Pathogenic cryptic variants detectable through exome data reanalysis significantly increase the diagnostic yield in Joubert syndrome.

Joubert syndrome (JS) is a genetically heterogeneous neurodevelopmental ciliopathy. Despite exome sequencing (ES), several patients remain undiagnosed. This study aims to increase the diagnostic yield by uncovering cryptic variants through targeted ES reanalysis. We first focused on 26 patients in whom ES only disclosed heterozygous pathogenic coding variants in a JS gene. We reanalyzed raw ES data searching for copy number variants (CNVs) and intronic variants affecting splicing. We validated CNVs through real-time PCR or chromosomal microarray, and splicing variants through RT-PCR or minigenes. Cryptic variants were then searched in additional 44 ES-negative JS individuals. We identified cryptic "second hits" in 14 of 26 children (54%) and biallelic cryptic variants in 3 of 44 (7%), reaching a definite diagnosis in 17 of 70 (overall diagnostic gain 24%). We show that CNVs and intronic splicing variants are a common mutational mechanism in JS; more importantly, we demonstrate that a significant proportion of such variants can be disclosed simply through a focused reanalysis of available ES data, with a significantly increase of the diagnostic yield especially among patients previously found to carry heterozygous coding variants in the KIAA0586, CC2D2A and CPLANE1 genes.

Burden re-analysis of neurodevelopmental disorder cohorts for prioritization of candidate genes.

This study aimed to uncover novel genes associated with neurodevelopmental disorders (NDD) by leveraging recent large-scale de novo burden analysis studies to enhance a virtual gene panel used in a diagnostic setting. We re-analyzed historical trio-exome sequencing data from 745 individuals with NDD according to the most recent diagnostic standards, resulting in a cohort of 567 unsolved individuals. Next, we designed a virtual gene panel containing candidate genes from three large de novo burden analysis studies in NDD and prioritized candidate genes by stringent filtering for ultra-rare de novo variants with high pathogenicity scores. Our analysis revealed an increased burden of de novo variants in our selected candidate genes within the unsolved NDD cohort and identified qualifying de novo variants in seven candidate genes: RIF1, CAMK2D, RAB11FIP4, AGO3, PCBP2, LEO1, and VCP. Clinical data were collected from six new individuals with de novo or inherited LEO1 variants and three new individuals with de novo PCBP2 variants. Our findings add additional evidence for LEO1 as a risk gene for autism and intellectual disability. Furthermore, we prioritize PCBP2 as a candidate gene for NDD associated with motor and language delay. In summary, by leveraging de novo burden analysis studies, employing a stringent variant filtering pipeline, and engaging in targeted patient recruitment, our study contributes to the identification of novel genes implicated in NDDs.

Novel molecular, structural and clinical findings in an Italian cohort of congenital cataract.

The current genetic diagnostic workup of congenital cataract (CC) is mainly based on NGS panels, whereas exome sequencing (ES) has occasionally been employed. In this multicentre study, we investigated by ES the detection yield, mutational spectrum and genotype-phenotype correlations in a CC cohort recruited between 2020 and mid-2022. The cohort consisted of 67 affected individuals from 51 unrelated families and included both non-syndromic (75%) and syndromic (25%) phenotypes, with extra-CC ocular/visual features present in both groups (48% and 76%, respectively). The functional effect of variants was predicted by 3D modelling and hydropathy properties changes. Variant clustering was used for the in-depth assessment of genotype-phenotype correlations. A diagnostic (pathogenic or likely pathogenic) variant was identified in 19 out of 51 probands/families (~37%). In a further 14 probands/families a candidate variant was identified: in 12 families a VUS was detected, of which 9 were considered plausibly pathogenic (i.e., 4 or 5 points according to ACMG criteria), while in 2 probands ES identified a single variant in an autosomal recessive gene associated with CC. Eighteen probands/families, manifesting primarily non-syndromic CC (15/18, 83%), remained unsolved. The identified variants (8 P, 12 LP, 10 VUS-PP, and 5 VUS), half of which were unreported in the literature, affected five functional categories of genes involved in transcription/splicing, lens formation/homeostasis (i.e., crystallin genes), membrane signalling, cell-cell interaction, and immune response. A phenotype-specific variant clustering was observed in four genes (KIF1A, MAF, PAX6, SPTAN1), whereas variable expressivity and potential phenotypic expansion in two (BCOR, NHS) and five genes (CWC27, KIF1A, IFIH1, PAX6, SPTAN1), respectively. Finally, ES allowed to detect variants in six genes not commonly included in commercial CC panels. These findings broaden the genotype-phenotype correlations in one of the largest CC cohorts tested by ES, providing novel insights into the underlying pathogenetic mechanisms and emphasising the power of ES as first-tier test.

LZTFL1, a rare cause of Bardet-Biedl syndrome: A new patient with severe short stature and moderate intellectual disability, more than casual associations?

Bardet-Biedl syndrome (BBS) is an inherited ciliopathy affecting multiple organs and systems with wide clinical and genetic heterogeneity. To date, biallelic variants of the LZTFL1 gene have been reported only in six patients with BBS. We identified a homozygous LZTFL1 nonsense variant in a boy presenting with classical BBS features. In addition, he showed a more pronounced cognitive impairment than previously reported subjects and severe short stature, matching the phenotype displayed by some other patients with LZTFL1 variants and lztfl1 knock-out mice. This case report contributes to a better understanding of the clinical spectrum associated with LZTFL1 pathogenic variants, and highlights possible genotype-phenotype correlations.

Functional Study of SNCA p.V15A Variant: Further Linking α-Synuclein and Glucocerebrosidase.

BACKGROUND: SNCA p.V15A was reported in five families. In vitro models showed increased aggregation and seeding activity, mitochondrial damage, and apoptosis. Mutant flies had reduced flying ability and survival. OBJECTIVES: To clinically and functionally evaluate SNCA p.V15A in a large Italian family with Parkinson's disease (PD). METHODS: Genetic diagnosis was reached through next-generation sequencing. Pathogenicity was assessed by molecular dynamics simulation and biochemical studies on peripheral blood mononuclear cells (PBMCs). RESULTS: Five siblings carried SNCA p.V15A; three developed bradykinetic-rigid PD in their 50s with rapid motor progression and variable cognitive impairment. A fourth sibling had isolated mood disturbance, whereas the fifth was still unaffected at age 47. The mutant protein showed decreased stability and an unstable folded structure. Proband's PBMCs showed elevated total and phosphorylated α-synuclein (α-syn) levels and significantly reduced glucocerebrosidase activity. CONCLUSION: This study demonstrates accumulation of α-synV15A in PBMCs and strengthens the link between α-syn pathophysiology and glucocerebrosidase dysfunction. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Cerebellar heterotopia in an 11-year-old child with KDM6B-related neurodevelopmental disorder: A case report and review of the literature.

Heterozygous pathogenic variants in KDM6B have recently been associated to a rare neurodevelopmental disorder referred to as "Neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities" and characterized by non-pathognomonic facial and body dysmorphisms, a wide range of neurodevelopmental and behavioral disorders and nonspecific neuroradiological findings. KDM6B encodes a histone demethylase, expressed in different tissues during development, which regulates gene expression through the modulation of chromatin accessibility by RNA polymerase. We herein describe a 11-year-old male patient carrying a novel de novo pathogenic variant in KDM6B exhibiting facial dysmorphisms, dysgraphia, behavioral traits relatable to oppositional defiant, autism spectrum, and attention deficit hyperactivity disorders, a single seizure episode, and a neuroimaging finding of a single cerebellar heterotopic nodule, never described to date in this genetic condition. These findings expand the phenotypic spectrum of this syndrome, highlighting the potential role for KDM6B in cerebellar development and providing valuable insights for genetic counseling.

PGAP2-Related Hyperphosphatasia-Mental Retardation Syndrome: Report of a Novel Patient, Toward a Broadening of Phenotypic Spectrum and Therapeutic Perspectives.

PGAP2 gene has been known to be the cause of "hyperphosphatasia, mental retardation syndrome-3" (HPMRS3). To date, 14 pathogenic variants in PGAP2 have been identified as the cause of this syndrome in 24 patients described in single-case reports or small clinical series with pan-ethnic distribution. We aim to present a pediatric PGAP2-mutated case, intending to further expand the clinical phenotype of the syndrome and to report our experience on a therapeutic approach to drug-resistant epilepsy.We present the clinical, neuroradiological, and genetic characterization of a Caucasian pediatric subject with biallelic pathogenic variants in the PGAP2 gene revealed by next generation sequencing analysis.We identified a subject who presented with global developmental delay and visual impairment. Brain magnetic resonance imaging showed mild hypoplasia of the inferior cerebellar vermis and corpus callosum and mild white matter reduction. Laboratory investigations detected an increase in alkaline phosphatase. At the age of 13 months, he began to present epileptic focal seizures with impaired awareness, which did not respond to various antiseizure medications. Electroencephalogram (EEG) showed progressive background activity disorganization and multifocal epileptic abnormalities. Treatment with high-dose pyridoxine showed partial benefit, but the persistence of seizures and the lack of EEG amelioration prompted us to introduce ketogenic diet treatment.Our case provides a further phenotypical expansion of HPMRS3 to include developmental and epileptic encephalopathy. Due to the limited number of patients reported so far, the full delineation of the clinical spectrum of HPMRS3 and indications for precision medicine would benefit from the description of new cases and their follow-up evaluations.

Electroclinical Improvement in a Patient with Ring Chromosome 20 Syndrome Treated with Zonisamide: A Case Report.

Ring chromosome 20 or r(20) syndrome is a rare chromosomal disorder, mainly characterized by childhood-onset drug-resistant epilepsy with typical electroencephalographic findings, followed by mild to severe cognitive-behavioral decline. Recent studies support a possible role of the dopaminergic system in the epileptogenesis of this syndrome. We report the case of a 13-year-old female with mosaic r(20) who showed typical disease onset and evolution and a remarkable electroclinical improvement with zonisamide. Epilepsy related to r(20) is often medically intractable. When valproate and lamotrigine are not effective, zonisamide could be further investigated as a therapeutic option, since it acts as antifocal and it has a potential role in the prevention of dopamine depletion.

Marked intrafamilial variability of clinical and neuroimaging manifestations in NFIB-related developmental disorder.

NFIB belongs to the nuclear factor I (NFI) family of transcription factors that, by activating or repressing gene expression during embryogenesis, has a relevant role in the development of several organs including the brain. Heterozygous pathogenic variants of NFIB have recently been associated with developmental delay and mild-to-moderate intellectual disability, macrocephaly, nonspecific facial dysmorphisms, and corpus callosum dysgenesis. We identified a heterozygous missense variant in the NFIB gene in a 15-year-old boy with neurodevelopmental disorder and brain malformations, who inherited the variant from his substantially healthy mother presenting only minor physical and neuroanatomical defects.

Recurrent, founder and hypomorphic variants contribute to the genetic landscape of Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a neurodevelopmental ciliopathy characterised by a distinctive mid-hindbrain malformation, the 'molar tooth sign'. Over 40 JS-associated genes are known, accounting for two-thirds of cases. METHODS: While most variants are novel or extremely rare, we report on 11 recurring variants in seven genes, including three known 'founder variants' in the Ashkenazi Jewish, Hutterite and Finnish populations. We evaluated variant frequencies in ~550 European patients with JS and compared them with controls (>15 000 Italian plus gnomAD), and with an independent cohort of ~600 JS probands from the USA. RESULTS: All variants were markedly enriched in the European JS cohort compared with controls. When comparing allele frequencies in the two JS cohorts, the Ashkenazim founder variant (TMEM216 c.218G>T) was significantly enriched in American compared with European patients with JS, while MKS1 c.1476T>G was about 10 times more frequent among European JS. Frequencies of other variants were comparable in the two cohorts. Genotyping of several markers identified four novel European founder haplotypes.Two recurrent variants (MKS1 c.1476T>G and KIAA0586 c.428delG), have been detected in homozygosity in unaffected individuals, suggesting they could act as hypomorphic variants. However, while fibroblasts from a MKS1 c.1476T>G healthy homozygote showed impaired ability to form primary cilia and mildly reduced ciliary length, ciliary parameters were normal in cells from a KIAA0586 c.428delG healthy homozygote. CONCLUSION: This study contributes to understand the complex genetic landscape of JS, explain its variable prevalence in distinct geographical areas and characterise two recurrent hypomorphic variants.

A novel variant in NEUROD2 in a patient with Rett-like phenotype points to Glu130 codon as a mutational hotspot.

BACKGROUND: NEUROD2, encoding the neurogenic differentiation factor 2, is essential for neurodevelopment. To date, heterozygous missense variants in this gene have been identified in eight patients (from six unrelated families) with epileptic encephalopathy and developmental delay. CASE REPORT: We describe a child with initial clinical suspicion of Rett/Rett-like syndrome, in whom exome sequencing detected a novel de novo variant (c.388G > A, p.Glu130Lys) in NEUROD2. Interestingly, a missense change affecting the same codon, c.388G > C (p.Glu130Gln), was previously identified in other two patients. CONCLUSIONS: Our results suggest that Glu130 might represent a potential mutational hotspot of NEUROD2. Furthermore, the clinical findings (especially the absence of clinically overt seizures) strengthen the NEUROD2-phenotypic spectrum, implying that developmental delay may also manifest isolatedly. We suggest inclusion of NEUROD2-associated developmental and epileptic encephalopathies (DEEs) in the differential diagnosis of atypical Rett syndrome as well as gene panels related to autism spectrum disorder.

Genotype-phenotype correlates in Joubert syndrome: A review.

Joubert syndrome (JS) is a genetically heterogeneous primary ciliopathy characterized by a pathognomonic cerebellar and brainstem malformation, the "molar tooth sign," and variable organ involvement. Over 40 causative genes have been identified to date, explaining up to 94% of cases. To date, gene-phenotype correlates have been delineated only for a handful of genes, directly translating into improved counseling and clinical care. For instance, JS individuals harboring pathogenic variants in TMEM67 have a significantly higher risk of liver fibrosis, while pathogenic variants in NPHP1, RPGRIP1L, and TMEM237 are frequently associated to JS with renal involvement, requiring a closer monitoring of liver parameters, or renal functioning. On the other hand, individuals with causal variants in the CEP290 or AHI1 need a closer surveillance for retinal dystrophy and, in case of CEP290, also for chronic kidney disease. These examples highlight how an accurate description of the range of clinical symptoms associated with defects in each causative gene, including the rare ones, would better address prognosis and help guiding a personalized management. This review proposes to address this issue by assessing the available literature, to confirm known, as well as to propose rare gene-phenotype correlates in JS.

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum.

BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.

PUS3-related disorder: Report of a novel patient and delineation of the phenotypic spectrum.

PUS3 encodes the pseudouridylate synthase 3, an enzyme catalyzing the formation of tRNA pseudouridine, which plays a critical role in tRNA structure, function, and stability. Biallelic pathogenic variants of PUS3 have been previously associated with severe intellectual disability, microcephaly, epilepsy, and short stature. We identified a novel homozygous PUS3 frameshift variant in a child with facial dysmorphisms, growth failure, microcephaly, retinal dystrophy, cerebellar hypoplasia, congenital heart defect, and right kidney hypoplasia. This patient further expands the phenotypic spectrum of PUS3-related disorders to include a more severe syndromic presentation.

ARF1 haploinsufficiency causes periventricular nodular heterotopia with variable clinical expressivity.

The primary anatomical defect leading to periventricular nodular heterotopia occurs within the neural progenitors along the neuroepithelial lining of the lateral ventricles and results from a defect in the initiation of neuronal migration, following disruption of the neuroependyma and impaired neuronal motility. Growing evidence indicates that the FLNA-dependent actin dynamics and regulation of vesicle formation and trafficking by activation of ADP-ribosylation factors (ARFs) can play an important role in this cortical malformation. We report the first inherited variant of ARF1 in a girl with intellectual disability and periventricular nodular heterotopia who inherited the variant from the father with previously undiagnosed single nodular heterotopia and mild clinical expression. Additionally, both patients presented some features suggestive of hypohidrotic ectodermal dysplasia. These clinical features showed similarities to those of three previously reported cases with ARF1 missense variants, confirming that haploinsufficiency of this gene causes a recognisable neurological disorder with abnormal neuronal migration and variable clinical expressivity.

Diagnostic Yield and Cost-Effectiveness of "Dynamic" Exome Analysis in Epilepsy with Neurodevelopmental Disorders: A Tertiary-Center Experience in Northern Italy.

BACKGROUND: The advent of next-generation sequencing (NGS) techniques in clinical practice led to a significant advance in gene discovery. We aimed to describe diagnostic yields of a "dynamic" exome-based approach in a cohort of patients with epilepsy associated with neurodevelopmental disorders. METHODS: We conducted a retrospective, observational study on 72 probands. All patients underwent a first diagnostic level of a 135 gene panel, a second of 297 genes for inconclusive cases, and finally, a whole-exome sequencing for negative cases. Diagnostic yields at each step and cost-effectiveness were the objects of statistical analysis. RESULTS: Overall diagnostic yield in our cohort was 37.5%: 29% of diagnoses derived from the first step analysis, 5.5% from the second step, and 3% from the third. A significant difference emerged between the three diagnostic steps (p < 0.01), between the first and second (p = 0.001), and the first and third (p << 0.001). The cost-effectiveness plane indicated that our exome-based "dynamic" approach was better in terms of cost savings and higher diagnostic rate. CONCLUSIONS: Our findings suggested that "dynamic" NGS techniques applied to well-phenotyped individuals can save both time and resources. In patients with unexplained epilepsy comorbid with NDDs, our approach might maximize the number of diagnoses achieved.

WITHDRAWN: Schuurs-Hoeijmakers syndrome: Severe expression of the recurrent PACS1 c.607C>T mutation.

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