HUWE1 mutation explains phenotypic severity in a case of familial idiopathic intellectual disability

The advent of next-generation sequencing has proven to be a key force in the identification of new genes associated with intellectual disability. In this study, high-throughput sequencing of the coding regions of the X-chromosome led to the identification of a missense variant in the HUWE1 gene. The same variant has been reported before by Froyen et al. (2008). We compare the phenotypes and demonstrate that, in the present family, the HUWE1 mutation segregates with the more severe ID phenotypes of two out of three brothers. The third brother has a milder form of ID and does not carry the mutation

De novo SPAST mutations may cause a complex SPG4 phenotype

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Discovery of Novel Genes for Intellectual Disability and Multiple Congenital Anomalies in the Next Generation Sequencing Era

This study was aimed at exploring the utility of next-generation sequencing (NGS) in the discovery of novel genes causing intellectual disability (ID) and/or multiple congenital anomalies (MCA) (chapter 1 & 2). It was started in 2010, around the same time when NGS was initiated at the KU Leuven Center for Human Genetics. Our starting point were patients with either syndromic or non-syndromic forms of ID, including both sporadic and familial cases, without a molecular diagnosis. NGS was performed on the Illumina HiSeq 2000 platform (chapter 3), with exception of the family described in chapter 5.3. In order to investigate the benefit of NGS in sporadic ID, we collected DNA samples of patients sharing the same clinical phenotype of Circumferential skin creases Kunze type (CSC-KT), which thus far had an unknown genetic origin (chapter 4.1). After applying whole-exome sequencing (WES), we demonstrated the presence of causal MAPRE2 or TUBB mutations in the patient cohort, which was validated by Sanger sequencing and further confirmed in additional patients. Functional analysis of the mutations revealed their influence on microtubule dynamics. Therefore, CSC-KT can be categorized as a tubulinopathy. Pathogenicity of all mutations was further confirmed using a zebrafish model that showed perturbed craniofacial development. On the whole, the study highlighted the presence of multiple inheritance paradigms resulting in the same clinical phenotype. The role of NGS in familial ID was investigated in three families with separate entities. Family 1 (chapter 5.1) consisted of a consanguineous couple with six children, three of which had intellectual disability, seizures and behavioural problems. WES combined with linkage analysis on all family members, led to the identification of a homozygous missense variant in STYXL1 as the most likely candidate. We demonstrated a significant reduction of transcript levels of STYXL1 in the proband versus his carrier father, further supporting pathogenicity of the variant. A drawback of this study was the lack of additional cases with a mutation in STYXL1, which may be explained by the differing ethnicity and the extreme heterogeneity of the phenotype. The approach in family 2 (chapter 5.2) consisted of applying WES to two affected siblings, their unaffected parents as well as their unaffected sibling. Although it was the most likely inheritance pattern, we did not detect a causal recessive variant in the affected patients. Rather, we identified a heterozygous variant in the FHF1 gene, which turned out to be the result of germline mosaicism in either one of the parents. We established FHF1 as a novel gene associated with a severe phenotype of early-onset epileptic encephalopathy and cerebellar atrophy. Pathogenic effects of the mutation were further verified in a zebrafish model replicating epileptiform discharges through a gain-of-function mechanism. Finally, in family 3 we used an X-exome-based approach based on the pedigree (chapter 5.3). two out of three brothers had a severe ID and minor dysmorphic features. They carried a previously described pathogenic HUWE1 missense mutation. Through this additional family, we emphasize the importance of HUWE1 point mutations in cases with severe ID in males, variably associated with minor dysmorphism. In addition, we discuss the challenging interpretation of these findings in a familial setting, where other family members - including a sibling and their mother - also had some level of cognitive impairment. In conclusion, our study explored the application of NGS in the field of human genetics and ID (chapter 6). The value of NGS versus conventional techniques is illustrated by its benefit when it comes to a growing number of genetic heterogeneous disorders. Also, the constant drop in NGS costs makes it a more attractive choice when the clinical diagnosis is not very clear. On the other hand, clinical evaluation remains an indispensable part of a genetic workup and it's very important to remember the limitations of NGS techniques. Finally, we provide an overview of the challenges that emerge during diagnostic implementation of NGS and discuss some future perspectives on NGS.List of abbreviations Chapter 1: General introduction to rare diseases and next-generation sequencing Chapter 2: Research objectives Chapter 3: Materials and methods Chapter 4: Sporadic cases of syndromic intellectual disability Chapter 5: Familial cases of intellectual disability Chapter 6: General discussion, clinical implications and future perspectives Appendicesnrpages: 159status: publishe

Haploinsufficiency of ANKRD11 causes mild cognitive impairment, short stature and minor dysmorphisms

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Gain-of-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy

Voltage-gated sodium channel (Nav)-encoding genes are among early-onset epileptic encephalopathies (EOEE) targets, suggesting that other genes encoding Nav-binding proteins, such as fibroblast growth factor homologous factors (FHFs), may also play roles in these disorders. Methods: To identify additional genes for EOEE, we performed whole-exome sequencing in a family quintet with 2 siblings with a lethal disease characterized by EOEE and cerebellar atrophy. The pathogenic nature and functional consequences of the identified sequence alteration were determined by electrophysiologic studies in vitro and in vivo. Results: A de novo heterozygous missense mutation was identified in the FHF1 gene (FHF1AR114H, FHF1BR52H) in the 2 affected siblings. The mutant FHF1 proteins had a strong gain-of-function phenotype in transfected Neuro2A cells, enhancing the depolarizing shifts in Nav1.6 voltage-dependent fast inactivation, predicting increased neuronal excitability. Surprisingly, the gain-of-function effect is predicted to result from weaker interaction of mutant FHF1 with the Nav cytoplasmic tail. Transgenic overexpression of mutant FHF1B in zebrafish larvae enhanced epileptiform discharges, demonstrating the epileptic potential of this FHF1 mutation in the affected children. Conclusions: Our data demonstrate that gain-of-function FHF mutations can cause neurologic disorder, and expand the repertoire of genetic causes (FHF1) and mechanisms (altered Nav gating) underlying EOEE and cerebellar atrophy

STXBP1 Syndrome Is Characterized by Inhibition-Dominated Dynamics of Resting-State EEG

STXBP1 syndrome is a rare neurodevelopmental disorder caused by heterozygous variants in the STXBP1 gene and is characterized by psychomotor delay, early-onset developmental delay, and epileptic encephalopathy. Pathogenic STXBP1 variants are thought to alter excitation-inhibition (E/I) balance at the synaptic level, which could impact neuronal network dynamics; however, this has not been investigated yet. Here, we present the first EEG study of patients with STXBP1 syndrome to quantify the impact of the synaptic E/I dysregulation on ongoing brain activity. We used high-frequency-resolution analyses of classical and recently developed methods known to be sensitive to E/I balance. EEG was recorded during eyes-open rest in children with STXBP1 syndrome (n = 14) and age-matched typically developing children (n = 50). Brain-wide abnormalities were observed in each of the four resting-state measures assessed here: (i) slowing of activity and increased low-frequency power in the range 1.75\u20134.63 Hz, (ii) increased long-range temporal correlations in the 11\u201318 Hz range, (iii) a decrease of our recently introduced measure of functional E/I ratio in a similar frequency range (12\u201324 Hz), and (iv) a larger exponent of the 1/f-like aperiodic component of the power spectrum. Overall, these findings indicate that large-scale brain activity in STXBP1 syndrome exhibits inhibition-dominated dynamics, which may be compensatory to counteract local circuitry imbalances expected to shift E/I balance toward excitation, as observed in preclinical models. We argue that quantitative EEG investigations in STXBP1 and other neurodevelopmental disorders are a crucial step to understand large-scale functional consequences of synaptic E/I perturbations

Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.Genetics of disease, diagnosis and treatmen

Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1