Ali K. Ozturk

Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers. An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development. Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62) as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

Genome-wide association study of intracranial aneurysm identifies three new risk loci

Saccular intracranial aneurysms are balloon-like dilations of the intracranial arterial wall; their hemorrhage commonly results in severe neurologic impairment and death. We report a second genome-wide association study with discovery and replication cohorts from Europe and Japan comprising 5,891 cases and 14,181 controls with ∼832,000 genotyped and imputed SNPs across discovery cohorts. We identified three new loci showing strong evidence for association with intracranial aneurysms in the combined dataset, including intervals near RBBP8 on 18q11.2 (odds ratio (OR) = 1.22, P = 1.1 × 10−12), STARD13-KL on 13q13.1 (OR = 1.20, P = 2.5 × 10−9) and a gene-rich region on 10q24.32 (OR = 1.29, P = 1.2 × 10−9). We also confirmed prior associations near SOX17 (8q11.23–q12.1; OR = 1.28, P = 1.3 × 10−12) and CDKN2A-CDKN2B (9p21.3; OR = 1.31, P = 1.5 × 10−22). It is noteworthy that several putative risk genes play a role in cell-cycle progression, potentially affecting the proliferation and senescence of progenitor-cell populations that are responsible for vascular formation and repair.

Mapping a Mendelian Form of Intracranial Aneurysm to 1p34.3-p36.13

The identification of pathways that underlie common disease has been greatly impacted by the study of rare families that segregate single genes with large effect. Intracranial aneurysm is a common neurological problem; the rupture of these aneurysms constitutes a frequently catastrophic neurologic event. The pathogenesis of these aneurysms is largely unknown, although genetic and environmental factors are believed to play a role. Previous genomewide studies in affected relative pairs have suggested linkage to several loci, but underlying genes have not been identified. We have identified a large kindred that segregates nonsyndromic intracranial aneurysm as a dominant trait with high penetrance. Genomewide analysis of linkage was performed using a two-stage approach: an analysis of ~10,000 single-nucleotide polymorphisms in the 6 living affected subjects, followed by the genotyping of simple tandem repeats across resulting candidate intervals in all 23 kindred members. Analysis revealed significant linkage to a single locus, with a LOD score of 4.2 at 1p34.3-p36.13 under a dominant model with high penetrance. These findings identify a Mendelian form of intracranial aneurysm and map the location of the underlying disease locus.

Genetics of intracranial aneurysms.

DESPITE ADVANCES IN the treatment of intracranial aneurysms (IA) in recent years, the overall outcome of patients with aneurysmal subarachnoid hemorrhage has shown only modest improvement. Given this poor prognosis, diagnosis of IA before rupture is of paramount importance. Currently, there are no reliable methods other than screening imaging studies of high-risk individuals to diagnose asymptomatic patients. Multiple levels of evidence suggest that environmental factors acting in concert with genetic susceptibilities lead to the formation, growth, and rupture of aneurysms in these patients. Epidemiological studies have already identified aneurysm-specific risk factors such as size and location, as well as patient-specific risk factors, such as age, sex, and presence of medical comorbidities, such as hypertension. In addition, exposure to certain environmental factors such as smoking have been shown to be important in the formation of IA. Furthermore, substantial evidence proves that certain loci contribute genetically to IA pathogenesis. Genome-wide linkage studies using relative pairs or rare families that are affected with the Mendelian forms of IA have already shown genetic heterogeneity of IA, suggesting that multiple genes, alone or in combination, are important in the disease pathophysiology. The linkage results, along with association studies, will ultimately lead to the identification of IA susceptibility genes. Identification of the genes important in IA pathogenesis will not only provide novel insights into the primary determinants of IA, but will also result in new opportunities for early diagnosis in the preclinical setting. Ultimately, novel therapeutic strategies based on biology will be developed, which will target these newly elucidated genetic susceptibilities.

Mutations in Apoptosis-related Gene, PDCD10, Cause Cerebral Cavernous Malformation 3

OBJECTIVE:To identify the CCM3 gene in a population of 61 families with a positive family history of cerebral cavernous malformations (CCM), 8 of which had suggestive linkage to the CCM3 locus. METHODS:We searched for mutations within the CCM3 interval using a high-throughput screening technique, temperature-gradient capillary electrophoresis. Mutations detected by this device were subsequently sequenced, and the results were analyzed. RESULTS:A recent study by Bergametti et al. established Programmed Cell Death 10 (PDCD10) as the gene responsible for CCM3. We hereby confirm PDCD10 as the CCM3 gene by reporting four novel mutations in 61 CCM families. Two of these mutations were identical and produced a stop codon in exon 7. Another two resulted in frameshift mutations in exon 6, although the mutations occurred at different points along the exon. The last mutation caused a frameshift in exon 9. Of note, mutations in these families completely cosegregated with the trait. Three of the five families had prior linkage data suggestive of the CCM3 locus, whereas the remaining two were identified in index patients with a positive family history but no linkage data. CONCLUSION:Our data establish PDCD10 as the gene responsible for CCM in families linking to the CCM3 locus. The discovery of the third gene involved in inherited forms of CCM, after KRIT1 and Malcavernin, is an important step toward dissecting the molecular pathophysiology of this disease.

Common variant near the endothelin receptor type A (EDNRA) gene is associated with intracranial aneurysm risk

The pathogenesis of intracranial aneurysm (IA) formation and rupture is complex, with significant contribution from genetic factors. We previously reported genome-wide association studies based on European discovery and Japanese replication cohorts of 5,891 cases and 14,181 controls that identified five disease-related loci. These studies were based on testing replication of genomic regions that contained SNPs with posterior probability of association (PPA) greater than 0.5 in the discovery cohort. To identify additional IA risk loci, we pursued 14 loci with PPAs in the discovery cohort between 0.1 and 0.5. Twenty-five SNPs from these loci were genotyped using two independent Japanese cohorts, and the results from discovery and replication cohorts were combined by meta-analysis. The results demonstrated significant association of IA with rs6841581 on chromosome 4q31.23, immediately 5′ of the endothelin receptor type A with P = 2.2 × 10−8 [odds ratio (OR) = 1.22, PPA = 0.986]. We also observed substantially increased evidence of association for two other regions on chromosomes 12q22 (OR = 1.16, P = 1.1 × 10−7, PPA = 0.934) and 20p12.1 (OR = 1.20, P = 6.9 × 10−7, PPA = 0.728). Although endothelin signaling has been hypothesized to play a role in various cardiovascular disorders for over two decades, our results are unique in providing genetic evidence for a significant association with IA and suggest that manipulation of the endothelin pathway may have important implications for the prevention and treatment of IA.

Recessive LAMC3 mutations cause malformations of occipital cortical development

The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin γ3 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations.

Molecular Genetic Analysis of Two Large Kindreds With Intracranial Aneurysms Demonstrates Linkage to 11q24-25 and 14q23-31

Background and Purpose— Both environmental and genetic factors contribute to the formation, growth, and rupture of intracranial aneurysms (IAs). To search for IA susceptibility genes, we took an outlier approach, using parametric genome-wide linkage analysis in extended IA kindreds in which IA is inherited as a simple Mendelian trait. We hereby present the molecular genetic analysis of 2 such families. Methods— For genome-wide linkage analysis, we used a 2-stage approach. First, using gene chips in affected-only analysis, we identified genomic regions that provide maximum theoretical logarithm of odds (lod) scores. Next, to confirm or exclude these candidate loci, we genotyped all available family members, both affected and unaffected, using polymorphic microsatellite markers located within these regions. Results— We obtained significant lod scores of 4.3 and 3.00 for linkage to chromosomes 11q24-25 and 14q23-31, respectively. Conclusions— Molecular genetic analysis of 2 large IA kindreds confirms linkage to chromosome 11q and 14q, which were suggested to contain IA susceptibility genes in a previous study of Japanese sib pairs. Independent identification of these 2 loci strongly suggests that IA susceptibility genes lie within these regions. While demonstrating the genetic heterogeneity of IA, these results are also an important step toward cloning IA genes and ultimately understanding its pathophysiology.

CCM2 Expression Parallels That of CCM1

BACKGROUND AND PURPOSE Mutations in CCM2 (MGC4607 or malcavernin) cause familial cerebral cavernous malformation (CCM), an autosomal dominant neurovascular disease. Both the function of this molecule and the pathogenesis of the disease remain elusive. METHODS We analyzed the mRNA expression of Ccm1 and Ccm2 in the embryonic and postnatal mouse brain by in situ hybridization. Subsequently, we generated CCM2-specific polyclonal antibodies and tested their specificity using transient transfection experiments in various cell lines. We then investigated CCM2 protein expression in cerebral and extracerebral tissues by Western blot analysis as well as immunohistochemistry and compared these results with CCM1 (KRIT1) protein expression. RESULTS In situ analysis shows similar temporal and spatial expression patterns for Ccm1 and Ccm2, although Ccm1 expression appears more widespread. Immunohistochemical analysis shows that CCM2 is expressed in various human organs, most noticeably in the arterial vascular endothelium. As is the case with CCM1, CCM2 is not expressed in other vascular wall elements such as smooth muscle cells or the venous circulation. Within cerebral tissue, it is also expressed in pyramidal neurons, astrocytes, and their foot processes. In extracerebral tissues, CCM2 is present in various epithelial cells necessary for blood-organ barrier formation. CONCLUSIONS CCM1 and CCM2 have similar expression patterns during development and postnatally thereafter. Given the fact that the disease phenotypes caused by mutations in either gene are clinically and pathologically indistinguishable, the significant overlap in expression pattern supports the hypothesis that both molecules are involved in the same pathway important for central nervous system vascular development.

Novel VLDLR microdeletion identified in two Turkish siblings with pachygyria and pontocerebellar atrophy

Congenital ataxia with cerebellar hypoplasia is a heterogeneous group of disorders that presents with motor disability, hypotonia, incoordination, and impaired motor development. Among these, disequilibrium syndrome describes a constellation of findings including non-progressive cerebellar ataxia, mental retardation, and cerebellar hypoplasia following an autosomal recessive pattern of inheritance and can be caused by mutations in the Very Low Density Lipoprotein Receptor (VLDLR). Interestingly, while the majority of patients with VLDL-associated cerebellar hypoplasia in the literature use bipedal gait, the previously reported patients of Turkish decent have demonstrated similar neurological sequelae, but rely on quadrupedal gait. We present a consanguinous Turkish family with two siblings with cerebellar atrophy, predominantly frontal pachygyria and ataxic bipedal gait, who were found to have a novel homozygous deletion in the VLDLR gene identified by using high-density single nucleotide polymorphism microarrays for homozygosity mapping and identification of CNVs within these regions. Discovery of disease causing homozygous deletions in the present Turkish family capable of maintaining bipedal movement exemplifies the phenotypic heterogeneity of VLDLR-associated cerebellar hypoplasia and ataxia.