Identifizierung und Charakterisierung der Kandidatengene FAR1, EDC3, FRRS1L und HMG20A bei Individuen mit autosomal rezessiver mentaler Retardierung

Intellectual disability (ID) has a frequency of 2-3% of the population and thus poses huge and still often unresolved health problem. In the course of this thesis I have examined the causes of intellectual disability in 16 large consanguineous families with several affected individuals using autozygositiy mapping and whole exome sequencing. Then I characterized the identified candidate mutations in more detail to establish their impact on the pathogenesis. With this approach 3 mutations in previously reported genes involved in ID (C12orf65, ALDH5A1, GPR56) were identified and confirmed as disease causing mutations. This result supports the use of massive parallel sequencing in genetic diagnostics, since these diagnoses could not have been suspected by the clinical appearance. In one family with profound intellectual disability, epilepsy and cataracts a mutation in FAR1, which has a function in plasmalogen synthesis, could be identified. Following in vitro analysis of the mutation showed a strong impact on enzyme activity. The identification of another in-dividual with a similar phenotype and compound heterozygous mutations in FAR1 confirmed FAR1 as causative for the newly established peroxisomal FAR1 deficiency disorder. In a further family with non-specific ID a homozygous missense mutation in EDC3 could be identified. EDC3 is an enhancer of mRNA decapping and in decapping assays we could show a strong impairment of the decapping activity likely due to decreased domain stability. Whole-transcriptome sequencing of patient cells in comparison to unaffected family members showed a significant change in RNA levels of target genes. In another family with profound intellectual disability a homozygous mutation in FRRS1L was identified. FRRS1L is part of the AMPA receptor and thus plays a prominent role in syn-aptic plasticity. The identification of 2 further families with a similar phenotype and loss-of function or missense mutations in FRRS1L supports FRRS1L as a good candidate gene for ID. A candidate mutation in HMG20A was identified in a further family. HMG20A plays a role in gene regulation of especially neuronal genes and functional studies showed a slight, yet not significant change in expression levels of target genes by the identified variant. Thus, this variant could not be confirmed as causative, but could not be excluded either. In total the genetic causes of intellectual disability could be solved in 6 families during this project and 3 new ID-genes were identified and further characterized.Mentale Retardierung (MR) stellt mit einer Frequenz von etwa 2-3% der Bevölkerung ein großes und dennoch oft ungelöstes Gesundheitsproblem dar. In dieser Arbeit habe ich mittels positioneller Kartierung und massiv-paralleler Sequenzierung 16 große konsanguine Familien mit mehreren Betroffenen auf die genetischen Ursachen der MR hin untersucht. Identifizierte Kandidatenmutationen habe ich daraufhin funktionell weiter charakterisiert, um die Pathoge-nese weiter zu klären. Auf diese Weise konnten 3 Mutationen in bereits bekannten Genen (C12orf65, ALDH5A1, GPR56) für mentale Retardierung identifiziert und als ursächlich bestätigt werden. Dieses Ergebnis unterstützt die Anwendung von massiv-paralleler Sequenzierung in der genetischen Diagnostik, denn die gestellten Diagnosen konnten aufgrund unspezifischer Ausprägung des Krankheitsbildes nicht differential-diagnostisch in Erwägung gezogen werden. In einer Familie mit schwerer mentaler Retardierung, Epilepsie und Katarakten konnte eine Mutation in FAR1 identifiziert werden, dessen Genprodukt eine Funktion in der Plasmalogen-synthese hat. Nachfolgende in vitro Analysen zeigten eine starke Einschränkung der Enzym-aktivität durch die Mutation. Die Identifikation eines weiteren Patienten mit ähnlichem Krankheitsbild und compound heterozygoten Mutationen in FAR1 bestätigte FAR1 als ursächlich für die neu etablierte peroxisomale FAR1 Funktionsstörung. In einer weiteren Familie mit nicht-spezifischer MR konnte eine homozygote missense Muta-tion in EDC3 identifiziert werden. EDC3 ist ein enhancer des RNA decappings und in vitro Decapping-Assays zeigten eine starke Einschränkung der Decapping-Aktivität, vermutlich aufgrund von einer verringerten Domänen-Stabilität durch die identifizierte Veränderung. Eine Transkriptomanalyse von Patientenzellen zeigte weiterhin eine signifikante Veränderung der RNA Expression von Zielgenen. In einer weiteren Familie mit schwerer mentaler Retardierung wurde eine homozygote Muta-tion in FRRS1L identifiziert. FRRS1L ist Teil des AMPA-Rezeptors und spielt somit eine prominente Rolle in der synaptischen Plastizität. Die Identifikation von 2 weiteren Familien mit ähnlichem Krankheitsbild und missense bzw. trunkierenden Mutationen in FRRS1L unter-stützt FRRS1L als ein weiteres MR-Gen. Eine Kandidatenmutation in HMG20A wurde in einer weiteren Familie identifiziert. HMG20A spielt eine Rolle in der Genregulation insbesondere von neuronalen Genen. Funkti-onelle Analysen ergaben eine Veränderung der Expression von Zielgenen durch die identifi-zierte Variante, welche jedoch nicht signifikant war. Daher konnte die Variante in diesem Gen nicht als ursächlich bestätigt, jedoch auch nicht ausgeschlossen werden. Insgesamt konnten im Rahmen dieser Arbeit die genetischen Grundlagen der mentalen Retar-dierung in 6 Familien gelöst werden und dabei 3 neue MR-Gene identifiziert und weiter cha-rakterisiert werden

Unraveling the genetic cause of hereditary ophthalmic disorders in Arab societies from Israel and the Palestinian Authority

Visual impairment due to inherited ophthalmic disorders is amongst the most common disabilities observed in populations practicing consanguineous marriages. Here we investigated the molecular genetic basis of an unselected broad range of ophthalmic disorders in 20 consanguineous families from Arab villages of Israel and the Palestinian Authority. Most patients had little or very poor prior clinical workup and were recruited in a field study. Homozygosity mapping followed by candidate gene sequencing applying conventional Sanger sequencing or targeted next generation sequencing was performed in six families. In the remaining 14 families, one affected subject per family was chosen for whole exome sequencing. We discovered likely disease-causing variants, all homozygous, in 19 of 20 independent families (95%) including a previously reported novel disease gene for congenital nystagmus associated with foveal hypoplasia. Moreover, we found a family in which disease-causing variants for two collagenopathies — Stickler and Knobloch syndrome — segregate within a large sibship. Nine of the 19 distinct variants observed in this study were novel. Our study demonstrated a very high molecular diagnostic yield for a highly diverse spectrum of rare ophthalmic disorders in Arab patients from Israel and the Palestinian Authority, even with very limited prior clinical investigation. We conclude that ‘genetic testing first' may be an economic way to direct clinical care and to support proper genetic counseling and risk assessment in these families

iSAW: Integrating Structure, Actors, and Water to Study Socio-Hydro-Ecological Systems

Urbanization, climate, and ecosystem change represent major challenges for managing water resources. Although water systems are complex, a need exists for a generalized representation of these systems to identify important components and linkages to guide scientific inquiry and aid water management. We developed an integrated Structure-Actor-Water framework (iSAW) to facilitate the understanding of and transitions to sustainable water systems. Our goal was to produce an interdisciplinary framework for water resources research that could address management challenges across scales (e.g., plot to region) and domains (e.g., water supply and quality, transitioning, and urban landscapes). The framework was designed to be generalizable across all human–environment systems, yet with sufficient detail and flexibility to be customized to specific cases. iSAW includes three major components: structure (natural, built, and social), actors (individual and organizational), and water (quality and quantity). Key linkages among these components include: (1) ecological/hydrologic processes, (2) ecosystem/geomorphic feedbacks, (3) planning, design, and policy, (4) perceptions, information, and experience, (5) resource access and risk, and (6) operational water use and management. We illustrate the flexibility and utility of the iSAW framework by applying it to two research and management problems: understanding urban water supply and demand in a changing climate and expanding use of green storm water infrastructure in a semi-arid environment. The applications demonstrate that a generalized conceptual model can identify important components and linkages in complex and diverse water systems and facilitate communication about those systems among researchers from diverse disciplines

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations; however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESCs), including a knockout and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-sequencing analyses in Zscan10−/− mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10

Heterozygous frameshift variants in HNRNPA2B1 cause early-onset oculopharyngeal muscular dystrophy

Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families

Clinical presentation and natural history of infantile-onset ascending spastic paralysis from three families with an ALS2 founder variant.

Biallelic mutations of the alsin Rho guanine nucleotide exchange factor (ALS2) gene cause a group of overlapping autosomal recessive neurodegenerative disorders including infantile-onset ascending hereditary spastic paralysis (IAHSP), juvenile primary lateral sclerosis (JPLS), and juvenile amyotrophic lateral sclerosis (JALS/ALS2), caused by retrograde degeneration of the upper motor neurons of the pyramidal tracts. Here, we describe 11 individuals with IAHSP, aged 2-48 years, with IAHSP from three unrelated consanguineous Iranian families carrying the homozygous c.1640+1G>A founder mutation in ALS2. Three affected siblings from one family exhibit generalized dystonia which has not been previously described in families with IAHSP and has only been reported in three unrelated consanguineous families with JALS/ALS2. We report the oldest individuals with IAHSP to date and provide evidence that these patients survive well into their late 40s with preserved cognition and normal eye movements. Our study delineates the phenotypic spectrum of IAHSP and ALS2-related disorders and provides valuable insights into the natural disease course

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Identification and characterization of the candidate genes FAR1, EDC3, FRRS1L and HMG20A in individuals with autosomal recessive intellectual disability

Mentale Retardierung (MR) stellt mit einer Frequenz von etwa 2-3% der Bevölkerung ein großes und dennoch oft ungelöstes Gesundheitsproblem dar. In dieser Arbeit habe ich mittels positioneller Kartierung und massiv-paralleler Sequenzierung 16 große konsanguine Familien mit mehreren Betroffenen auf die genetischen Ursachen der MR hin untersucht. Identifizierte Kandidatenmutationen habe ich daraufhin funktionell weiter charakterisiert, um die Pathoge-nese weiter zu klären. Auf diese Weise konnten 3 Mutationen in bereits bekannten Genen (C12orf65, ALDH5A1, GPR56) für mentale Retardierung identifiziert und als ursächlich bestätigt werden. Dieses Ergebnis unterstützt die Anwendung von massiv-paralleler Sequenzierung in der genetischen Diagnostik, denn die gestellten Diagnosen konnten aufgrund unspezifischer Ausprägung des Krankheitsbildes nicht differential-diagnostisch in Erwägung gezogen werden. In einer Familie mit schwerer mentaler Retardierung, Epilepsie und Katarakten konnte eine Mutation in FAR1 identifiziert werden, dessen Genprodukt eine Funktion in der Plasmalogen-synthese hat. Nachfolgende in vitro Analysen zeigten eine starke Einschränkung der Enzym-aktivität durch die Mutation. Die Identifikation eines weiteren Patienten mit ähnlichem Krankheitsbild und compound heterozygoten Mutationen in FAR1 bestätigte FAR1 als ursächlich für die neu etablierte peroxisomale FAR1 Funktionsstörung. In einer weiteren Familie mit nicht-spezifischer MR konnte eine homozygote missense Muta-tion in EDC3 identifiziert werden. EDC3 ist ein enhancer des RNA decappings und in vitro Decapping-Assays zeigten eine starke Einschränkung der Decapping-Aktivität, vermutlich aufgrund von einer verringerten Domänen-Stabilität durch die identifizierte Veränderung. Eine Transkriptomanalyse von Patientenzellen zeigte weiterhin eine signifikante Veränderung der RNA Expression von Zielgenen. In einer weiteren Familie mit schwerer mentaler Retardierung wurde eine homozygote Muta-tion in FRRS1L identifiziert. FRRS1L ist Teil des AMPA-Rezeptors und spielt somit eine prominente Rolle in der synaptischen Plastizität. Die Identifikation von 2 weiteren Familien mit ähnlichem Krankheitsbild und missense bzw. trunkierenden Mutationen in FRRS1L unter-stützt FRRS1L als ein weiteres MR-Gen. Eine Kandidatenmutation in HMG20A wurde in einer weiteren Familie identifiziert. HMG20A spielt eine Rolle in der Genregulation insbesondere von neuronalen Genen. Funkti-onelle Analysen ergaben eine Veränderung der Expression von Zielgenen durch die identifi-zierte Variante, welche jedoch nicht signifikant war. Daher konnte die Variante in diesem Gen nicht als ursächlich bestätigt, jedoch auch nicht ausgeschlossen werden. Insgesamt konnten im Rahmen dieser Arbeit die genetischen Grundlagen der mentalen Retar-dierung in 6 Familien gelöst werden und dabei 3 neue MR-Gene identifiziert und weiter cha-rakterisiert werden.Intellectual disability (ID) has a frequency of 2-3% of the population and thus poses huge and still often unresolved health problem. In the course of this thesis I have examined the causes of intellectual disability in 16 large consanguineous families with several affected individuals using autozygositiy mapping and whole exome sequencing. Then I characterized the identified candidate mutations in more detail to establish their impact on the pathogenesis. With this approach 3 mutations in previously reported genes involved in ID (C12orf65, ALDH5A1, GPR56) were identified and confirmed as disease causing mutations. This result supports the use of massive parallel sequencing in genetic diagnostics, since these diagnoses could not have been suspected by the clinical appearance. In one family with profound intellectual disability, epilepsy and cataracts a mutation in FAR1, which has a function in plasmalogen synthesis, could be identified. Following in vitro analysis of the mutation showed a strong impact on enzyme activity. The identification of another in-dividual with a similar phenotype and compound heterozygous mutations in FAR1 confirmed FAR1 as causative for the newly established peroxisomal FAR1 deficiency disorder. In a further family with non-specific ID a homozygous missense mutation in EDC3 could be identified. EDC3 is an enhancer of mRNA decapping and in decapping assays we could show a strong impairment of the decapping activity likely due to decreased domain stability. Whole-transcriptome sequencing of patient cells in comparison to unaffected family members showed a significant change in RNA levels of target genes. In another family with profound intellectual disability a homozygous mutation in FRRS1L was identified. FRRS1L is part of the AMPA receptor and thus plays a prominent role in syn-aptic plasticity. The identification of 2 further families with a similar phenotype and loss-of function or missense mutations in FRRS1L supports FRRS1L as a good candidate gene for ID. A candidate mutation in HMG20A was identified in a further family. HMG20A plays a role in gene regulation of especially neuronal genes and functional studies showed a slight, yet not significant change in expression levels of target genes by the identified variant. Thus, this variant could not be confirmed as causative, but could not be excluded either. In total the genetic causes of intellectual disability could be solved in 6 families during this project and 3 new ID-genes were identified and further characterized