDs. To broaden the knowledge on this population, deep exome sequencing and consanguinity-based analysis was performed in order to elucidate the genetic etiology in patients with IRD.

In this study, 10 homozygous disease-causing mutations were successfully identified. As for the genetic pathogenesis of four typical RP families, USH2A and EYS mutations accounted for 50%, which was consistent with our previous study showing that USH2A and EYS are among the most common disease-causing genes of ARRP in Chinese population [14].

F1-III:4 was diagnosed with RP and high myopia and was identified to harbor a novel homozygous mutation c.1654_1655delAG (p. R552Afs*5) within FAM161A. To date, FAM161A mutations have only been linked to ARRP, and mostly are in the form of null mutations [41]. A previous study did observe that RP patients associated with FAM161A mutation manifested moderate-high myopia, which was consistent with our observation [42]. The protein encoded by FAM161A is evolutionarily conserved in vertebrates such as Pan troglodytes, Macaca mulatta, Equus asinus and Sarcophilus harrisii. It localizes to photoreceptor connecting cilia in human and ciliary basal body in mammalian ciliated cells. Recent research has demonstrated that this protein is involved in microtubule acetylation and stabilization by maintaining transport processes in photoreceptors as a component of the cilia-basal body complex [43, 44]. FAM161A directly interacts with cilia proteins CEP290, SDCCAG8, OFD1 and lebercilin, all of which are implicated in heterogeneous IRDs [45].

CEP78 has been reported to be related to the pathogenesis of Usher syndrome or cone dystrophy along with sensory hearing loss in recent studies [46, 47]. Patient F8-III:5 in our study had a homozygous missense mutation c.830T > C (p.L277P) in CEP78 and presented cone and rod dystrophy without hearing impairment. The mutation is very likely to be the genetic cause based on the evidences above, but the phenotype is unusual as all patients reported to harbor CEP78 mutations have hearing impairment. Our report of the clinical manifestation indicates that CEP78-related IRDs may have a high clinical heterogeneity. However, further research and clinical observation are needed.

A frameshift mutation c.445-448delCTCT (p. Leu149Serfs*31) in gene IQCB1 was determined in F10-III:1, a patient who was diagnosed with LCA. This mutation was pathogenic according to ACMG guidelines, and has been reported in two unrelated German families to be related to Senior-Loken syndrome [40]. Senior-Loken syndrome is a rare autosomal recessive ciliopathy characterized by the association of nephronophthisis (NPHP) and early onset retinal dystrophy. Age of onset, severity and progression of symptoms can vary greatly among affected individuals. Patient F10-III:1 suffered from severe vision impairment in the first year of her life and no kidney disorder was found in the following 30 years. This may be a novel correlation between IQCB1 mutation and complex clinical manifestations. However, regular follow-up at nephrology department is needed.

F2-III:3 was characterized by a rare type of RP associated with macular and choroid coloboma, and had a homozygous mutation c.437T > A (p.V146D) in RDH12. RDH12 coded protein is predominantly expressed in the inner segment of rod and cone photoreceptors as an NADPH-dependent reductase involved in the conversion of all-trans-retinal and 11-cis-retinal to the corresponding retinols [48]. This p.V146D mutation was previously reported in severe early-onset ARRP family [34]. In contrast, the proband in this current study exhibited additional phenotypes. A prior study also indicated that RDH12 mutation could lead to LCA with macular coloboma [49]. However, in our study, characteristic features of LCA, including nystagmus, photophobia, amaurotic pupils and flat or nondetectable ERG, were all absent [50].

F7-III:4 was diagnosed with CORD and harbored a homozygous mutation c.3991C > T (p.R1331C) in CRB1. CRB1 localizes to the outer limiting membrane of the retina and is expressed in adherens junction in Muller glia cells, which may control retinogenesis via signaling pathway such as mTOR, Hippo, Wnt and Notch1 [51].The p.R1331C mutation was reported to have caused unusual maculopathy in an Irish family [38]. In contrast, F7-III:4 had a definite diagnosis of CORD with a wider range of fundus atrophy and evident ERG performance. Furthermore, the patient suffered from more severe binocular glaucoma and cataract. Our findings have verified the implication of the RP or LCA-related CRB1 mutation in maculopathy in a Chinese individual.

To date, more than 110 mutations within RDH12 have been classified to be responsible for either RP, LCA or CORD. And more than 150 CRB1 mutations are shown to be associated with specific fundus features other than RP or LCA, such as preserved para-arteriole retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (Coats-like exudative vasculopathy) [52, 53]. Moreover, some mutations within CRB1 were also causative of Familial foveal Retinoschisis (FFR) [54]. We reviewed RDH12 and CRB1-related disorders from public databases such as ClinVar (https://​www.​ncbi.​nlm.​nih.​gov/​clinvar/​) and HGMD (http://​www.​hgmd.​cf.​ac.​uk), and summarized the proportion of heterogeneous phenotypes correlated with RDH12 and CRB1 mutations in Fig. 5. Some researches indicate that additional modifying factors could be responsible for the absence of clear correlation between mutations and phenotypes. For example, a previous study has demonstrated an earlier onset and more severe retinal phenotype when Mthfr and rd8 mutations coexist in mice, indicating that Mthfr is a modify factors of Crb1 [55].

The strategy we use in this study would help achieving quick and accurate localization of the possible disease-causing mutations, thus identifying the genetic cause of diseases efficiently and cost-effectively. However, there are some limitations. For example, focusing only on homozygous variations may lead to missing of de novo mutations, as well as the underlying disease-causing mutation in an AD or X linked pattern of hereditary. And due to the extreme genetic and clinical heterogeneity of IRD, as well as the existence of de novo mutation, copy number variation and structural variation, this approach is not 100% successful. In order to solve the genetic causes of patients with negative results, further mining is needed. Homozygosity mapping or detection of runs of homozygosity (ROH) through WES data can be performed to detect novel candidate genes using several practical and reliable tools. AutoMap, for example, was conducted and tested to be one of the best homozygosity mapping tools by a similar recent study in Iranian families. With AutoMap, the researchers predicted ROHs from WES data with high specificity and sensitivity, and also obtain a high detection rate in their IRD cohort [56]. De novo mutations should arouse great attention and whole genome sequencing (WGS) and long-read sequencing can play reliable roles in detecting large structural variations.

Our study employed a consanguinity-based exome sequencing analysis approach to dissect the genetic causes of IRDs in ten consanguineous Chinese families. We revealed two novel mutations and genotype–phenotype correlations, thereby expanding our understanding of the underlying heterogeneous mechanism of IRDs. Moreover, this current study attested to the value of molecular genetic testing using a consanguinity-based approach in the diagnosis of recessive genetic defects, providing a perspective for determining novel pathogenic genes in a more cost-effective and efficient way.