The proband was a Chinese full-term boy who was first hospitalized for tachypnea, cleft palate, and short trunk shortly after birth. The gestational age was 38 + 2 weeks and the birth weight was 2,850 g. The Apgar score was 9 points for 1 min, 5 min, and 10 min (all were breathing-1 scores). His mother was healthy during the pregnancy and had no history of pregnancy-induced hypertension, diabetes, or viral infection. His parents were unrelated and denied any family history of genetic diseases. The proband was the second child of these unrelated parents, who had a healthy older daughter. Ultrasound examination in the third trimester of pregnancy showed spinal column disorder, low position of the spinal cord, unclear display of the aortic arch, and bilateral renal pelvis separation.

Physical examination on admission: heart rate 140 beats/min, respiration 50 breaths/min, SpO₂ 92% (oxygen concentration 30%), blood pressure 65/32 mmHg, body weight 2,850 g (10–25th percentile), head circumference 33 cm (10th percentile), and body length 42 cm (<3rd percentile). The proband's cry was loud and the response was good. A positive inspiratory trident sign was observed. There was no jaundice, cyanosis, or other skin abnormalities. The soft palate and part of the hard palate were cracked in the oral cavity. The thorax was short, but no pathological murmur was heard on auscultation. The abdomen was soft, and the liver and spleen were unpalpable under the ribs. The primitive reflexes were intact.

Auxiliary examinations: routine laboratory testing was normal. Chest x-ray confirmed a short rib cage, with a length of approximately 3.4 cm, showing distorted and fused ribs (Figure 1A). Chest CT demonstrated skeletal deformities, including half cones, butterfly vertebrae, rib fusion, and spinal canal enlargement (Figures 1B–D). Kidney color Doppler ultrasound revealed left hydronephrosis (approximately 13 mm), and right renal collection system separation (approximately 4.9 mm) (Figure 1E). Furthermore, a patent foramen ovale was found on cardiac color Doppler ultrasound. No abnormalities were reported by cranial ultrasound and magnetic resonance.

After 44 days of treatment with special pacifier feeding, anti-infection agents, and oxygen support, the child was discharged from the hospital and sucked well without choking. At the 3-month follow-up, the child sucked well without choking but still showed poor growth, with a body weight of 4,530 g (<3rd percentile), head circumference of 37 cm (<3rd percentile), and body length of 59 cm (10th percentile). A mild inspiratory three-concave sign was observed with SpO₂ 95% (on room air). He underwent surgery for congenital hydronephrosis at 5 months old and underwent cleft palate repair when he was 1 year old. At 1 year old, his body weight was 7,280 g (3rd percentile), with a head circumference of 43.5 cm (3rd–10th percentile), and a body length of 70 cm (3rd percentile). The boy could stand while holding things and could pronounce “Dad, Mom”. He had a good diet without coughing after eating rice porridge and drinking milk. At this age, the inhalation three-concave sign was negative. Mild scoliosis was observed on the chest x-ray (Figure 1F). At the age of 18 months, the body weight was 9,000 g (3rd percentile), the head circumference was 45.5 cm (3rd percentile), and the body length was 77 cm (3rd percentile). The proband could walk alone and could speak words clearly. The scoliosis showed no progression. Due to the absence of other abnormal symptoms, no treatment was given. At the time of submitting this manuscript, the child was 2 years and 7 months old with a body weight of 12,400 g (25–50th percentile), head circumference of 48.5 cm (25–50th percentile), and body length of 89 cm (10th percentile). He could run and play normally, distinguish colors and partial shapes, and express his meaning in sentences, such as “Mom, we play hide-and-seek” etc. The scoliosis still did not progress, but the boy was required to attend regular follow-ups. A developmental assessment was performed using a neurodevelopmental scale for children aged 0–6 years. The results were gross motor 30, fine motor 33, adaptive ability 31.5, language 30, social behavior 30, intellectual age 30.9, developmental quotient 99.6, and moderate intelligence. No adverse or unanticipated events occurred. The parents were satisfied with the development of the proband. Long-term follow-up was needed for further observation.

Spondylocostal dysostosis may be caused by at least six distinct gene variants. Whole-exome sequencing (WES) and copy number variation (CNV) detection were conducted to discover the pathogenic genetic variations responsible for the symptoms in the proband. Written informed consent was obtained from the patient's parents. This project was approved by the Ethics Committee of Bethune International Peace Hospital (Approval Nos. 20180023 and 2022-KY-26). Trio-WES and trio-CNV detection were performed on an Illumina Novaseq6000 sequencing system in Beijing Berry Hekang Medical Laboratory. Sequencing data were compared with the human reference genome (hg19/GRCh37) using Burrows–Wheeler Aligner (BWA, CA, USA) software to align the original mapping result. Picard was used to mark and remove duplicate reads. Based on the results of the alignments, the obtained set of candidate variants was functionally annotated by Variant Effect Predictor (VEP) using a variety of bioinformatic databases. In brief, the detected variants fulfilled the following strategies: variants of single-nucleotide polymorphisms, splicing-sites variants, inframe indels, and frameshift insertions and deletions; variants with a minor allele frequency <0.5% in the genome Aggregation Database (gnomAD), 1,000 Genomes Project, Exome Aggregation Consortium (ExAC), or Exome Sequencing Project (ESP6500si); the retained missense variants were submitted to PolyPhen-2, SIFT or M-CAP for functional prediction and scored as “deleterious” by SIFT, “damaging” or “possibly damaging” by Polyphen-2, and “probably damaging” or “possibly pathogenic” by M-CAP. CNVs were evaluated by an in-house pipeline using read counts based on a smoothness model (Berry Genomics, Beijing, China) according to a previous study (9). The pathogenicity of each variant was assessed according to the American College of Medical Genetics and Genomics (ACMG) guidelines (10, 11).

A homozygous variant HES7 c.226+1G>A was identified by trio-WES, which was inherited from both parents (Figures 2A,B). This novel variant was located at the splicing region, which was predicted to influence the splicing of HES7 mRNA (PVS1). Nevertheless, this variant was not found in reference databases such as 1,000 genomes, ExAC, gnomAD, or the local Berry database with only Chinese samples (PM2_supporting). As the parents of the proband denied consanguinity, kinship analysis was performed using King software with WES data (12). The kinship value between the couple was 0.0721 which indicated that they were third degree relatives (Supplementary Figure S1). The couple have no common relatives, but they come from the same town. Founder effect may be an explanation for this non-consanguineous couple carrying the same rare variant. No suspicious variants were found in the trio-CNV detection results. The proband was finally diagnosed as SCDO4 based on the WES results.

HES7 is located at chromosome 17p13.1 and has two transcripts, namely, NM_001165967 and NM_3032580 (Figure 2C). The two transcripts share the same transcription initiation point, transcription endpoint, and exon number. The only difference between the two transcripts is that the fourth exon of NM_001165967 has five more amino acids than that of NM_3032580. By the end of April 2023, only six pathogenic variants of HES7 have been reported in public databases (Figure 2D, Table 1), including four missense variants, one frameshift variant, and one variant in the 3′-UTR. These variants were distributed dispersedly in the whole HES7 region. Including the patient in this article, a total of 13 cases have been reported with detailed clinical symptoms (Table 1). Overall, 84.6% (11/13) of patients showed short stature, 53.8% (7/13) had dextrocardia, 30.8% (4/13) presented vertebral artery hypoplasia, 83.3% (10/12) presented a restrictive ventilatory defect, and 84.6% (11/13) demonstrated chest deformities such as situs inversus, short thorax, rib deformity, and pectus excavatum. Furthermore, 23.1% (3/13) of patients presented abdomen situs inversus, 61.5% (8/13) exhibited spine abnormities such as hemivertebrae, butterfly vertebra, and abnormal odontoid process, and 50% (6/12) showed spina bifida occulta or myelomeningocele. 76.9% (10/13) of patients were alive when their case was reported (Table 1). The proband in this report presented short stature, short thorax, rib and spine deformities, which are the typical symptoms of SCDO4 (PP4).

To investigate the effect of variant c.226+1G>A on the HES7 gene, minigene plasmids were constructed and transfected into 293 T cells (Figure 3A). The wild-type DNA fragments were amplified using genomic DNA from healthy controls according to the manufacturer's instructions. Subsequently, mutant plasmids were constructed by PCR site-directed mutagenesis. Electrophoresis results revealed that the full-length amplification product was 295 bp in 293 T cells transfected with the wild-type minigene plasmids, while the amplification products were 600 bp in 293 T cells transfected with the mutant plasmids (Figure 3B). Sanger sequencing of gel-purified products demonstrated that the wild-type plasmid-transcribed mRNA sequences contained complete exon3 and exon4. However, the RT-PCR products of the mutant minigene plasmids also contained a complete intron3 sequence (Figure 3C), which proved that the variant HES7 c.226+1G>A resulted in the retention of intron3 in the mutant mRNA (PVS1_strong, Figure 3D). The retention of intron3 would lead to a frameshift of HES7 protein (HES7 p.Ala76AspfsTer168, NP_001159439.1). However, we could not confirm this effect in the patient cells because his parents refused to do so. According to ACMG guidelines, we confirmed the variant to be likely pathogenic (PVS1_strong + PM2_ supporting + PP4).