De novo EIF2AK1 and EIF2AK2 Variants Are Associated with Developmental Delay, Leukoencephalopathy, and Neurologic Decompensation.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder.

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.

Type I Alexander disease: Update and validation of the clinical evolution-based classification.

BACKGROUND AND OBJECTIVES: Alexander disease (AxD) is a rare progressive leukodystrophy caused by autosomal dominant mutations in the Glial Fibrillary Acidic Protein (GFAP) gene. Three main disease classifications are currently in use, the traditional one defined by the age of onset, and two other based on clinical features at onset and brain MRI findings. Recently, we proposed a new classification, which is based on taking into consideration not only the presenting features, but also data related to the clinical course. In this study, we tried to apply this modified classification system to the cases of pediatric-onset AxD described in literature. METHODS: A literature review was conducted in PubMed for articles published between 1949 to date. Articles that reported no patient's medical history and the articles about Adult-onset AxD were excluded. We included patients with a confirmed diagnosis of pediatric-onset AxD and of whom information about age and symptoms at onset, developmental milestones and loss of motor and language skills was available. RESULTS: Clinical data from 205 patients affected with pediatric-onset AxD were retrospectively reviewed. Among these, we identified 65 patients, of whom we had enough information about the clinical course and developmental milestones, and we assessed their disease evolutionary trajectories over time. DISCUSSION: Our results confirm that patients with Type I AxD might be classified into four subgroups (Ia, Ib, Ic, Id) basing on follow up data. In fact, despite the great variability of phenotypes in AxD, there are some shared trajectories of the disease evolution over time.

Expanding the Spectrum of NUBPL-Related Leukodystrophy.

Mitochondrial leukodystrophies constitute a group of different conditions presenting with a wide range of clinical presentation but with some shared neuroradiological features. Genetic defects in NUBPL have been recognized as cause of a pediatric onset mitochondrial leukodystrophy characterized by onset at the end of the first year of life with motor delay or regression and cerebellar signs, followed by progressive spasticity. Early magnetic resonance imagings (MRIs) show white matter abnormalities with predominant involvement of frontoparietal regions and corpus callosum. A striking cerebellar involvement is usually observed. Later MRIs show spontaneous improvement of white matter abnormalities but worsening of the cerebellar involvement evolving to global atrophy and progressive involvement of brainstem. After the 7 cases initially described, 11 more subjects were reported. Some of them were similar to patients from the original series while few others broadened the phenotypic spectrum. We performed a literature review and report on a new patient who further expand the spectrum of NUBPL-related leukodystrophy. With our study we confirm that the association of cerebral white matter and cerebellar cortex abnormalities is a feature commonly observed in early stages of the disease but beside the original and so far prevalent presentation, there are also uncommon phenotypes: clinical onset can be earlier and more severe than previously thought and signs of extraneurological involvement can be observed. Brain white matter can be diffusely abnormal without anteroposterior gradient, can progressively worsen, and cystic degeneration can be present. Thalami can be involved. Basal ganglia can also become involved during disease evolution.

FDXR-associated disease: a challenging differential diagnosis with inflammatory peripheral neuropathy.

BACKGROUND AND AIMS: Mutations in FDXR gene, involved in mitochondrial pathway, cause a rare recessive neurological disorder with variable severity of phenotypes. The most common presentation includes optic and/or auditory neuropathy, variably associated to developmental delay or regression, global hypotonia, pyramidal, cerebellar signs, and seizures. The review of clinical findings in previously described cases from literature reveals also a significant incidence of sensorimotor peripheral polyneuropathy (22.72%) and ataxia (43.18%). To date, 44 patients with FDXR mutations have been reported. We describe here on two new patients, siblings, who presented with a quite different phenotype compared to previously described patients. METHODS: Clinical, neurophysiological, and genetic features of two siblings and a systematic literature review focused on the clinical spectrum of the disease are described. RESULTS: Both patients presented with an acute-sub-acute onset of peripheral neuropathy and only in later stages of the disease developed the typical features of FDXR-associated disease. INTERPRETATION: The peculiar clinical presentation at onset and the evolution of the disease in our patients and in some cases revised from the literature shed lights on a new possible phenotype of FDXR-associated disease: a peripheral neuropathy which can mimic an acute inflammatory disease.

The 2021 European Alliance of Associations for Rheumatology/American College of Rheumatology points to consider for diagnosis and management of autoinflammatory type I interferonopathies: CANDLE/PRAAS, SAVI and AGS.

OBJECTIVE: Autoinflammatory type I interferonopathies, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature/proteasome-associated autoinflammatory syndrome (CANDLE/PRAAS), stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) and Aicardi-Goutières syndrome (AGS) are rare and clinically complex immunodysregulatory diseases. With emerging knowledge of genetic causes and targeted treatments, a Task Force was charged with the development of 'points to consider' to improve diagnosis, treatment and long-term monitoring of patients with these rare diseases. METHODS: Members of a Task Force consisting of rheumatologists, neurologists, an immunologist, geneticists, patient advocates and an allied healthcare professional formulated research questions for a systematic literature review. Then, based on literature, Delphi questionnaires and consensus methodology, 'points to consider' to guide patient management were developed. RESULTS: The Task Force devised consensus and evidence-based guidance of 4 overarching principles and 17 points to consider regarding the diagnosis, treatment and long-term monitoring of patients with the autoinflammatory interferonopathies, CANDLE/PRAAS, SAVI and AGS. CONCLUSION: These points to consider represent state-of-the-art knowledge to guide diagnostic evaluation, treatment and management of patients with CANDLE/PRAAS, SAVI and AGS and aim to standardise and improve care, quality of life and disease outcomes.

Movement disorders in MCT8 deficiency/Allan-Herndon-Dudley Syndrome.

BACKGROUND AND OBJECTIVES: MCT8 deficiency is a rare genetic leukoencephalopathy caused by a defect of thyroid hormone transport across cell membranes, particularly through blood brain barrier and into neural cells. It is characterized by a complex neurological presentation, signs of peripheral thyrotoxicosis and cerebral hypothyroidism. Movement disorders (MDs) have been frequently mentioned in this condition, but not systematically studied. METHODS: Each patient recruited was video-recorded during a routine outpatient visit according to a predefined protocol. The presence and the type of MDs were evaluated. The type of MD was blindly scored by two child neurologists experts in inherited white matter diseases and in MD. Dystonia was scored according to Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). When more than one MD was present, the predominant one was scored. RESULTS: 27 patients were included through a multicenter collaboration. In many cases we saw a combination of different MDs. Hypokinesia was present in 25/27 patients and was the predominant MD in 19. It was often associated with hypomimia and global hypotonia. Dystonia was observed in 25/27 patients, however, in a minority of cases (5) it was deemed the predominant MD. In eleven patients, exaggerated startle reactions and/or other paroxysmal non-epileptic events were observed. CONCLUSION: MDs are frequent clinical features of MCT8 deficiency, possibly related to the important role of thyroid hormones in brain development and functioning of normal dopaminergic circuits of the basal ganglia. Dystonia is common, but usually mild to moderate in severity, while hypokinesia was the predominant MD in the majority of patients.

How to look for intracranial calcification in children with neurological disorders: CT, MRI, or both of them?

BACKGROUND: Intracranial calcification (ICC) is an important diagnostic clue in pediatric neurology. Considering the radiation-induced cancer risk associated with computed tomography (CT), we aim to define the diagnostic value of magnetic resonance imaging (MRI) sequences sensitive to paramagnetic/diamagnetic substances in the detection of ICC, comparing with CT scanning. MATERIALS AND METHODS: We selected MRI and CT scans performed in children affected by neurological conditions associated with ICC referred to the participating centers between 2005 and 2018. Inclusion criteria were age at neuroradiological investigation < 18 years, availability of good quality CT positive for calcification, and MRI scan that included GE or/and SWI sequences, performed no more than 6 months apart. RESULTS: Eighty-one patients were included in the study. CT and MRI scans were reviewed by consensus. MRI failed to detect ICC in 14% of the cases. Susceptibility-weighted imaging (SWI) was the best MRI sequence to use in this setting, followed by gradient echo imaging. In 19% of the cases, CT could have been avoided because the identification or monitoring of ICC has not been necessary for the clinical management of the patient. CONCLUSION: In the diagnostic workup of pediatric-onset neurological disorders of unknown cause, the first step to look for ICC should be an MRI that includes SWI and GE sequences. If ICC is absent on MRI, brain CT scanning should be performed at least once. When the identification or monitoring of ICC is unlikely to add information useful for patient's follow-up or treatment, we recommend not performing CT scanning.

Alexander disease evolution over time: data from an Italian cohort of pediatric-onset patients.

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by dominant variants in the Glial Fibrillary Acidic Protein gene. Three main classifications are currently used, the traditional one defined by the age of onset, and two more recent ones based on both clinical features at onset and brain MRI findings. In this study, we retrospectively included patients with genetically confirmed pediatric-onset AxD. Twenty-one Italian patients were enrolled, and we revised all their clinical and radiological data. Participants were divided according to the current classification systems. We qualitatively analyzed data on neurodevelopment and neurologic decline in order to identify the possible trajectories of the evolution of the disease over time. One patient suffered from a Neonatal presentation and showed a rapidly evolving course which led to death within the second year of life (Type Ia). 16 patients suffered from the Infantile presentation: 5 of them (here defined Type Ib) presented developmental delay and began to deteriorate by the age of 5. A second group (Type Ic) included patients who presented a delay in neuromotor development and started deteriorating after 6 years of age. A third group (Type Id) included patients who presented developmental delay and remained clinically stable beyond adolescence. In 4 patients, the age at last evaluation made it not possible to ascertain whether they belonged to Type Ic or Id, as they were too young to evaluate their neurologic decline. 4 patients suffered from the Juvenile presentation: they had normal neuromotor development with no or only mild cognitive impairment; the subsequent clinical evolution was similar to Type Ic AxD in 2 patients, to Id group in the other 2. In conclusion, our results confirm previously described findings about clinical features at onset; based on follow-up data we might classify patients with Type I AxD into four subgroups (Ia, Ib, Ic, Id). Further studies will be needed to confirm our results and to better highlight the existence of clinical and neuroradiological prognostic factors able to predict disease progression.

Spinal cord involvement and paroxysmal events in "Infantile Onset Transient Hypomyelination" due to TMEM63A mutation.

Monoallelic mutations on TMEM63A have been recently reported as cause of a previously unrecognized disorder named "infantile-onset transient hypomyelination". Clinical and neuroradiological presentation is described as highly similar to Pelizaeus-Merzbacher Disease but evolution over time was surprisingly benign with a progressive spontaneous improving course. We report on a new TMEM63A-mutated girl. The clinical picture was similar to the one already described except for the presence of recurrent episodes of unilateral eyelid twitching, and for the evidence of spinal cord involvement on MRI. These are interesting findings helping in distinguishing this condition from classic PMD since early disease stages. However, additional observations are needed to confirm if these are common features of this condition.

Placental features of fetal vascular malperfusion and infant neurodevelopmental outcomes at 2 years of age in severe fetal growth restriction.

BACKGROUND: Placental pathologic lesions suggesting maternal or fetal vascular malperfusion are common among pregnancies complicated by intrauterine growth restriction. Data on the relationship between pathologic placental lesions and subsequent infant neurodevelopmental outcomes are limited. OBJECTIVE: This study aimed to assess the relationship between placental pathologic lesions and infant neurodevelopmental outcomes at 2 years of age in a cohort of pregnancies complicated by intrauterine growth restriction. STUDY DESIGN: An observational cohort study included singleton intrauterine growth restriction pregnancies delivered at ≤34 weeks' gestation and with a birthweight of ≤1500 g at a single institution in the period between 2007 and 2016. Maternal and neonatal data were collected at discharge from the hospital. Infant neurodevelopmental assessment was performed every 3 months during the first year of life and every 6 months in the second year. Penalized logistic regression was used to test the association of maternal vascular malperfusion and fetal vascular malperfusion with infant outcomes adjusting for confounders. RESULTS: Of the 249 pregnancies enrolled, neonatal mortality was 8.8% (22 of 249). Severe and overall maternal vascular malperfusion were 16.1% (40 of 249) and 31.7% (79 of 249), respectively. Severe maternal vascular malperfusion was associated with an increased risk of neonatal mortality (adjusted odds ratio, 3.3; 95% confidence interval, 1.2-9.5). Among the 198 survivors after a 2-year neurodevelopmental follow-up evaluation, the rate of major and minor neurodevelopmental sequelae was 57.1% (4 of 7) among severe fetal vascular malperfusion (adjusted odds ratio, 24.5; 95% confidence interval, 4.1-146), 44.8% (13 of 29) among overall fetal vascular malperfusion (adjusted odds ratio, 5.8; 95% confidence interval, 5.1-16.2), and 7.1% (12 of 169) in pregnancies without fetal vascular malperfusion. Infants born from pregnancies with fetal vascular malperfusion also had lower 2-year general quotient, personal-social, hearing and speech, and performance subscales scores than those without fetal vascular malperfusion. Finally, in the presence of fetal vascular malperfusion, the likelihood of a 2-year infant survival with normal neurodevelopmental outcomes was reduced by more than 70% (adjusted odds ratio, 0.29; 95% confidence interval, 0.14-0.63). Noticeably, 10 of the 20 subjects with a 2-year major neurodevelopmental impairment (3 of 4 with severe fetal vascular malperfusion) had little or no abnormal neurologic findings at discharge from neonatal intensive care unit. CONCLUSION: In preterm intrauterine growth restriction, placental fetal vascular malperfusion is correlated with an increased risk of abnormal infant neurodevelopmental outcomes at 2 years of age even in the absence of brain lesions or neurologic abnormalities at discharge from the neonatal intensive care unit. In the case of a diagnosis of fetal vascular malperfusion, pediatricians and neurologists should be alerted to an increased risk of subsequent infant neurodevelopmental problems.

Ruxolitinib in Aicardi-Goutières syndrome.

Aicardi-Goutières Syndrome (AGS) is a monogenic leukodystrophy with pediatric onset, clinically characterized by a variable degree of neurologic impairment. It belongs to a group of condition called type I interferonopathies that are characterized by abnormal overproduction of interferon alpha, an inflammatory cytokine which action is mediated by the activation of two of the four human Janus Kinases. Thanks to an ever-increasing knowledge of the molecular basis and pathogenetic mechanisms of the disease, Janus Kinase inhibitors (JAKIs) have been proposed as a treatment option for selected interferonopathies. Here we reported the 24 months follow-up of the fifth AGS patient treated with ruxolitinib described so far in literature. The treatment was globally well tolerated; clinical examinations and radiological images demonstrated a progressively improving course. It is however to note that patients presenting with mild and spontaneously improving course have been reported. Large natural history studies on AGS spectrum are strongly required in order to get a better understanding of the results emerging from ongoing therapeutic trials on such rare disease.

Genetic and phenotypic spectrum associated with IFIH1 gain-of-function.

IFIH1 gain-of-function has been reported as a cause of a type I interferonopathy encompassing a spectrum of autoinflammatory phenotypes including Aicardi-Goutières syndrome and Singleton Merten syndrome. Ascertaining patients through a European and North American collaboration, we set out to describe the molecular, clinical and interferon status of a cohort of individuals with pathogenic heterozygous mutations in IFIH1. We identified 74 individuals from 51 families segregating a total of 27 likely pathogenic mutations in IFIH1. Ten adult individuals, 13.5% of all mutation carriers, were clinically asymptomatic (with seven of these aged over 50 years). All mutations were associated with enhanced type I interferon signaling, including six variants (22%) which were predicted as benign according to multiple in silico pathogenicity programs. The identified mutations cluster close to the ATP binding region of the protein. These data confirm variable expression and nonpenetrance as important characteristics of the IFIH1 genotype, a consistent association with enhanced type I interferon signaling, and a common mutational mechanism involving increased RNA binding affinity or decreased efficiency of ATP hydrolysis and filament disassembly rate.

MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

Spontaneous MRI improvement and absence of cerebral calcification in Aicardi-Goutières syndrome: Diagnostic and disease-monitoring implications.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a rare genetic leukoencephalopathy related to inappropriate activation of type I interferon. Neuroradiological findings are typically characterized by white matter abnormalities, cerebral atrophy and cerebral calcification. The disease usually manifests itself during the first year of life in the form of an initial "encephalitic-like" phase followed by a chronic phase of stabilization of the neurological signs. Recently new therapeutic strategies have been proposed aimed at blocking the abnormal activation of the interferon cascade. MATERIALS AND METHODS: We reviewed clinical and MRI findings in three young RNASEH2B-mutated patients studied with serial CT and MRI studies. RESULTS: All three patients presented clinical and MRI features consistent with AGS but, very unexpectedly, an improving neuroradiological course. In patient 1, the MRI improvement was noted some months after treatment with high-dose steroid and IVIg treatment; in patients 2 and 3 it occurred spontaneously. Patient 2 did not show cerebral calcification on CT images. CONCLUSIONS: Our series highlights the possibility of spontaneous neuroradiological improvement in AGS2 patients, as well as the possibility of absence of cerebral calcification in AGS. The study underlines the need for extreme caution when using MRI as an outcome measure in therapeutic trials specific for this disease. MRI follow-up studies in larger series are necessary to describe the natural course of AGS.

Expanding the phenotypic spectrum of Allan-Herndon-Dudley syndrome in patients with SLC16A2 mutations.

The aim of the study was to redefine the phenotype of Allan-Herndon-Dudley syndrome (AHDS), which is caused by mutations in the SLC16A2 gene that encodes the brain transporter of thyroid hormones. Clinical phenotypes, brain imaging, thyroid hormone profiles, and genetic data were compared to the existing literature. Twenty-four males aged 11 months to 29 years had a mutation in SLC16A2, including 12 novel mutations and five previously described mutations. Sixteen patients presented with profound developmental delay, three had severe intellectual disability with poor language and walking with an aid, four had moderate intellectual disability with language and walking abilities, and one had mild intellectual disability with hypotonia. Overall, eight had learned to walk, all had hypotonia, 17 had spasticity, 18 had dystonia, 12 had choreoathetosis, 19 had hypomyelination, and 10 had brain atrophy. Kyphoscoliosis (n=12), seizures (n=7), and pneumopathies (n=5) were the most severe complications. This study extends the phenotypic spectrum of AHDS to a mild intellectual disability with hypotonia. Developmental delay, hypotonia, hypomyelination, and thyroid hormone profile help to diagnose patients. Clinical course depends on initial severity, with stable acquisition after infancy; this may be adversely affected by neuro-orthopaedic, pulmonary, and epileptic complications. WHAT THIS PAPER ADDS: Mild intellectual disability is associated with SLC16A2 mutations. A thyroid hormone profile with a free T3 /T4 ratio higher than 0.75 can help diagnose patients. Patients with SLC16A2 mutations present a broad spectrum of neurological phenotypes that are also observed in other hypomyelinating disorders. Axial hypotonia is a consistent feature of Allan-Herndon-Dudley syndrome and leads to specific complications.

Novel mutations in SLC16A2 associated with a less severe phenotype of MCT8 deficiency.

Mutations in the thyroid hormone transporter MCT8 cause severe intellectual and motor disability and abnormal serum thyroid function tests, a syndrome known as MCT8 deficiency (or: Allan-Herndon-Dudley syndrome, AHDS). Although the majority of patients are unable to sit or walk independently and do not develop any speech, some are able to walk and talk in simple sentences. Here, we report on two cases with such a less severe clinical phenotype and consequent gross delay in diagnosis. Genetic analyses revealed two novel hemizygous mutations in the SLC16A2 gene resulting in a p.Thr239Pro and a p.Leu543Pro substitution in the MCT8 protein. In vitro studies in transiently transfected COS-1 and JEG-3 cells, and ex vivo studies in patient-derived fibroblasts revealed substantial residual uptake capacity of both mutant proteins (Leu543Pro > Thr239Pro), providing an explanation for the less severe clinical phenotype. Both mutations impair MCT8 protein stability and interfere with proper subcellular trafficking. In one of the patients calcifications were observed in the basal ganglia at the age of 29 years; an abnormal neuroradiological feature at this age that has been linked to untreated (congenital) hypothyroidism and neural cretinism. Our studies extend on previous work by identifying two novel pathogenic mutations in SLC16A2 gene resulting in a mild clinical phenotype.

Compound heterozygous missense and deep intronic variants in NDUFAF6 unraveled by exome sequencing and mRNA analysis.

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions.

Neonatal detection of Aicardi Goutières Syndrome by increased C26:0 lysophosphatidylcholine and interferon signature on newborn screening blood spots.

BACKGROUND: Aicardi Goutières Syndrome (AGS) is a heritable interferonopathy associated with systemic autoinflammation causing interferon (IFN) elevation, central nervous system calcifications, leukodystrophy and severe neurologic sequelae. An infant with TREX1 mutations was recently found to have abnormal C26:0 lysophosphatidylcholine (C26:0 Lyso-PC) in a newborn screening platform for X-linked adrenoleukodystrophy, prompting analysis of this analyte in retrospectively collected samples from individuals affected by AGS. METHODS: In this study, we explored C26:0 Lyso-PC levels and IFN signatures in newborn blood spots and post-natal blood samples in 19 children with a molecular and clinical diagnosis of AGS and in the blood spots of 22 healthy newborns. We used Nanostring nCounter™ for IFN-induced gene analysis and a high-performance liquid chromatography with tandem mass spectrometry (HPLC MS/MS) newborn screening platform for C26:0 Lyso-PC analysis. RESULTS: Newborn screening cards from patients across six AGS associated genes were collected, with a median disease presentation of 2months. Thirteen out of 19 (68%) children with AGS had elevations of first tier C26:0 Lyso-PC (>0.4µM), that would have resulted in a second screen being performed in a two tier screening system for X-linked adrenoleukodystrophy (X-ALD). The median (95%CI) of first tier C26:0 Lyso-PC values in AGS individuals (0.43µM [0.37-0.48]) was higher than that seen in controls (0.21µM [0.21-0.21]), but lower than X-ALD individuals (0.72µM [0.59-0.84])(p<0.001). Fourteen of 19 children had elevated expression of IFN signaling on blood cards relative to controls (Sensitivity 73.7%, 95%CI 51-88%, Specificity 95%, 95% CI 78-99%) including an individual with delayed disease presentation (36months of age). All five AGS patients with negative IFN signature at birth had RNASEH2B mutations. Consistency of agreement between IFN signature in neonatal and post-natal samples was high (0.85). CONCLUSION: This suggests that inflammatory markers in AGS can be identified in the newborn period, before symptom onset. Additionally, since C26:0 Lyso-PC screening is currently used in X-ALD newborn screening panels, clinicians should be alert to the fact that AGS infants may present as false positives during X-ALD screening.