Clinical features and genotype-phenotype correlations in epilepsy patients with de novo DYNC1H1 variants.

OBJECTIVE: DYNC1H1 variants are involved on a disease spectrum from neuromuscular disorders to neurodevelopmental disorders. DYNC1H1-related epilepsy has been reported in small cohorts. We dissect the electroclinical features of 34 patients harboring de novo DYNC1H1 pathogenic variants, identify subphenotypes on the DYNC1H1-related epilepsy spectrum, and compare the genotype-phenotype correlations observed in our cohort with the literature. METHODS: Patients harboring de novo DYNC1H1 pathogenic variants were recruited through international collaborations. Clinical data were retrospectively collected. Latent class analysis was performed to identify subphenotypes. Multivariable binary logistic regression analysis was applied to investigate the association with DYNC1H1 protein domains. RESULTS: DYNC1H1-related epilepsy presented with infantile epileptic spasms syndrome (IESS) in 17 subjects (50%), and in 25% of these individuals the epileptic phenotype evolved into Lennox-Gastaut syndrome (LGS). In 12 patients (35%), focal onset epilepsy was defined. In two patients, the epileptic phenotype consisted of generalized myoclonic epilepsy, with a progressive phenotype in one individual harboring a frameshift variant. In approximately 60% of our cohort, seizures were drug-resistant. Malformations of cortical development were noticed in 79% of our patients, mostly on the lissencephaly-pachygyria spectrum, particularly with posterior predominance in a half of them. Midline and infratentorial abnormalities were additionally reported in 45% and 27% of subjects. We have identified three main classes of subphenotypes on the DYNC1H1-related epilepsy spectrum. SIGNIFICANCE: We propose a classification in which pathogenic de novo DYNC1H1 variants feature drug-resistant IESS in half of cases with potential evolution to LGS (Class 1), developmental and epileptic encephalopathy other than IESS and LGS (Class 2), or less severe focal or genetic generalized epilepsy including a progressive phenotype (Class 3). We observed an association between stalk domain variants and Class 1 phenotypes. The variants p.Arg309His and p.Arg1962His were common and associated with Class 1 subphenotype in our cohort. These findings may aid genetic counseling of patients with DYNC1H1-related epilepsy.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency.

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we found that neurodegeneration resistant regions accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the hippocampal dentate gyrus, which shows a progressive neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 deficient neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant, impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation in neurons with available GTP precursor molecules, providing resistance to neurodegeneration. Our findings open the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

Heterozygous nonsense variants in the ferritin heavy-chain gene FTH1 cause a neuroferritinopathy.

Ferritin, the iron-storage protein, is composed of light- and heavy-chain subunits, encoded by FTL and FTH1, respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole-exome sequencing, with a recurrent variant (p.Phe171∗) identified in four unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia, and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminal variants in FTH1 truncate ferritin's E helix, altering the 4-fold symmetric pores of the heteropolymer, and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a disorder in the spectrum of NBIA. Targeted knockdown of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this pediatric neurodegenerative disorder.

Expanding genotype-phenotype correlations in FOXG1 syndrome: results from a patient registry.

BACKGROUND: We refine the clinical spectrum of FOXG1 syndrome and expand genotype-phenotype correlations through evaluation of 122 individuals enrolled in an international patient registry. METHODS: The FOXG1 syndrome online patient registry allows for remote collection of caregiver-reported outcomes. Inclusion required documentation of a (likely) pathogenic variant in FOXG1. Caregivers were administered a questionnaire to evaluate clinical severity of core features of FOXG1 syndrome. Genotype-phenotype correlations were determined using nonparametric analyses. RESULTS: We studied 122 registry participants with FOXG1 syndrome, aged < 12 months to 24 years. Caregivers described delayed or absent developmental milestone attainment, seizures (61%), and movement disorders (58%). Participants harbouring a missense variant had a milder phenotype. Compared to individuals with gene deletions (0%) or nonsense variants (20%), missense variants were associated with more frequent attainment of sitting (73%). Further, individuals with missense variants (41%) achieved independent walking more frequently than those with gene deletions (0%) or frameshift variants (6%). Presence of epilepsy also varied by genotype and was significantly more common in those with gene deletions (81%) compared to missense variants (47%). Individuals with gene deletions were more likely to have higher seizure burden than other genotypes with 53% reporting daily seizures, even at best control. We also observed that truncations preserving the forkhead DNA binding domain were associated with better developmental outcomes. CONCLUSION: We refine the phenotypic spectrum of neurodevelopmental features associated with FOXG1 syndrome. We strengthen genotype-driven outcomes, where missense variants are associated with a milder clinical course.

Heterozygous Nonsense Variants in the Ferritin Heavy Chain Gene FTH1 Cause a Novel Pediatric Neuroferritinopathy.

Ferritin, the iron storage protein, is composed of light and heavy chain subunits, encoded by FTL and FTH1 , respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole exome sequencing, with a recurrent de novo variant (p.F171*) identified in three unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminus variants in FTH1 truncate ferritin's E-helix, altering the four-fold symmetric pores of the heteropolymer and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a novel disorder in the spectrum of NBIA. Targeted knock-down of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this novel pediatric neurodegenerative disorder.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

Pathogenic variants in CASK: Expanding the genotype-phenotype correlations.

Pathogenic variants in CASK, an X-linked gene that plays a role in brain development and synaptic function, are the cause of both microcephaly with pontine and cerebellar hypoplasia (MICPCH), and X-linked intellectual disability (XLID) with or without nystagmus. MICPCH is caused by loss of function variants in CASK, typically affects females, and is associated with moderate-to-severe intellectual disability (ID). Additional findings, present in about one-third of individuals, include feeding difficulties, ophthalmologic issues, hypertonicity, epilepsy, and sensorineural hearing loss. Only a few affected males with MICPCH phenotype have been reported and most have had profound developmental disability and intractable epilepsy. The XLID phenotype is typically caused by missense variants and most often manifests in males; carrier females are mildly affected or unaffected. Nystagmus is often present. In total, over 175 patients have been reported in the literature. We now report an additional 11 patients with pathogenic variants in CASK that expand these phenotypes and reported genotype-phenotype correlations.

Lysosomal dysfunction impairs mitochondrial quality control and is associated with neurodegeneration in TBCK encephaloneuronopathy.

Biallelic variants in the TBCK gene cause intellectual disability with remarkable clinical variability, ranging from static encephalopathy to progressive neurodegeneration (TBCK-Encephaloneuronopathy). The biological factors underlying variable disease penetrance remain unknown. Since previous studies had suggested aberrant autophagy, we tested whether mitophagy and mitochondrial function are altered in TBCK -/- fibroblasts derived from patients exhibiting variable clinical severity. Our data show significant accumulation of mitophagosomes, reduced mitochondrial respiratory capacity and mitochondrial DNA content, suggesting impaired mitochondrial quality control. Furthermore, the degree of mitochondrial dysfunction correlates with a neurodegenerative clinical course. Since mitophagy ultimately depends on lysosomal degradation, we also examined lysosomal function. Our data show that lysosomal proteolytic function is significantly reduced in TBCK -/- fibroblasts. Moreover, acidifying lysosomal nanoparticles rescue the mitochondrial respiratory defects in fibroblasts, suggesting impaired mitochondrial quality control secondary to lysosomal dysfunction. Our data provide insight into the disease mechanisms of TBCK Encephaloneuronopathy and the potential relevance of mitochondrial function as a biomarker beyond primary mitochondrial disorders. It also supports the benefit of lysosomal acidification strategies for disorders of impaired lysosomal degradation affecting mitochondrial quality control.

Mitochondrial Dysfunction: A Common Denominator in Neurodevelopmental Disorders?

Mitochondria, the organelles classically seen as the powerhouse of the cell, are increasingly associated with a wide variety of neurodevelopmental disorders. Although individually rare, a myriad of pediatric neurogenetic disorders have been identified in the last few years, thanks to advances in clinical genetic sequencing and data analysis. As this exponential growth continues, mitochondrial dysfunction is increasingly implicated in childhood neurodevelopmental disorders, with clinical presentations ranging from syndromic autism, intellectual disability, and epileptic encephalopathies to childhood onset neurodegeneration. Here we review recent evidence demonstrating mitochondrial involvement in neurodevelopmental disorders, identify emerging mechanistic trends, and reconsider the long-standing question of the role of mitochondria in light of new evidence: causation versus mere association.

Phenotypic expansion of CACNA1C-associated disorders to include isolated neurological manifestations.

PURPOSE: CACNA1C encodes the alpha-1-subunit of a voltage-dependent L-type calcium channel expressed in human heart and brain. Heterozygous variants in CACNA1C have previously been reported in association with Timothy syndrome and long QT syndrome. Several case reports have suggested that CACNA1C variation may also be associated with a primarily neurological phenotype. METHODS: We describe 25 individuals from 22 families with heterozygous variants in CACNA1C, who present with predominantly neurological manifestations. RESULTS: Fourteen individuals have de novo, nontruncating variants and present variably with developmental delays, intellectual disability, autism, hypotonia, ataxia, and epilepsy. Functional studies of a subgroup of missense variants via patch clamp experiments demonstrated differential effects on channel function in vitro, including loss of function (p.Leu1408Val), neutral effect (p.Leu614Arg), and gain of function (p.Leu657Phe, p.Leu614Pro). The remaining 11 individuals from eight families have truncating variants in CACNA1C. The majority of these individuals have expressive language deficits, and half have autism. CONCLUSION: We expand the phenotype associated with CACNA1C variants to include neurodevelopmental abnormalities and epilepsy, in the absence of classic features of Timothy syndrome or long QT syndrome.

Assessing seizure burden in pediatric epilepsy using an electronic medical record-based tool through a common data element approach.

OBJECTIVE: Improvement in epilepsy care requires standardized methods to assess disease severity. We report the results of implementing common data elements (CDEs) to document epilepsy history data in the electronic medical record (EMR) after 12 months of clinical use in outpatient encounters. METHODS: Data regarding seizure frequency were collected during routine clinical encounters using a CDE-based form within our EMR. We extracted CDE data from the EMR and developed measurements for seizure severity and seizure improvement scores. Seizure burden and improvement was evaluated by patient demographic and encounter variables for in-person and telemedicine encounters. RESULTS: We assessed a total of 1696 encounters in 1038 individuals with childhood epilepsies between September 6, 2019 and September 11, 2020 contributed by 32 distinct providers. Childhood absence epilepsy (n = 121), Lennox-Gastaut syndrome (n = 86), and Dravet syndrome (n = 42) were the most common epilepsy syndromes. Overall, 43% (737/1696) of individuals had at least monthly seizures, 17% (296/1696) had a least daily seizures, and 18% (311/1696) were seizure-free for >12 months. Quantification of absolute seizure burden and changes in seizure burden over time differed between epilepsy syndromes, including high and persistent seizure burden in patients with Lennox-Gastaut syndrome. Individuals seen via telemedicine or in-person encounters had comparable seizure frequencies. Individuals identifying as Hispanic/Latino, particularly from postal codes with lower median household incomes, were more likely to have ongoing seizures that worsened over time. SIGNIFICANCE: Standardized documentation of clinical data in childhood epilepsies through CDE can be implemented in routine clinical care at scale and enables assessment of disease burden, including characterization of seizure burden over time. Our data provide insights into heterogeneous patterns of seizure control in common pediatric epilepsy syndromes and will inform future initiatives focusing on patient-centered outcomes in childhood epilepsies, including the impact of telemedicine and health care disparities.

ANKRD11 variants: KBG syndrome and beyond.

Mutations affecting the transcriptional regulator Ankyrin Repeat Domain 11 (ANKRD11) are mainly associated with the multisystem developmental disorder known as KBG syndrome, but have also been identified in individuals with Cornelia de Lange syndrome (CdLS) and other developmental disorders caused by variants affecting different chromatin regulators. The extensive functional overlap of these proteins results in shared phenotypical features, which complicate the assessment of the clinical diagnosis. Additionally, re-evaluation of individuals at a later age occasionally reveals that the initial phenotype has evolved toward clinical features more reminiscent of a developmental disorder different from the one that was initially diagnosed. For this reason, variants in ANKRD11 can be ascribed to a broader class of disorders that fall within the category of the so-called chromatinopathies. In this work, we report on the clinical characterization of 23 individuals with variants in ANKRD11. The subjects present primarily with developmental delay, intellectual disability and dysmorphic features, and all but two received an initial clinical diagnosis of either KBG syndrome or CdLS. The number and the severity of the clinical signs are overlapping but variable and result in a broad spectrum of phenotypes, which could be partially accounted for by the presence of additional molecular diagnoses and distinct pathogenic mechanisms.

Understanding the phenotypic spectrum of ASXL-related disease: Ten cases and a review of the literature.

Over the past decade, pathogenic variants in all members of the ASXL family of genes, ASXL1, ASXL2, and ASXL3, have been found to lead to clinically distinct but overlapping syndromes. Bohring-Opitz syndrome (BOPS) was first described as a clinical syndrome and later found to be associated with pathogenic variants in ASXL1. This syndrome is characterized by developmental delay, microcephaly, characteristic facies, hypotonia, and feeding difficulties. Subsequently, pathogenic variants in ASXL2 were found to lead to Shashi-Pena syndrome (SHAPNS) and in ASXL3 to lead to Bainbridge-Ropers syndrome (BRPS). While SHAPNS and BRPS share many core features with BOPS, there also seem to be emerging clear differences. Here, we present five cases of BOPS, one case of SHAPNS, and four cases of BRPS. By adding our cohort to the limited number of previously published patients, we review the overlapping features of ASXL-related diseases that bind them together, while focusing on the characteristics that make each neurodevelopmental syndrome unique. This will assist in diagnosis of these overlapping conditions and allow clinicians to more comprehensively counsel affected families.

Boricua Founder Variant in FRRS1L Causes Epileptic Encephalopathy With Hyperkinetic Movements.

OBJECTIVE: To describe a founder mutation effect and the clinical phenotype of homozygous FRRS1L c.737_739delGAG (p.Gly246del) variant in 15 children of Puerto Rican (Boricua) ancestry presenting with early infantile epileptic encephalopathy (EIEE-37) with prominent movement disorder. BACKGROUND: EIEE-37 is caused by biallelic loss of function variants in the FRRS1L gene, which is critical for AMPA-receptor function, resulting in intractable epilepsy and dyskinesia. METHODS: A retrospective, multicenter chart review of patients sharing the same homozygous FRRS1L (p.Gly246del) pathogenic variant identified by clinical genetic testing. Clinical information was collected regarding neurodevelopmental outcomes, neuroimaging, electrographic features and clinical response to antiseizure medications. RESULTS: Fifteen patients from 12 different families of Puerto Rican ancestry were homozygous for the FRRS1L (p.Gly246del) pathogenic variant, with ages ranging from 1 to 25 years. The onset of seizures was from 6 to 24 months. All had hypotonia, severe global developmental delay, and most had hyperkinetic involuntary movements. Developmental regression during the first year of life was common (86%). Electroencephalogram showed hypsarrhythmia in 66% (10/15), with many older children evolving into Lennox-Gastaut syndrome. Six patients demonstrated progressive volume loss and/or cerebellar atrophy on brain magnetic resonance imaging (MRI). CONCLUSIONS: We describe the largest cohort to date of patients with epileptic encephalopathy. We estimate that 0.76% of unaffected individuals of Puerto Rican ancestry carry this pathogenic variant due to a founder effect. Children homozygous for the FRRS1L (p.Gly246del) Boricua variant exhibit a very homogenous phenotype of early developmental regression and epilepsy, starting with infantile spasms and evolving into Lennox-Gastaut syndrome with hyperkinetic movement disorder.

Intracranial calcifications in childhood: Part 1.

This article is the first of a two-part series on intracranial calcification in childhood. Intracranial calcification can be either physiological or pathological. Physiological intracranial calcification is not an expected neuroimaging finding in the neonatal or infantile period but occurs, as children grow older, in the pineal gland, habenula, choroid plexus and occasionally the dura mater. Pathological intracranial calcification can be broadly divided into infectious, congenital, endocrine/metabolic, vascular and neoplastic. The main goals in Part 1 are to discuss the chief differences between physiological and pathological intracranial calcification, to discuss the histological characteristics of intracranial calcification and how intracranial calcification can be detected across neuroimaging modalities, to emphasize the importance of age at presentation and intracranial calcification location, and to propose a comprehensive neuroimaging approach toward the differential diagnosis of the causes of intracranial calcification. Finally, in Part 1 the authors discuss the most common causes of infectious intracranial calcification, especially in the neonatal period, and congenital causes of intracranial calcification. Various neuroimaging modalities have distinct utilities and sensitivities in the depiction of intracranial calcification. Age at presentation, intracranial calcification location, and associated neuroimaging findings are useful information to help narrow the differential diagnosis of intracranial calcification. Intracranial calcification can occur in isolation or in association with other neuroimaging features. Intracranial calcification in congenital infections has been associated with clastic changes, hydrocephalus, chorioretinitis, white matter abnormalities, skull changes and malformations of cortical development. Infections are common causes of intracranial calcification, especially neonatal TORCH (toxoplasmosis, other [syphilis, varicella-zoster, parvovirus B19], rubella, cytomegalovirus and herpes) infections.

Intracranial calcifications in childhood: Part 2.

This article is the second of a two-part series on intracranial calcification in childhood. In Part 1, the authors discussed the main differences between physiological and pathological intracranial calcification. They also outlined histological intracranial calcification characteristics and how these can be detected across different neuroimaging modalities. Part 1 emphasized the importance of age at presentation and intracranial calcification location and proposed a comprehensive neuroimaging approach toward the differential diagnosis of the causes of intracranial calcification. Pathological intracranial calcification can be divided into infectious, congenital, endocrine/metabolic, vascular, and neoplastic. In Part 2, the chief focus is on discussing endocrine/metabolic, vascular, and neoplastic intracranial calcification etiologies of intracranial calcification. Endocrine/metabolic diseases causing intracranial calcification are mainly from parathyroid and thyroid dysfunction and inborn errors of metabolism, such as mitochondrial disorders (MELAS, or mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; Kearns-Sayre; and Cockayne syndromes), interferonopathies (Aicardi-Goutières syndrome), and lysosomal disorders (Krabbe disease). Specific noninfectious causes of intracranial calcification that mimic TORCH (toxoplasmosis, other [syphilis, varicella-zoster, parvovirus B19], rubella, cytomegalovirus, and herpes) infections are known as pseudo-TORCH. Cavernous malformations, arteriovenous malformations, arteriovenous fistulas, and chronic venous hypertension are also known causes of intracranial calcification. Other vascular-related causes of intracranial calcification include early atherosclerosis presentation (children with risk factors such as hyperhomocysteinemia, familial hypercholesterolemia, and others), healed hematoma, radiotherapy treatment, old infarct, and disorders of the microvasculature such as COL4A1- and COL4A2-related diseases. Intracranial calcification is also seen in several pediatric brain tumors. Clinical and familial information such as age at presentation, maternal exposure to teratogens including viruses, and association with chromosomal abnormalities, pathogenic genes, and postnatal infections facilitates narrowing the differential diagnosis of the multiple causes of intracranial calcification.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes.

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband's fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.