Copy-Number-Variations (CNVs) in der Ätiologie von Hirnfehlbildungen

I.Introduction 1\. Development of the brain 1.1 Development of the anterior neural plate 1.2 Development of the forebrain 1.3 Development of the cortex 1.4 Development of the cerebellum 1.5 Development of corpus callosum 2\. Brain malformations 2.1 Classification of brain malformations 2.1.1. Midline defects 2.1.1.1 Holoprosencephaly 2.1.1.2 Corpus callosum agenesis 2.1.2 Neuronal migration disorders 2.1.2.1 Abnormal neuronal migration 2.1.2.1.1 Gray Matter Heterotopia 2.1.2.1.2 Lissencephaly-Agyria (type 1 lissencephaly) 2.1.2.1.3 Reelin-Type Pachygyria 2.1.2.1.4 Cobblestone cortex (type 2 lissencephaly) 2.1.2.2 Disorders of neuronal organization 2.1.2.2.1 Focal cortical dysplasia- microdysgenesis 2.1.2.2.2 polymicrogyria 2.1.2.3 Abnormal glial or neural proliferation 2.1.2.3.1 Microcephaly 2.1.2.4 Malformations of the posterior fossa 2.1.2.4.1 Dandy Walker syndrome (DWS) 2.2 Molecular classification of brain malformations 2.2.1 Peroxisome biogenesis disorders 2.2.2 Glycosylation abnormalities 2.2.3 Ciliopathies 3\. Identification of genes 3.1 Historical overview 3.2 Array comparative genomic hybridization II. Materials and Methods 1\. Materials 1.1 Equipment 1.2Chemicals 1.3Solutions 1.4DNA microarrays 1.4.1BAC- arrays 1.4.2Oligo-arrays 1.5 Cell culture media and reagents 1.6 DNA purification and labeling kits 1.7 Samples 1.7.1 Reference samples 1.7.2 Test samples 1.8 Information management and resources 1.8.1 Softwares 1.8.2 Databases 2\. Methods 2.1 Selection of cases 2.2 Documentation 2.3 Chromosomal study 2.4 Sample preparation 2.4.1 DNA extraction 2.4.2 DNA preparation for BAC array 2.5 BAC array 2.5.1 Labelling 2.5.2 DNA precipitation 2.5.3 Pre- Hybridization 2.5.4 Hybridization 2.5.5 Post hybridization wash 2.5.6 Scanning 2.5.7 Analysis 2.6 Oligo-array 2.7 Interphase/metaphase FISH 2.7.1 Probe preparation by nick translation 2.7.2 EtOH precipitation 2.7.3 Pre- Hybridization 2.7.4 FISH hybridization 2.7.5 Post hybridization wash III. Results 1\. Clinical findings in patients with CNVs 1.1 Brain malformations 1.2 Other clinical findings 2\. Array CGH results 3\. Alternate techniques for confirmation 3.1 Confirmation by oligoarray 3.2 Confirmation by FISH 4\. Gene enrichment analysis IV. Discussion 1\. Cohort selection criteria 1.1 Array CGH in patients with seizures 1.2 Array CGH in intellectual disability 2\. The size and nature of the CNVs 2.1 Comparison of hereditary and de novo CNVs 2.2 Size of de novo CNVs 2.3 Size of hereditary CNVs 3\. CNVs in specific brain malformations 3.1 Corpus Callosum Agenesis 3.2 Cerebellar hypoplasia: 3.3 Microcephaly 3.4 Posterior fossa malformations: 3.5 Lissencephaly/Pachygyria 4\. Gene Content of CNVs 4.1 Network analysis for Brain Development genes 4.2 Brain development genes 4.3 Gene content of de novo CNVs 4.3.1 PAFAHB1, YWHAE in 17p13.3 deletions 4.3.2 AKT3 and ZNF238 in corpus callosum agenesis 4.3.3. Known syndromes 4.3.3.1 Tetrasomy of 12p 4.3.3.2 Microdeletion 1p36 4.3.3.3 Microdeletion 22q11.2 4.3.4 Novel genomic imbalances 4.3.4.1 Deletion 2p and duplication 19q 4.3.4.2 Duplication 16p13.2 4.3.4.3 Deletion 9q and duplication 9q 4.4. Gene content of hereditary CNVs 4.4.1 The 16p13.11 duplication 4.4.2 Other genes 4.4.2.1 VLDLR 4.4.2.2. SLC19A3 4.4.2.3 Genes with brain expression 4.5. GO Term analysis 4.5.1 Biological processes 4.5.2 Protein-protein interactions V. Conclusion Genome and gene studies: an outlookThe proper spatio-temporal expression of structural and regulatory genes is essential to brain development. In an attempt to identify underlying mechanisms, we investigated the role of CNVs in the etiology of brain malformations. We studied genomic variations in 100 prospective patients with various brain malformations by array CGH on a 200 kb overlapping BAC array platform. We considered only those copy number variations that encompassed at least 3 overlapping BAC clones and differed by more than a total of 100 kbs difference on either or both sides from formerly reported CNVs in the Database of Genomic Variants. These CNVs were confirmed by parental studies or by a second alternate technique such as oligoarray or FISH and in a few cases by both. We find 27 CNVs in 22 patients, where 3 patients have two and one patient has 3 CNVs. There are a total of 13 de novo CNVs in 10 patients and 14 hereditary CNVs in one of the above and 12 other patients. The CNVs range from 150 kb to 10.2 Mb in size. Seven of the de novo CNVs, (microdeletion 1p36, microdeletion 1q43q44, two 12p tetrasomy, 2 deletions of 17p, and one microdeletion 22q11.21), one of the inherited CNVs (microdeletion 16p13.2), directly established the diagnosis of known deletion/duplication syndromes. In three patients one or more de novo aberrations not corresponding to know syndromes were identified. We found that 22 of 100 patients (22%) with various forms of brain malformations, ID, and symptomatic epilepsy carried one or more rare CNVs. This frequency, albeit the stringent exclusion criteria, is higher than frequencies detected in studies of patients with ID or idiopathic generalized epilepsy, emphasizing the importance of testing for CNVs in patients with structural brain malformations. Tetrasomy12p tetrasomy is the single CNV with the highest occurrence (10%) and PAFAH1B1 is the single gene with the highest actual (10%) and assumed (20%) involvement in specific structural brain malformations. We find that among the structural brain malformations in our patients, midline defects (28%) and more specifically corpus callosum agenesis has the highest association with CNVs. We discuss the significance of our findings in the further elaboration of the role of previously candidated genes ZNF238, AKT3, NTAN1, NDE1 in the phenotype of these patients. The five novel imbalances (as of yet unreported) in three patients, may provide insight into genes involved in structural brain malformations within these regions and we propose that APBA1 might be a novel candidate gene for brain malformations. Our analysis of GO Terms and PPI networks suggest that genes involved in “axonal transport,” “cation transmembrane transporter activity,” and in the “JNK cascade” play a significant role in the etiology of brain malformations. To the best of our knowledge, this is the first systematic study of CNVs in a cohort of patients selected based upon the presence of a detectable structural brain malformation. The genomic regions detected in this study include potential novel candidate genes and possible susceptibility loci for predisposition to structural brain malformations, epilepsy and intellectual disability.Die richtige räumliche und zeitliche Expression struktureller und regulatorischer Gene ist für die Hirnentwicklung von essentieller Bedeutung. Als Beitrag zur Aufklärung der daran beteiligten Mechanismen haben wir untersucht, welche Rolle submikroskopische Deletionen und Duplikationen (sog. Copy Number Variants, CNVs) in der Ätiologie von Hirnfehlbildungen spielen. Dazu haben wir bei 100 Patienten mit Hilfe der Array CGH-Technologie nach genomischen Varianten gesucht. Für diese Untersuchungen wurde ein Raster aus überlappenden BAC Klonen verwendet, welches das ganze Genom lückenlos überspannt. Dabei wurden nur solche Genomveränderungen berücksichtigt, die mindestens drei BAC Klone überspannten und sich an einer oder beiden Seiten um mehr als 100 kb von früher geschriebenen und in der Datenbank genomischer Varianten aufgeführten CNVs unterschieden. Diese CNVs wurden durch Untersuchung der Eltern mithilfe von Oligonukleotid-basierter Array CGH oder durch FISH validiert. In 22 Patienten wurden insgesamt 27 CNVs gefunden. Drei Patienten wiesen zwei und einer drei verschiedene CNVs auf. 13 dieser CNVs wurden nur bei Patienten gefunden, während 14 andere auch bei einem Elternteil vorkamen. Die Größe der CNVs variierte von 150 kb bis 10,2 Mb. Sieben der neuen CNVs (Mikrodeletion 1p36, Mikrodeletion 1q43q44, Tetrasomie 12p (2 mal), zwei 17p-Deletionen und eine Mikrodeletion 22q11.21) und eine der vererbten CNVs (Mikrodeletion 16p13.2) korrespondierten mit bereits bekannten Deletions- bzw. Duplikationssyndromen. Bei drei Patienten wurden de novo Veränderungen gefunden, für die vorher kein Krankheitsbezug bekannt war. Wir fanden, daß 22 von 100 Patienten mit verschiedenen Hirnfehlbildungen, geistiger Behinderung und Epilepsie einen oder mehrere CNVs aufwiesen. Diese unter stringenten Ausschlußkriterien ermittelte Häufigkeit ist deutlich höher als die Häufigkeit von CNVs bei Patienten mit geistiger Behinderung oder idiopathischer generalisierter Epilepsie, was die Bedeutung von CNVs in der Ätiologie struktureller Hirnfehlbildungen unterstreicht. Zehn Prozent aller Patienten wiesen eine Tetrasomie 12p auf. Damit ist diese Störung die häufigste bei Patienten mit strukturellen Hirnfehlbildungen gefundene Veränderung, und das PAFAH1B1-Gen ist am häufigsten an klinisch relevanten CNVs beteiligt. Unter den strukturellen Hirnfehlbildungen sind Mittelliniendefekte und insbesondere die Corpus Callosum-Agenesie am engsten mit CNVs assoziiert. Wir diskutieren außerdem die Rolle der früher beschriebenen Kandidatengene ZNF238, AKT3, NTAN1 und NDE1 beim Zustandekommen des Phänotyps dieser Patienten. Bei drei Patienten wurden fünf neue, bisher noch nicht beschriebene genomische Imbalanzen gefunden, die möglicherweise Rückschlüsse auf pathogenetische relevante Gene erlauben. Auf diese Weise haben wir das APBA1-Gen als neues plausibles Kandidatengen für angeborene Hirnfehlbildungen identifiziert. Überdies sprechen unsere Untersuchungen dafür, daß Defekte des axonalen Transports, des Kationen-Transports und der JunK-Kaskade bei der Entstehung von Hirnfehlbildungen eine bedeutende Rolle spielen. Nach unserer Kenntnis ist dies die erste systematische Suche nach CNVs in einer Kohorte Patienten mit Fehlbildungen der Hirnstruktur. Die im Rahmen dieser Studie definierten deletierten oder duplizierten Genomabschnitte sind ein Schlüssel zur Identifizierung weiterer Kandidatengene und Risikofaktoren für strukturelle Hirnfehlbildungen, Epilepsie und geistige Behinderung

5p13 microduplication in a malformed fetus and his unaffected father

The 5p13 microduplication syndrome is a contiguous gene syndrome characterized by developmental delay intellectual disability, hypotonia, unusual facies with marked variability, mild limb anomalies, and in some cases brain malformations. The duplication ranges in size from 0.25 to 1.08 Mb and encompasses five genes (NIPBL, SLC1A3, CPLANE1, NUP155, and WDR70), of which NIPBL has been suggested to be the main dose sensitive gene. All patients with duplication of the complete NIPBL gene reported thus far have been de novo. Here, we report a 25-week-old male fetus with hypertelorism, wide and depressed nasal bridge, depressed nasal tip, low-set ears, clenched hands, flexion contracture of elbows, knees, and left wrist, and bilateral clubfeet, bowing and shortening of long bones and brain malformation of dorsal part of callosal body. The fetus had a 667 kb gain at 5p13.2 encompassing SLC1A3, NIPBL and exons 22–52 of CPLANE1. The microduplication was inherited from the healthy father, in whom no indication for mosaicism was detected. The family demonstrates that incomplete penetrance of 5p13 microduplication syndrome may occur which is important in genetic counseling of families with this entity

Familial Case of Pelizaeus-Merzbacher Disorder Detected by Oligoarray Comparative Genomic Hybridization: Genotype-to-Phenotype Diagnosis

Introduction. Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive hypomyelinating leukodystrophy characterized by nystagmus, spastic quadriplegia, ataxia, and developmental delay. It is caused by mutation in the PLP1 gene. Case Description. We report a 9-year-old boy referred for oligoarray comparative genomic hybridization (OA-CGH) because of intellectual delay, seizures, microcephaly, nystagmus, and spastic paraplegia. Similar clinical findings were reported in his older brother and maternal uncle. Both parents had normal phenotypes. OA-CGH was performed and a 436 Kb duplication was detected and the diagnosis of PMD was made. The mother was carrier of this 436 Kb duplication. Conclusion. Clinical presentation has been accepted as being the mainstay of diagnosis for most conditions. However, recent developments in genetic diagnosis have shown that, in many congenital and sporadic disorders lacking specific phenotypic manifestations, a genotype-to-phenotype approach can be conclusive. In this case, a diagnosis was reached by universal genomic testing, namely, whole genomic array

Pericentric inversion of chromosome 18 in parents leading to a phenotypically normal child with segmental uniparental disomy 18

In this study, we report a familial inversion of chromosome 18, inv(18)(p11.31q21.33), in both members of a consanguineous couple. Their first child had inherited one balanced pericentric inversion along with a recombinant chromosome 18 resulting in dup(18q)/del(18p), and had mild dysmorphic features in the absence of mental and developmental retardation. The second child had received two recombinant chromosomes 18, from the mother a derivative chromosome 18 with dup(18p)/del(18q) and from the father a derivative chromosome 18 with dup(18q)/del(18p). The aberration was prenatally detected; however, as the two opposite aneuploidies were thought to compensate each other, the family decided to carry on with the pregnancy, knowing that uniparental disomy for the segments outside the inversion could have an adverse influence on the development of the child. Uniparental disomy was confirmed by SNP arrays. The child, who has been followed up until the age of 20 months, is healthy and normal. It seems to be the first reported case with two opposite recombinant chromosomes that compensate each other and lead to segmental uniparental disomy for two segments on the chromosome, one maternal and the other paternal

Next generation sequencing in a family with autosomal recessive Kahrizi syndrome (OMIM 612713) reveals a homozygous frameshift mutation in SRD5A3

As part of a large-scale, systematic effort to unravel the molecular causes of autosomal recessive mental retardation, we have previously described a novel syndrome consisting of mental retardation, coloboma, cataract and kyphosis (Kahrizi syndrome, OMIM 612713) and mapped the underlying gene to a 10.4-Mb interval near the centromere on chromosome 4. By combining array-based exon enrichment and next generation sequencing, we have now identified a homozygous frameshift mutation (c.203dupC; p.Phe69LeufsX2) in the gene for steroid 5α-reductase type 3 (SRD5A3) as the disease-causing change in this interval. Recent evidence indicates that this enzyme is required for the conversion of polyprenol to dolichol, a step that is essential for N-linked protein glycosylation. Independently, another group has recently observed SRD5A3 mutations in several families with a type 1 congenital disorder of glycosylation (CDG type Ix, OMIM 212067), mental retardation, cerebellar ataxia and eye disorders. Our results show that Kahrizi syndrome and this CDG Ix subtype are allelic disorders, and they illustrate the potential of next-generation sequencing strategies for the elucidation of single gene defects

A Defect in the TUSC3 Gene Is Associated with Autosomal Recessive Mental Retardation

Recent studies have shown that autosomal recessive mental retardation (ARMR) is extremely heterogeneous, and there is reason to believe that the number of underlying gene defects goes into the thousands. To date, however, only four genes have been implicated in nonsyndromic ARMR (NS-ARMR): PRSS12 (neurotrypsin), CRBN (cereblon), CC2D1A, and GRIK2. As part of an ongoing systematic study aiming to identify ARMR genes, we investigated a large consanguineous family comprising seven patients with nonsyndromic ARMR in four sibships. Genome-wide SNP typing enabled us to map the relevant genetic defect to a 4.6 Mbp interval on chromosome 8. Haplotype analyses and copy-number studies led to the identification of a homozygous deletion partly removing TUSC3 (N33) in all patients. All obligate carriers of this family were heterozygous, but none of 192 unrelated healthy individuals from the same population carried this deletion. We excluded other disease-causing mutations in the coding regions of all genes within the linkage interval by sequencing; moreover, we verified the complete absence of a functional TUSC3 transcript in all patients through RT-PCR. TUSC3 is thought to encode a subunit of the endoplasmic reticulum-bound oligosaccharyltransferase complex that catalyzes a pivotal step in the protein N-glycosylation process. Our data suggest that in contrast to other genetic defects of glycosylation, inactivation of TUSC3 causes nonsyndromic MR, a conclusion that is supported by a separate report in this issue of AJHG. TUSC3 is only the fifth gene implicated in NS-ARMR and the first for which mutations have been reported in more than one family

An autosomal recessive syndrome of severe mental retardation, cataract, coloboma and kyphosis maps to the pericentromeric region of chromosome 4

We report on three siblings with a novel mental retardation (MR) syndrome who were born to distantly related Iranian parents. The clinical problems comprised severe MR, cataracts with onset in late adolescence, kyphosis, contractures of large joints, bulbous nose with broad nasal bridge, and thick lips. Two patients also had uni- or bilateral iris coloboma. Linkage analysis revealed a single 10.4 Mb interval of homozygosity with significant LOD score in the pericentromeric region of chromosome 4 flanked by SNPs rs728293 (4p12) and rs1105434 (4q12). This interval contains more than 40 genes, none of which has been implicated in MR so far. The identification of the causative gene defect for this syndrome will provide new insights into the development of the brain and the eye