Haploinsufficiency as a Foreground Pathomechanism of Poirer-Bienvenu Syndrome and Novel Insights Underlying the Phenotypic Continuum of CSNK2B-Associated Disorders

CSNK2B encodes for the regulatory subunit of the casein kinase II, a serine/threonine kinase that is highly expressed in the brain and implicated in development, neuritogenesis, synaptic transmission and plasticity. De novo variants in this gene have been identified as the cause of the Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) characterized by seizures and variably impaired intellectual development. More than sixty mutations have been described so far. However, data clarifying their functional impact and the possible pathomechanism are still scarce. Recently, a subset of CSNK2B missense variants affecting the Asp32 in the KEN box-like domain were proposed as the cause of a new intellectual disability-craniodigital syndrome (IDCS). In this study, we combined predictive functional and structural analysis and in vitro experiments to investigate the effect of two CSNK2B mutations, p.Leu39Arg and p.Met132LeufsTer110, identified by WES in two children with POBINDS. Our data prove that loss of the CK2beta protein, due to the instability of mutant CSNK2B mRNA and protein, resulting in a reduced amount of CK2 complex and affecting its kinase activity, may underlie the POBINDS phenotype. In addition, the deep reverse phenotyping of the patient carrying p.Leu39Arg, with an analysis of the available literature for individuals with either POBINDS or IDCS and a mutation in the KEN box-like motif, might suggest the existence of a continuous spectrum of CSNK2B-associated phenotypes rather than a sharp distinction between them

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain- hindbrain malformation

Abstract Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain- hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder. Author summary During brain development, localized gene expression is crucial for the formation and function of specific brain regions. Various groups of proteins are known to regulate segmentation through controlled gene expression, among them, the Rho GTPase regulator family. In this study, we identified a frameshift mutation in the Rho guanine nucleotide exchange factor 2 gene (ARHGEF2) in two children presenting with intellectual disability, mild microcephaly, and a midbrain- hindbrain malformation. This phenotype is also observed in Arhgef2 mutant mice, highlighting the importance of ARHGEF2 across development of distinct mammalian species. We show that loss of Arhgef2 affects neurogenesis and also cell migration. In addition, we extended the current knowledge of ARHGEF2 expression and its role in early central nervous system development, with special reference to the formation of the precerebellar system. In addition to extensive literature on ARHGEF2, we now provide evidence for its significant role in neuronal migration in brain development and link the gene to human neurodevelopmental disease

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1-3% of the world's population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorde

Redefining the MED13L syndrome

Congenital cardiac and neurodevelopmental deficits have been recently linked to the mediator complex subunit 13-like protein MED13L, a subunit of the CDK8-associated mediator complex that functions in transcriptional regulation through DNA-binding transcription factors and RNA polymerase II. Heterozygous MED13L variants cause transposition of the great arteries and intellectual disability (ID). Here, we report eight patients with predominantly novel MED13L variants who lack such complex congenital heart malformations. Rather, they depict a syndromic form of ID characterized by facial dysmorphism, ID, speech impairment, motor developmental delay with muscular hypotonia and behavioral difficulties. We thereby define a novel syndrome and significantly broaden the clinical spectrum associated with MED13L variants. A prominent feature of the MED13L neurocognitive presentation is profound language impairment, often in combination with articulatory deficits

Molecular characterization of Noonan Syndrome

1. INTRODUCTION..............................................................................................................5 1.1 HISTORY...........................................................................................................................5 1.2 CLINICAL FEATURES...........................................................................................................6 1.2.1 Craniofacial features..............................................................................................6 1.2.2 Cardiovascular anomalies......................................................................................7 1.2.3 Growth....................................................................................................................8 1.2.4 Skeletal findings......................................................................................................8 1.2.5 Genitourinary system..............................................................................................9 1.2.6 Haematology............................................................................................................9 1.2.7 Lymphatics...............................................................................................................9 1.2.8 Development and behaviour..................................................................................10 1.2.9 Hearing anomalies.................................................................................................10 1.2.10 Ocular anomalies...................................................................................................10 1.2.11 Ectoderm................................................................................................................10 1.3 GENETIC STUDIES............................................................................................................12 1.4 GENETIC HETEROGENEITY................................................................................................15 1.4.1 Noonan syndrome 2 (MIM 605275)......................................................................15 1.4.2 Neurofibromatosis-Noonan syndrome (NF-NS) (MIM 601321)...........................16 1.4.3 LEOPARD syndrome (MIM #151100)..................................................................18 1.4.4 Cardio-facio-cutaneous syndrome (CFC)(MIM 115150).....................................18 1.5 CHROMOSOMAL REARRANGEMENTS IN HUMANS.................................................................20 1.5.1 Numerical chromosomal abnormalities................................................................22 1.5.2 Structural chromosomal rearrangements.............................................................21 1.5.3 Disease-associated balanced chromosome rearrangements: a genetic tool for gene identification and cloning..........................................................................................22 1.6 AIMS AND OBJECTIVE.......................................................................................................24 2. MATERIALS AND METHODS....................................................................................25 2.1 MATERIALS......................................................................................................................25 2.1.1 Chemicals and equipment.....................................................................................25 2.1.2 Solutions and media..............................................................................................26 2.1.3 Buffer solutions.....................................................................................................27 2.1.4 Enzymes.................................................................................................................28 2.1.5 Kits........................................................................................................................29 2.1.6 Vectors..................................................................................................................29 2.1.7 Primers..................................................................................................................29 2.1.8 Genomic material..................................................................................................34 2.1.9 Bacterial material.................................................................................................34 2.2 METHODS........................................................................................................................35 2.2.1 Patients' sample collection...................................................................................35 2.2.2 Bioinformatic analysis..........................................................................................35 2.2.3 DNA isolation........................................................................................................36 2.2.4 RNA isolation........................................................................................................36 2.2.5 Fluorescence in Situ Hybridisation (FISH)..........................................................36 2.2.6 Polymerase Chain Reaction (PCR)......................................................................37 2.2.7 Allele-Specific PCR amplification (ASO).............................................................38 2.2.8 RT-PCR.................................................................................................................38 2.2.9 Semiquantitative RT-PCR.....................................................................................38 2.2.10 Purification of PCR products................................................................................39 2.2.11 Recombinant DNA techniques..............................................................................39 2.2.12 Sequencing............................................................................................................39 2.2.13 5'-RACE (Rapid Amplification of cDNA Ends)....................................................39 2.2.14 Labelling of DNA probes for hybridisations.........................................................40 2.2.15 Library screening..................................................................................................41 2.2.16 Southern blot hybridisations.................................................................................42 2.2.17 Northern blot hybridisations.................................................................................42 2.2.18 Mutation screening by Denaturing High Performance Liquid Chromatography (DHPLC)...........................................................................................................................43 3. RESULTS........................................................................................................................44 3.1 MOLECULAR CHARACTERIZATION OF THE BREAKPOINT REGIONS ASSOCIATED WITH A CONSTITUTIONAL T(2;12)(Q37;Q24)PAT IN A PATIENT WITH NOONAN SYNDROME ..............44 3.1.1 Case report............................................................................................................44 3.1.2 Identification of chromosome-specific BAC clones that spanned the translocation breakpoints........................................................................................................................45 3.1.3 Chromosome 12: cosmid library screening and Southern blot experiments........48 3.1.4 Chromosome 2: cosmid library screening and Southern blot experiments..........49 3.1.5 In silico analysis of the breakpoint-spanning BAC clone on chromosome 12.....51 3.1.6 Molecular analysis of the breakpoint region on chromosome 12.........................52 3.1.7 Identification and characterization of the TRAP240-like gene............................54 3.1.8 Expression analysis of the TRAP240-like gene....................................................58 3.1.9 Expression analysis of the TRAP240-like gene in the patient with t(2;12)(q37;q24)pat...........................................................................................................59 3.1.10 Mutation analysis of the candidate gene TRAP240-like.......................................60 3.1.11 Analysis of the breakpoint region on chromosome 2............................................65 3.2 PTPN11 GENE ANALYSIS....................................................................................................67 3.2.1 Mutation analysis of the PTPN11 gene in 96 NS and 5 CFC patients.................67 3.2.2 Mutation analysis of the PTPN11 gene in patients with LEOPARD syndrome...71 4. DISCUSSION...................................................................................................................74 4.1 ROLE OF PTPN11 GENE IN THE AETIOLOGY OF NOONAN SYNDROME, CARDIO-FACIO-CUTANEOUS SYNDROME, AND LEOPARD SYNDROME..........................................................................................74 4.1.1 Spectrum of PTPN11 mutation in NS patients......................................................77 4.1.2 Genotype-phenotype correlation..........................................................................82 4.1.3 Cosegregation.......................................................................................................86 4.1.4 Cardio-facio-cutaneous syndrome........................................................................86 4.1.5 LEOPARD syndrome............................................................................................87 4.2 ROLE OF TRAP240-LIKE GENE IN THE AETIOLOGY OF NOONAN SYNDROME.............................89 4.3 SELENOCYSTEINE LYASE GENE................................................................................................94 4.4 CONCLUSION........................................................................................................................98 5. REFERENCES...............................................................................................................100 6. SUMMARY....................................................................................................................116 6.1 RIASSUNTO....................................................................................................................116 6.2 SUMMARY......................................................................................................................119 7. APPENDIX.....................................................................................................................124 7.1 PUBLICATIONS...............................................................................................................124 7.2 ACKNOWLEDGMENTS.....................................................................................................12

Challenging Occam’s Razor: Dual Molecular Diagnoses Explain Entangled Clinical Pictures

Dual molecular diagnoses are defined as the presence of pathogenic variants at two distinct and independently segregating loci that cause two different Mendelian conditions. In this study, we report the identification of double genetic disorders in a series of patients with complex clinical features. In the last 24 months, 342 syndromic patients have been recruited and clinically characterised. Whole Exome Sequencing analysis has been performed on the proband and on both parents and identified seven patients affected by a dual molecular diagnosis. Upon a detailed evaluation of both their clinical and molecular features, subjects are able to be divided into two groups: (A) five patients who present distinct phenotypes, due to each of the two different underlying genetic diseases; (B) two patients with overlapping clinical features that may be underpinned by both the identified genetic variations. Notably, only in one case a multilocus genomic variation was already suspected during the clinical evaluation. Overall, our findings highlight how dual molecular diagnoses represent a challenging model of complex inheritance that should always be considered whenever a patient shows atypical clinical features. Indeed, an accurate genetic characterisation is of the utmost importance to provide patients with a personalised and safe clinical management

The Genetic Diagnosis of Ultrarare DEEs: An Ongoing Challenge

Epileptic encephalopathies (EEs) and developmental and epileptic encephalopathies (DEEs) are a group of severe early-onset neurodevelopmental disorders (NDDs). In recent years, next-generation equencing (NGS) technologies enabled the discovery of numerous genes involved in these conditions. However, more than 50% of patients remained undiagnosed. A major obstacle lies in the high degree of genetic heterogeneity and the wide phenotypic variability that has characterized these disorders. Interpreting a large amount of NGS data is also a crucial challenge. This study describes a dynamic diagnostic procedure used to investigate 17 patients with DEE or EE with previous negative or inconclusive genetic testing by whole-exome sequencing (WES), leading to a definite diagnosis in about 59% of participants. Biallelic mutations caused most of the diagnosed cases (50%), and a pathogenic somatic mutation resulted in 10% of the subjects. The high diagnostic yield reached highlights the relevance of the scientific approach, the importance of the reverse phenotyping strategy, and the involvement of a dedicated multidisciplinary team. The study emphasizes the role of recessive and somatic variants, new genetic mechanisms, and the complexity of genotype–phenotype associations. In older patients, WES results could end invasive diagnostic procedures and allow a more accurate transition. Finally, an early pursued diagnosis is essential for comprehensive care of patients, precision approach, knowledge of prognosis, patient and family planning, and quality of life

Common Pathological Mutations in PQBP1 Induce Nonsense-Mediated mRNA Decay and Enhance Exclusion of the Mutant Exon

The polyglutamine binding protein 1 (PQBP1) gene plays an important role in X-linked mental retardation (XLMR). Nine of the thirteen PQBP1 mutations known to date affect the AG hexamer in exon 4 and cause frameshifts introducing premature termination codons (PTCs). However, the phenotype in this group of patients is variable. To investigate the pathology of these PQBP1 mutations, we evaluated their consequences on mRNA and protein expression. RT-PCRs revealed mutation-specific reduction of PQBP1 mRNAs carrying the PTCs that can be partially restored by blocking translation, thus indicating a role for the nonsense-mediated mRNA decay pathway. In addition, these mutations resulted in altered levels of PQBP1 transcripts that skipped exon 4, probably as a result of altering important splicing motifs via nonsense-associated altered splicing (NAS). This hypothesis is supported by transfection experiments using wild-type and mutant PQBP1 minigenes. Moreover, we show that a truncated PQBP1 protein is indeed present in the patients. Remarkably, patients with insertion/deletion mutations in the AG hexamer express significantly increased levels of a PQBP1 isoform, which is very likely encoded by the transcripts without exon 4, confirming the findings at the mRNA level. Our study provides significant insight into the early events contributing to the pathogenesis of the PQBP1 related XLMR disease. Hum Mutat 31:90–98, 2010

Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutanesous syndrome

Noonan syndrome (NS) is a relatively common, but genetically heterogeneous autosomal dominant malformation syndrome. Characteristic features are proportionate short stature, dysmorphic face, and congenital heart defects. Only recently, a gene involved in NS could be identified. It encodes the non-receptor protein tyrosine phosphatase SHP-2, which is an important molecule in several intracellular signal transduction pathways that control diverse developmental processes, most importantly cardiac semilunar valvulogenesis. We have screened this gene for mutations in 96 familial and sporadic, well-characterised NS patients and identified 15 different missense mutations in a total of 32 patients (33%), including 23 index patients. Most changes clustered in one exon which encodes parts of the N-SH2 domain. Five of the mutations were recurrent. Interestingly, no mutations in the PTPN11 gene were detected in five additional patients with cardio-facio-cutaneous (CFC) syndrome, which shows clinical similarities to NS