Figure 3. Western blot analysis of POLR3A and RNA expression levels of POL III targets in patient fibroblasts.

(A) SDS-PAGE for POLR3A, HA, and GAPDH in control (Ctrl) and proband (P) fibroblasts expressing empty vector (P^EV), POLR3A-HA wild type (P^POLR3A_WT_1 and P^POLR3A_WT_2), and POLR3A-HA mutant (P^{POLR3A_V1241M_1}, P^{POLR3A_V1241M_2}, and P^{POLR3A_V1241M_3}); qRT-PCR measurement in fibroblasts isolated RNA showing levels of (B) HNRNPH2, (C) UBB, (D) LTF, and (E) HSP90AA1 normalized per GAPDH. (F) Intersection among the patient features (caudate nucleus atrophy, dysphagia, dystonia, finger clubbing, hypodontia, hypotonia, lactate peak, leukocytosis, lipid peak, low weight, metabolic acidosis, milestone regression, putamen atrophy, and recurrent respiratory infection) and symptoms described for NPS (aged face, agenesis of the corpus callosum, apparent macrocephaly, ataxia, beak-shaped nose, blue sclerae, broad eyebrows, Chiari malformation, cryptorchidism, Dandy-Walker malformation, decreased subcutaneous fat, deep-set eyes, dental delayed eruption, developmental delay, downslanting palpebral fissures, downturned corners of mouth, ectropion, endocrine abnormalities, entropion, failure to thrive, fat accumulation around the buttocks, dysphagia, frontal bossing, generalized lipoatrophy, gynecomastia, hydrocephalus, hypertelorism, hypertonia, hypodontia, hypoplasia of the facial bones, hypoplastic ilia, hypotonia, hypotrichosis, increased triglycerides, intention tremor, intrauterine growth retardation, irregular metaphyseal endplates, joint contractures, lagophthalmos, large feet, large hands, long fingers, long thin bones with enlarged metaphyseal endplates, long toes, lower eyelid covering part of the cornea, malar hypoplasia, mandibular hypoplasia, mental retardation, micrognathia, muscle atrophy, nasal high-pitched voice, natal teeth, nystagmus, parietal bossing, partly unossified atlas at birth, persistent fontanelles, pinched nose, pointed chin, poor postnatal growth, progeroid appearance, prominent abdomen, prominent forehead, prominent scalp veins, pseudohydrocephalus, recurrent respiratory infections, scoliosis, short stature, small mouth, sparse eyebrows, sparse eyelashes, thin diaphyses, thin ribs, thin translucent skin, thin upper vermilion, triangular face, trident configuration of acetabula, upslanting palpebral fissures, and widely open sutures); and HLD7 (abnormal smooth pursuit, ataxia, bladder dysfunction, cerebellar atrophy, cerebellar signs, cognitive regression, cortical atrophy, decreased vibratory and positional sense, delayed dentition, dental delayed eruption, delayed puberty, developmental delay, drooling, dysarthria, dysmetria, dysphagia, dystonia, extensor plantar responses, hyperreflexia, hypodontia, hypogonadotropic, hypogonadism hypomyelination, leukodystrophy, motor regression, myopia, nystagmus, oligodontia, optic atrophy, peripheral neuropathy, postural tremor, seizures, short stature, spasticity, thinning of the corpus callosum, upper motor neuron signs, vertical gaze limitation, and white matter rarefaction).