Historically, the diagnosis of CSS was based entirely on clinical features before the establishment of molecular analyses. Important diagnostic criteria include absent/hypoplastic fifth distal phalanx, psychomotor developmental delay, and coarse facial features1. variants in ARID1B are associated with several clinical phenotypes of intellectual disability, which are also recognized as: ARID1B-related conditions19. Morphological abnormalities in the little finger or fingernails, which are the characteristic features of CSS, were not apparent when we performed surface body clinical examination in our case. At the age of 2 years and 11 months, the length of the patient’s fifth finger was 3.0 cm and the middle finger was 4.3 cm. Thus, both were similarly (−1 SD) shorter compared to those of the Japanese population normals20. Given the patient’s short stature of 89.5 cm, which is also rated as −1 SD, it can be concluded that the fifth fingers of the proband are proportional to its length and are not particularly short. However, the radiographic examination of the metacarpophalangeal bones, performed at 3 years and 6 months of age, suggested hypoplastic distal phalanges and fifth middle phalanges. Recently, a metacarpophalangeal profile in three patients with ARID1B variants was reported21. In this report, patients showed different patterns of brachydactyly, not only the shortening of the fifth distal phalanges but also the fifth middle phalanges, or a combination of shortened multiple bones. Our patient can be described as a case of mild brachydactyly with a novel ARID1B variant. Initially, it was difficult for doctors to diagnose our case as CSS, as the complication of fingers was not detected by visual inspection and palpation. A radiographic examination may be useful and considered for the accurate diagnosis of brachydactyly. Both CSS and ARID1Brelated conditions have a broad phenotypic spectrum and cannot always be easily divided into two categories. This study suggests that molecular assays, such as WES or gene sequence panels, are powerful tools for the diagnosis of CSS or ARID1B-related disorders.
In this study, we identified a novel nonsense variant c.4282C>T in the ARID1B gene and demonstrated the occurrence of both NMD and NAS. The amount of mRNA was quantified based on the Sanger sequencing chromatograms of cDNA (Fig. 2e). mRNA of the proband contained the sequence derived from the WT allele and two alternative sequences derived from the mutated allele. We amplified these three products by PCR and read the sequence simultaneously using the same primer sets (Supplementary Figure 1a). We suggest that these two mRNAs are the remnant of NMD and the product of NAS because the amounts of these products are altered by CHX treatment, which inhibits NMD and NAS.16,17,18.
Since the height of the Sanger chromatogram is influenced by several factors:22, we tested a single base primer extension assay using the SNaPshot Primer Extension kit (Thermo Fisher) for the quantification of the RNA. In the SNaPshot method, primers are designed just next to the variant and react with fluorescence-labeled ddNTPs (not dNTPs). The SNaPshot reaction extends one base and stops. Detecting the fluorescence reveals the elongated base, which is complementary to the variant. When we apply the SNaPshot assay to quantify our three RNA products, we need to perform two experiments independently and calculate these results to estimate the amount of three RNAs (Supplementary Figure 1b). This is done because the detection of these three RNA products must be performed at different positions. In our case, the “C” at the position of c.4282 is from the wild-type allele and the “T” is from the nonsense variant of the mutant allele. However, the “A” signal at that position is not from c.4282 but from the base of the same distance in exon 18. Assay #1 can quantify the amounts of WT and mutated RNA; however, it cannot detect exon 17 skipped RNA. Assay #2 can detect exon 17 skipped RNA; however, it cannot discriminate between WT and mutated RNA. When we compared the chromatograms of genomic DNA obtained from Sanger sequencing and the SNaPshot assay, the SNaPshot chromatogram required standardization (Supplementary Figure 1c). We tested the nonsense variant with Assay #1 from SNaPshot and the results (Supplementary Figure 1d) were similar to those obtained with Sanger chromatography (Figure 2e). With these results, we think that Sanger sequencing, as a semi-quantitative conventional method, is still useful in special situations if we can produce clean data with a deep understanding of its limitations.
The total amount of RNA of the mutant allele increased from 52% (25% + 27%) to ~100% (83% + 17%) with CHX treatment (Fig. 2e), suggesting that NMD was completely inhibited by CHX -therapy. However, NAS was partially inhibited by CHX treatment because the mRNA with exon 17 skipping remained at ~17%. The restoration of the T signal at the c.4282 position by CHX treatment was caused not only by the inhibition of NMD of mRNA containing nonsense variants, but also by the inhibition of NAS from exon 17 skipping (as result, the nonsense variant with mRNA is produced). The combined effect of the co-occurring NMD and NAS is difficult to discuss. If a nonsense variant exists in an exon containing the number of bases in multiples of three, NAS can produce a splice variant RNA without frameshift. In those cases, we cannot simply conclude that the effect of the “nonsense variant” is “pathogenic”. This is because the effect of the DNA variant on the phenotype is influenced by the sensitivity of pre-mRNA to NMD and NAS and the stability or functional retention of the exon-skipped short protein. In our case, both transcripts, the “nonsense variant containing” and “exon 17 skipped” mRNAs, had PTC and became targets of NMD. Moreover, even if they are translated, functional ARID1B proteins are unlikely to be produced. This is because both prematurely terminated transcripts lack a nuclear localization signal and BAF250C domain that are important for nuclear localization and BAF complex formation, respectively. Overall, we concluded that the nonsense variant c.4282C > T is a pathogenic variant. Our in vitro analyzes using LCLs do not necessarily recapitulate early developmental conditions. There is a report that the skipping of exon 17 caused by a synonymous variant is associated with CSS8; however, we cannot test how our patient’s clinical symptoms were altered with concurrent NMD and NAS. These are limitations of this study.
In conclusion, we identified a new nonsense variant, c.4282C > T, in the ARID1B gene. It is suggested that this variant is “pathogenic” and causes the clinical phenotype. This nonsense variant, c.4282C > T, was found to cause NMD and NAS simultaneously.