CHARGE syndrome is a rare autosomal dominant disease that is typically caused by heterozygous CHD7 mutations. A de novo variant in a CHD7 splicing acceptor site (NM_017780.3:c.7165–4A>G) was identified in a Japanese boy with CHARGE syndrome. This variant has been considered to be an “unclassified variant” due to its position outside the consensus splicing sites. In this study, abnormal splicing derived from this known variant was confirmed by cDNA sequencing.
CHARGE syndrome (CS) (OMIM 214800) is a rare, autosomal dominant syndromic disease that is characterized by a variety of anomalies, including choanal atresia and malformations of the heart, ears and eyes. These malformations include coloboma, microphthalmia, septal defects in the heart, growth retardation and delayed mental development, genital hypoplasia, external and middle ear abnormalities, and deafness. Furthermore, facial palsy, cleft palate and dysphagia are commonly associated with CS.1,
This study was approved by the Institutional Review Board Committees of the National Research Institute for Child Health and Development and Kurume University. The patient was a 7-year-old Japanese boy diagnosed with CS on the basis of the phenotypic expression of the disease, namely, choanal atresia, cleft palate, dysphagia, cryptorchidism, advanced hearing loss and cardiac septal defects. No coloboma or microphthalmia has been observed thus far. An apparent retardation of growth or development has also not been identified. The clinical details of the patient will be described in a subsequent report. Informed consent was obtained from the patient’s parents.
Exome sequencing was performed as described earlier.9 Using leukocytes obtained from the patient and his parents, genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). DNA fragments corresponding to protein-coding sequences were enriched using commercially available oligonucleotide libraries followed by next-generation sequencing (HiSeq 2500; Illumina, San Diego, CA, USA). The read lengths of the single- and paired-end libraries were 125 bases. The sequences obtained from the patient and his parents were aligned to the reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner version 0.6.2 (Geeknet, Mountain View, CA, USA). Removal of potential PCR duplicates, recalibration of base quality values, local realignments and variation calls were performed using Samtools, Picard (http://broadinstitute.github.io/picard/) and GATK version 2.3–9. Filtration of putative false-positive variations was executed using optimization criteria, quality value recalibration, indel realignment, variant quality score recalibration, variant quality score recalibration PASS filter and GATK-recommended hard filters (GATK best practice v3/v4). Nucleotide substitutions with allele frequencies above 1% in the normal population were excluded as polymorphisms. A rare CHD7 variant detected by exome sequencing was confirmed by Sanger sequencing using the CHD7-intron 33 FW1 (5′- CAGCTCTGTGCACCAGTCAT-3′) and CHD7-exon 34 RV1-2 (5′- AAGGCATCAGACACTGGTGT-3′) primer pair. We retrieved the mutation from previous reports or from public databases of normal populations (The ExAC browser, http://exac.broadinstitute.org/; 1000 Genome Browser, https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/).
Total RNA was extracted from lymphoblastoid cells from the patient’s blood sample using the RNeasy RNA extraction kit (Qiagen). cDNA was reverse-transcribed from total RNA with the EMD Millipore Novagen oligo(dT) primer (Thermo Fisher Scientific, Waltham, MA, USA) and Applied Biosystems High-Capacity RNA-to-cDNA kit (Foster City, CA, USA). cDNA was PCR-amplified using CHD7 coding sequence amplification with the CHD7-exon 33 FW2 (5′- GGCCTCATCCCAGGTTACAC-3′) and CHD7-exon 34 RV1-2 (5′- AAGGCATCAGACACTGGTGT-3′) primer pair. Amplified cDNA fragments were sub-cloned into the TOPO vector (Life Technologies, Carlsbad, CA, USA), and sequences were confirmed by Sanger sequencing.
Mutation Taster (http://www.mutationtaster.org/index.html), NNSPLICE (Berkeley Drosophila Genome Project) (http://fruitfly.org/seq_tools/splice.html), Human Splicing Finder (http://www.umd.be/HSF3/index.html) and MaxEntScan::score3ss (http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html) were used for predicting splice site alterations.
We detected a rare substitution that led to an alteration in the splicing acceptor and an ectopic premature termination within the thirty-third intron of CHD7 (GRCh37, Chr8:61769000, NM_017780.3:c.7165–4A>G). This genomic substitution has been reported in patients with CS (HGMD number CS061264), but has been referred to as an “unclassified variant”.6,10 Exome trio analyses using leukocyte genomic DNA derived from the patient and his parents were performed. The NM_017780.3:c.7165–4A>G substitution was not detected in the parents; thus, this variant was considered to be a de novo mutation. Genomic DNA from the patient was sequenced directly to confirm the NM_017780.3:c.7165–4A>G substitution (Figure 1a).
To identify the allele-specific sequence, the rs2272727 NM_017780.3:c.7356A>G polymorphism was used as a marker for each allele. Because the nucleotide position of rs2272727 is 195 bp away from the NM_017780.3:c.7165–4A>G substitution, the next-generation sequencing library could not determine the genomic linkage of these nucleotide variations. Thus, the genomic linkage between the c.7165–4G substitution and A polymorphism of rs2272727 (NM_017780.3:c.7356A/G) was confirmed by direct sequencing of genomic DNA sub-cloned into the TOPO vector (Life Technologies; Figure 1b). Exome sequencing indicated that the patient’s mother possessed the G polymorphism of rs2272727, while the A polymorphism was assumed to be of paternal origin.
Moreover, to verify the single-nucleotide substitution-induced activation of cryptic splice sites, cDNA was reverse-transcribed using total RNA from lymphoblastoid cells derived from the patient’s blood sample, and the PCR-amplified cDNA was sub-cloned and sequenced. Of 12 cDNA clones, 5 contained the cryptic splice acceptor site three bases upstream of the original site, and the remaining 7 had the wild-type (WT) sequence (Figure 2a). All of the WT cDNA clones had the G polymorphic variation at c.7356, whereas all of the mutated cDNA clones had an A residue at this site (Figure 2b). These data indicate that most of the RNA transcribed from the mutated allele possessed the cryptic splice acceptor. Furthermore, the available cryptic splice site prediction methods11,
We detected the NM_017780.3:c.7165–4A>G substitution four bases upstream of the WT splice acceptor site in exon 34 of CHD7. According to cDNA analyses, this substitution leads to the loss of the normal acceptor site and gain of a cryptic splicing acceptor three bases upstream of the WT splice acceptor site. Moreover, the variant RNA contains a termination codon UAG (TAG) immediately after the cryptic splicing acceptor, leading to a loss of 610 C-terminal amino acids from the full-length CHD7 protein (2,998 amino acids). The WT exons at positions 34–38 encode a nuclear localization signal and the BRK domain, a protein module associated with the CHROMO domain and DEAD/DEAH box helicase domain.14,15 More than 20 nonsense and frameshift mutations have been reported in patients with CS in this genomic region.8,10 Notably, de novo heterozygous frameshift mutations in exon 38 (last exon), which lead to the C-terminal truncation of CHD7 (1–2,988), are reported to be pathogenic.10,16,17 Therefore, we propose that premature termination of CHD7 due to the NM_017780.3:c.7165–4A>G substitution, adversely affects normal growth and development.
Sequencing analyses of individual cDNA clones from the patient’s RNA indicated that the activity of nonsense-mediated mRNA decay is probably subtle, presumably because of extremely long 3′ untranslated regions.18,
In conclusion, we report a case of CS in a 7-year-old Japanese boy with a pathogenic de novo CHD7 mutation at an atypical splice site, which was previously described as an “unclassified variant”. Experimental assessment of splicing variants is critical and contributes to the clinical diagnosis of certain unclassified mutations.
Availability of data and material
The data and materials described in this report are available on request. The identified mutation will be uploaded on the ClinVar website https://www.ncbi.nlm.nih.gov/clinvar/ (the accession SCV000599799). Supplementary Information (Supplementary Table S1; NM_017780.3:c.7165–4A>G evaluation using a splice site predictor) is available on the Human Genome Variation website.
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Katoh-Fukui, Yuko HGV Database http://dx.doi.org/10.6084/m9.figshare.hgv.1890 (2018)
We thank Dr Keiko Hayashi (Department of Genome Medicine, National Research Institute for Child Health and Development, Tokyo, Japan) for technical support. This report was supported by the Foundation for Growth Science. The study was approved by the Ethics Committees of the National Research Institute for Child Health and Development and Kurume University School of Medicine, and written informed consent to participate was obtained from the patient’s parents.