Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

most daily activities, and developed severe headaches.Neurological examination revealed a combination of severe bradykinesia, rigidity, dystonic postures in the neck and limbs, broken pursuit, reduced up-gaze, dysarthria, weak cough with poorly protruding tongue.Levodopa treatment had a modest benefit at the onset of the disease, but limited benefit was recorded at this stage.On the last examination, 17 years into the disease, she presented with a combination of cognitive deficit, severe extrapyramidal and cerebellar syndrome, severe speech and swallowing difficulties, becoming anarthric and unable to swallow, requiring percutaneous endoscopic gastrostomy (PEG) feeding.At this stage, there were extensive brain calcifications on brain imaging.
Her younger brother, case F1-II-2, had a normal development and health until his twenties.His symptom at onset was severe psychosis requiring hospitalization.He continued to have visual and auditory hallucinations and was subsequently diagnosed with Schizophrenia.Despite medical treatment, the hallucinations were refractory to treatment.A CT scan performed at the age of 39 showed extensive symmetrical brain calcifications involving bilateral basal ganglia.Despite a different onset, the disease progressed in a similar way to his sibling.He started having walking difficulties with a combination of cerebellar and parkinsonian syndrome.Ten years from onset of psychiatric symptoms he needed walking aids and three years after that, he became bed bound.On last examination, 15 years from onset of the disease, the patient required full-time care, was anarthric, unable to swallow and being PEG-fed, presenting very limited movement due to severe rigidity and bradykinesia.

Family 2. NAA60, c.338-1G>C, p.(Gly113Valfs*32)
Family 2 includes two siblings from a consanguineous family of Algerian descent.The proband had normal development until her early thirties.She worked as a restaurant manager.At the age of thirty she presented generalized seizures which led to the discovery of symmetric and extensive brain calcifications involving basal ganglia, cerebellar hemispheres, and frontoparieto-temporo-occipital cortex.Despite these widespread calcifications, neurological examination was normal.There was no cerebellar ataxia.She could stand more than 10s on monopodial support and walk a tightrope.She had neither pyramidal tract irritation signs nor extrapyramidal symptoms.EEG was normal.Acquired and metabolic causes of brain calcifications were excluded, as well as variants of POLG, MELAS, MERRF, SLC20A2, PDGFB, PDGFRB, XPR1 and TREX1.CSF study and eye examination were normal.During follow up, she presented insular and temporal seizures easily controlled with lamotrigine.
Though she did not develop cerebellar, extrapyramidal, or sensorimotor symptoms, she complained of progressive memory and impulse control disorders.Neuropsychological examination revealed frontal with attention deficit and dysexecutive syndrome, and visuospatial impairment.Two years later, she insidiously developed slight bradykinesia without abnormal gait, which did not require dopamine medication nor physiotherapy.Five years after the initial diagnosis, she is still under lamotrigine monotherapy and does not present significant disability except frontal impairment.
Since childhood, her brother presented an intellectual disability with a delay in speech development since childhood.Nevertheless, he followed a normal schooling until the age of 14. Afterward, he worked as a mechanic in a centre for people with disabilities.In his late twenties he developed a progressive cerebellar syndrome, seizures and extrapyramidal signs which required physiotherapy, lamotrigine, and dopamine medication.A CT-scan performed at the age of 43 showed extensive symmetrical brain calcifications similar to those of his sister.
After four years of disease, there is no significant worsening of the clinical signs.Both siblings had macrocrania with an oblong face.Metabolic screen revealed no calcium-phosphate metabolism disorder.In addition, she had a history of intellectual disability.Intellectual quotient was not assessed.Minimental state evaluation scored 18/30 at age 33.Physical examination revealed macrocrania, long face, agenesis of second, third and fourth right toes and hypoplasia of second, third and fourth left toes as well as hypopigmented regions on both upper limbs.
Her sibling was referred to an expert neurological center at the age of 24 years for the etiological assessment of extensive brain calcifications identified in the context of cephalalgia.
A diagnosis of migraine without aura was performed.She complained of sadness and anxiety and of unsteadiness and fatigue.At ages 24 and 25, neurological examination was normal.
However, at the age of 31, neurological examination showed a right Babinski sign and upper limb hypermetria with unsteadiness, indicating a cerebellar involvement.CT-scan revealed extensive calcifications (Total Calcification Score = 50).Metabolic screen revealed no calciumphosphate metabolism disorder and normal blood lactate and pyruvate levels.Electroencephalography was normal.In addition, she had a history of mild intellectual disability.
Neuropsychological assessment revealed a total Intellectual Quotient of 63 (verbal IQ=63, performance IQ=64).She presented some dysmorphic features, including low-set ears and high arched palate.She also presented with coxa valga and underwent surgery in childhood for aortic coarctation and suffered from hearing impairment.
Both parents were reported to be unaffected, but could not be examined.

Family 4. NAA60, c.391C>T, p.His131Tyr
This family of Gujarati origin have two children and 5 miscarriages.During the pregnancy the baby was noted to be small for gestational age.There was oligohydramnios and she was born by caesarean section at 37 weeks due to breech presentation weighing 2kg.The proband had congenital cataracts, early feeding difficulties, constipation, glue ears and hyperacusis.She was referred to genetics age 10 years with ADHD (diagnosed at age 6 years), short stature (<9 th centile), learning difficulties, delayed speech and language development, and dyspraxia.She exhibited sensory seeking behaviour.On examination she licked her hands repeatedly, presented poor social skills and struggled to hold a conversation.When concentrating on tasks, she made tongue movements and had abnormal posturing.She also had low tone, sweaty palms, hypopigmented linear birthmark on her right upper arm, almond shapes eyes, and low set ears.She presented dystonia and chorea.Parents observed that she had difficulty making friends and engaging in conversations.When concentrating on tasks she would protrude her tongue and have posturing.She was treated with methylphenidate for her ADHD and had a positive response on the medication.
Her brain MRI showed symmetrical low signal in globus pallidus bilaterally and supratentorially some scattered dots and small patches of T2/FLAIR hyperintensity predominantly in the frontoparietal subcortical white matter bilaterally.She also presents generalised pruritis, multiple allergies, and congenital cataracts.

Family 5. NAA60 c.130C>T, p.Arg44Cys
The proband was born to consanguineous parents and presented delayed motor milestones, pyramidal signs, seizures, and dystonia.He also had an abnormal EEG and dysmorphic features.Antiepileptic treatment was started due to seizures.He used multiple antiepileptics over the years due to ongoing seizures and he still does.At the last physical examination, he was microcephalic, had impaired motor development, no head control and no unassisted sitting.There were joint deformities with spasticity in the extremities.He speaks in single words, unable to form sentences.The brain MRI showed multiple T2 and Flair intense foci in the subcortical and cortical regions of the bilateral cerebral hemispheres, atrophy in the left cerebral peduncle, and cortical thickening areas compatible with polymicrogyria in the bilateral perisivian regions, temporal and parietal areas of the left cerebral hemisphere.There was no calcification in brain CT.

Family 6. NAA60 c.50T>G, p.Leu17Arg
The proband is a male from consanguineous parents from Turkey.He presented at 12 years old with motor and cognitive developmental delay, first observed at 6 months old.He has a history of seizures without fever at the age of 8-9 months and was since treated with sodium valproate.Due to hyperactivity, a child psychiatrist was consulted when he was 2 years old, and methyl phenidate was started.At 3 years old, a shift in the eyes was noticed and glasses were given due to severe astigmatism and hyperopia.At last examination, he could speak but was not clearly understood.On examination, he presents with mild intellectual disability, microcephaly, high palate, strabismus, spasticity, pyramidal syndrome (brisk reflexes, upgoing plantars), Pes planus, assisted in long-distance walking.

Family 7. NAA60 c.428A>C, p.Asn143Thr
A single affected Saudi male born to non-consanguineous parents, both from the same tribe is the only affected member in kindred of nine siblings.The index had normal development until the age of 27 years when he presented with upper respiratory tract infection (high-grade fever and cough) and later developed facial asymmetry with slurred speech and decreased concentration.Past medical history included two head traumas, at 1 year old after a fall without any complication and at 7 years old after an accidental head injury that required suturing.His physical examination revealed mild cognitive impairment with MoCA score of 22/30.
Neurological examination showed a combination of pyramidal and cerebellar syndrome.Brain MRI revealed multifocal ischemic changes including chronic and subacute ischemic changes, and extensive intracranial calcifications.There was also evidence of multifocal narrowing of the distal intracranial arteries compatible with intracranial vasculopathy.On CT scan, multifocal scattered intracranial coarse calcification was seen.In addition, PET-CT brain with dopa challenge of the patient gave a very unexpected pattern where dopa uptake by the basal ganglia was normal despite extensive calcification.Other investigations showed normal transthoracic echocardiography and neurophysiology examination showed no electrophysiological evidence of peripheral neuropathy in the upper or lower extremities, and EEG was normal for awake and drowsy EEG.

Family 3 .
NAA60, c.338-1G>C, p.(Gly113Valfs*32) Two sisters were affected by extensive brain calcifications.They were born from consanguineous parents and originated from Morocco.The older sister, suffered from migraine without aura.Upon neurological examination at the age of 31, bilateral Babinski sign was noticed along with mild akineto-hypertonic syndrome, slight dysmetria in the context of unsteadiness, suggesting mild cerebellar syndrome.SARA scale scored 16/40 and UPDRS-III 10/108.CT-scan revealed extensive calcifications (Total Calcification Score = 46).

a,
GTEx gene expression of NAA60 and SLC20A2 and b, BRANIAC NAA60 gene expression in different brain areas in adult pathologically normal human brains show that NAA60 is expressed in all ten brain regions with highest expression detected in the putamen, cerebellum, and occipital regions (PUTM -putamen, HIPP -hippocampus, CRBLcerebellum, OCTX -occipital cortex, THAL -thalamus, MEDU -medulla, FCTX -frontal cortex, TCTX -temporal cortex, SNIG -substantia nigra, WHMT -white matter).c, NAA60 gene expression in the developing brain (NCX -neocortex; STR -striatum, HPhippocampus, AMY -amygdala, MD -midbrain, CBC -cerebellum).d-f, Eigengenes correlation.In the three brain tissues of interest (cerebellum, nucleus accumbens and caudate), NAA60and SLC20A2 belong to different modules within each tissue-specific network.However, for the nucleus accumbens network, these two modules are very close, and these can be visualised in the corresponding dendrogram.In each dendrogram, the modules of interest are highlighted in red.g, Top-Down plot of the NAA60 module genes in the caudate tissue.Only the most connected genes are shown.Size of gene nodes reflect their connectivity with the rest of genes in the module.Proximity of genes in the plot reflects their similarity in terms of shared connections with other genes.

Table 1 .
In silico prediction of reported NAA60 variants.