Hande Kaymakçalan

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.

Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1, and SMO

Genetic Clues to Meningioma Meningiomas are the most common primary brain tumors in adults. Located within the layer of tissue covering the brain, these tumors are usually slow-growing and benign but can cause serious neurological complications. About half of these tumors have mutations in the neurofibromin 2 gene (NF2). To identify other genes that contribute to meningioma pathogenesis, Clark et al. (p. 1077, published online 24 January) performed genome sequence analysis on 300 tumors. Meningiomas fell into two general classes: benign tumors located at the skull base—which tend to harbor mutations in the TRAF7, KLF4, AKT1, and SMO genes—and higher-grade tumors located in the cerebral and cerebellar hemispheres harbor mutations in NF2. The mutational profiles of meningiomas, a common type of brain tumor, correlate with their anatomical location and clinical status. We report genomic analysis of 300 meningiomas, the most common primary brain tumors, leading to the discovery of mutations in TRAF7, a proapoptotic E3 ubiquitin ligase, in nearly one-fourth of all meningiomas. Mutations in TRAF7 commonly occurred with a recurrent mutation (K409Q) in KLF4, a transcription factor known for its role in inducing pluripotency, or with AKT1E17K, a mutation known to activate the PI3K pathway. SMO mutations, which activate Hedgehog signaling, were identified in ~5% of non-NF2 mutant meningiomas. These non-NF2 meningiomas were clinically distinctive—nearly always benign, with chromosomal stability, and originating from the medial skull base. In contrast, meningiomas with mutant NF2 and/or chromosome 22 loss were more likely to be atypical, showing genomic instability, and localizing to the cerebral and cerebellar hemispheres. Collectively, these findings identify distinct meningioma subtypes, suggesting avenues for targeted therapeutics.

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.

Mutations in LAMB1 Cause Cobblestone Brain Malformation without Muscular or Ocular Abnormalities

Cobblestone brain malformation (COB) is a neuronal migration disorder characterized by protrusions of neurons beyond the first cortical layer at the pial surface of the brain. It is usually seen in association with dystroglycanopathy types of congenital muscular dystrophies (CMDs) and ocular abnormalities termed muscle-eye-brain disease. Here we report homozygous deleterious mutations in LAMB1, encoding laminin subunit beta-1, in two families with autosomal-recessive COB. Affected individuals displayed a constellation of brain malformations including cortical gyral and white-matter signal abnormalities, severe cerebellar dysplasia, brainstem hypoplasia, and occipital encephalocele, but they had less apparent ocular or muscular abnormalities than are typically observed in COB. LAMB1 is localized to the pial basement membrane, suggesting that defective connection between radial glial cells and the pial surface mediated by LAMB1 leads to this malformation.

NGLY1 mutation causes neuromotor impairment, intellectual disability, and neuropathy.

N-glycanase 1 (NGLY1) is a conserved enzyme that is responsible for the deglycosylation of misfolded N-glycosylated proteins in the cytoplasm prior to their proteasome-mediated degradation. Disruption of this degradation process has been associated with various neurologic diseases including amyotrophic lateral sclerosis and Parkinsons disease. Here, we describe two siblings with neuromotor impairment, apparent intellectual disability, corneal opacities, and neuropathy who were found to possess a novel homozygous frame-shift mutation due to a 4 base pair deletion in NGLY1 (c.1533_1536delTCAA, p.Asn511LysfsX51). We hypothesize that this mutation likely limits the capability of neuronal cells to respond to stress due to accumulation of misfolded proteins, thereby impairing their survival and resulting in progressive loss of neurological function.

Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly

Cell division terminates with cytokinesis and cellular separation. Autosomal-recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a reduction in brain and head size at birth in addition to non-progressive intellectual disability. MCPH is genetically heterogeneous, and 16 loci are known to be associated with loss-of-function mutations predominantly affecting centrosomal-associated proteins, but the multiple roles of centrosomes in cellular function has left questions about etiology. Here, we identified three families affected by homozygous missense mutations in CIT, encoding citron rho-interacting kinase (CIT), which has established roles in cytokinesis. All mutations caused substitution of conserved amino acid residues in the kinase domain and impaired kinase activity. Neural progenitors that were differentiated from induced pluripotent stem cells (iPSCs) derived from individuals with these mutations exhibited abnormal cytokinesis with delayed mitosis, multipolar spindles, and increased apoptosis, rescued by CRISPR/Cas9 genome editing. Our results highlight the importance of cytokinesis in the pathology of primary microcephaly.

Prenatal Health, Educational Attainment, and Intergenerational Inequality : The Northern Finland Birth Cohort 1966 Study

In this article, we study the effects of prenatal health on educational attainment and on the reproduction of family background inequalities in education. Using Finnish birth cohort data, we analyze several maternal and fetal health variables, many of which have not been featured in the literature on long-term socioeconomic effects of health despite the effects of these variables on birth and short-term health outcomes. We find strong negative effects of mother’s prenatal smoking on educational attainment, which are stronger if the mother smoked heavily but are not significant if she quit during the first trimester. Anemia during pregnancy is also associated with lower levels of attained education. Other indicators of prenatal health (pre-pregnancy obesity, mother’s antenatal depressed mood, hypertension and preeclampsia, early prenatal care visits, premature birth, and small size for gestational age) do not predict educational attainment. Our measures explain little of the educational inequalities by parents’ class or education. However, smoking explains 12%—and all health variables together, 19%—of the lower educational attainment of children born to unmarried mothers. Our findings point to the usefulness of proximate health measures in addition to general ones. They also point to the potentially important role played by early health in intergenerational processes.

Brain Malformations Associated With Knobloch Syndrome—Review of Literature, Expanding Clinical Spectrum, and Identification of Novel Mutations

BACKGROUND Knobloch syndrome is a rare, autosomal recessive, developmental disorder characterized by stereotyped ocular abnormalities with or without occipital skull deformities (encephalocele, bone defects, and cutis aplasia). Although there is clear heterogeneity in clinical presentation, central nervous system malformations, aside from the characteristic encephalocele, have not typically been considered a component of the disease phenotype. METHODS Four patients originally presented for genetic evaluation of symptomatic structural brain malformations. Whole-genome genotyping, whole-exome sequencing, and confirmatory Sanger sequencing were performed. Using immunohistochemical analysis, we investigated the protein expression pattern of COL18A1 in the mid-fetal and adult human cerebral cortex and then analyzed the spatial and temporal changes in the expression pattern of COL18A1 during human cortical development using the Human Brain Transcriptome database. RESULTS We identified two novel homozygous deleterious frame-shift mutations in the COL18A1 gene. On further investigation of these patients and their families, we found that many exhibited certain characteristics of Knobloch syndrome, including pronounced ocular defects. Our data strongly support an important role for COL18A1 in brain development, and this report contributes to an enhanced characterization of the brain malformations that can result from deficiencies of collagen XVIII. CONCLUSIONS This case series highlights the diagnostic power and clinical utility of whole-exome sequencing technology-allowing clinicians and physician scientists to better understand the pathophysiology and presentations of rare diseases. We suggest that patients who are clinically diagnosed with Knobloch syndrome and/or found to have COL18A1 mutations via genetic screening should be investigated for potential structural brain abnormalities even in the absence of an encephalocele.

Novel compound heterozygous mutations in GPT2 linked to microcephaly, and intellectual developmental disability with or without spastic paraplegia

We here describe novel compound heterozygous missense variants, NM_133443:c.[400C>T] and NM_133443:[1435G>A], in the glutamic‐pyruvic transaminase 2 (GPT2) gene in a large consanguineous family with two affected siblings diagnosed with microcephaly intellectual disability and developmental delay (IDD). In addition to these clinical phenotypes, the male sibling has spastic paraplegia, and the female sibling has epilepsy. Their four extended family members have IDD and microcephaly. Both of these variants, c.400C>T (p.R134C) and c.1435G>A (p.V479M), reside in the pyridoxal phosphate‐dependent aminotransferase domain. The missense variants affect highly conserved amino acids and are classified to be disease‐causing by meta‐SVM. The candidate variants were not found in the Exome Aggregation Consortium (ExAC) dataset or in dbSNP. Both GPT2 variants have an allele frequency of 0% (0/ ∼ 600) in the whole‐exome sequenced Turkish cohort. Upon Sanger sequencing, we confirmed these mutations in all affected family members and showed that the index patient and his affected sister inherited one mutant allele from each unaffected parent. To the best of our knowledge, this is the first family in which a novel compound heterozygous variant in the GPT2 gene was identified.

ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features

Primary cardiomyopathy is one of the most common inherited cardiac diseases and harbors significant phenotypic and genetic heterogeneity. Because of this, genetic testing has become standard in treatment of this disease group. Indeed, in recent years, next-generation DNA sequencing has found broad applications in medicine, both as a routine diagnostic tool for genetic disorders and as a high-throughput discovery tool for identifying novel disease-causing genes. We describe a male infant with primary dilated cardiomyopathy who was diagnosed using intrauterine echocardiography and found to progress to hypertrophic cardiomyopathy after birth. This proband was born to a nonconsanguineous family with a past history of a male fetus that died because of cardiac abnormalities at 30 wk of gestation. Using whole-exome sequencing, a novel homozygous frameshift mutation (c.2018delC; p.Gln675SerfsX30) in ALPK3 was identified and confirmed with Sanger sequencing. Heterozygous family members were normal with echocardiographic examination. To date, only two studies have reported homozygous pathogenic variants of ALPK3, with a total of seven affected individuals with cardiomyopathy from four unrelated consanguineous families. We include a discussion of the patients phenotypic features and a review of relevant literature findings.