2.1 Subjects

From a large cohort of unrelated Iranian families with ARID who were referred to the Genetics Research Center (GRC) at the University of Social Welfare and Rehabilitation Sciences (USWR) and Kariminejad - Najmabadi Pathology & Genetics Center (KNPGC), Tehran, Iran from 2006 to 2017 for NGS, families with detected variants in the EXOSC gene family were selected. The study was approved by the Ethics Committee of USWR, Tehran, Iran. After obtaining informed consent, all patients underwent comprehensive clinical evaluation and their intelligence quotient (IQ) scores were assessed using the Wechsler Adult Intelligence Scales (WAIS) and The Wechsler Intelligence Scales for Children (WISC), if applicable. Detailed clinical and neurological examinations comprising brain MRI, whenever possible, have been carried out.

2.2 Targeted and whole-exome sequencing

Based on instructions from the referring physicians, genomic DNA from families 1, 2 and 5, were subjected to WES and samples from families 3 and 4 underwent TES using two targeted gene panels (Methods in Appendix S1 and Table S1).

3.1 Family1

An 18-month-old male proband (IV:1) was born to healthy first-degree cousins (Figure S1.1-1A, Table 1) originating from Fars province of Iran following uncomplicated and term pregnancy with normal delivery. Birth measurements showed a normal range for head circumference (HC: 34 cm; −0.8 SD), weight (3100 g; −0.7 SD), and length (50 cm; −0.06 SD). By age 18 months, his weight, head circumference and height had reached 10 kg (−1.5 SD), 52 cm (+3.2 SD), and 83 cm (+0.4 SD), respectively. His parents noticed their infant's inattention at 58 days of age. He gradually manifested hypotonia and his developmental milestones were retarded. His speech did not develop and he had a prominent forehead, long philtrum, short nose, broad nasal tip, broad columella, and thin upper lip. Upon clinical examination, increased tone in the form of spasticity was revealed in the upper and lower limbs with no contractures (Figure S1.1-1C). Ophthalmic examination showed myopia and strabismus; however, his retina and hearing status remained unexamined. A reported brain MRI at 10 months old was suggestive of a delay in the myelination process in the anterior limb of the internal capsule and anterior commissure. With a diagnosis of developmental delay, his physician referred the family to KNPGC for WES. We identified a disease-causing homozygous variant (c.673-1G > T) at the splice acceptor site of intron 7 of EXOSC2 gene (NM_014285.6) (Figure S1.1-1B, Figure 3A, Table 1).

3.2 Family 2

A 6-year-old male proband (IV:1) from first cousin healthy parents (Figure S1.1-2A, Table 1) originating from Kerman province of Iran was referred to GRC. He was born with a small HC (33 cm; −1.3 SD), low weight for gestational age (2200 g; −2.2 SD) and short height (48 cm; −0.8 SD). He never developed head control, sitting, standing, walking or speaking abilities. He showed a lack of eye contact, generalized hypotonia and feeding difficulties. His facial features included a prominent forehead, broad nasal tip, broad columella, and thin upper lip (Figure S1.1-2C). He developed generalized tonic-clonic seizures at infancy, but responded successfully to medication. Although his vision showed impairment, we were unable to exam his retina. He had a history of unilateral inguinal hernia and was operated at the age of 4 years. By 6 years of age, his height was more significantly affected (97 cm; −3.6 SD) than his occipitofrontal circumference (OCF: 51 cm; −0.4 SD), and his weight (15 kg; −2.5 SD). He also showed myopathic face and dysphagia. Under detailed examination, upward gaze, vertical nystagmus, quadriparesis, and hyperreflexia were noted. He did not have the ability to communicate or pursue topics. Although his IQ was not formally tested, all his adaptive functions and behavior including conceptual, social and practical skills were impaired. Brain MRI showed cerebellar hypoplasia and abnormal white matter suggestive of leukodystrophy (Figure S1.1-2C). From WES, a homozygous disease-causing variant was identified at the splice acceptor site of intron 7 of EXOSC2 (NM_014285.6; c.673-1G > T)5 (Figure S1.1-2B, Figure 3A, Table 1).

3.3 Family 3

A 38-month-old female proband (V:1) was born to consanguineous parents (Figure S1.1-3A, Table 1) originating from Fars province of Iran following an uneventful pregnancy with uncomplicated term delivery. Recorded measurements at birth showed normal HC, normal weight, and length, while her OCF and weight dropped to below the 2 percentile (OCF: 45.5 cm; −2.0 SD and weight: 9 kg; −3.7 SD) by the age of 38 months. She showed motor developmental and speech delay. Deep tendon reflexes (DTR) were very brisk with no clonus. Some dysmorphic features including a prominent forehead, short nose, long philtrum, thin upper lip, and sparse hair were noted (Figure S1.1-3C). Clinical evaluation showed spastic cerebral palsy and strabismus. MRI of the brain showed cerebellar hypoplasia with incomplete myelination. The electroencephalogram (EEG) was within normal limits. The case was successively endorsed by TES testing in KNPGC detecting a homozygous disease-causing variant in exon 2 of the EXOSC3 (NM_016042.4; c.395A > C; p.[Asp132Ala]) (Figure S1.1-3B, Figure 3B, Table 1).

3.4 Family 4

A 15-month-old male proband (V:1) was born to first cousin healthy parents (Figure S1.1-4A, Table 1) from Shahrud district of Iran after an uneventful pregnancy but with pre-term delivery and hypoxia. Birth measurements showed a normal HC (35 cm; +0.1 SD), low weight (2500 g; −1.7 SD), and length (42 cm; −3.3 SD) but HC had dropped to below the first percentile (43 cm; −3.2 SD) by age 15 months. Clinical investigation had begun at birth because of severe hypotonia. He never achieved head control, crawling, sitting or walking. Muscle imaging was unremarkable while nerve conduction velocity (NCV) was suggestive of neurogenic changes.

Vision and hearing status remained unexamined. Findings from brain MRI indicated suspected incomplete myelination. Using TES at KNPGC, we found a homozygous disease-causing variant (c.395A > C; p.[Asp132Ala]) in exon 2 of EXOSC3 (NM_016042.4) (Figure S1.1-4B, Figure 3B, Table 1).

3.5 Family 5

Three affected children [V:1, V:2 and V:3] (Figure S1.1-5A, Table 1) from healthy second cousin parents from the northern-east of Iran were referred to GRC to undergo WES. The family had two other healthy female children. The three probands were born after uneventful pregnancies. Delayed motor milestones were apparent at infancy, at which time their parents noticed muscular hypotonia and weakness. Gradually, after starting to walk, they developed an unbalanced gait. Dysphagia, early nystagmus and remarkable dysarthria in these three affected children were reported by their parents. At examination, aged 27, 17, and 6 years, respectively had heights 160 cm; − 0.5 SD (V:2), 157 cm; − 0.9 SD (V:3) and 120 cm; + 0.9 SD (V:1), and HC 55 cm; + 0.6 SD (V:2), 53 cm; − 1.2 SD (V:3) and 50 cm; − 1.1 SD (V:1). Myopathic faces, horizontal nystagmus, cerebellar ataxia, spasticity, and increased DTR have been noted in their examination. In addition to speech delay, their pronunciation remained inarticulate. Cranial MRI of V:3 showed pachygyria, cortical atrophy and cerebellar hypoplasia (Figure S1.1-5C). Ophthalmological examinations, including fundoscopy of patient V:1 revealed myopia, strabismus, and slightly reduced macular reflexes because of myopia (−2 diopters). At adolescent age, the patients were diagnosed with valvular heart disease including mitral and tricuspid regurgitation, Right Bundle Branch Block and bradycardia requiring cardiac pacemakers. Cognitive status assessment using WAIS-IV revealed moderate ID ranging from 40 to 50. Using WES, a homozygous disease-causing variant (NM_020158.4; c.341C > T; p.[Thr114Ile])6 in EXOSC5 was identified in all patients (Figure S1.1-5B, Figure 3C, Table 1).

Comparisons between clinical data and genomic changes in the EXOSC genes which led to ID in our investigated Iranian patients and which have been reported are summarized in Table 1, Figure S2 and 3.