3.1 Clinical information
The patients were from a family in Quanzhou, Fujian province, China (Fig. 1a). The proband and her identical twin sister were born without complications by caesarean delivery at 38 weeks and three days, with Apgar scores of 10 at one, five, and ten min. Birth weight of the proband and her sister was 2,650 g and 2,300 g, respectively. Their mother had no fever, no infection, and no intrauterine hypoxia in late pregnancy. After birth, the proband suffered from shortness of breath, groaning, and cyanosis, and was transferred to the neonatal intensive care unit (NICU). The proband was treated with non-invasive positive pressure ventilation, invasive mechanical ventilation, and high frequency oscillation ventilation. They were also treated with a combination of PS supplementation, antibiotics, inhalation of nitric oxide (NO) to reduce persistent pulmonary hypertension, and the anti-inflammatory methylprednisolone. The echocardiogram did not reveal any structural abnormalities of the heart. The chest radiograph of the proband at birth showed reticular granular blur of both lungs (Fig 1b), and then progressed to bilateral "white lungs" (Fig 1c) at 6 days of admission. Despite the above treatments, the proband’s condition worsened rapidly, presenting with refractory dyspnoea, hypoxemia, persistent pulmonary hypertension, eventually leading to death at the age of 23 days. Her identical twin sister presented a similar profile and died of the same cause 23 days after birth.
Inquiring about family history revealed that the proband had an older sister born from her mother’s first pregnancy more than a year earlier. The older sister was delivered by vaginal delivery at 38 weeks and six days after an uncomplicated pregnancy with Apgar score of 10 at one, five and ten min and birth weight of 3,200 g. Her condition was similar to that of the proband. However, she had a relatively long clinical treatment process. Fifteen days after the birth, the patient's breathing improved after supplementation with PS, and she was out of oxygen therapy for eight days. However, she still showed symptoms of respiratory failure and she was unable to leave the oxygen support until her death. From 35 to 42 days after birth, she was continuously treated with methylprednisolone and azithromycin, but no significant improvement was observed. Airway CT reconstruction at 56 days showed right side tracheal bronchus and bronchopulmonary dysplasia (BPD) (Fig. 1d,e). She died 109 days after birth due to refractory dyspnoea and hypoxemia.
The clinical presentations and suspicious family history led us to hypothesise that there was a genetic cause. Peripheral blood samples were obtained from the infants and parents for WES and further genetic analysis. However, we were unable to obtain pathological specimens because the parents refused fiberoptic bronchoscopy, lung biopsy, and autopsy.
3.2 Sequencing and qPCR results: ABCA3 mutations identified by WES
Two ABCA3 variants were identified by WES of the proband’s DNA sample, namely a heterozygous deletion of exons 4-7 (Fig. 2a) and a novel heterozygous synonymous variant c.G873A (p.Lys291Lys) in exon 8 (Fig. 2d). The ABCA3 gene variants were subsequently determined to be in trans in the proband, with the unaffected father found to be carrying the heterozygous deletion of exon 4-7 (Fig. 2b) and the unaffected mother carrying the heterozygous synonymous variant c.G873A (p.Lys291Lys) (Fig. 2e).
The deletion of exon 4-7 of the ABCA3 gene was identified by bioinformatic analysis of single-gene copy number variants using NGS data. This loss-of-function variant was absent from the gnomAD and ExAC population database. It was interpreted as pathogenic according to the ACMG guidelines [10]. The c.G873A (p.Lys291Lys) novel variant was synonymous, however, it was also absent from gnomAD, HGMD, 1,000 Genomes and EXAC gene databases. Furthermore, it was consistently predicted to be a novel cryptic splice donor site by three different splice site algorithms (dbscSNV11_AdaBoost, dbscSNV11_RandomForest, and HSF) within exon 8 of the ABCA3 gene, which may lead to aberrant splicing of the pre-mRNA. Given the consistent in silico prediction of a splicing site, the absence in control populations, and presence of in trans with the pathogenic variant (deletion of exon 4-7) in two patients, both the proband and her identical twin sister, this variant was also interpreted as likely pathogenic according to the ACMG guidelines (PM2_supporing, PP3, PM3_strong). We further confirmed the ABCA3 mutations in DNA sample of the proband’s identical twin sister. The result of real-time quantitative PCR experiment revealed a heterozygous deletion mutation of exon 4-7 (Fig. 3a). The heterozygous c.G873A (p.Lys291Lys) mutation of the ABCA3 gene was identified by Sanger sequencing (Fig. 2g). These results were consistent with the proband. Therefore, the evidence above supported the diagnosis of autosomal recessive PS metabolism dysfunction caused by deficiency of ABCA3.
Table 2. Identified ABCA3 variants identified
Gene
|
Chromosome
|
Nucleotide Change
|
Protein Change
|
Variant type
|
In silico prediction
|
Genotype
|
Parent of origin
|
ABCA3
|
Chr16:2390573−2326806
|
NM_001089:(Chr16:2390573−2326806)X1
|
Exon 4-7 deletion
|
Deletion
|
LOF
|
Heterozygous
|
Paternal
|
ABCA3
|
Chr16:2369582
|
NM_001089:
exon8:c.873G>A
|
p.Lys291Lys
|
Synonymous
|
Cryptic splice site activation
|
Heterozygous
|
Maternal
|
Table 3. In silico prediction of the ABCA3 synonymous variant c.873G>A
Software
|
Score
|
Interpretation
|
AdaBoosta
|
1
|
splice-altering
|
Random Foresta
|
0.976
|
splice-altering
|
HSFb
|
-10.08
(87>76.92=>-11.59%)
|
Alteration of the WT donor site, most probably affecting splicing.
|
aAdaBoost and Random Forest: A score of more than 0.6 is predicted as splice-altering. [11]
bHSF: A score of less than 0 is determined as splice site broken. (https://hsf.genomnis.com/)