Biallelic ASCC1 variants including a novel intronic variant result in expanded phenotypic spectrum of spinal muscular atrophy with congenital bone fractures 2 (SMABF2)

Abstract

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2), a type of arthrogryposis multiplex congenita (AMC), is characterized by congenital joint contractures, prenatal fractures of long bones, and respiratory distress and results from biallelic variants in ASCC1. Here, we describe an infant with severe, diffuse hypotonia, congenital contractures, and pulmonary hypoplasia characteristic of SMABF2, with the unique features of cleft palate, small spleen, transverse liver, and pulmonary thromboemboli with chondroid appearance. This infant also had impaired coagulation with diffuse petechiae and ecchymoses which has only been reported in one other infant with AMC. Using trio whole genome sequencing, our proband was identified to have biallelic variants in ASCC1. Using deep next generation sequencing of parental cDNA, we characterized alteration of splicing encoded by the novel, maternally inherited ASCC1 variant (c.297–8 T > G) which provides a mechanism for functional pathogenicity. The paternally inherited ASCC1 variant is a rare nonsense variant (c.466C > T; p.Arg156*) that has been previously identified in one other infant with AMC. This report extends the phenotypic characteristics of ASCC1-associated AMC (SMABF2) and describes a novel intronic variant that partially disrupts RNA splicing.

1 |. INTRODUCTION

Arthrogryposis multiplex congenita (AMC) is one of three subtypes of arthrogryposis (amyoplasia, distal arthrogryposis, and AMC) that are differentiated by location of joint contractures and the presence of underlying central nervous system or neuromuscular etiologies (Bamshad et al., 2009). Arthrogryposis is associated with decreased fetal movement, and affected infants have increased connective tissue and atrophy of muscles around the joints as well as skin dimpling (Hall, 2014). The pathogenesis of arthrogryposis is diverse and includes primary neuropathic, myopathic, neuromuscular junction, connective tissue, or metabolic disorders, as well as secondary etiologies including in utero space limitation, maternal illness or environmental exposures, or placental vascular disorders (Hall, 2014).

Biallelic variants in ASCC1 (MIM# 616867) have been recently identified as the underlying cause of a rare form of AMC known as spinal muscular atrophy with congenital bone fractures 2 (SMABF2) (Bamshad et al., 2009; Giuffrida et al., 2020; Knierim et al., 2016; Lu et al., 2020; Oliveira et al., 2017). ASCC1 encodes a subunit of the tetrameric ASC-1 transcriptional co-integrator complex that includes ASCC1, ASCC2, ASCC3, and TRIP4 (Jung et al., 2002). The ASC-1 transcriptional co-integrator complex regulates transcription of genes involved in neurogenesis and bone formation and participates in transcriptional co-activation and RNA processing (Knierim et al., 2016). ASCC1 and TRIP4 are highly expressed in dorsal root ganglia, paraspinal sympathetic, and trigeminal ganglia, as well as the thyroid and submandibular glands in murine embryos (Knierim et al., 2016).

Here, we report a novel intronic variant in ASCC1 that partially disrupts RNA splicing in trans with a rare nonsense variant identified in an infant with AMC, whose phenotype included profound, diffuse hypotonia, cleft palate, small spleen, transverse liver, pulmonary hypoplasia, and cardiac dysfunction. This report confirms and extends the SMABF2 phenotypic characteristics associated with biallelic variants in ASCC1, describes a novel intronic variant that partially disrupts RNA splicing, and suggests that more than 25% of wild-type ASCC1 transcript is required for normal neuromuscular development.

2 |. METHODS

2.1 |. Editorial policies and ethical considerations

The proband was enrolled as a participant in a prospective multicenter, time-delayed, clinical trial to investigate the impact of WGS on acute care infants (ClinicalTrial.gov NCT03290469). This study was approved by the Washington University School of Medicine Human Research Protection Office.

2.2 |. Patient consent

Written informed consent was obtained from the parents of the affected infant.

2.3 |. Subject

A female infant was born by cesarean section at 36 weeks’ estimated gestation to a 25-year-old gravida 2 para 2 mother, whose pregnancy was complicated by fetal arthrogryposis with decreased fetal movements, contractures, increased proportion of subcutaneous fat to muscle, breech presentation, progressive polyhydramnios treated with amnioreduction, and preterm spontaneous rupture of membranes. The family history was negative for neuromuscular disorders but notable for a full female sibling and a paternal female half-sibling with unilateral developmental hip dysplasia at birth and a maternal great-grandfather with clubfoot.

At delivery, the infant required intubation for insufficient respiratory effort. Her birth weight was 3.1 kg (75th percentile), length 49 cm (75th percentile), and head circumference 35 cm (95th percentile) (Fenton & Kim, 2013). Her physical examination was notable for low-set and mildly posteriorly rotated ears, flattened and low nasal bridge, cleft palate, short neck with mildly increased skin folds, anteriorly displaced anus, arachnodactyly, equinovarus deformity bilaterally, and bilateral joint contractures of her ankles, knees, hips, wrists, fingers, and elbows. She had profound, diffuse axial and appendicular hypotonia and areflexia, minimal spontaneous movements, and bilateral dorsiflexion of ankles to noxious stimuli. She grimaced to bright light but had no spontaneous eye opening. She had diffuse petechiae most pronounced on her head and upper trunk, ecchymoses of her right upper extremity and both ears, and prolonged capillary refill, which improved after volume resuscitation. Her laboratory studies were notable for prolonged prothrombin time (18.6 s, normal 12.0–16.1) and normal international normalized ratio.

Chest and abdominal radiographs were notable for small lung volumes consistent with pulmonary hypoplasia, diffuse interstitial opacities consistent with hyaline membrane disease, and markedly thin ribs, clavicles, and long bones with poor differentiation between muscles and extremity fat (Figure 1(a)). The inferior portion of the body of the ilium (pelvis) appeared narrow. These findings were consistent with muscle weakness and contractures, although direct and intrinsic effects on bone remodeling could not be excluded. A very small corner fracture of the proximal right humerus (white arrow) was noted without evidence of other fractures. Transthoracic echocardiogram demonstrated a dilated and hypertrophied right ventricle with severely decreased function, moderately to severely decreased left ventricular function, and flattened interventricular septum with decreased motion. Abdominal ultrasound was notable for a small left-sided spleen (6.7 standard deviations below the mean), transversely positioned liver, small portal vein, bilateral slightly enlarged kidneys, and thickening of the lining of the left ureter. Ultrasound of her muscles showed subcutaneous soft tissue edema in the right arm and leg and increased echointensity of the right biceps brachii. Electromyography showed severe myopathy with absent compound muscle action potentials and myopathic motor units seen in the tibialis anterior most consistent with an irritable myopathy with evidence of fibrillation potentials (Lyu, Cornblath, & Chaudhry, 1999).

FIGURE 1.

Chest and abdomen radiograph and lung histology from an infant with biallelic ASCC1 variants. (a) Chest and abdominal radiograph shows small lung volumes consistent with pulmonary hypoplasia and diffuse interstitial opacities consistent with hyaline membrane disease. The ribs, clavicles, and long bones are markedly thin. A tiny corner fracture (white arrow) of the proximal right humerus is noted. (b) Hematoxylin and eosin (H&E) stain demonstrates lung parenchyma with congested, but relatively normal alveolar morphology. There are numerous intra-alveolar aspirated squamous cells (white arrows) and patchy intra-alveolar neutrophils (black arrowheads). Arterial medial wall thickening is present (black arrows). (c) Pulmonary artery with endothelialized thromboembolus consistent with chondroid tissue (arrow) (H&E)

The proband’s condition deteriorated despite mechanical ventilation, surfactant administration, empiric antibiotics, and ionotropic support with dobutamine. She required thoracostomy tube placement for a right-sided pneumothorax. Due to her poor prognosis for recovery and after a thorough discussion with the care team and her family, she was compassionately extubated and died on day of life 3.

Autopsy confirmed the congenital anomalies of the spleen, liver, and kidneys as described above. Also noted were histologic features of hepatic and renal congestion and pseudofollicular changes in the left adrenal cortex consistent with perinatal stress and adrenal congestion. Gross examination of her lungs revealed normal lobation, congestion, and uneven expansion of the lung parenchyma. Pulmonary hypoplasia was noted based on lung/body weight ratio of 1.16% (less than 10th percentile). Histologically, her lungs demonstrated normal alveolar morphology for gestational age (Figure 1(b)) but had focal approximation of the terminal airways to the pleura consistent with pulmonary hypoplasia with decreased numbers of alveoli. Numerous intra-alveolar desquamated squamous cells and meconium were present consistent with aspiration. Also noted were changes of pulmonary hypertension with medial wall thickening of small arteries and arterioles greater than expected in the first week of life. Scattered, focally numerous paucicellular thromboemboli were noted in arteries and arterioles, some with surface endothelialization (Figure 1(c)), strongly suggestive of embolization in utero. The thromboemboli had a chondroid appearance, with morphology and staining similar to cartilage with periodic acid-Schiff diastase and pentachrome stains (special stains not shown). Nerve and quadriceps muscle pathology showed intramuscular nerves with a reduced number of myelinated axons, normal to reduced size of neuromuscular junctions, diffusely small muscle fibers, disorganized myofibrils, and enlarged Z-bands (Figure 2).

FIGURE 2.

Nerve and muscle from an infant with biallelic ASCC1 variants. While sural nerve demonstrated a normal number of myelinated and nonmyelinated axons (A, red = myelin protein zero and green = neurofilament, B = neurofilament plus Gomori trichrome [GT]), there were multiple intramuscular nerves (arrows in C and D) with a reduced number of myelinated axons on Verhoeff-Von Gieson staining (C), and neuromuscular junctions showed normal staining intensity but varied from normal (black circle) to reduced (white circle) in size on nonspecific esterase (d). H&E staining of quadriceps muscle demonstrated diffuse muscle fiber smallness (e) with marked type 2 predominance and rare scattered, often larger type 1 fibers (arrows) on ATPase pH 4.3 (f). While there were no clear morphologic abnormalities or rods on GT (g), ultrastructure analysis of muscle showed disorganized myofibrils and a few instances of Z-band enlargement (arrows, h). Black scale bar (panel e) = 50 μM in b, e, f; 20 μM in c, d, g; and 800 nM in h

2.4 |. Whole genome sequencing

DNA was extracted from blood samples from the proband and her unaffected parents. Trio WGS was performed at the Illumina Clinical Services Laboratory using the TruGenome Undiagnosed Disease test. Variants were aligned to the Human Reference Genome (v. 37.1). Over 99% of the genome was covered by 10x coverage or more (mean greater than 30x coverage) and at least 97% of the genome passed all quality filters. A list of 3140 candidate genes was generated using the Online Mendelian Inheritance in Man (OMIM) database to identify genes associated with key elements of the proband’s phenotype.

2.5 |. Functional analysis

RNA was extracted from parental blood (PaxGene RNA tubes and blood RNA kit [Qiagen, Germantown, MD]) and used to synthesize cDNA with SuperScript III (Invitrogen, Carlsbad, CA). A sample for RNA extraction from the proband was not available. To assess RNA splicing, we designed PCR primers that would specifically amplify cDNA product from exons 2 through 7 of the ASCC1 gene. We gel purified PCR bands and used Sanger sequencing of the individual PCR products to determine the splicing effects of the c.297–8 T > G variant. To characterize RNA splicing more specifically, we ligated Illumina adaptors to our PCR products and performed deep next generation sequencing on an Illumina Miseq instrument (Illumina, Carlsbad, CA).

3 |. RESULTS

Trio WGS results demonstrated that the proband carried biallelic variants in ASCC1 (NM_001198799.2): a paternally inherited nonsense variant c.466C > T;p.Arg156* (minor allele frequency 0.000035 in the GnomAd database; gnomad.broadinstitute.org, v2.1.1, accessed March 2021) (Karczewski et al., 2019) and a maternally inherited, novel intronic variant c.297–8 T > G predicted to alter splicing (Alamut Software, Sofia Genetics, Boston, MA). Splicing predictions suggested alteration of the canonical 3′ (or acceptor) splice site of exon 5 of ASCC1 (NM_001198799.2) as well as creation of a new 3′ (or acceptor) splice site 7 base pairs (bp) upstream of the canonical splice site (Figure S1A). Both alternate transcripts are predicted to produce premature termination codons and likely nonsense mediated decay. Exon 5 is 98 bp, so skipping is predicted to result in a frameshift and stop codon 18 amino acids downstream. The upstream cryptic splice site would add 7 bp to the beginning of exon 5, also causing a frameshift and a premature termination codon 5 amino acids downstream.

Using primers that amplified exons 2 through 7, we observed a higher molecular weight cDNA in both parents, which upon gel extraction and Sanger sequencing was found to be a 373 bp product which corresponds to canonical (wild type) splicing (Figure S1B). In addition, sequencing of the maternal higher molecular weight cDNA also revealed a 380 bp product, which corresponds to aberrant splicing from the predicted alternate upstream splice site. We also observed a faint lower molecular weight cDNA band in the maternal sample, which was identified to be a 290 bp product that corresponds to skipping of exon 5. These results suggest that the c.297–8 T > G variant causes leaky splicing, producing transcripts spliced from both the canonical splice site and the alternate splice site created by this variant, as well as exon 5 skipping. Deep next generation sequencing of the maternal cDNA-derived PCR product (577x mean coverage) confirmed our observations by demonstrating the presence of transcripts from the canonical splice site (75.2%, assuming ~50% is derived from the wild-type allele), from the alternate upstream splice site (13.3%), and with exon 5 skipping (11.5%) (Figure S1C).

4 |. DISCUSSION

Our proband was found to have biallelic variants in ASCC1 and her clinical phenotype is consistent with SMABF2 as reported in 11 other infants (Table 1). (Bohm et al., 2019; Giuffrida et al., 2020; Knierim et al., 2016; Lu et al., 2020; Oliveira et al., 2017) The findings in our proband confirm the association of biallelic variants in ASCC1 with SMABF2 characterized by severe diffuse hypotonia and congenital fractures and extends the phenotypic spectrum with her unique features of cleft palate, small spleen, transverse liver, cardiac abnormalities, and pulmonary vasculature with thromboemboli with chondroid appearance. The muscle histology findings of fiber atrophy, myofibrillar disorganization, and enlarged Z-bands are typical for SMABF2. The skin findings of petechiae and ecchymoses, possibly linked to abnormal coagulation studies, have been described in one other infant with biallelic ASCC1 variants (Lu et al., 2020). The chondroid thromboemboli identified in the small pulmonary arteries is a unique finding and may be related to congenital fractures (Davignon et al., 2016). The possibility of other genetic etiologies for her additional unique clinical features cannot be excluded. The c.466C > T;p.Arg156* variant has been identified in one other infant (Bohm et al., 2019 (S6, Table 1)) with SMABF2 while the intronic variant is novel. Of the 11 infants previously reported with SMABF2 due to biallelic ASCC1 variants, 9 were homozygous or compound heterozygous for loss-of-function (frameshift, nonsense) variants and 2 were compound heterozygous for nonsense variants in trans with a deletion of exon 5 or a 64 kb microdeletion (Table 1). We confirmed that the novel, maternally inherited c.297–8 T > G intronic variant identified in this infant partially disrupted RNA splicing. While tissue-specific RNA isoforms in neurons may differ from the peripheral blood, quantitation of transcript expression in parental peripheral blood suggests that more than 25% of wild-type ASCC1 transcript is required for normal neuromuscular development. In this case, we speculate that the proband had ~25% wild-type transcript and manifested AMC, whereas the mother has ~75% wild-type transcript (50% from the wild-type allele and 25% from the maternal splice variant) and the father has 50% wild-type transcript due to the paternal nonsense variant. These data suggest that more than 25% wild-type transcript is required for normal neuromuscular development, but 50% wild-type transcript is sufficient as indicated by the father’s results.

TABLE 1.

Reported infants with arthrogryposis multiplex congenital (AMC) due to biallelic ASCC1 variants

Subjects (S)	S1 (Proband in this report)	S2 (Knierim et al., 2016)	S3 (Knierim et al., 2016)	S4 (Bohm et al., 2019)	S5 (Bohm et al., 2019)	S6 (Bohm et al., 2019)	S7 (Bohm et al., 2019)	S8 (Bohm et al., 2019)	S9 (Bohm et al., 2019)	S10 (Oliveira et al., 2017)	S11 (Lu et al., 2020)	S12 (Giuffrida et al., 2020)	Prevalence of clinical features
ASCC1 Variants	c.466C > T; p.Arg156*/c.297–8 T > G	c.157dupG; p.Glu53fs19* (homozygous)	c.157dupG; p.Glu53fs19* (homozygous)	c.157dupG; p.Glu53fs19* (homozygous)	c.157dupG; p.Glu53fs19* (homozygous)	c.157dupG; p.Glu53fs19*/c.466C > T; p.Arg156* (compound heterozygous)	c.412C > T; p.Arg138* (homozygous)	c.412C > T; p.Arg138* (homozygous)	c.412C > T; p.Arg138* (homozygous)	c.157dupG; p.Glu53fsl9* (homozygous)	c.932C > G; p.Ser311*/Exon 5 deletion (compound heterozygous)	c.1027C > T; p.Arg343*/10q22.1 microdeletion 64 kb (compound heterozygous)
Sex	Female	Female	Female	Male	Male	Female	Male	Female	N.R.	Female	Male	Female
Family History	Negative	Affected sibling (S3)	Affected sibling (S2)	Affected sibling (S5)	Affected sibling (S4)	Negative	Affected siblings (S8,S9)	Affected siblings (S7,S9)	Affected siblings (S7,S8)	One similarly affected sibling and one sibling intrauterine fetal death	Negative	Negative
Ethnicity	European	Turkish	Turkish	Tunisian	Tunisian	Moroccan	Sri Lankan	Sri Lankan	Sri Lankan	Portuguese	Chinese	European
Consanguinity	No	Yes	Yes	Yes	Yes	No	Yes	Yes	Yes	No	No	N.R.
Pregnancy/delivery	36 weeks	<37 weeks	<37 weeks	N.R.	N.R.	Normal pregnancy	N.R.	N.R.	Pregnancy terminated	N.R.	Normal pregnancy	Cesarean section at 34 weeks due to fetal distress
Status	Deceased 3 days of life	N.R.	N.R.	Deceased shortly after birth	Deceased shortly after birth	Deceased at 13 days of life	Deceased shortly after birth	Deceased shortly after birth	Pregnancy terminated	N.R.	Deceased at two days of life	Stillbirth
Hypotonia	Diffuse axial and appendicular hypotonia, areflexia	Generalized severe muscle atrophy, absent deep tendon reflexes	Generalized severe muscle atrophy, absent deep tendon reflexes	Hypotonia, fetal hypokinesia	Hypotonia, fetal hypokinesia	Severe hypotonia	Profound hypotonia, fetal akinesia, minimal respiratory effort	Profound hypotonia, fetal akinesia, minimal respiratory effort	Fetal akinesia	Severe neonatal hypotonia, lack of spontaneous movements	Reduced fetal movements, hypotonia, muscle atrophy	Bilateral muscular atrophy	12/12 (100%)
Skeletal abnormalities	Joint contractures, arachnodactyly, equinovarus deformity bilaterally	Distal and proximal joint contractures	Distal and proximal joint contractures	Arthrogryposis	Arthrogryposis	Arthrogryposis	Arthrogryposis	Arthrogryposis		Arthrogryposis, thin gracile ribs	Joint contractures, talipes equinovarus	Hyperextension of both lower limbs, bilateral club foot, thin and gracile ribs and clavicles, camptodactyly of hands	11/12 (92%)
Congenital bone fractures	Corner fracture of right proximal humerus	Multiple prenatal fractures of long bones	Multiple prenatal fractures of long bones	Humeral fractures	Humeral fractures	Femoral and humeral fractures	Multiple bone fractures	Multiple bone fractures		Bilateral femoral fractures	Humeral fractures	Corner metaphyseal fractures of distal right femur, bilateral humeral shaft fractures	11/12 (92%)
Dysmorphic features	Low-set and mildly posteriorly rotated ears, flattened and low nasal bridge, short neck with mildly increased skin tissue folds, anteriorly displaced anus									High arched palate, tent-shaped mouth and microretrognathia	Cryptorchidism, posteriorly rotated and low-set ears	Nasal bridge depression, narrowing of bregmatic fontanelle, large nasal bone, advanced maturation of teeth	4/12 (33%)
Neonatal pulmonary findings	Pulmonary hypoplasia	Pulmonary hypoplasia	Pulmonary hypoplasia								Pneumonia		4/12 (33%)
Neurologic findings		Simplified cortical gyration on brain MRI	Simplified cortical gyration on brain MRI								Mild bilateral lateral ventricle dilation		3/12 (25%)
Skin findings	Ecchymoses and petechiae										Ecchymoses		2/12 (17%)
Cardiac	Dilated and hypertrophied right ventricle with severely decreased function, moderate to severe decreased left ventricular function, and flattened interventricular septum with decreased motion												1/12 (8%)
Other	Transverse liver, prolonged prothrombin time, cleft palate, small spleen

Abbreviation: N.R, not reported.

Biallelic pathogenic variants in both ASCC1 and TRIP4 (MIM# 604501), which encode subunits of the tetrameric ASC-1 transcriptional co-integrator complex, have been identified among infants with AMC and respiratory failure. Variants in ASCC2 (MIM# 614216) and ASCC3 (MIM# 614217) have not been associated with human disease. In RNA expression studies of zebrafish, the ASC-1 transcriptional co-integrator complex was shown to downregulate genes involved in neurogenesis, neuronal projection, pathfinding, migration, and suppression of neuronal apoptosis, as well as genes involved in the negative regulation of bone resorption, ossification, and regulation of osteoblast differentiation, collagen fibril organization, and calcium ion homeostasis (Knierim et al., 2016). Expression studies also demonstrated upregulation of genes that reduce neuronal plasticity and neuron projection (Knierim et al., 2016). Disruption of these neuronal and osseous developmental pathways may be the mechanisms by which pathogenic variants in ASCC1 and TRIP4 result in the AMC phenotype, which includes impaired neuronal maturation and increased propensity for congenital fractures. Zebrafish with genetically abrogated ASCC1 or TRIP4 demonstrate severe impairment of axonal outgrowth of alpha-motoneurons, impaired formation of the neuromuscular junction, and abnormal organization of the myotome, and mutant zebrafish larvae demonstrate impaired swimming (Knierim et al., 2016).

Our report extends the phenotypic characteristics of SMABF2 resulting from biallelic variants in ASCC1, describes a novel ASCC1 intronic variant that alters RNA splicing associated with SMABF2, and suggests that more than 25% of wild-type ASCC1 transcript is required for normal neuromuscular development.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.