3. Results

3.1. Clinical Data

3.1.1. Family 1

The proband is a 7‐year‐old girl hospitalized for fatigue. Physical examination revealed that the child had shortness of breath and mild cyanosis at the fingertips (Figure 1A), with the peripheral oxygen saturation ranging from 83% to 85%. There was also significant growth and developmental delay: weight was 18 kg (−1.3 SD, 5th percentile); height was 116 cm (−1.4 SD, 10th percentile). Her complete blood count revealed a hemoglobin of 168 g/L, hematocrit of 0.488 L/L. Contrast‐enhanced pulmonary angiography (CTPA) suggested the presence of PAVMs (Figure 1B,C). The patient underwent successful percutaneous endovascular embolotherapy of the arteriovenous fistula (Figure 1D). Follow‐up at 3 months showed the child was catching up in growth and development: Weight was 18.6 kg (−1.2 SD, 10th percentile); height was 118 cm (−0.8 SD, 25th percentile).

Figure 1: Imaging of the proband in Family 1. (A) Clubbing of fingers, slight cyanosis at the fingertip. (B) Oblique sagittal MIP shows supplying pulmonary artery and draining vein. (C) Axial plane shows tortuous and enlarged vascular shadow in the posterior segment of the left lower lobe. (D) Arterial DSA angiography confirms it as a cystic PAVF.

Further history revealed a family history of recurrent nosebleeds in the mother, sister, and brother (Table 3).

 | Family 1 | Family 2 | Family 3 | Family 4
Proband | Mother | Sister | Brother | Proband | Mother | Proband | Mother | Proband | Mother | Aunt | Cousin
Nosebleed | + | + | + | + | + |  |  | + |  |  |  | +
Capillary dilation |  |  |  |  | + |  |  |  |  |  |  | 
Pulmonary arteriovenous malformation | + |  |  |  | + | + | + | + |  |  | + | 
Cerebral arteriovenous malformation |  |  |  |  | + |  | + |  |  |  | + | 
Hepatic arteriovenous malformation |  |  |  |  |  | + |  |  |  |  |  | 
Dyspnea | + |  |  |  |  |  |  |  |  |  |  | 
Cyanosis | + |  |  |  |  |  |  |  |  |  |  | 
Intracerebral hemorrhage |  |  |  |  |  |  |  |  | + |  |  | 
Cerebral infarction |  |  |  |  | + |  |  |  |  |  | + | 
Anemia |  |  |  |  |  |  | + |  |  |  |  | 
Erythrocytosis | + |  |  |  |  |  |  |  |  |  |  | 
Clubbing of fingers | + |  |  |  |  |  |  | + |  |  |  | 
Epilepsy |  |  |  |  |  |  |  |  | + |  |  | 
Spontaneous abortion |  |  |  |  |  |  |  | + |  |  |  | 
HOHF |  |  |  |  |  |  |  |  |  |  |  | 
Growth and development delay | + |  |  |  |  |  | + |  | + |  |  | 

3.1.2. Family 2

The proband is a 13‐year‐old boy who has a history of recurrent spontaneous epistaxis. Nasal endoscopy revealed telangiectases on the left nasal septum. He was admitted for transient syncope accompanied by facial asymmetry. Physical examination on admission revealed left facial palsy (nasolabial flattening with rightward oral commissure deviation), left hemiparesis (upper limb 0/5, lower limb 4–5/5 by MRC scale), left‐sided hypesthesia, and preserved right‐sided strength (5/5). Cranial magnetic resonance imaging (MRI) demonstrated a cerebral infarction in the right hemisphere. Chest CT revealed a large tubular shadow and nodules in the right middle lobe, with smaller vascular dilatations in the right lower lobe. CTPA showed an arteriovenous malformation in the right middle lobe and bilateral lower lobe infections (Figure 2A,B). The patient underwent cerebral angiography and intracranial mechanical thrombectomy, with intraoperative diagnosis of right middle cerebral artery (MCA) occlusion (Figure 2C). Posttreatment, he reported mild weakness during ambulation, with near‐complete recovery of left limb muscle strength. At the 1‐month and 3‐year follow‐ups, he underwent partial percutaneous embolotherapy of the right pulmonary artery.

Figure 2: Imaging of Family 2. (A) Proband: Axial plane shows a large ductal shadow and nodular shadow in the right middle lobe of the lung. (B) Proband: VR shows an aneurysm sac, supplying pulmonary artery, and draining vein. (C) Proband: Cranial DWI shows high signal in the right corona radiata. (D) The proband′s mother: Axial plane shows tortuous and enlarged vascular shadow in the right middle lobe of the lung.

Further history revealed a family history of PAVM in the mother (Table 3). Chest CT indicated dense linear opacities and abnormal vasculature in the anterior segment of the right upper lobe (Figure 2D). Abdominal ultrasound revealed a hyperechoic mass in the right liver, suggestive of hemangioma, with concomitant calcifications. The mother also underwent percutaneous embolotherapy. The father has no clinical manifestations.

3.1.3. Family 3

The proband is a 2.5‐year‐old girl who was admitted for evaluation of growth and developmental delay. At 21 weeks of gestation, fetal ultrasound demonstrated a 1‐week growth discrepancy. She was delivered at the 39th week weighing 2200 g (<−2 SD, small for gestational age infant), accompanied by a thin umbilical cord and small placenta. She achieved gross motor milestones as follows: head control at 4 months, rolling over at 5 months, independent sitting at 8–9 months, crawling at 10 months, and independent walking at 1.5 years. Language development was delayed, with no meaningful vocalizations before 1 year, and only 2–3 word combinations at 2.5 years. There was significant growth and developmental delay: Weight was 10.8 kg (<−2 SD, < 3rd percentile) and height was 87 cm (<−2 SD, < 3rd percentile). CTPA indicated a suspicious local vascular shunt in the right middle lobe with distal inflammatory lesions (Figure 3A). Cerebral magnetic resonance angiography (MRA) revealed that the A3–4 segments of the left anterior cerebral artery appeared slender, with mild focal stenosis in the M2 segment of the right MCA, and a fetal‐type left posterior cerebral artery (Figure 3B). Abdominal ultrasound revealed splenomegaly, whereas cardiac echocardiography showed no abnormalities.

Figure 3: Imaging of Family 3. (A) Proband: Oblique coronal MIP shows suspected arteriovenous malformation in the right middle lobe of the lung. (B) Proband: MRA indicates that the A3–4 segment of the left anterior cerebral artery appears slender, whereas the M2 segment of the right middle cerebral artery shows localized mild stenosis. (C) Proband′s mother: Sagittal plane shows an aneurysm sac in the right lung, with supplying pulmonary artery and draining vein. (D) Proband′s mother: Postpulmonary embolism surgery: MIP shows a dense shadow in part of the arterial branches in the right lower lobe of the lung, with strip‐shaped filling defects in the distal areas.

Further history revealed that the mother had experienced two spontaneous abortions and recurrent nosebleeds (Table 3). Physical examination showed finger clubbing. CTPA demonstrated arteriovenous malformations in both lower lung lobes (Figure 3C,D). She also underwent percutaneous embolotherapy. The father has no clinical manifestations.

3.1.4. Family 4

The proband is an 11‐month‐old boy who was delivered vaginally at 37+6 weeks. At birth, the infant had a weight of 2470 g (−1 SD). At 34 weeks of gestation, the mother noticed abnormal fetal movements. Prenatal MRI demonstrated extensive subdural hematomas in the left cerebral hemisphere and left posterior fossa, with bilateral cerebral hemispheric encephalomalacia. The infant presented with dysmorphic facial features, followed by a weakness in sucking and necessitating nasal feeding. Subsequent follow‐ups revealed significant developmental delay and cognitive impairment.

The proband′s aunt was hospitalized for left‐sided limb weakness. CTPA indicated arteriovenous malformations in the lateral segment of the right middle lobe and the basal segment of the right lower lobe, with filling defects in the pulmonary veins and branches of the right middle lobe, indicating thrombosis (Figure 4A,B). Cerebral angiography revealed a fetal‐type left posterior cerebral artery and mild hypoplasia of the right MCA (M2–M3 segments) (Figure 4C). Cranial MRI demonstrated an acute infarction in the right fronto‐parieto‐insular periventricular basal ganglia region (Figure 4D). The aunt underwent cerebral angiography with intracranial mechanical thrombectomy and embolotherapy of the pulmonary artery.

Figure 4: Images of the proband′s aunt in Family 4. (A) Sagittal plane MIP shows communication between the posterior basal segment of the right lower lobe and the right inferior pulmonary vein. (B) Axial plane MIP shows a vascular‐like enhancing nodule in the lateral segment of the right middle lobe. (C) Right intracranial artery DSA shows partial occlusion of the M2 segment of the right middle cerebral artery with formation of abnormal collateral vessels. (D) Head MRI, T2‐FLAIR shows abnormal signal in the right basal ganglia region.

The maternal grandfather died prematurely due to cerebral infarction (no WES performed). The aunt′s son has recurrent nosebleeds, whereas her daughter shows no clinical manifestations (Table 3). The proband′s father has no clinical manifestations.

3.2. Variant Detection

3.2.1. Family 1

A heterozygous deletion variant in exon 5 of ENG (c.613del; NM_000118.3) was identified in the proband (II‐1; Figure 5A) by PCR‐based Sanger sequencing (Figure 5B). DNA analysis revealed that the mother, brother, and sister (I‐2, II‐2, and II‐3) all carry the heterozygous variant.

Figure 5: Pedigree and the sequencing result of the studied families. □ represents the normal male; ○ represents the normal female; ■ represents the affected male; ● represents the affected female. The black arrow indicates the proband. The red arrow symbol indicates the mutation site. (A, B) Family 1. (C, D) Family 2. (E, F) Family 3. (G, H) Family 4.

3.2.2. Family 2

A heterozygous point variant (c.1428 + 2 T > C; NM_000118) in exon 11 of ENG was identified in the proband (II‐1; Figure 5C) by PCR‐based Sanger sequencing (Figure 5D). DNA analysis confirmed that the mother (I‐2) also carries the heterozygous variant.

3.2.3. Family 3

A heterozygous duplication variant (c.1498dup; NM_001114753.2) in exon 12 of ENG was identified in the proband (III‐1; Figure 5E) by PCR‐based Sanger sequencing (Figure 5F). DNA analysis revealed that the mother (II‐2) carries the heterozygous variant, indicating that the variant is a de novo variant originating from the mother (II‐2).

3.2.4. Family 4

A heterozygous deletion variant (c.322del; NM_000118.4) in exon 3 of ENG was identified in the proband (III‐1; Figure 5G) by PCR‐based Sanger sequencing (Figure 5H). DNA analysis showed that the mother, aunt, and cousin (II‐2, II‐3, and III‐2) all carry the heterozygous variant.

3.3. In Silico Assay

In the GnomAD database, the ENG variants (c.1428 + 2 T > C, c.1498dup, and c.322del) are not included, indicating that these are novel variants (Table 4). We conducted functional prediction for the ENG variants by utilizing the RDDC platform. The assessment revealed the ENG variant (c.1428 + 2 T > C) may affect mRNA splicing (Figure 6A,B). For c.1428 + 2 T > C, the splice AI algorithm returned a high value (Δ Score = 0.99). This value, which was above the high precision threshold (Δ Score ≥ 0.8), was used to detect higher sensitivity of splice change variants (Table 5).

Family | Variant ID | HGVS Consequence | VEP Annotation
Family 1 (c.613del: p.Arg205Glyfs∗17) | 9‐127825770‐C‐T | p.Arg205Arg | Synonymous
9‐127825770‐C‐A | p.Arg205Leu | Missense
9‐127825770‐C‐T | p.Arg205Gln | Missense
9‐127825770‐C‐C | p.Arg205Gly | Missense
9‐127825770‐C‐A | p.Arg205Trg | Missense
9‐127825770‐C‐T | p.Arg205Arg | Synonymous

Family 2 (c.1428 + 2 T > C) | 9‐127818707‐G‐A | c.1428 + 9C > T | Intron
9‐127818709‐G‐T | c.1428 + 7G > A | Splice region
9‐127818711‐G‐C | c.1428 + 5C > G | Intron
9‐127818711‐G‐T | c.1428 + 5C > A | Intron
9‐127818718‐G‐T | p.Gln476Lys | Missense

Family 3 (c.1498dup: p.Glu500Glyfs∗28) | 9‐127818305‐C‐G | p.Gly501Arg | Missense
9‐127818305‐C‐T | p.Glu500Glu | Synonymous
9‐127818305‐C‐G | p.Glu500Gln | Missense
9‐127818305‐C‐T | p.Glu500Lys | Missense
9‐127818305‐A‐G | p.Pro499Pro | Synonymous

Family 4 (c.322del: p.His108Ilefs∗55) | 9‐127829722‐G‐A | p.Leu109Phe | Missense
9‐127829724‐T‐C | p.His108Arg | Missense
9‐127829725‐G‐A | p.His108Tyr | Missense
9‐127829725‐G‐T | p.His108Asn | Missense
9‐127829726‐C‐A | p.Leu107Leu | Synonymous

Figure 6: Family 2′s results of in silico assay. (A, B) Patterns of RNA splicing prediction. (A) Deleting 117 bp, exon skipping. (B) Insert 338 bp, frameshift mutation, and premature termination.

Acceptor loss | Donor loss | Acceptor gain | Donor gain
0.42 | 0.99 | 0.00 | 0.16
118 bp | 2 bp | 43 bp | −205 bp

3.4. Minigene Assay

In vitro transcriptional assays were performed to analyze the effect of intron donor locus variation on ENG mRNA splicing. The pcDNA3.1‐ENG small gene was 1541 bp long and covered DNA regions, including Exon10 (3 bp), Intron10 (789 bp), Exon11 (117 bp), Intron 11 (338 bp), and Exon12 (25 bp, Figure 7A,B). A 533‐bp complete reverse transcription PCR product, which includes a 119‐bp segment of plasmid DNA and a 414‐bp segment of the target gene, was observed in HEK293T and HeLa cells after transfection with the pcDNA3.1‐ENG minigene construct (Figure 7C). Sanger sequencing verified the 414‐bp fragment as normal ENG mRNA with exons 10–12 spliced and introns 10–11 excluded (Figure 7D). The pcDNA3.1‐ENG‐mut minigene containing the c.1428 + 2 T > C variant was transfected into the HeLa and HEK293T cell lines. Conversely, WT ENG mRNA was absent in the mutant (MUT) swim channel, a smaller band (416 bp, Figure 7C). In the MUT lanes, there was an observation of an ENG aberrantly spliced transcript manifesting as a shorter band of 416 bp, encompassing solely exons 10 and 12, with exon 11 completely skipped. Figure 7D displays the Sanger DNA chromatogram of this misspliced ENG transcript. The outcomes obtained from the experiment performed using the pcMINI‐C vector aligned with prior findings, demonstrating the skipping of exon 11. Analysis of the complete sequence of the improperly spliced transcript, as determined by small gene analysis, indicated that this variant caused complete skipping of exon 11 without altering the subsequent reading frame. Due to the loss of 117 bp of nucleotides (c.1312_1428del), it resulted in an internal deletion of 39 amino acids (p.[Lys438_Gln476del]) in the protein, potentially leading to a shorter protein. These findings align with predictions from in silico assays, and the RNA splicing pattern corresponds to the outlined splicing pattern I (Figure 6A).

Figure 7: Functional analysis of the effect of the ENG c.1428 + 2 T > C variant on mRNA splicing. (A, B) Construction strategy of pcDNA3.1 minigene vector, ∗ representative of the variant site. (C) Gel electrophoresis of reverse transcription polymerase chain reaction products displayed a single band a (estimated 533 bp) from the wild type (wt) and a smaller band b (estimated 416 bp) in the mutant (mut) type. (D) Illustration of the sequencing of band a (wild type in HEK293T and HeLa cells) and band b (variant c.1428 + 2 T > C in HEK293T and HeLa cells) products lead to a shorter transcript with deletion of exon 11 including 117 bp.

3.5. Western Blot Analysis

Western blot results revealed that the MUT protein exhibited a shorter form (smaller molecular weight) compared with the WT (Figure 8). Both WT and MUT lanes showed two bands: the upper band (glycosylated form) and the lower band (less/nonglycosylated form). Bioinformatics analysis using NetNGlyc predicted that Asn444 (WT sequence: Asn444‐Met445‐Asp446) and Asn465 (WT sequence: Asn465‐Thr466‐Ile467), located within the Lys438‐Gln476 region, are potential glycosylation sites. The minigene assay demonstrated that the c.1428 + 2 T > C mutation disrupted normal splicing, leading to exon 11 skipping (117‐bp deletion) and resulting in the p.Lys438_Gln476del alteration at the protein level. Therefore, the two predicted glycosylation sites at Asn444 and Asn465 are likely deleted in the MUT protein. Western blot results demonstrated that the MUT lane exhibited significantly enhanced accumulation of the lower band compared with the WT lane. This is likely attributable to the loss of N‐glycosylation sites at Asn444 and Asn465 caused by the p.Lys438_Gln476 deletion, which consequently led to elevated expression of the less/nonglycosylated protein. These findings suggest that the ENG c.1428 + 2 T > C variant causes exon 11 deletion (117 bp), which corresponds to the p.Lys438_Gln476del mutation and results in a shorter protein (618 aa) and impaired glycosylation.

Figure 8: Western blot results on total proteins extracted from transfected 293T cells with the pLV3‐FLAG vector and the p.Lys438_Gln476del construct. Two bands of different molecular weights are observed for ENG proteins likely corresponding to more glycosylated (upper band) and less/nonglycosylated (lower band) ENG monomers. GAPDH corresponds to the used antibody for the reference proteins. kDa, kilodalton; M, protein ladder; WT, wild type; CT, negative control corresponding to pLV3‐empty vector.

3.6. Three‐Dimensional Structure of the Protein

Compared with the WT sequence, the variant protein exhibited an altered amino acid sequence, leading to a shorter protein variant with a deletion of 39 native amino acids p.(Lys438_Gln476del). The variants in the four families differentially affect the functional domains of ENG (Figure 9).

Figure 9: The domain of ENG proteins. Structural representation of endoglin. OR: an NH2‐terminal orphan domain. ZP, zona pellucida domain; TM, the transmembrane domain. The cytoplasmic (CYT) domain can be phosphorylated (P) at Ser/Thr/Tyr residues. The scheme is not to scale.