"True" microcephaly with dominant-inheritance chorioretinal dysplasia

Within the heterogeneous group of microcephalies, a syndrome can be defined characterized by microcephaly, mental retardation, and chorioretinal dysplasia, often also with microphtalmia and embryonic remnants such as persistance of the primitive vitreum. Although this condition is usually considered autosomal recessive, the authors report a family observation consistent with dominant transmission.     

Mutations in TUBGCP4 Alter Microtubule Organization via the γ-Tubulin Ring Complex in Autosomal-Recessive Microcephaly with Chorioretinopathy

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.     

Cellular and clinical impact of haploinsufficiency for genes involved in ATR signaling

Ataxia telangiectasia and Rad3-related (ATR) protein, a kinase that regulates a DNA damage-response pathway, is mutated in ATR-Seckel syndrome (ATR-SS), a disorder characterized by severe microcephaly and growth delay. Impaired ATR signaling is also observed in cell lines from additional disorders characterized by microcephaly and growth delay, including non-ATR-SS, Nijmegen breakage syndrome, and MCPH1 (microcephaly, primary autosomal recessive, 1)-dependent primary microcephaly. Here, we examined ATR-pathway function in cell lines from three haploinsufficient contiguous gene-deletion disorders--a subset of blepharophimosis-ptosis-epicanthus inversus syndrome, Miller-Dieker lissencephaly syndrome, and Williams-Beuren syndrome--in which the deleted region encompasses ATR, RPA1, and RFC2, respectively. These three genes function in ATR signaling. Cell lines from these disorders displayed an impaired ATR-dependent DNA damage response. Thus, we describe ATR signaling as a pathway unusually sensitive to haploinsufficiency and identify three further human disorders displaying a defective ATR-dependent DNA damage response. The striking correlation of ATR-pathway dysfunction with the presence of microcephaly and growth delay strongly suggests a causal relationship.     

Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.     

Socioeconomic disparities associated with symptomatic Zika virus infections in pregnancy and congenital microcephaly: A spatiotemporal analysis from Goiânia,Brazil (2016 to 2020)

The Zika virus (ZIKV) epidemic, which was followed by an unprecedented outbreak of congenital microcephaly, emerged in Brazil unevenly, with apparent pockets of susceptibility. The present study aimed to detect high-risk areas for ZIKV infection and microcephaly in Goiania, a large city of 1.5 million inhabitants in Central-West Brazil. Using geocoded surveillance data from the Brazilian Information System for Notifiable Diseases (SINAN) and from the Public Health Event Registry (RESP-microcefalia), we analyzed the spatiotemporal distribution and socioeconomic indicators of laboratory confirmed (RT-PCR and/or anti-ZIKV IgM ELISA) symptomatic ZIKV infections among pregnant women and clinically confirmed microcephaly in neonates, from 2016 to 2020. We investigated temporal patterns by estimating the risk of symptomatic maternal ZIKV infections and microcephaly per 1000 live births per month. We examined the spatial distribution of maternal ZIKV infections and microcephaly cases across the 63 subdistricts of Goiania by manually plotting the geographical coordinates. We used spatial scan statistics estimated by discrete Poisson models to detect high clusters of maternal ZIKV infection and microcephaly and compared the distributions by socioeconomic indicators measured at the subdistrict level. In total, 382 lab-confirmed cases of maternal ZIKV infections, and 31 cases of microcephaly were registered in the city of Goiania. More than 90% of maternal cases were reported between 2016 and 2017. The highest incidence of ZIKV cases among pregnant women occurred between February and April 2016. A similar pattern was observed in the following year, although with a lower number of cases, indicating seasonality for ZIKV infection, during the local rainy season. Most congenital microcephaly cases occurred with a time-lag of 6 to 7 months after the peak of maternal ZIKV infection. The highest estimated incidence of maternal ZIKV infections and microcephaly were 39.3 and 2.5 cases per 1000 livebirths, respectively. Districts with better socioeconomic indicators and with higher proportions of self-identified white inhabitants were associated with lower risks of maternal ZIKV infection. Overall, the findings indicate heterogeneity in the spatiotemporal patterns of maternal ZIKV infections and microcephaly, which were correlated with seasonality and included a high-risk geographic cluster. Our findings identified geographically and socio-economically underprivileged groups that would benefit from targeted interventions to reduce exposure to vector-borne infections.     

Ornithine delta-aminotransferase mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.

Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate. Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina. We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations. Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles. We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts. The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA. By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen. Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients.     

Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration associated with hyperornithinemia with an autosomal recessive mode of inheritance. Cultured skin fibroblasts from five affected patients showed a virtual absence of ornithine ketoacid transaminase (OKT) (L-ornithine:2-oxoacid aminotransferase E.C.2.6.1.13) activity. Fibroblasts from four carrier parents showed a 42%-65% reduction in OKT activity. Increasing the concentration of pyridoxal phosphate (vitamin B6 in the assay media resulted in partial restoration of OKT activity in fibroblasts from one out of five patients studied. We conclude that OKT deficiency is closely associated with the genetic defect in gyrate atrophy of the choroid and retina and that genetic heterogeneity exists in this disease.     

Exome Analysis Identified a Novel Mutation in the RBP4 Gene in a Consanguineous Pedigree with Retinal Dystrophy and Developmental Abnormalities

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5^th decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism.     

Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in colombian patients with acute and chronic ocular toxoplasmosis

We studied the frequency of specific anti-Toxoplasma IgM, IgA and IgE antibodies in serum of 28 immunocompetent Colombian patients, selected by ophthalmologists and with lesions that were compatible with ocular toxoplasmosis. Patients were classified in three groups: (i) group 1 consisted of ten patients with a first episode; (ii) group 2, with seven patients with a recurrence and (iii) group 3, consisted of eleven patients with chronic chorioretinal lesion without uveitis. We found that 10/28 (35%) of Colombian patients with ocular toxoplasmosis possessed at least one serological marker for Toxoplasma infection different from IgG. In group 1 (first episode), we found simultaneous presence of specific IgM plus IgA plus IgE in 1/10 (10%). In group 2 (recurrences) in 1/7 (14%) we found IgM and IgA test positives and in 1/7 (14%) we found IgM and IgE tests positives. In group 3 (toxoplasmic chorioretinal scar) the IgA serological test was positive in 2/11 (18%). These results show that serum IgM or IgA or IgE can be present during recurrences.     

Human microcephaly

Microcephaly is defined as a reduction in head circumference and this clinical finding infers that an individual has a significant diminution in brain volume. Microcephaly can be usefully divided into primary microcephaly, in which the brain fails to grow to the correct size during pregnancy, and secondary microcephaly, in which the brain is the expected size at birth but subsequently fails to grow normally. Current work suggests that primary microcephaly is caused by a decrease in the number of neurones generated during neurogenesis, but that in secondary microcephaly it is the number of dendritic processes and synaptic connections that is reduced. Important insights into human neurogenesis are being revealed by the study of rare genetic diseases that involve primary microcephaly, illustrated by the identification of the Microcephalin, abnormal spindle in microcephaly and ataxia-telangiectasia and Rad3-related genes. Furthermore, these findings facilitate the search for the evolutionary changes that have lead to the human brain being so much larger than that of any other primates.     

Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch''s membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM–choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM–choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-μm-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM. (J Histochem Cytochem 57:731–739, 2009)     

A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy

Gyrate atrophy (GA) is an autosomal recessive eye disease involving a progressive loss of vision due to chorioretinal degeneration in which the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is defective. Two sisters with GA are described in this study in whom an A-to-G substitution at the 3 splice acceptor site of intron 4 in one allele of the OAT gene results in a truncated OAT mRNA devoid of exon 5 sequence. The mutation in the other allele was identified to be a missense mutation at codon 318 by denaturing gradient gel electrophoresis and direct sequencing of the polymerase chain reaction (PCR)-amplified DNA. Thus, these GA patients are compound heterozygotes with respect to mutations in the OAT gene that result in inactivation of OAT.     

Congenital abnormalities associated with Zika virus infection–Dengue as potential co-factor? A systematic review

Zika virus (ZIKV) emerged in Brazil during 2013–2014 causing an epidemic of previously unknown congenital abnormalities. The frequency of severe congenital abnormalities after maternal ZIKV infection revealed an unexplained geographic variability, especially between the Northeast and the rest of Brazil. Several reasons for this variability have been discussed. Prior immunity against Dengue virus (DENV) affecting ZIKV seems to be the most likely explanation. Here we summarise the current evidence regarding this prominent co-factor to potentially explain the geographic variability.This systematic review followed the PRISMA guidelines. The search was conducted up to May 15th, 2020, focussing on immunological interactions from Zika virus with previous Dengue virus infections as potential teratogenic effect for the foetus.Eight out of 339 screened studies reported on the association between ZIKV, prior DENV infection and microcephaly, mostly focusing on antibody-dependent enhancement (ADE) as potential pathomechanism. Prior DENV infection was associated with enhancement for ZIKV infection and increased neurovirulence in one included in vitro study only. Interestingly, the seven in vivo studies exhibited a heterogeneous picture with three studies showing a protective effect of prior DENV infections and others no effect at all. According to several studies, socio-economic factors are associated with increased risk for microcephaly.Very few studies addressed the question of unexplained variability of infection-related microcephaly. Many studies focussed on ADE as mechanism without measuring microcephaly as endpoint. Interestingly, three of the included studies reported a protective effect of prior DENV infection against microcephaly. This systematic review strengthens the hypothesis that immune priming after recent DENV infection is the crucial factor for determining protection or enhancement activity. It is of high importance that the currently ongoing prospective studies include a harmonised assessment of the potential candidate co-factors.     

Microcephalin: A Causal Link Between Impaired Damage Response Signalling and Microcephaly

Seckel Syndrome (SS) and Primary Microcephaly (MCPH) are disorders exhibiting marked microcephaly with a head circumference less than 3 standard deviations below the mean. ATR-Seckel Syndrome is conferred by mutations in ataxia and telangiectasia and Rad3 related (ATR), a kinase that activates a DNA damage signalling response. Cell lines from additional SS patients, who are normal for ATR, show defective ATR signalling, suggesting that they carry mutations in other components of the ATR pathway. Primary Microcephaly is distinct from SS since patients displaying solely microcephaly without accompanying growth delay. MCPH1, the first Primary Microcephaly causative gene identified, encodes three BRCT domains, similar to other damage response proteins. Recent studies employing MCPH1 siRNA or exploiting cell lines from MCPH1 patients have shown that MCPH1 functions in the ATR-dependent DNA damage response pathway. Additionally, MCPH1 has a function in the regulation of mitotic entry that is ATR-independent and confers a characteristic phenotype of premature chromosome condensation. Recent studies will be reviewed and their relationship to the aetiology of microcephaly discussed.     

Anterograde and retrograde degenerative reactions in caudate nucleus and putamen after experimental lesion of the pallidum in squirrel monkey (Saimiri sciureus)

After stereotactic lesions in the pallidum in 4 squirrel monkeys, electron microscopic material from the striatum was examined for anterograde and retrograde degenerative changes. In the experiment with pallidum internum lesion, only degenerated striatal fibers were observed, more than likely thalamostriatal fibers that pass through the site of the lesion. The three experiments with pallidum externum lesion revealed that the two types of striatal aspiny neurons react with a penumbral degeneration to interruption of their axons. Also, the axospinous type IV striatal synapses, which originate in the center median or parafascicular nucleus of the the thalamus, react to interruption of their axons in the pallidum externum with the dark or crystalloid forms of degeneration. The plump axospinous type III synapses, which have previously not been differentiated, were the most frequently altered, showing dark, crystalloid, or pale forms of degeneration. Their degeneration can be attributed directly to the lesions of the pallidum externum nerve cells; thus, an immediate connection between the pallidum externum and the striatum has been demonstrated. A comparison of the retrograde degeneration of striated nerve cells after pallidum externum lesions with that following columnar isolation of striatal tissue revealed two overlapping forms of penumbral degeneration of the aspiny neurons.     

Microcephaly genes and the evolution of sexual dimorphism in primate brain size

Microcephaly genes are amongst the most intensively studied genes with candidate roles in brain evolution. Early controversies surrounded the suggestion that they experienced differential selection pressures in different human populations, but several association studies failed to find any link between variation in microcephaly genes and brain size in humans. Recently, however, sex‐dependent associations were found between variation in three microcephaly genes and human brain size, suggesting that these genes could contribute to the evolution of sexually dimorphic traits in the brain. Here, we test the hypothesis that microcephaly genes contribute to the evolution of sexual dimorphism in brain mass across anthropoid primates using a comparative approach. The results suggest a link between selection pressures acting on MCPH1 and CENPJ and different scores of sexual dimorphism.     

Calvarial sutural abnormalities: metopic synostosis and coronal deformation--an anatomic, three-dimensional radiographic, and pathologic study

The purposes of this study were (1) to evaluate the histologic differences between synostotic versus deformational suture abnormalities and (2) to correlate these histologic findings with anatomic and three-dimensional computed tomographic (CT) scans. We examined three infants with premature metopic synostosis; one infant also had microcephaly trisomy 13 and curious overriding of the coronal sutures. The three-dimensional CT scans demonstrated obliteration of the metopic suture inferiorly. Histologic sections of this suture showed complete bony stenosis. The same pattern was found in all three infants, including the two infants with trigonocephaly who did not have trisomy 13 or microcephaly. In the trisomy 13 infant, the overlapped inferior coronal suture was obliterated on CT examination. However, histologic sections in this region showed a merging of bone; there was no synostosis. In summary, three-dimensional CT re-formation correlated with metopic suture histology. "Stenotic" fusion existed in all infants with trigonocephaly, those with normal and abnormal karyotypes, with and without microcephaly. However, three-dimensional CT re-formation of the trisomic infant showed opacification of the coronal suture in the areas of greatest overlap, whereas histology revealed a curious bone remodeling pattern, possibly a precursor to "deformational" craniosynostosis.     

Evidence for a second gene for primary microcephaly at MCPH5 on chromosome 1

Primary microcephaly has been mapped to five loci on different chromosomes. We present here the fine mapping of one of the loci, MCPH5, to a region of only 0.58 Mb located at the 1q31.3-1q32.1 junction. A genome scan was performed on five families from the Netherlands and Jordania, with 14 patients affected by microcephaly. A maximum LOD score of 4.78 was found for marker D1S1660 at the MCPH5 locus. Haplotype analysis suggests that the gene causing microcephaly is located between markers D1S3469 and D1S1660, which excludes the previously reported ASPM gene.