Clinical findings
A female patient was referred for genetic assessment at 16 years of age for hearing impairment, scoliosis, and left side facial palsy. The proband was the second child of healthy unrelated Lithuanian parents aged 29 at the proband’s birth. The proband had neither a history of prenatal exposure to teratogenic agents nor any family history of congenital malformations. During the pregnancy, polyhydramnios was diagnosed. Congenital heart defect of the foetus was detected at 35 weeks of gestation. The proband was born at 38 weeks by Caesarian section due to congenital heart defect of the foetus. At birth, her weight was 2800 g (3rd centile), her length was 49 cm (10th centile), the circumference of her head was 35 cm (50th centile), and her Apgar score was 6 at 1 min and 8 at 5 min. Complete atrioventricular communication with a small ventricular septal defect, a moderately sized atrial septal defect with an aneurysm in the interatrial septum, and insufficiency of the mitral valve were diagnosed. Additionally, left side choanal atresia, left side facial palsy, a short neck, dysplasia of the sacrum, and dysplasia of the hip joint were observed. As a neonate, the patient had feeding difficulties and received a nasogastric tube for 1 month. Poor weight gain and cardiac insufficiency manifested at the age of 7 months. Auditory Brainstem Response Audiometry was performed and unilateral deafness in the left was diagnosed during infancy. Bilateral aplasia of the semicircular canals and cochlear nerve canal atresia on the left were found on temporal bone CT scans (Fig. 1). At the age of 1 year, radical surgical correction of atrioventricular communication was performed. Pyelonephritis and grade IV vesicoureteral reflux were first diagnosed at 1 year of age, and recurrent episodes of pyelonephritis continued until the age of 7 years. At age of 4 years, surgical repair of choanal atresia on the left was performed and a tympanostomy tube was placed in the same side due to recurrent otitis media. The choanal atresia operation was performed again at the age of 5–6 years. Bone-anchored hearing aid (BAHA) surgery was performed at 7 years of age. Due to a skin reaction at the location of the implant, another operation to implant a new BAHA attract system was performed after the device had been used for 4 years. Adenoidectomy was performed at 11 years of age. The proband had hypermetropia, for which glasses were prescribed. Scoliosis of 17° was evaluated at the age of 16 years.
Motor development of the proband was delayed; she walked alone at the age of 27 months. According to the DISC scale, at the age of 29 months her psychomotor development was evaluated at the range of 21 (gross motor) to 29 (fine motor, visual attention and memory) months. Eruption of permanent teeth was also mildly delayed. The girl attended a regular school and had no behavioural problems. Her psychological evaluation at the age of 16 years yielded a verbal score of 90, a performance score of 97, and a full score of 94 on the Wechsler Intelligence Scale for Children (WISC-III).
During an examination at the age of 16 years, her height was 162 cm (25th centile), her weight was 48.5 kg (3rd–10th centile), and the circumference of her head was 53.5 cm (3rd–10th centile). Her phenotype was remarkable for left side facial palsy, low forehead, deformed nasal bridge, synophrys, protruding ears, auricular pits, low posterior hairline, short neck, limited movements of the neck, pectus excavatum, hirsutism, and Tanner PH-4, B-3. Additionally, primary amenorrhoea and premature ovarian insufficiency were diagnosed at 18 years of age. She had low levels of estradiol and near normal concentrations of prolactin and follicle-stimulating hormone. A pelvic ultrasound visualised a uterus (49x15x31 mm) and ovaries (26-27 × 12 mm).
Whole exome sequencing
Whole genomic DNA (gDNA) was extracted from peripheral blood of our proband and her healthy parents following the standard phenol-chloroform extraction protocol.
Whole exome sequencing (WES) using the high throughput next generation sequencing (NGS) technique was used to sequence the sample of the proband (Illumina, Inc., USA). DNA libraries generated using TruSeq Rapid Exome Library Prep kit (8x3plex) (Illumina, Inc., USA). In order to precisely measure the concentration of DNA libraries, Qubit dsDNA BR Assay kit (ThermoFisher Scientific, USA) and Qubit fluorimeter (ThermoFisher Scientific, USA) were used. Clusters amplificated using cBot system (Illumina, Inc., USA), TruSeq PE Cluster Kit v3-HS (Illumina, Inc., USA) and TruSeq Dual Index Sequencing Primer Box – Paired End (Illumina, Inc., USA). WES was performed using TruSeq SBS Kit v3-HS (Illumina, Inc., USA) and by employing the HiScanSQ (Illumina, Inc., USA) genetic analyzer.
Analyses of high throughput sequencing data started with alignment against The Human NCBI Build GRCh37 (hg19/2009) reference genome. The annotation of NGS data was made using the ANNOVAR v.2018Apr16 program. The pathogenicity of variants was assessed using ACMG criteria, taking into account the data provided by the ANNOVAR program, the available databases (ExAC Browser, 1000 Genome Project, NCBI dbSNP, NCBI dbVar, HGMD, NCBI OMIM, NCBI ClinVAR, Leiden Open Variation Database, NCBI Genome, Deafness Variation Database), and the relevant scientific literature. The pathogenic or probably pathogenic sequence variants were checked by analyzing proband’s BAM files using the visualization tool Integrative Genomics Viewer (IGV).
Reverse transcription reaction
Total RNA of the proband and her healthy parents’ samples was extracted from whole blood using a Tempus™ Blood RNA- Tube and Tempus™ Spin RNA Isolation Kit (Thermo Fisher Scientific, USA) according to the optimized manufacturer’s protocols. Total RNA concentration and quality were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was reverse-transcribed (RT) from a total RNA using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, USA) and a ProFlex PCR system (Thermo Fisher Scientific, USA) following manufacturer’s protocol and recommended conditions.
Polymerase chain reactions and Sanger sequencing
Polymerase chain reactions (PCR) of gDNA and cDNA sequences flanking splice site c.5535-1G > A variant of CHD7 were performed using specific primers designed with Primer Blast tool [21, 22]. gDNA was PCR amplified using primer pair designed on exon 26 (forward primer) and on intron 27 (reverse primer), while PCR amplifications of cDNA sequence were performed using the same forward primer, but different reverse primers, which were designed on the 28–29 exon junction as well as on the 29–30 exon junction (Additional file 1: Table S1). PCR products were fractioned according to standard agarose gel electrophoresis.
gDNA samples was used for the segregation analysis. In order to elucidate the pathogenicity of detected variant, the cDNA samples were analysed. The PCR products were sequenced with BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) and ABI 3130xL Genetic Analyser (Thermo Fisher Scientific, USA). The sequences were aligned with the reference sequence of the CHD7 (NCBI: NM_017780.4) gene.
In silico analysis
Mutation Taster [23, 24] and Human Splicing Finder [25] databases were used for predicting splice site alterations. Sequences of evolutionary distinct species were obtained from the Ensembl genome browser [26, 27], while a sequence alignment was produced using Clustal Omega tool [28]. Possible splice site c.5535-1G > A variant’s effect on the CHD7 protein (UniProtKB: Q9P2D1) was predicted using different tools and databases, such as ExPASy Bioinformatics Resource Portal [29, 30] and Pfam 32.0 database [31, 32].
Genetic findings
Heterozygous CHD7 variant NC_000008.11(NM_017780.4):c.5535-1G > A, located in the acceptor splice site of intron 26, was identified in the proband’s gDNA sample after analysis of WES data.
The acceptor splice site variant is novel and not recorded in the CHD7 database, HGMD, or other databases. The variant was confirmed by Sanger sequencing on the proband’s gDNA sample. Segregation analysis by Sanger sequencing on the gDNA samples of her parents confirmed the de novo origin of the variant. The splice site variant was evaluated by in silico analysis.
A variant effect prediction method, Mutation Taster, predicted the variant to be likely pathogenic and disease causing. Moreover, the alteration of the acceptor splice site was predicted by the Human Splicing Finder database. The variant was predicted to affect pre-mRNA splicing by the altering acceptor splice site at − 1 position and the possible use of a cryptic splice site. Sequence alignment of the CHD7 protein in seven species revealed that the region containing the splice site variant is highly conserved (Fig. 2b).
To study the potential pathogenicity of the CHD7 gene c.5535-1G > A variant, the analysis of the cDNA sequence of the patient and healthy controls was performed. Sanger sequencing of the patient’s cDNA sample revealed that the c.5535-1G > A variant causes the loss of an original acceptor splice site at position − 1 in the intron 26 of CHD7 and consequently activates a cryptic splice site only one nucleotide downstream of the pathogenic variant site (Fig. 2a). Meanwhile, Sanger sequencing of cDNA samples from healthy controls did not expose any alteration in the splicing mechanism. In silico, this variant leads to the frameshift of 23 new amino acids and therefore a truncated 1869 amino acid protein NP_060250.2:p.(Gly1846Glufs*23) (Fig. 2d). The truncated CHD7 protein contains two functionally important chromodomains, the SNF2 domain and helicase domain, but there is a loss of two BRK domains (Fig. 2c).