[Epilepsy with PCDH19 mutation: polypharmacy as a consequence of the complexity and diversity of pathogenesis mechanisms]. / Epilepsiya pri mutatsii gena PCDH19: polipragmaziya kak sledstvie slozhnosti i mnogoobraziya mekhanizmov patogeneza.

Mutations in the human PCDH19 gene lead to epileptic encephalopathy of early childhood. It is characterized by the early onset of serial seizures, cognitive impairment and behavioral disorders (including autistic personality traits). In most cases, difficulties arise in selecting therapy due to pharmacoresistance. The pathogenesis of the disease is complex. The data available to us at the moment from numerous studies present the pathogenesis of «PCDH19 syndrome¼ as multi-level, affecting both the epigenetic support of cell life, and development of stem cells and progenitor cells in the process of neuroontogenesis, and the influence on the neurotransmitter mechanisms of the brain, and disruption of the formation of neural networks with an inevitable increase in the excitability of the cerebral cortex as a whole, and local changes in the highly labile regulatory structures of the hippocampal region. And it is not surprising that all these changes entail not only (and perhaps not so much) epileptization, but a profound disruption of the regulation of brain activity, accompanied by autism spectrum disorders, more profound disorders in the form of schizophrenia or cyclothymia, and the formation of delayed psychomotor development. A «side branch¼ of these pathogenetic processes can also be considered the participation of PCDH19 dysfunctions in certain variants of oncogenesis. The need for polypharmacy (in most cases) confirms the diversity of mechanisms involved in the pathogenesis of the disease and makes the prospects for the development of effective and rational treatment regimens very vague. Cautious optimism is caused only by attempts at relatively specific treatment with ganaxolone.

Protocadherin 19 regulates axon guidance in the developing Xenopus retinotectal pathway.

Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons.

Dravet-like syndrome with PCDH19 mutations in Taiwan - A multicenter study.

OBJECTIVE: Protocadherin-19 (PCDH19) epilepsy is a rare female restricted epilepsy syndrome with early onset seizures and developmental delay caused by a change or mutation of the PCDH19 gene on the X chromosome. SCN1A-negative patients with a Dravet-like phenotype may have a gene mutation in PCDH19. The aim of this case series was to characterize the phenotype of epileptic patients according to PCDH19 mutations, antiseizure medications, brain images and mutation types in Taiwan. METHODS: We retrospectively reviewed the medical records of patients with PCDH19 epilepsy from July 2017 to December 2021 from multiple centers in Taiwan. We analyzed the patients' clinical data and genetic reports. RESULTS: Fifteen female patients (age 3-23 years) were enrolled. Seizure onset was at 4 months to 2 years 7 months of age with generalized tonic-clonic or focal seizures. Seizure frequency tended to be in clusters rather than single longer seizures. The patients had varying degrees of intellectual disability, however 3 had no impairment. Two patients had abnormal brain images including mesial temporal sclerosis, subcortical and periventricular white matter lesions. On average, the patients received 4 antiseizure medications (range 3-6), including 9 patients who were seizure free, and 3 who received sodium channel blockers without aggravation. Missense and truncating variants (frameshift and nonsense variants) accounted for 40% and 46.7% of all mutations. The mutations of 13 patients were located on EC1 to EC4, and EC5 to cytoplasmic domain in 2 patients. SIGNIFICANCE: PCDH19 epilepsy has distinct phenotypes and an unusual X-linked pattern of expression in which females manifest core symptoms. Psychiatric and behavioral problems are frequently part of the clinical picture. Patients are usually treated with a wide array of standard antiseizure medications, with no preferred antiseizure medication class. No strong correlations between phenotype and location of variant mutations were found in our patients.

Phenotypic and genotypic characteristics of children with PCDH19 clustering epilepsy in China.

PURPOSE: PCDH19 gene variants, termed PCDH19 clustering epilepsy, represent a distinct etiology of epilepsy. This study aimed to elucidate the clinical manifestations and explore the genotypes and phenotypes of children affected by PCDH19 clustering epilepsy. METHODS: This retrospective study included medical history, magnetic resonance imaging, video-electroencephalography, and genetic analysis of patients diagnosed with PCDH19 Clustering Epilepsy at the Neurology Department of Beijing Children's Hospital from 2015 to 2023. Chi-square tests and logistic regression analyses were conducted to study the factors associated with developmental delay in patients. RESULTS: The age at seizure onset ranged from 5 to 61 months among all 30 patients (median 14 months; IQR 9.25-22.5 months). Among the 30 patients, 29 were female and 1 was male. Clusters of seizures and fever-triggered seizures were observed, with the most prevalent seizure types being focal to bilateral tonic-clonic seizures (FBTCS). Seizures were successfully controlled in 15 patients. Unfortunately, one patient experienced a sudden unexpected death in epilepsy (SUDEP). Additionally, 14 patients had hereditary mutations, 14 had de novo mutations, 1 had both hereditary and de novo mutations, and 1 male patient had a mosaic component mutation of 0.64 due to a somatic mutation. Developmental delays were identified in 17 patients (56.7 %), and 6 patients (20 %) were diagnosed with autism spectrum disorder (ASD). Among the 17 patients, 9 experienced developmental delays before the onset of epilepsy, while 8 were initially normal but later developed developmental delays during disease progression. Statistical analysis revealed that the presence of drug-resistant epilepsy was an independent risk factor for the occurrence of developmental delays (P = 0.020, OR = 9.758, 95 % CI (1.440-66.111)). CONCLUSION: In this study, 13 new potential rare pathogenic variations in PCDH19 clustering epilepsy were identified. The clinical features observed in patients are consistent with known phenotypic features, and we found that patients with drug-resistant epilepsy are more likely to have developmental delays. The severity of the phenotype in patients with PCDH19 variants ranged from drug-responsive seizures to refractory epilepsy.

Multicenter retrospective study of patients with PCDH19-related epilepsy: The first Hungarian cohort.

OBJECTIVE: PCDH19-related epilepsy occurs predominantly in girls and is caused by pathogenic variant of the protocadherin-19 gene. The initial seizures usually develop in association with fever, begin on average at 15 months of age, and often occur in clusters. Autistic symptoms, intellectual disability, and sleep disturbance are often associated. METHODS: In our retrospective, multicenter study, we reviewed clinical data of nine children with epilepsy genetically confirmed to be associated with PCDH19. RESULTS: In the Hungarian patient population aged 0-18 years, the prevalence of PCDH19-related epilepsy was found to be lower (1/100000 live births in females) than the reported international data (4-5/100000 live births in females). Four of our nine patients had positive family history of epilepsy (cousins, sister, and mother). We assessed brain anomalies in three patients (in one patient focal cortical dysplasia and left anterior cingulate dysgenesis, and in two children right or left hippocampal sclerosis) and in another three cases incidentally identified benign alterations on brain MRI were found. The first seizure presented as a cluster in seven out of nine children. In seven out of nine cases occurred status epilepticus. Six out of nine children had autistic symptoms and only one child had normal intellectual development. Seven of our patients were seizure free with combined antiseizure medication (ASM). The most effective ASMs were levetiracetam, valproate, and clobazam. SIGNIFICANCE: The prevalence of PCDH19-related epilepsy is presumably underestimated because of the lack of widely performed molecular genetic evaluations. Molecular genetic testing including PCDH19 pathogenic variants is recommended for female patients with an onset of seizures before the age of 3 years.

The impact of developmental and epileptic encephalopathies on families: a qualitative study.

Developmental and epileptic encephalopathies (DEEs) cause disability and dependence affecting both children and the family. The aim of the study was to describe the perspective of parents of children with DEEs regarding the impact of the disease on the family. We carried out a qualitative study based on the interpretivist paradigm. Twenty-one participants were selected using purposive sampling. Parents of children with DEEs of SCN1A, KCNQ2, CDKL5, PCDH19, and GNAO1 variants were included. In-depth interviews and researcher notes were used for data collection. A thematic analysis was performed on the data. Three themes were identified in the results: (a) Assuming conflicts and changes within the couple, causing them to distance themselves, reducing their time and intimacy and leading them to reconsider having more children; (b) impact of the disorder on siblings and grandparents, where siblings perceived DEE as a burden in their lives, felt neglected, and needed to grow and mature alone; conversely, the grandparents suffered for their grandchildren and the parents, in addition to perceiving that their health worsened, and (c) reconciling the care of the child with family life and work; this led the parents to share tasks, abandon or reduce working hours and ask for help.Conclusions: Caring for a child with DEE can result in neglect of social, psychological, emotional, recreational, educational, or occupational needs and obligations that ultimately impact all family members. What is Known: • Children with DEE may develop seizures and experience developmental and cognitive problems. • Caring for a child with DEE has a social and psychological impact on the entire family.

NGS-Based Identification of Two Novel PCDH19 Mutations in Female Patients with Early-Onset Epilepsy.

Developmental and epileptic encephalopathy-9 (DEE9) is characterized by seizure onset in infancy, mild to severe intellectual impairment, and psychiatric features and is caused by a mutation in the PCDH19 gene on chromosome Xq22. The rare, unusual X-linked type of disorder affects heterozygous females and mosaic males; transmitting males are unaffected. In our study, 165 patients with epilepsy were tested by Next Generation Sequencing (NGS)-based panel and exome sequencing using Illumina technology. PCDH19 screening identified three point mutations, one indel, and one 29 bp-long deletion in five unrelated female probands. Two novel mutations, c.1152_1180del (p.Gln385Serfs*6) and c.830_831delinsAA (p.Phe277*), were identified and found to be de novo pathogenic. Moreover, among the three inherited mutations, two originated from asymptomatic mothers and one from an affected father. The PCDH19 c.1682C>T and c.1711G>T mutations were present in the DNA samples of asymptomatic mothers. After targeted parental testing, X chromosome inactivation tests and Sanger sequencing were carried out for mosaicism examination on maternal saliva samples in the two asymptomatic PCDH19 mutation carrier subjects. Tissue mosaicism and X-inactivation tests were negative. Our results support the opportunity for reduced penetrance in DEE9 and contribute to expanding the genotype-phenotype spectrum of PCDH19-related epilepsy.

A Male Child with Infantile Epilepsy due to a Mosaic Missense Variant of PCDH19.

Background: Pathogenic variants of PCDH19, located on the X-chromosome (Xq22.1), cause a rare epileptic encephalopathy with speech and development delay, seizures, behavioral and psychiatric problems. The specific underlying pathogenic mechanism is known as "cellular interference" that results in affected heterozygous females, normal hemizygous males and affected mosaic males but its functioning is not yet clear. Objectives: Reporting new cases of affected males is considered useful to a deeper insight. Subject and Method: We present the case of a three-year-old boy with early-onset seizures at 3 months of age, mild cognitive impairment, partial control of seizures with levetiracetam, normal brain imaging. Results: The patient has a mosaic pathogenic variant c.698A>G (p.Asp233Gly) in PCDH19 assessed by Next Generation Sequencing analysis. We have compared his characteristics with the genotypes and phenotypes of 34 PCDH19 mosaic males earlier reported in the literature. Finally, we have summarized today's knowledge about phenotype-genotype correlation and pharmacological response in these patients. Conclusions: Our report confirms that the clinical picture of mosaic affected males, resembling that of females, can show a wide variability in severity of disease and underlines a stringent need to improve therapeutic approaches and to collect data on long-term follow-up.

Mapping combinatorial expression of non-clustered protocadherins in the developing brain identifies novel PCDH19-mediated cell adhesion properties.

Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequencing, we demonstrate combinatorial inter- and intra-familial expression of ncPcdhs in the developing mouse cortex and hippocampus, at single-cell resolution. We discovered the combinatorial expression of Protocadherin-19 (Pcdh19), a protein involved in PCDH19-clustering epilepsy, with Pcdh1, Pcdh9 or Cadherin 13 (Cdh13) in excitatory neurons. Using aggregation assays, we demonstrate a code-specific adhesion function of PCDH19; mosaic PCDH19 absence in PCDH19+9 and PCDH19 + CDH13, but not in PCDH19+1 codes, alters cell-cell interaction. Interestingly, we found that PCDH19 as a dominant protein in two heterophilic adhesion codes could promote trans-interaction between them. In addition, we discovered increased CDH13-mediated cell adhesion in the presence of PCDH19, suggesting a potential role of PCDH19 as an adhesion mediator of CDH13. Finally, we demonstrated novel cis-interactions between PCDH19 and PCDH1, PCDH9 and CDH13. These observations suggest that there is a unique combinatorial code with a cell- and region-specific characteristic where a single molecule defines the heterophilic cell-cell adhesion properties of each code.

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Introduction: Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. Methods: To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). Results: We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Discussion: Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

Prenatal diagnosis of developmental and epileptic encephalopathy 9 with a 10.05-Mb microdeletion at Xq21.31q22.1 inherited from mother: A case report and literature review.

BACKGROUND: Developmental and epileptic encephalopathy 9 (DEE9) is characterized by early infantile seizures and mild-to-severe neuropsychiatric symptoms. Despite being an X-linked dominant disorder, DEE9 mainly affects heterozygous females or mosaic males, while hemizygous males are less affected. PCDH19 gene has been documented as the causative gene. METHODS: Karyotyping analysis and copy number variation sequencing (CNV-seq) were performed on a pregnant woman with epilepsy, together with her husband, son, and fetus. RESULTS: A disease-causing microdeletion, seq[GRCh37] del(X)(q21.31q22.1) (90310001-100360000), was identified in the pregnant woman and her female fetus. The microdeletion includes the entire PCDH19 gene and is classified as "pathogenic" according to the American College of Medical Genetics and Genomics guidelines. CONCLUSION: In this case study, we have not only identified the epilepsy type of the woman as DEE9 but have also made an unfavorable prognosis for her fetus. Our findings from this prenatal case provide valuable clinical resources for prenatal diagnosis and genetic counseling, while also implying the potential of CNV-seq as a viable method for uncovering PCDH19-related epilepsy.

Early onset developmental and epileptic encephalopathy and Rett-like phenotype in a 15-year-old girl affected by Cornelia de Lange syndrome type 2 due to a SMC1A gene mutation.

Developmental and epileptic encephalopathies (DEE) are conditions in which a mutated gene may cause abnormal functioning of the central nervous system, resulting in both encephalopathy and epileptogenesis. We present a case of a girl with a DEE characterized by a Rett-like phenotype in association with febrile and afebrile clusters of focal seizures. The girl presented typical development until the age of 18 months, followed by regression. The first febrile bilateral tonic-clonic seizure was observed at 30 months of age, and the following month seizures recurred in clusters of several episodes per day every 10 days. These seizures were characterized by behavioural arrest, emotional symptoms, head turning, and followed by bilateral tonic-clonic seizures. The administration of valproic acid and levetiracetam led to prolonged seizure control. However, from the age of 7 years, she had monthly recurrent clusters of focal seizures and non-convulsive status epilepticus which occurred at different ages. Brain and spinal cord MRI showed mild non-progressive hemispheric cerebellar atrophy. A next generation sequencing panel for epilepsy identified the de novo splicing mutation c.2973+1G>A of the SMC1A gene.

Epilepsy surgery as a treatment option for select patients with PCDH19-related epilepsy.

PCDH19 is a common epilepsy gene causing medication resistant epilepsy with fever-related seizures. Traditionally, patients with PCDH19-related epilepsy have not been considered surgical candidates. This retrospective review evaluated three patients with pathogenic variants in PCDH19 who presented with seizures in childhood, had one seizure semiology, became medication resistant, and had concordant imaging, seizure semiology and electrographic findings. All three patients ultimately underwent temporal lobectomy, resulting in seizure freedom. These findings suggest epilepsy surgery can be an effective treatment option for select patients with PCDH19-related epilepsy and a single seizure semiology.

Deriving early single-rosette brain organoids from human pluripotent stem cells.

Brain organoid methods are complicated by multiple rosette structures and morphological variability. We have developed a human brain organoid technique that generates self-organizing, single-rosette cortical organoids (SOSR-COs) with reproducible size and structure at early timepoints. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D developmental transition from neural plate to neural tube. Most monolayer fragments form spheres with a single central lumen. Over time, the SOSR-COs develop appropriate progenitor and cortical laminar cell types as shown by immunocytochemistry and single-cell RNA sequencing. At early time points, this method demonstrates robust structural phenotypes after chemical teratogen exposure or when modeling a genetic neurodevelopmental disorder, and should prove useful for studies of human brain development and disease modeling.

Novel variants in established epilepsy genes in focal epilepsy.

INTRODUCTION: Next generation sequencing (NGS) has greatly expanded our understanding of genetic contributors in multiple epilepsy syndromes, including focal epilepsy. Describing the genetic architecture of common syndromes promises to facilitate the diagnostic process as well as aid in the identification of patients who stand to benefit from genetic testing, but most studies to date have been limited to examining children or adults with intellectual disability. Our aim was to determine the yield of targeted sequencing of 5 established epilepsy genes (DEPDC5, LGI1, SCN1A, GRIN2A, and PCHD19) in an extensively phenotyped cohort of focal epilepsy patients with normal intellectual function or mild intellectual disability, as well as describe novel variants and determine the characteristics of variant carriers. PATIENTS AND METHODS: Targeted panel sequencing was performed on 96 patients with a strong clinical suspicion of genetic focal epilepsy. Patients had previously gone through a comprehensive diagnostic epilepsy evaluation in The Neurology Clinic, University Clinical Center of Serbia. Variants of interest (VOI) were classified using the American College of Medical Genetics and the Association for Molecular Pathology criteria. RESULTS: Six VOI in eight (8/96, 8.3%) patients were found in our cohort. Four likely pathogenic VOI were determined in six (6/96, 6.2%) patients, two DEPDC5 variants in two patients, one SCN1A variant in two patients and one PCDH19 variant in two patients. One variant of unknown significance (VUS) was found in GRIN2A in one (1/96, 1.0%) patient. Only one VOI in GRIN2A was classified as likely benign. No VOI were detected in LGI1. CONCLUSION: Sequencing of only five known epilepsy genes yielded a diagnostic result in 6.2% of our cohort and revealed multiple novel variants. Further research is necessary for a better understanding of the genetic basis in common epilepsy syndromes in patients with normal intellectual function or mild intellectual disability.

Modeling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells.

BACKGROUND: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease. METHODS: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. RESULTS: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. CONCLUSIONS: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.

Genetic variants and phenotype analysis in a five-generation Chinese pedigree with PCDH19 female-limited epilepsy.

Objective: Albeit the gene of PCDH19-FE was ascertained, the correlation of gene mutation, PCDH19 protein structure, and phenotype heterogeneity remained obscure. This study aimed to report a five-generation pedigree of seven female patients of PCDH19-FE and tried to explore whether two variants were correlated with PCDH19 protein structure and function alteration, and PCDH19-FE phenotype. Methods: We analyzed the clinical data and genetic variants of a PCDH19-FE pedigree, to explore the phenotype heterogeneity of PCDH19-FE and underlying mechanisms. In addition to the clinical information of family members, next-generation sequencing was adopted to detect the variant sites of probands with validation by sanger sequencing. And the sanger sequencing was conducted in other patients in this pedigree. The biological conservation analysis and population polymorphism analysis of variants were also performed subsequently. The structure alteration of mutated PCDH19 protein was predicted by AlphaFold2. Results: Based on a five-generation pedigree of PCDH19-FE, missense variants of c.695A>G and c.2760T>A in the PCDH19 gene were found in the heterozygous proband (V:1), which resulted in the change of amino acid 232 from Asn to Ser (p.Asn232Ser) and amino acid 920 from Asp to Glu (p.Asp920Glu) influencing PCDH19 function. The other six females in the pedigree (II:6, II:8, IV:3, IV:4, IV:5, IV:11) exhibited different clinical phenotypes but shared the same variant. Two males with the same variant have no clinical manifestations (III:3, III:10). The biological conservation analysis and population polymorphism analysis demonstrated the highly conservative characteristics of these two variants. AlphaFold2 predicted that the variant, p.Asp920Glu, led to the disappearance of the hydrogen bond between Asp at position 920 and His at position 919. Furthermore, the hydrogen bond between Asp920 and His919 also disappeared when the Asn amino acid mutated to Ser at position 232. Conclusion: A strong genotype-phenotype heterogeneity was observed among female patients with the same genotype in our PCDH19-FE pedigree. And two missense variants, c.695A > G and c.2760T>A in the PCDH19 gene, have been identified in our pedigree. The c.2760T>A variant was a novel variant site probably related to the PCDH19-FE.

PCDH19 in Males: Are Hemizygous Variants Linked to Autism?

BACKGROUND: Autism spectrum disorder (ASD) is a complex developmental disability that impairs the social communication and interaction of affected individuals and leads to restricted or repetitive behaviors or interests. ASD is genetically heterogeneous, with inheritable and de novo genetic variants in more than hundreds of genes contributing to the disease. However, these account for only around 20% of cases, while the molecular basis of the majority of cases remains unelucidated as of yet. MATERIAL AND METHODS: Two unrelated Lebanese patients, a 7-year-old boy (patient A) and a 4-year-old boy (patient B), presenting with ASD were included in this study. Whole-exome sequencing (WES) was carried out for these patients to identify the molecular cause of their diseases. RESULTS: WES analysis revealed hemizygous variants in PCDH19 (NM_001184880.1) as being the candidate causative variants: p.Arg787Leu was detected in patient A and p.Asp1024Asn in patient B. PCDH19, located on chromosome X, encodes a membrane glycoprotein belonging to the protocadherin family. Heterozygous PCDH19 variants have been linked to epilepsy in females with mental retardation (EFMR), while mosaic PCDH19 mutations in males are responsible for treatment-resistant epilepsy presenting similarly to EFMR, with some reported cases of comorbid intellectual disability and autism. Interestingly, a hemizygous PCDH19 variant affecting the same amino acid that is altered in patient A was previously reported in a male patient with ASD. CONCLUSION: Here, we report hemizygous PCDH19 variants in two males with autism without epilepsy. Reporting further PCDH19 variants in male patients with ASD is important to assess the possible involvement of this gene in autism.

PCDH19-related epilepsy in mosaic males: The phenotypic implication of genotype and variant allele frequency.

Objective: To analyze the genotypes and phenotypes of mosaic male patients with PCDH19-related epilepsy (PCDH19-RE) and explore the correlation between genotype, variant allele frequency (VAF), and phenotypic severity. Methods: Clinical data and peripheral blood samples of 11 male mosaic patients were collected and analyzed in our study. The VAF of the PCDH19 gene from peripheral blood was quantified using amplicon-based deep sequencing. Additional 20 mosaic male patients with PCDH19-RE were collected from the published literature, with 10 patients whose VAFs of the PCDH19 gene were available for analytic purposes. Results: In our cohort of 11 patients, 10 variants were identified, and four were novel. The VAF of the PCDH19 gene from peripheral blood ranged from 27 to 90%. The median seizure onset age was 6 months (range: 4-9 months). Clinical manifestations included cluster seizures (100%), fever sensitivity (73%), focal seizures (91%), developmental delay/intellectual disability (DD/ID, 82%), and autistic features (45%). Thirty-one mosaic male patients collected from our cohort and the literature developed seizures mostly (87%) within one year of age. Variant types included missense variants (42%), truncating variants (52%), splice variants (3%), and whole PCDH19 deletion (3%). Among 21 patients with a definite VAF from our cohort and the literature, nine had a low VAF ( ≤ 50%) and 12 had a high VAF (> 50%). Seventy-five percent of variants from the high VAF group were missense, whereas 89% of those from the low VAF group were truncations. The median seizure onset age was 6 months in the low VAF group and 9 months in the high VAF group (p = 0.018). Forty-four percent (4/9) of patients from the low VAF group achieved seizure-free for ≥1 year, whereas none of the 12 patients from the high VAF group did (p = 0.021). DD/ID was present in 83% (10/12) of the high VAF group and 56% (5/9) of the low VAF group (p = 0.331). Conclusion: The predominant variant types were truncating and missense variants. Missense variants tended to have higher VAFs. Patients with a high VAF were more likely to have a more severe epileptic phenotype. Our findings shed light on the phenotypic implications of VAF in mosaic males with PCDH19-RE.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.