Background

 With the increasing clinical implementation of next-generation sequencing, the gene diagnostic yield for monogenic diseases has improved but still rarely exceeds 50%^1-3. For the majority of unsolved patients, this may be explained by limited power to resolve complex structural variations (SVs) of the short-read sequencing platform and incomplete knowledge of the functional consequences of most noncoding variants⁴. Although long-read sequencing technology improves the identification of complex SVs, the analytical challenge around variant interpretation persists, especially for balanced SVs with noncoding breakpoints^5-7. Noncoding regions, representing approximately 98% of the human genome, have recently attracted attention in relation to genetically unsolved patients with Mendelian disorders^8-10. To date, a number of noncoding variants have been identified, most of which were recognized according to a physical location in or adjacent to the known disease-causing gene, such as intronic, regulatory, upstream or downstream regions of known genes. These noncoding variants are reasonably linked with the aberrant expression of their adjacent known genes^11,12. However, for variants at intergenic regions, evidence, both at the genetic level and in functional analysis, is necessary to confirm the pathogenicity of these variants.

 Key findings

 Identification of a unique condition of central iris hypoplasia in a large family

 In our study, we identified the central iris hypoplasia (CIH) as a unique condition in a large family (Figure 1a). All the six affected patients of the large family had a history of photophobia since birth and a central absence of the iris pupillary zone alone in both eyes with a pseudo-enlarged pupil. They had clear cornea and normal-like fundus with foveal reflex without nystagmus or any other obvious abnormality. The CIH in this family is different from aniridia, which is characterized by iris coloboma involving both pupillary and peripheral zones, foveal hypoplasia, congenital nystagmus, and low visual acuity (Figure 1b).  

Figure 1. The CIH phenotype in a large family

Identification of a balanced translocation with intergenic breakpoints in the family

 A genome-wide linkage scan was performed on genomic DNA from all 12 individuals, and two-point linkage analysis mapped two loci on chromosomes 6q15-q23.3 and 18.p11.31-q12.1 (Figure 1a). After short-read sequencing excluded SNVs and InDels inside the linkage intervals, long-read genome sequencing identified a balanced translocation t (6;18) (q22.31;p11.22) with both breakpoints at intergenic regions, chr6:121619304 and chr18:10432739 (Figure 2a). Sanger sequencing confirmed that the noncoding translocation variation completely co-segregated with CIH within the family, further supporting it as the disease-causing change for this family (Figure 2b). 

The translocation led to reorganizations of chromatin domains over the breakpoints

 To further explore the molecular mechanism underlying CIH caused by the balanced structural variation, we generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of one patient and one unaffected individual in the family. Hi-C sequencing was conducted on the iPSCs samples from the two individuals, and it showed that the translocation disrupted the two chromatin topologically associating domains (TADs) on chromosome 6 and 18 in the normal control but led to the formation of neo-TADs over the breakpoints on the derivative chromosomes in the patient (Figure 2c). CUT&Tag analysis identified the presence of a tissue-specific enhancer cluster on chromosome 6, which was driven next to the APCDD1 promoter in a neo-TAD by the translocation (Figure 2c). RNA-seq analysis identified that APCCD1 was the only gene with a statistically significant expression change among 40 genes within 2.5 Mb of each breakpoint (Figure 2d). These findings suggest that the translocation rewired the three-dimensional chromatin structure, leading to aberrant interact between enhancers and APCDD1 promoter and thereby upregulating its expression. 

Figure 2. Noncoding translocation generated neo-TAD and upregulated APCDD1 expression

APCDD1 play a role in development and maintain of the iris

To assess the potential roles of APCDD1 in the iris, we conducted immunofluorescence staining in eyes of human adult as well as mice at different stages. The results demonstrated that APCDD1 was specifically expressed in the fibroblasts of the human iris  (Figure 3a), whereas the expression pattern in mouse eyes transferred from the peripheral inner layer of the neural retina (E11.5 ~ E14.5) to constriction in the boundary of the ciliary body and the retina (E17.5 ~ P3M) (Figure 3b). These results confirmed that APCDD1 plays a role in development and maintenance of the iris. 

Figure 3. Expression pattern of APCDD1 in the human iris and embryonic mouse eyes 

Conclusion 

Our research underscores several implications in the area of medical genetics. First, we provide evidence for the identification of complex SVs at noncoding intergenic regions relevant to Mendelian diseases. Although such complex SVs can be identified by cytogenetic methods, they are rarely concerned in routine genetic diagnosis for Mendelian diseases¹³. A uniform phenotype in large families with definite mapped loci based on genome-wide linkage analysis provides unique opportunities to explore noncoding SVs for Mendelian diseases, as in the current study’s analysis of the large family with CIH. Second, pathogenicity of noncoding SVs is achieved through disrupting the three-dimensional chromatin structure^14,15, which provide an avenue to interpreting the function and pathogenicity of noncoding variants, such as the balanced translocation with noncoding breakpoints in this study. Third, the discovery of disease-associated genes via increased dosage effects has been particularly challenging, as in the case of APCDD1 in this study. The majority of genes causing human diseases are identified as a result of loss-of-function variants and occasionally via gain-of-function^16,17. However, variants with increased dosage effects are easily missed by current variant prioritization strategies in clinical genetic pipelines¹⁸. Additional studies aimed at understanding the upregulation of functional genes through coding or noncoding elements, as in the current study, may improve the prediction probabilities for variants with an effect on the upregulation of the genes that contribute to human diseases, which may in turn elucidate the genetic cause of diseases in approximately half of unsolved patients with inherited disease^17,19,20.

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