Impact of genetic and non-genetic factors on phenotypic diversity in NBAS-associated disease.

Biallelic pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause a pleiotropic multisystem disorder. Three clinical subgroups have been defined correlating with the localisation of pathogenic variants in the NBAS gene: variants affecting the C-terminal region of NBAS result in SOPH syndrome (short stature, optic atrophy, Pelger-Huët anomaly), variants affecting the Sec 39 domain are associated with infantile liver failure syndrome type 2 (ILFS2) and variants affecting the ß-propeller domain give rise to a combined phenotype. However, there is still unexplained phenotypic diversity across the three subgroups, challenging the current concept of genotype-phenotype correlations in NBAS-associated disease. Therefore, besides examining the genetic influence, we aim to elucidate the potential impact of pre-symptomatic diagnosis, emergency management and other modifying variables on the clinical phenotype. We investigated genotype-phenotype correlations in individuals sharing the same genotypes (n = 30 individuals), and in those sharing the same missense variants with a loss-of-function variant in trans (n = 38 individuals). Effects of a pre-symptomatic diagnosis and emergency management on the severity of acute liver failure (ALF) episodes also were analysed, comparing liver function tests (ALAT, ASAT, INR) and mortality. A strong genotype-phenotype correlation was demonstrated in individuals sharing the same genotype; this was especially true for the ILFS2 subgroup. Genotype-phenotype correlation in patients sharing only one missense variant was still high, though at a lower level. Pre-symptomatic diagnosis in combination with an emergency management protocol leads to a trend of reduced severity of ALF. High genetic impact on clinical phenotype in NBAS-associated disease facilitates monitoring and management of affected patients sharing the same genotype. Pre-symptomatic diagnosis and an emergency management protocol do not prevent ALF but may reduce its clinical severity.

De novo variants in PLCG1 are associated with hearing impairment, ocular pathology, and cardiac defects.

Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol, important signaling molecules involved in many cellular processes. PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here we describe three unrelated individuals with de novo heterozygous missense variants in PLCG1 (p.Asp1019Gly, p.His380Arg, and p.Asp1165Gly) who exhibit variable phenotypes including hearing loss, ocular pathology and cardiac septal defects. To model these variants in vivo, we generated the analogous variants in the Drosophila ortholog, small wing (sl). We created a null allele slT2A and assessed the expression pattern. sl is broadly expressed, including in wing discs, eye discs, and a subset of neurons and glia. Loss of sl causes wing size reductions, ectopic wing veins and supernumerary photoreceptors. We document that mutant flies exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in slT2A mutant rescues the loss-of-function phenotypes whereas expressing the variants causes lethality. Ubiquitous overexpression of the variants also reduces viability, suggesting that the variants are toxic. Ectopic expression of an established hyperactive PLCG1 variant (p.Asp1165His) in the wing pouch causes severe wing phenotypes, resembling those observed with overexpression of the p.Asp1019Gly or p.Asp1165Gly variants, further arguing that these two are gain-of-function variants. However, the wing phenotypes associated with p.His380Arg overexpression are mild. Our data suggest that the PLCG1 de novo heterozygous missense variants are pathogenic and contribute to the features observed in the probands.

Assortative mating and parental genetic relatedness contribute to the pathogenicity of variably expressive variants.

We examined more than 97,000 families from four neurodevelopmental disease cohorts and the UK Biobank to identify phenotypic and genetic patterns in parents contributing to neurodevelopmental disease risk in children. We identified within- and cross-disorder correlations between six phenotypes in parents and children, such as obsessive-compulsive disorder (R = 0.32-0.38, p < 10-126). We also found that measures of sub-clinical autism features in parents are associated with several autism severity measures in children, including biparental mean Social Responsiveness Scale scores and proband Repetitive Behaviors Scale scores (regression coefficient = 0.14, p = 3.38 × 10-4). We further describe patterns of phenotypic similarity between spouses, where spouses show correlations for six neurological and psychiatric phenotypes, including a within-disorder correlation for depression (R = 0.24-0.68, p < 0.001) and a cross-disorder correlation between anxiety and bipolar disorder (R = 0.09-0.22, p < 10-92). Using a simulated population, we also found that assortative mating can lead to increases in disease liability over generations and the appearance of "genetic anticipation" in families carrying rare variants. We identified several families in a neurodevelopmental disease cohort where the proband inherited multiple rare variants in disease-associated genes from each of their affected parents. We further identified parental relatedness as a risk factor for neurodevelopmental disorders through its inverse relationship with variant pathogenicity and propose that parental relatedness modulates disease risk by increasing genome-wide homozygosity in children (R = 0.05-0.26, p < 0.05). Our results highlight the utility of assessing parent phenotypes and genotypes toward predicting features in children who carry rare variably expressive variants and implicate assortative mating as a risk factor for increased disease severity in these families.

NFIA haploinsufficiency: case series and literature review.

Background: NFIA-related disorder (OMIM #613735) is an autosomal dominant neurodevelopmental disorder characterized by a variable degree of cognitive impairment and non-specific dysmorphic features. To date, fewer than thirty patients affected by this disorder have been described. Methods: Our study included three children with NFIA haploinsufficiency recruited from three medical genetics centers. Clinical presentations were recorded on a standardized case report form. Results: All patients presented a variable degree of intellectual disability. None of the individuals in our cohort had urinary tract malformations. Three novel mutations, c.344G>A, c.261T>G, and c.887_888del are reported here. Conclusion: NFIA haploinsufficiency can be suspected through careful observation of specific dysmorphisms, including macrocephaly and craniofacial abnormalities. Instrumental tests such as MRI and renal ultrasound provide further diagnostic clues, while genetic testing can confirm the diagnosis.

Assortative mating and parental genetic relatedness drive the pathogenicity of variably expressive variants.

We examined more than 38,000 spouse pairs from four neurodevelopmental disease cohorts and the UK Biobank to identify phenotypic and genetic patterns in parents associated with neurodevelopmental disease risk in children. We identified correlations between six phenotypes in parents and children, including correlations of clinical diagnoses such as obsessive-compulsive disorder (R=0.31-0.49, p<0.001), and two measures of sub-clinical autism features in parents affecting several autism severity measures in children, such as bi-parental mean Social Responsiveness Scale (SRS) scores affecting proband SRS scores (regression coefficient=0.11, p=0.003). We further describe patterns of phenotypic and genetic similarity between spouses, where spouses show both within- and cross-disorder correlations for seven neurological and psychiatric phenotypes, including a within-disorder correlation for depression (R=0.25-0.72, p<0.001) and a cross-disorder correlation between schizophrenia and personality disorder (R=0.20-0.57, p<0.001). Further, these spouses with similar phenotypes were significantly correlated for rare variant burden (R=0.07-0.57, p<0.0001). We propose that assortative mating on these features may drive the increases in genetic risk over generations and the appearance of "genetic anticipation" associated with many variably expressive variants. We further identified parental relatedness as a risk factor for neurodevelopmental disorders through its inverse correlations with burden and pathogenicity of rare variants and propose that parental relatedness drives disease risk by increasing genome-wide homozygosity in children (R=0.09-0.30, p<0.001). Our results highlight the utility of assessing parent phenotypes and genotypes in predicting features in children carrying variably expressive variants and counseling families carrying these variants.

DNA methylation episignatures are sensitive and specific biomarkers for detection of patients with KAT6A/KAT6B variants.

Accurate diagnosis for patients living with neurodevelopmental disorders is often met with numerous challenges, related to the ambiguity of findings and lack of specificity in genetic variants leading to pathology. Genome-wide DNA methylation analysis has been used to develop highly sensitive and specific 'episignatures' as biomarkers capable of differentiating and classifying complex neurodevelopmental disorders. In this study we describe distinct episignatures for KAT6A syndrome, caused by pathogenic variants in the lysine acetyltransferase A gene (KAT6A), and for the two neurodevelopmental disorders associated with lysine acetyl transferase B (KAT6B). We demonstrate the ability of our models to differentiate between highly overlapping episignatures, increasing the ability to effectively identify and diagnose these conditions.

Epm2aR240X knock-in mice present earlier cognitive decline and more epileptic activity than Epm2a-/- mice.

Lafora disease is a rare recessive form of progressive myoclonic epilepsy, usually diagnosed during adolescence. Patients present with myoclonus, neurological deterioration, and generalized tonic-clonic, myoclonic, or absence seizures. Symptoms worsen until death, usually within the first ten years of clinical onset. The primary histopathological hallmark is the formation of aberrant polyglucosan aggregates called Lafora bodies in the brain and other tissues. Lafora disease is caused by mutations in either the EPM2A gene, encoding laforin, or the EPM2B gene, coding for malin. The most frequent EPM2A mutation is R241X, which is also the most prevalent in Spain. The Epm2a-/- and Epm2b-/- mouse models of Lafora disease show neuropathological and behavioral abnormalities similar to those seen in patients, although with a milder phenotype. To obtain a more accurate animal model, we generated the Epm2aR240X knock-in mouse line with the R240X mutation in the Epm2a gene, using genetic engineering based on CRISPR-Cas9 technology. Epm2aR240X mice exhibit most of the alterations reported in patients, including the presence of LBs, neurodegeneration, neuroinflammation, interictal spikes, neuronal hyperexcitability, and cognitive decline, despite the absence of motor impairments. The Epm2aR240X knock-in mouse displays some symptoms that are more severe that those observed in the Epm2a-/- knock-out, including earlier and more pronounced memory loss, increased levels of neuroinflammation, more interictal spikes and increased neuronal hyperexcitability, symptoms that more precisely resemble those observed in patients. This new mouse model can therefore be specifically used to evaluate how new therapies affects these features with greater precision.

FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

IRF2BPL: A new genotype for progressive myoclonus epilepsies.

The progressive myoclonus epilepsies (PMEs) are a heterogeneous group of neurodegenerative disorders, typically presenting in late childhood. An etiologic diagnosis is achieved in about 80% of patients with PME, and genome-wide molecular studies on remaining, well-selected, undiagnosed cases can further dissect the underlying genetic heterogeneity. Through whole-exome sequencing (WES), we identified pathogenic truncating variants in the IRF2BPL gene in two, unrelated patients presenting with PME. IRF2BPL belongs to the transcriptional regulators family and it is expressed in multiple human tissues, including the brain. Recently missense and nonsense mutations in IRF2BPL were found in patients presenting with developmental delay and epileptic encephalopathy, ataxia, and movement disorders, but none with clear PME. We identified 13 other patients in the literature with myoclonic seizures and IRF2BPL variants. There was no clear genotype-phenotype correlation. With the description of these cases, the IRF2BPL gene should be considered in the list of genes to be tested in the presence of PME, in addition to patients with neurodevelopmental or movement disorders.

Genome-Wide DNA Methylation Profiling Solves Uncertainty in Classifying NSD1 Variants.

BACKGROUND: Inactivating NSD1 mutations causing Sotos syndrome have been previously associated with a specific genome-wide DNA methylation (DNAm) pattern. Sotos syndrome is characterized by phenotypic overlap with other overgrowth syndromes, and a definite diagnosis might not be easily reached due to the high prevalence of variants of unknown significance (VoUS) that are identified in patients with a suggestive phenotype. OBJECTIVE: we performed microarray DNAm profiling in a set of 11 individuals with a clinical suspicion of Sotos syndrome and carrying an NSD1 VoUS or previously unreported variants to solve uncertainty in defining pathogenicity of the observed variants. The impact of the training cohort size on sensitivity and prediction confidence of the classifier was assessed. RESULTS: The Sotos syndrome-specific DNAm signature was validated in six individuals with a clinical diagnosis of Sotos syndrome and carrying bona fide pathogenic NSD1 variants. Applying this approach to the remaining 11 individuals with NSD1 variants, we succeeded in confirming pathogenicity in eight subjects and excluding the diagnosis of Sotos syndrome in three. The sensitivity and prediction confidence of the classifier based on the different sizes of the training sets did not show substantial differences, though the overall performance was improved by using a data balancing strategy. CONCLUSIONS: The present approach solved uncertainty in cases with NDS1 VoUS, further demonstrating the clinical utility of DNAm profiling.

Clinical and electroencephalographic features of epilepsy in patients with triple X syndrome: A case series.

PURPOSE: Triple X syndrome, is an often undiagnosed chromosomal abnormality with an incidence of 1/1000 females. Main associated disorders are urogenital malformations, premature ovarian failure or primary amenorrhea, gastrointestinal problems, psychiatric disorders and epilepsy. To date, triple X is not related to a specific epileptic syndrome. Therefore, the purpose of this clinical series is to analyze seizure semiology, electroencephalogram features and the long-term outcome of 13 patients with epilepsy and triple X syndrome. METHODS: We retrospectively evaluated the long-term seizure outcome in patients with triple X syndrome who had been referred to 11 Epilepsy Centers in Italy. A close electroclinical follow-up was made for at least 2 years and outcomes were reported. RESULTS: Our case series confirms that epilepsy is not an occasional finding but part of the phenotypic spectrum of this syndrome. The seizure semiology shows an higher prevalence of focal seizures in 62% of patients. EEG findings of focal epileptic activity were reported in 85% of patients. Anti-seizure medications were successful in all our patients whom in most cases were responsive to monotherapy. CONCLUSION: According to our case series most successful drugs were VPA and LEV. Long term prognosis of epilepsy in our case series was good. Our experience suggests that all triple X patients achieve good seizure control and in 69% of cases normalization of the EEG.

Expanding the genetic and clinical characteristics of Protocadherin 19 gene mutations.

BACKGROUND: PCDH19-related epilepsy is a rare X-linked type of epilepsy caused by genomic variants of the Protocadherin 19 (PCDH19) gene. The clinical characteristics of PCDH19-related epilepsy are epileptic and non-epileptic symptoms with highly variable severity among patients. CASE PRESENTATION: We present a case of a 4-year old female with PCDH19-related epilepsycaused by new variants in the PCDH19 gene. Our patient was admitted for the first time at the age of 12 months for seizure clusters arising under condition of apyrexia. The electroencephalography (EEG) showed frontal paroxysmal activity. The genetic analysis identified the two variants c.1006G > A (p.Val336Met) and c.1014C > A (p.Asp338Glu) in the gene PCDH19. The patient was treated with Carbamazepine and Clonazepam achieving the disappearance of seizures. During the follow-up, the neurological examination was persistently normal with neither cognitive impairment nor behavior disturbances. From 2 years of age EEG controls were persistently normal. CONCLUSION: This patient presents two novel variants of the PCDH19 gene associated with a mild form of epilepsy with normal cognitive development with an apparently better prognosis. According to our experience, the dual therapy with Carbamazepine and Clonazepam has led to a good control of seizures.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Novel genetic variants of KHDC3L and other members of the subcortical maternal complex associated with Beckwith-Wiedemann syndrome or Pseudohypoparathyroidism 1B and multi-locus imprinting disturbances.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) and Pseudohypoparathyroidism type 1B (PHP1B) are imprinting disorders (ID) caused by deregulation of the imprinted gene clusters located at 11p15.5 and 20q13.32, respectively. In both of these diseases a subset of the patients is affected by multi-locus imprinting disturbances (MLID). In several families, MLID is associated with damaging variants of maternal-effect genes encoding protein components of the subcortical maternal complex (SCMC). However, frequency, penetrance and recurrence risks of these variants are still undefined. In this study, we screened two cohorts of BWS patients and one cohort of PHP1B patients for the presence of MLID, and analysed the positive cases for the presence of maternal variants in the SCMC genes by whole exome-sequencing and in silico functional studies. RESULTS: We identified 10 new cases of MLID associated with the clinical features of either BWS or PHP1B, in which segregate 13 maternal putatively damaging missense variants of the SCMC genes. The affected genes also included KHDC3L that has not been associated with MLID to date. Moreover, we highlight the possible relevance of relatively common variants in the aetiology of MLID. CONCLUSION: Our data further add to the list of the SCMC components and maternal variants that are involved in MLID, as well as of the associated clinical phenotypes. Also, we propose that in addition to rare variants, common variants may play a role in the aetiology of MLID and imprinting disorders by exerting an additive effect in combination with rarer putatively damaging variants. These findings provide useful information for the molecular diagnosis and recurrence risk evaluation of MLID-associated IDs in genetic counselling.

SPECC1L Mutations Are Not Common in Sporadic Cases of Opitz G/BBB Syndrome.

Opitz G/BBB syndrome (OS) is a rare genetic developmental condition characterized by congenital defects along the midline of the body. The main clinical signs are represented by hypertelorism, laryngo-tracheo-esophageal defects and hypospadias. The X-linked form of the disease is associated with mutations in the MID1 gene located in Xp22 whereas mutations in the SPECC1L gene in 22q11 have been linked to few cases of the autosomal dominant form of this disorder, as well as to other genetic syndromes. In this study, we have undertaken a mutation screening of the SPECC1L gene in samples of sporadic OS cases in which mutations in the MID1 gene were excluded. The heterozygous missense variants identified are already reported in variant databases raising the issue of their pathogenetic meaning. Recently, it was reported that some clinical manifestations peculiar to OS signs are not observed in patients carrying mutations in the SPECC1L gene, leading to the proposal of the designation of 'SPECC1L syndrome' to refer to this disorder. Our study confirms that patients with diagnosis of OS, mainly characterized by the presence of hypospadias and laryngo-tracheo-esophageal defects, do not carry pathogenic SPECC1L mutations. In addition, SPECC1L syndrome-associated mutations are clustered in two specific domains of the protein, whereas the missense variants detected in our work lies elsewhere and the impact of these variants in the function of this protein is difficult to ascertain with the current knowledge and will require further investigations. Nonetheless, our study provides further insight into the SPECC1L syndrome classification.

DNA methylation episignature testing improves molecular diagnosis of Mendelian chromatinopathies.

PURPOSE: Chromatinopathies include more than 50 disorders caused by disease-causing variants of various components of chromatin structure and function. Many of these disorders exhibit unique genome-wide DNA methylation profiles, known as episignatures. In this study, the methylation profile of a large cohort of individuals with chromatinopathies was analyzed for episignature detection. METHODS: DNA methylation data was generated on extracted blood samples from 129 affected individuals with the Illumina Infinium EPIC arrays and analyzed using an established bioinformatic pipeline. RESULTS: The DNA methylation profiles matched and confirmed the sequence findings in both the discovery and validation cohorts. Twenty-five affected individuals carrying a variant of uncertain significance, did not show a methylation profile matching any of the known episignatures. Three additional variant of uncertain significance cases with an identified KDM6A variant were re-classified as likely pathogenic (n = 2) or re-assigned as Wolf-Hirschhorn syndrome (n = 1). Thirty of the 33 Next Generation Sequencing negative cases did not match a defined episignature while three matched Kabuki syndrome, Rubinstein-Taybi syndrome and BAFopathy respectively. CONCLUSION: With the expanding clinical utility of the EpiSign assay, DNA methylation analysis should be considered part of the testing cascade for individuals presenting with clinical features of Mendelian chromatinopathy disorders.

A Rare Case of Brachyolmia with Amelogenesis Imperfecta Caused by a New Pathogenic Splicing Variant in LTBP3.

In recent years, a rare form of autosomal recessive brachyolmia associated with amelogenesis imperfecta (AI) has been described as a novel nosologic entity. This disorder is characterized by skeletal dysplasia (e.g., platyspondyly, short trunk, scoliosis, broad ilia, elongated femoral necks with coxa valga) and severe enamel and dental anomalies. Pathogenic variants in the latent transforming growth factor-ß binding protein 3 (LTBP3) gene have been found implicated in the pathogenesis of this disorder. So far, biallelic pathogenic LTBP3 variants have been identified in less than 10 families. We here report a young boy born from consanguineous parents with a complex phenotype including skeletal dysplasia associated with aortic stenosis, hypertrophic cardiomyopathy, hypodontia and amelogenesis imperfecta caused by a previously unreported homozygous LTBP3 splice site variant. We also compare the genotypes and phenotypes of patients reported to date. This work provides further evidence that brachyolmia with amelogenesis imperfecta is a distinct nosologic entity and that variations in LTBP3 are involved in its pathogenesis.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

Childhood-onset dystonia-causing KMT2B variants result in a distinctive genomic hypermethylation profile.

BACKGROUND: Dystonia is a clinically and genetically heterogeneous movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements and/or postures. Heterozygous variants in lysine methyltransferase 2B (KMT2B), encoding a histone H3 methyltransferase, have been associated with a childhood-onset, progressive and complex form of dystonia (dystonia 28, DYT28). Since 2016, more than one hundred rare KMT2B variants have been reported, including frameshift, nonsense, splice site, missense and other in-frame changes, many having an uncertain clinical impact. RESULTS: We characterize the genome-wide peripheral blood DNA methylation profiles of a cohort of 18 patients with pathogenic and unclassified KMT2B variants. We resolve the "episignature" associated with KMT2B haploinsufficiency, proving that this approach is robust in diagnosing clinically unsolved cases, properly classifying them with respect to other partially overlapping dystonic phenotypes, other rare neurodevelopmental disorders and healthy controls. Notably, defective KMT2B function in DYT28 causes a non-random DNA hypermethylation across the genome, selectively involving promoters and other regulatory regions positively controlling gene expression. CONCLUSIONS: We demonstrate a distinctive DNA hypermethylation pattern associated with DYT28, provide an epigenetic signature for this disorder enabling accurate diagnosis and reclassification of ambiguous genetic findings and suggest potential therapeutic approaches.

Identification of a DNA Methylation Episignature in the 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20-40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome-wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS.