Clinical report: family 1, individual 1
Individual 1 (F1:IV-2) is a 19-year-old man hemizygous for a novel NAA10 c.16G>C p.(A6P) variant (Fig. 1A; Table 1). He has ID and hypertrophic cardiomyopathy with a family history suggesting an X-linked cause. The proband had a sister (F1:IV-1) who was born with an interrupted aortic arch and died at 10 days of life. Both of his living sisters and parents are healthy and had normal regular echocardiograms. A maternal uncle (F1:III-5) had ID, medullary sponge kidneys with renal failure, and died at age 40 from complications of an idiopathic restrictive cardiomyopathy. No one else in the family has been found to have medullary sponge kidneys or recurrent kidney stones, so this is not likely to be caused by NAA10 impairment. There were concerns about the proband’s development from about 15 months of age. His mother reported that he was saying “mama” and “dada” at that age but stopped and did not start using words again until almost age 4 years. There were no concerns about his early motor development. He had issues with attention, concentration, and could be oppositional with his parents, teachers, and providers. He received special education all through school. Formal neuropsychological testing was attempted a number of times but without completion due to a lack of cooperation and effort. The neuropsychologist provided an estimated IQ < 60 based on what he was able to complete. At the completion of high school, he was reading at a 2nd-grade level and doing math at a 3rd-grade level.
Hypertrophic cardiomyopathy was diagnosed at age 7 years. Regular follow-up since that time has confirmed persistent and stable asymmetric septal hypertrophy, most recently with a thickness of 18 mm without obstruction. He had no issues with fatigue, shortness of breath, chest pain, syncope, nor near syncope.
On physical examination at age 19 he had a height of 168 cm (10–25th percentile), weight of 52.7 kg (3rd percentile), and head circumference of 56.8 cm (50–75th percentile). He had bilateral indentations in the region of the lambdoid sutures. His nasal tip was broad with thickened alae. He had no additional facial dysmorphic features. His skin was normal without excessively laxity. He had normal muscle strength and tonus.
Previous clinical genetic testing included fragile-X testing, chromosome microarray, and an 18-gene hypertrophic cardiomyopathy gene panel. Furthermore, a brain magnetic resonance imaging (MRI) was normal.
Based on the family history suggesting an X-linked cause for ID and cardiomyopathy, genetic testing with a 116-gene next-generation sequencing panel for X-linked ID was carried out (Fulgent Genetics, Temple City, CA, USA) which identified the NAA10 variant. Subsequent genetic testing of the proband’s mother (F1:III-3) confirmed that the variant was maternally inherited.
Clinical report: family 2, individuals 2–5
Family 2 is a four-generation family with ID including four affected males across two generations (Fig. 1B; Table 1). Genetic testing was carried out in three individuals [individual 3 (F2:II-5), individual 4 (F2:III-3), and individual 5 (F2:III-4)], as the fourth individual 2 (F2:II-6) died before DNA was sampled. A novel NAA10 c.235C>T p.(R79C) variant was detected in all three family members by exome sequencing with a special focus on the X-chromosome due to the X-linked inheritance pattern. The female carriers in this family were asymptomatic.
Individual 2 (F2:II-6) died at 52 years of age. He was diagnosed with moderate degree of ID and high degree myopia (− 10). IQ testing revealed an IQ of 17 at 23 years of age and an IQ of 19 at age 28. Height measured at age 52 was 178 cm (50th percentile) and weight was 68 kg (25–50th percentile). Physical findings included full lips and high vaulted palate. Mobility and muscle strength were normal, but lively patellar reflexes were noted. He developed some speech, but it remained very incomprehensive. Expressive aphasia was suspected as he understood most of what was said to him. He had a friendly personality. Hearing was estimated to be normal, but he was unable to cooperate with formal audiological testing. At age 48, he suffered a grand mal seizure but a subsequent EEG did not reveal any abnormalities. Two weeks prior to his death, he developed hyperreflexia, athetosis and tremor. Autopsy showed cerebral haemorrhage in the right hemisphere and a tumour causing cortical compression. The tumour was very cell-rich and with partial degeneration and felt to be metastatic, but a primary tumour was not identified. The cortex and white matter were atrophic. A single cavernous haemangioma was identified at autopsy in the liver. Genetic testing was not carried out, but due to his medical and family history, it is likely that he also had the same familial variant.
Individual 3 (F2:II-5), the younger brother of individual 2, was a male born naturally with a weight of approximately 3500 g (50th percentile). At age 2, he still had no verbal language and at age 3, he started walking independently. He never attended school and had an IQ of 31 at age 10. His language was very limited, but his speech comprehension was good. His hearing was determined normal at age 37. He had tremor and restlessness and was treated with chlorprothixene. He was mentally regarded as having moderate degree of ID. He was able to feed and toilet independently. No striking dysmorphic facial features were noted except a high vaulted palate. He died at around 51 years of life. No autopsy was performed.
Individual 4 (F2:III-3) is a 66-year-old male. He sat unsupported at age 9 months and walked at age 23 months. He had delayed speech development with elements of aphasia but better speech comprehension. He has received speech therapy for a long time. His hearing is estimated to be normal. He is hyperactive and restless by nature but easy going in contact with others.
At age 14, his IQ was 43. He has small stature, hypermetropia, but no facial dysmorphic features. At age 24, a chromosome analysis revealed normal male 46, XY karyotype. He currently lives in a protected living setting.
Individual 5 (F2:III-4) is a 64-year-old male. As an infant, he required surgery for pyloric stenosis as well as umbilical and inguinal hernia. He started to walk at 10–12 months and had some speech from age 2. At age 12, his IQ was 56. He had delayed speech development (dysphasia/expressive aphasia), but has benefitted from speech therapy and has obtained useful verbal competence for communication and exhibits good social skills. Chromosome analysis conducted at age 22 showed normal male 46, XY karyotype. As an adult, he is married and lives with his wife in an apartment in a protected living setting.
Previous clinical genetic testing included fragile-X testing, chromosome microarray, and a screening for known microdeletions and duplications by MLPA analysis [MLPA p106 X-linked ID syndromes and MLPA p245 Microdeletion Syndromes-1 (MRC Holland)] of Individual 5 (F2:III-4). The NAA10 variant of the family was identified with exome sequencing as part of a research project to identify the underlying genetic factor in X-linked ID.
Clinical report: family 3, individual 6
Individual 6 is a 17-year-old female who was found to be heterozygous for the previously studied pathogenic NAA10 c.384T>G p.(F128L) variant (Saunier et al. 2016; Cheng et al. 2019) (Fig. 1C; Table 1). She was born at 34 weeks of gestation, with birth weight 2500 g (75th percentile). She was number one of fraternal twins. Her twin is healthy, with no health issues. In addition, she has two other healthy brothers. Early issues included respiratory distress, requiring supplemental oxygen for about 6 weeks. She was subsequently noted to have global DD and multiple contractures, including elbows and feet. There have been some issues with poor weight gain. She has central vision impairment, microcephaly and occasional tremors. She can walk with assistance but is frequently in a wheelchair. She was still incontinent at age 15 years. She was followed by paediatric cardiologists, and ECG and echocardiogram were unremarkable. At age 15 years, weight was 29.4 kg (< 3rd percentile, − 3 SD), height was 133.6 cm (< 3rd percentile, − 3 SD), and head circumference 48.2 cm (< 3rd percentile, − 3 SD). Findings at that time included contractures at the major joints, spasticity, and dysconjugate gaze. Laboratory evaluation included testing for Prader–Willi (OMIM #176270) and Angelman (OMIM #105830) syndromes, karyotyping, serum acylcarnitine profile and array CGH (comparative genome hybridisation), all of which were normal. The de novo NAA10 variant was identified using clinical exome sequencing.
Clinical report: family 4, individual 7
Individual 7 (F4:II-1) is a 33-month-old girl with a novel de novo NAA10 c.386A>C p.(Q129P) variant (Fig. 1D; Table 1). She was born at 41 weeks of gestation with birth weight of 2910 g (23rd percentile) and birth length of 47 cm (12th percentile). She had perinatal distress requiring resuscitation. Apgar scores were 1, 3, and 9. Prenatal MRI showed hypoplastic cerebellar vermis and hypoplastic pons. Subsequent imaging after birth showed cerebellar vermian hypoplasia with cystic dilatation of the 4th ventricle. She also had a left pneumothorax. A heart murmur was noted, and she was found to have a small patent ductus arteriosus, enlarged atrial septal defect and Ebstein anomaly of the tricuspid valve. Bilateral vocal cord paralysis was noted. She had nutritional issues which required feeding with first gastrostomy tube and subsequently with a gastro-jejunostomy tube. Physical findings at 33 months included a bowed upper lip, thickened gingiva, upper lid ptosis, prominent globes, a short columella, microcephaly, bilateral Sydney lines, and a heart murmur. At age 33 months, she “babbles” but has no words. She can use a standing device but is not walking independently. A chromosomal microarray showed a 47, XXX karyotype, but as this finding could not explain her clinical picture, exome sequencing was carried out detecting the NAA10 variant on one of her X chromosomes.
Clinical report: family 5, individual 8
Individual 8 (F5:III-1) has a novel, hemizygous NAA10 c.469G>A p.(E157K) variant (Fig. 1E; Table 1). He is the first child of healthy, unrelated parents and has one younger, healthy brother (III-2). The pregnancy and birth were uneventful and he had a normal head circumference at birth (34.5 cm, − 0.59 SD). Delayed psychomotor development was noted at age 1 year. He walked at age 21 months. Single words were spoken at age 1 year, but he did not put words together to form sentences until the age of 3. He displays autistic features like challenging interaction with other children, rigid behaviour, and loves sorting and systems. When excited, facial grimacing is observed. Mainstream school was started 1 year postponed. Formal testing at age 5 resulted in a diagnosis of a mixed specific developmental disorder with a cognitive level in the lower normal range. Muscular hypotonia was noted. His head circumference increased at a slow rate with decline to − 1.57 SD (46 cm) by 1 year of life and microcephaly by age 6 (− 2.67 SD, 48.5 cm). No cerebral MRI has been performed. He has a round face with full cheeks, narrow palpebral fissures, epicanthal folds, a smooth philtrum, bilateral single transverse palmar creases, and camptodactyly of the fourth fingers. The other finger joints are hypermobile. ECG and echocardiogram were performed at age 7 with normal results.
Genetic analyses including fragile-X testing and SNP array were normal. Diagnostic gene panel sequencing in a trio with his parents detected a NAA10 variant inherited from his mother (F5:II-2). Segregation studies in the family show that the variant was inherited from his healthy maternal grandmother (F5:I-1) to his mother (F5:II-2). His healthy brother (F5:III-2) does not carry the variant. His mother (F5:II-2) is healthy, and no abnormalities were noted in an ECG performed following the gene test.
NAA10 sequence conservation and structure
The evolutionary conservation of the five substituted NAA10 amino acids was investigated with multiple sequence alignment including nine species. NAA10 A6, R79 and F128 showed high conservation from yeast to human, whereas Q129 and E157 were mainly conserved in the animal kingdom (Fig. 2A). The high degree of conservation suggests an important role in maintaining NAA10 function. A three-dimensional NatA crystal structure (PDB ID: 6C9M) showed that A6 was located in the loop between β1 and α1, which is part of the NAA15 interaction domain (Fig. 2B). R79 was positioned close to the Ac-CoA-binding site; however, its side chain protruded towards NAA15 and was bioinformatically predicted to interact with several NAA15 residues (Fig. 2B). The missense variants p.(A6P) and p.(R79C) both affect amino acids situated near the NAA10–NAA15 interface and could potentially influence NatA complex formation and/or function. F128 was inward-pointing and part of a hydrophobic pocket at the core of NAA10. The side chains of Q129 and E157 were directed outwards of the NAA10 structure. Q129 was located at the start of β6, while E157 was located in α5 preceding the unstructured C-terminal tail. F128, Q129 and E157 were not located near the NAA10–NAA15-binding surface which suggests variants affecting these residues are more likely to impede monomeric NAA10 activity or stability rather than affecting the NatA complex itself.
Cellular stability of NAA10 variants
In order to investigate the cellular stability of the novel NAA10 missense variants, cycloheximide (CHX) chase assays were conducted in transfected HeLa cells. NAA10 A6P-V5, NAA10 Q129P-V5 and NAA10 E157K-V5 revealed a reduced stability compared to NAA10 WT-V5 throughout a 6-h time course (Fig. 3A, C, D). In contrast, NAA10 R79C-V5 displayed a similar turnover rate as for NAA10 WT-V5, indicating that this variant does not impact NAA10 stability (Fig. 3B).
NatA complex formation and catalytic activity
NatA complex formation with NAA15 of the NAA10 variants was investigated in HeLa cells by co-immunoprecipitation experiments. This revealed that fivefold less NAA15 co-immuno-precipitated with NAA10 A6P-V5 compared to NAA10 WT-V5, suggesting that NAA10 p.(A6P) is highly reduced in its capacity to form the NatA complex (Fig. 4A). On the other hand, there was no significant difference in co-immunoprecipitation of NAA15 with NAA10 WT-V5 and the three variants NAA10 R79C-V5, NAA10 Q129P-V5 and NAA10 E157K-V5 (Fig. 4B–D). Thus, these three NAA10 variants seemingly do not impair NAA10–NAA15 binding. The immuno-precipitated NAA10-V5 were further subjected to Nt-acetylation assays in order to investigate the catalytic activity of the different NAA10 variants. A serine–glutamate–serine–serine-starting peptide (SESS) derived from a canonical NatA substrate was used to test NatA activity and, since monomeric NAA10 has a high preference for acidic N-termini in vitro, a glutamate–glutamate–glutamate–isoleucine-starting peptide (EEEI) was used to measure monomeric NAA10 NAT activity (Arnesen et al. 2009; Damme et al. 2011). The measured product formation of Nt-acetylated SESS and EEEI was normalised to the amount of NAA15 (as a measure of NatA complex) and NAA10 present in the reaction, respectively. The only variant exhibiting reduced NatA catalytic activity after normalisation was NAA10 R79C-V5 (Fig. 4B). Moreover, all the four NAA10 variants tested displayed reduced Nt-Ac of the EEEI peptide as compared to WT, suggesting the monomeric NAA10 NAT function is attenuated (Fig. 4A–D). Notably, NAA10 Q129P-V5 had an almost abolished catalytic activity towards EEEI, whereas A6P, R79C and E157K displayed a more moderate reduction.