Microcephaly Syndrome Research Articles

A REPORT ABOUT ZIKA VIRUS IN BRAZIL

Zika virus, a Flavivirus first isolated in Africa, has spread to many countries around the world including Brazil. In this country, seven months after the football world cup, it was registered the occurrence of a epidemic, subsequently related to cases of microcephaly and Guillain-Barre syndrome. The virus is transmited via mosquito from Aedes genus, often following the bite from an infected vector. The febrile disease is self-limited, and there is no specific treatment. Few months after this outbreak in Brazil, the authorities recognized the relationship between the Zika virus and the occurrence of Guillain Barre syndrome and microcephaly in newborns, correponding to fetal cerebral malformation or brain lesions. This finding is being a worldwide alert for strengthening the preventive measures, restricted to prevent insect bites and care for the environment.

Zika Virus and Perinatal Outcomes: Beyond the Myth

The World Health Organization defines current outbreak of Zika infection as a Public Health Emergency of International Concern (PHEIC). An attempt was made to contribute in a comprehensive screening protocol for ZIKV in pregnancy, based on systematic assessment of the empirical data. A total of 34 published articles, conferring to the level I-IIA evidence, are randomly identified to inform clinical, immunology, imaging, and patho-histology findings from 8,389 singleton pregnancies. Results of a comparative analysis between the Zika-positive and Zikanegative pregnant women presented with macopapular rash suggest that active Zika virus infection is predictive to the adverse perinatal outcomes: prematurity, fetal growth restriction, microcephaly, and Guillain-Barre syndrome. Temporal analysis between the viral peak lags, the first acute rash in women, and perinatal outcomes, support such associations.

Zika Virus: The Emerging Global Health Challenge

Zika virus is a Flavi virus that is transmitted mostly by Aedes mosquitos. It was first isolated from a febrile rhesus monkey in the Zika forest of Uganda in 1947. Studies suggested wide spreads of the virus throughout Africa and Asia with over 40% of Nigerian adults and 25% of Nigerian children having antibodies to zika virus. The virus remained virtually unknown for over 70 years as the infections were “harmless”, sporadic, subclinical or asymptomatic in 80% of the cases. The current explosive spread of the virus, and its strong link to congenital microcephaly and Guillain-Barre Syndrome (GBS) in Brazil and other Americas made zika virus “a Public Health Emergency of International Concern” with emerging global health challenge. The challenges include non-availability of antiviral drugs or vaccines to combat the rapid viral spread, threat of worldwide spread of the “Brazilian” strain of the virus after the 2016 Olympics in Brazil, prevention of mosquito bites and zika virus infections especially in women of the reproductive age, and filling the gaps in knowledge about the virus vectors and reservoirs, and the pathogenesis. The objectives of this review are to update knowledge on the origin, spread and virology of zika virus, and the associated neurological complications sequel. We made recommendations for prevention of zika virus infections, and identified areas of future researches.

ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination.

We generate a mouse model for the human microcephaly syndrome by mutating the ASPM locus, and demonstrate a premature exhaustion of the neuronal progenitor pool due to dysfunctional self-renewal processes. Earlier studies have linked ASPM mutant progenitor excessive cell cycle exit to a mitotic orientation defect. Here, we demonstrate a mitotic orientation-independent effect of ASPM on cell cycle duration. We pinpoint the cell fate-determining factor to the length of time spent in early G1 before traversing the restriction point. Characterization of the molecular mechanism reveals an interaction between ASPM and the Cdk2/Cyclin E complex, regulating the Cyclin activity by modulating its ubiquitination, phosphorylation and localization into the nucleus, before the cell is fated to transverse the restriction point. Thus, we reveal a novel function of ASPM in mediating the tightly coordinated Ubiquitin- Cyclin E- Retinoblastoma- E2F bistable-signalling pathway controlling restriction point progression and stem cell maintenance.

Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the major facilitator superfamily domain-containing 2a (MFSD2A) protein. MFSD2A transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Affected individuals exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa-morphant zebrafish, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.

A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome.

The major pathway by which the brain obtains essential omega-3 fatty acids from the circulation is through a sodium-dependent lysophosphatidylcholine (LPC) transporter (MFSD2A), expressed in the endothelium of the blood-brain barrier. Here we show that a homozygous mutation affecting a highly conserved MFSD2A residue (p.Ser339Leu) is associated with a progressive microcephaly syndrome characterized by intellectual disability, spasticity and absent speech. We show that the p.Ser339Leu alteration does not affect protein or cell surface expression but rather significantly reduces, although not completely abolishes, transporter activity. Notably, affected individuals displayed significantly increased plasma concentrations of LPCs containing mono- and polyunsaturated fatty acyl chains, indicative of reduced brain uptake, confirming the specificity of MFSD2A for LPCs having mono- and polyunsaturated fatty acyl chains. Together, these findings indicate an essential role for LPCs in human brain development and function and provide the first description of disease associated with aberrant brain LPC transport in humans.

Brain malformations in a patient with deletion 2p16.1: A refinement of the phenotype to BCL11A

Microdeletions of 2p15-16.1 have been reported in 15 patients with a recognizable syndrome of dysmorphic features, intellectual disability and microcephaly. Facial features include telecanthus, short palpebral fissures, epicanthal folds, a broad nasal root, smooth and long philtrum and large ears. Brain malformations can be observed in this syndrome and include hypoplasia of the corpus callosum and a simplified cortical gyral pattern. Case reports have narrowed the critical region of the neurodevelopmental phenotype to a region that spans the B-cell CLL/lymphoma 11A (BCL11A) gene. Here we present a 3-year-old normocephalic girl with moderate development delay and dysmorphic features including a prominent forehead, telecanthus, depressed nasal bridge, thin upper vermilion and a small chin. Magnetic resonance imaging shows enlargement of the lateral, third and fourth ventricles and hypoplastic corpus callosum, cerebellar vermis and pons. Array CGH revealed a 0.875 Mb de novo deletion at 2p16.1 that includes only BCL11A. The moderate delays, hypoplastic and dysmorphic corpus callosum and hippocampi and the facial features are in keeping with the previously described 2p15-16.1 microdeletion syndrome. However, hypoplasia of the pons and cerebellum are not commonly recognized features and are reminiscent of the brain malformations observed in individuals with a mutation in CASK. CASK is known to interact with BCL11A in the normal growth of axons. This case report highlights the role of BCL11A in 2p15-16.1 microdeletion syndrome and the unique phenotype suggests a common pathway for BCL11A and other genes in neurodevelopment.

Mutations in PYCR2, encoding pyrroline-5-carboxylate reductase 2, cause microcephaly and hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes are yet to be identified. Here we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations in PYCR2 (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in PYCR2 level. When mutant cDNAs were transfected into HEK293FT cells, the mutant protein retained normal mitochondrial localization but the level was lower than the wild-type protein, suggesting that mutant protein is less stable. A PYCR2-deficient HEK293FT cell line generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of the zebrafish PYCR2 homolog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human central nervous system during development.

Genetic disorders associated with postnatal microcephaly.

Several genetic disorders are characterized by normal head size at birth, followed by deceleration in head growth resulting in postnatal microcephaly. Among these are classic disorders such as Angelman syndrome and MECP2-related disorder (formerly Rett syndrome), as well as more recently described clinical entities associated with mutations in CASK, CDKL5, CREBBP, and EP300 (Rubinstein-Taybi syndrome), FOXG1, SLC9A6 (Christianson syndrome), and TCF4 (Pitt-Hopkins syndrome). These disorders can be identified clinically by phenotyping across multiple neurodevelopmental and neurobehavioral realms, and enough data are available to recognize these postnatal microcephaly disorders as separate diagnostic entities in their own right. A second diagnostic grouping, comprised of Warburg MICRO syndrome, Cockayne syndrome, and Cerebral-oculo-facial skeletal syndrome, share similar features of somatic growth failure, ophthalmologic, and dysmorphologic features. Many postnatal microcephaly syndromes are caused by mutations in genes important in the regulation of gene expression in the developing forebrain and hindbrain, although important synaptic structural genes also play a role. This is an emerging group of disorders with a fascinating combination of brain malformations, specific epilepsies, movement disorders, and other complex neurobehavioral abnormalities.

A newly recognized syndrome of severe growth deficiency, microcephaly, intellectual disability, and characteristic facial features

Genetic syndromes with proportionate severe short stature are rare. We describe two sisters born to nonconsanguineous parents with severe linear growth retardation, poor weight gain, microcephaly, characteristic facial features, cutaneous syndactyly of the toes, high myopia, and severe intellectual disability. During infancy and early childhood, the girls had transient hepatosplenomegaly and low blood cholesterol levels that normalized later. A thorough evaluation including metabolic studies, radiological, and genetic investigations were all normal. Cholesterol metabolism and transport were studied and no definitive abnormality was found. No clinical deterioration was observed and no metabolic crises were reported. After due consideration of other known hereditary causes of post-natal severe linear growth retardation, microcephaly, and intellectual disability, we propose that this condition represents a newly recognized autosomal recessive multiple congenital anomaly-intellectual disability syndrome.

The supposed tumor suppressor gene WWOX is mutated in an early lethal microcephaly syndrome with epilepsy, growth retardation and retinal degeneration

BackgroundWWOX, encoding WW domain-containing oxidoreductase, spans FRA16D, the second most common chromosomal fragile site frequently altered in cancers. It is therefore considered a tumor suppressor gene, but its direct implication in cancerogenesis remains controversial.Methods and resultsBy whole-exome sequencing, we identified a homozygous WWOX nonsense mutation, p.Arg54*, in a girl from a consanguineous family with a severe syndrome of growth retardation, microcephaly, epileptic seizures, retinopathy and early death, a phenotype highly similar to the abormalities reported in lde/lde rats with a spontaneous functional null mutation of Wwox. As in rats, no tumors were observed in the patient or heterozygous mutation carriers.ConclusionsOur finding, a homozygous loss-of-function germline mutation in WWOX in a patient with a lethal autosomal recessive syndrome, supports an alternative role of WWOX and indicates its importance for human viability.

A complex microcephaly syndrome in a Pakistani family associated with a novel missense mutation in RBBP8 and a heterozygous deletion in NRXN1

We report on a consanguineous Pakistani family with a severe congenital microcephaly syndrome resembling the Seckel syndrome and Jawad syndrome. The affected individuals in this family were born to consanguineous parents of whom the mother presented with mild intellectual disability (ID), epilepsy and diabetes mellitus. The two living affected brothers presented with microcephaly, white matter disease of the brain, hyponychia, dysmorphic facial features with synophrys, epilepsy, diabetes mellitus and ID. Genotyping with a 250K SNP array in both affected brothers revealed an 18MB homozygous region on chromosome 18p11.21-q12.1 encompassing the SCKL2 locus of the Seckel and Jawad syndromes. Sequencing of the RBBP8 gene, underlying the Seckel and Jawad syndromes, identified the novel mutation c.919A>G, p.Arg307Gly, segregating in a recessive manner in the family. In addition, in the two affected brothers and their mother we have also found a heterozygous 607kb deletion, encompassing exons 13–19 of NRXN1. Bidirectional sequencing of the coding exons of NRXN1 did not reveal any other mutation on the other allele. It thus appears that the phenotype of the mildly affected mother can be explained by the NRXN1 deletion, whereas the more severe and complex microcephalic phenotype of the two affected brothers is due to the simultaneous deletion in NRXN1 and the homozygous missense mutation affecting RBBP8.

TRNA Methyltransferase Homolog Gene TRMT10A Mutation in Young Onset Diabetes and Primary Microcephaly in Humans

We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene TRMT10A (also called RG9MTD2). TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m1G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of β- and non-β-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human β-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects β-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.

Recent Advances in the Genetic Etiology of Brain Malformations

In the past few years, the increasing accessibility of next-generation sequencing technology has translated to a number of significant advances in our understanding of brain malformations. Genes causing brain malformations, previously intractable due to their complex presentation, rarity, sporadic occurrence, or molecular mechanism, are being identified at an unprecedented rate and are revealing important insights into central nervous system development. Recent discoveries highlight new associations of biological processes with human disease including the PI3K-AKT-mTOR pathway in brain overgrowth syndromes, the trafficking of cellular proteins in microcephaly-capillary malformation syndrome, and the role of the exosome in the etiology of pontocerebellar hypoplasia. Several other gene discoveries expand our understanding of the role of mitosis in the primary microcephaly syndromes and post-translational modification of dystroglycan in lissencephaly. Insights into polymicrogyria and heterotopias show us that these 2 malformations are complex in their etiology, while recent work in holoprosencephaly and Dandy-Walker malformation suggest that, at least in some instances, the development of these malformations requires "multiple-hits" in the sonic hedgehog pathway. The discovery of additional genes for primary microcephaly, pontocerebellar hypoplasia, and spinocerebellar ataxia continue to impress upon us the significant degree of genetic heterogeneity associated with many brain malformations. It is becoming increasingly evident that next-generation sequencing is emerging as a tool to facilitate rapid and cost-effective molecular diagnoses that will be translated into routine clinical care for these rare conditions in the near future.

MICrocephaly, disproportionate pontine and cerebellar hypoplasia syndrome: A clinico-radiologic phenotype linked to calcium/calmodulin-dependent serine protein kinase gene mutation

MICrocephaly, disproportionate pontine and cerebellar hypoplasia (MICPCH) syndrome, a rare X-linked disorder, generally seen in girls, is characterized by neurodevelopmental delay, microcephaly, and disproportionate pontine and cerebellar hypoplasia. It is caused by inactivating calcium/calmodulin-dependent serine protein kinase (CASK) gene mutations. We report a 2-year-old girl with severe neurodevelopmental delay, microcephaly, minimal pontine hypoplasia, cerebellar hypoplasia, and normal looking corpus callosum, with whom the conventional cytogenetic studies turned out to be normal, and an array-comparative genomic hybridization (a-CGH) analysis showed CASK gene duplication at Xp11.4. Our case highlights the importance of using clinico-radiologic phenotype to guide genetic investigation and it also confirms the role of a-CGH analysis in establishing the genetic diagnosis of MICPCH syndrome, when conventional cytogenetic studies are inconclusive.

Progressive cerebellar atrophy and polyneuropathy: expanding the spectrum of PNKP mutations

We present a neurodegenerative disorder starting in early childhood of two brothers consisting of severe progressive polyneuropathy, severe progressive cerebellar atrophy, microcephaly, mild epilepsy, and intellectual disability. The cause of this rare syndrome was found to be a homozygous mutation (c.1250_1266dup, resulting in a frameshift p.Thr424GlyfsX48) in PNKP, identified by applying homozygosity mapping and whole-genome sequencing. Mutations in PNKP have previously been associated with a syndrome of microcephaly, seizures and developmental delay (MIM 613402), but not with a neurodegenerative disorder. PNKP is a dual-function enzyme with a key role in different pathways of DNA damage repair. DNA repair disorders can result in accelerated cell death, leading to underdevelopment and neurodegeneration. In skin fibroblasts from both affected individuals, we show increased susceptibility to apoptosis under stress conditions and reduced PNKP expression. PNKP is known to interact with DNA repair proteins involved in the onset of polyneuropathy and cerebellar degeneration; therefore, our findings explain this novel phenotype.

RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion

Although neurogenic pathways have been described in the developing neocortex, less is known about mechanisms ensuring correct neuronal differentiation thus also preventing tumor growth. We have shown that RP58 (aka zfp238 or znf238) is highly expressed in differentiating neurons, that its expression is lost or diminished in brain tumors, and that its reintroduction blocks their proliferation. Mice with loss of RP58 die at birth with neocortical defects. Using a novel conditional RP58 allele here we show that its CNS-specific loss yields a novel postnatal phenotype: microencephaly, agenesis of the corpus callosum and cerebellar hypoplasia that resembles the chr1qter deletion microcephaly syndrome in human. RP58 mutant brains maintain precursor pools but have reduced neuronal and increased glial differentiation. Well-timed downregulation of pax6, ngn2 and neuroD1 depends on RP58 mediated transcriptional repression, ngn2 and neuroD1 being direct targets. Thus, RP58 may act to favor neuronal differentiation and brain growth by coherently repressing multiple proneurogenic genes in a timely manner.

Microcephaly with Simplified Gyration, Epilepsy, and Infantile Diabetes Linked to Inappropriate Apoptosis of Neural Progenitors

We describe a syndrome of primary microcephaly with simplified gyral pattern in combination with severe infantile epileptic encephalopathy and early-onset permanent diabetes in two unrelated consanguineous families with at least three affected children. Linkage analysis revealed a region on chromosome 18 with a significant LOD score of 4.3. In this area, two homozygous nonconserved missense mutations in immediate early response 3 interacting protein 1 (IER3IP1) were found in patients from both families. IER3IP1 is highly expressed in the fetal brain cortex and fetal pancreas and is thought to be involved in endoplasmic reticulum stress response. We reported one of these families previously in a paper on Wolcott-Rallison syndrome (WRS). WRS is characterized by increased apoptotic cell death as part of an uncontrolled unfolded protein response. Increased apoptosis has been shown to be a cause of microcephaly inanimal models. An autopsy specimen from one patient showed increased apoptosis in the cerebral cortex and pancreas beta cells, implicating premature cell death as the pathogenetic mechanism. Both patient fibroblasts and control fibroblasts treated with siRNA specific for IER3IP1 showed an increased susceptibility to apoptotic cell death under stress conditions in comparison to controls. This directly implicates IER3IP1 in the regulation of cell survival. Identification of IER3IP1 mutations sheds light on the mechanisms of brain development and on the pathogenesis of infantile epilepsy and early-onset permanent diabetes.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia

Feingold syndrome (FS) is a syndromic microcephaly entity for which MYCN is the major disease-causing gene. We studied the expression pattern of MYCN at different stages of human embryonic development and collected a series of 17 FS and 12 isolated oesophageal atresia (IOA) cases. An MYCN gene deletion/mutation was identified in 47% of FS cases exclusively. We hypothesized that mutations or deletions of highly conserved non-coding elements (HCNEs) at the MYCN locus could lead to its misregulation and thereby to FS and/or IOA. We subsequently sequenced five HCNEs at the MYCN locus and designed a high-density tiling path comparative genomic hybridization array of 3.3 Mb at the MYCN locus. We found no mutations or deletions in this region, supporting the hypothesis of genetic heterogeneity in FS.