Monogenic and Digenic Inheritance of Primary Microcephaly

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Monogenic and Digenic Inheritance

of Primary Microcephaly

Thesis submitted by Sarah DUERINCKX

in fulfilment of the requirements of the PhD Degree in medical science

(“Docteur en médecine”)

Academic year 2018-2019

Supervisor: Professor Marc ABRAMOWICZ

Co-supervisors: Professors Tom LENAERTS and Isabelle PIRSON

Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire

(IRIBHM)

Thesis jury:

Georges CASIMIR (Université libre de Bruxelles, Chair)

Marc ABRAMOWICZ (Université libre de Bruxelles, Secretary) Jean-Louis MANDEL (Université de Strasbourg)

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Monogenic and Digenic Inheritance

of Primary Microcephaly

Thesis submitted by Sarah DUERINCKX

in fulfilment of the requirements of the PhD Degree in medical science

(“Docteur en médecine”)

Academic year 2018-2019

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Acknowledgements

First of all, I wish to deeply thank my promotor, Marc Abramowicz, for giving me the fantastic opportunity to spend these last years in his lab. I discovered the field of genetics under his wings, and I developed a growing interest in it. He learned me so many things, answering questions like a reference book. I thank him for the very interesting project he designed for me, for the challenging environment he created, for all the stimulating interactions we had, for his inspiring ideas, for his ability to find solutions, for his patience, his optimism, and his reactivity in the more critical moments.

I thank my two co-promotors, Isabelle Pirson and Tom Lenaerts. I am grateful to Isabelle for generously sharing her expertise all along the years, for her availability, and for the big help she gave me during my first year when everything seemed so complicated. I thank Tom for his bioinformatics insights to this project that were so instructive, and for his careful advice. I thank Massimo Pandolfo and Vincent Detours for all their useful comments.

I thank Camille Perazzolo, our precious lab technician, for learning me almost everything in the lab, for her invaluable help, and for her patience, always remaining peaceful even when experiments didn’t work. I thank Valérie Jacquemin for our collaboration on microcephaly and hydrocephalus projects, and for taking care of my fishes when Camille and I both were on maternity leave. I thank Annick Massart for our scientific discussions, and for her diplomatic skills. I express all my gratitude to Camille, Valérie and Annick for their friendship, their support, and the pleasant working environment they created, that meant a lot for me during these years.

I thank Sabine Costagliola and Valérie Wittamer for managing the zebrafish platform and letting us use it, and for generously sharing their huge expertise. I thank Véronique Janssens and Marianne Caron, and all the PhD students working with zebrafish for patiently learning me what I needed to know and to do with the fish. I thank Anne Lefort and Frederick Libert for the design and generation of the TALENs.

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and for the discussions of protein interactions. I thank Viviane de Maertelaer and Judith Racapé for their help in statistics.

I thank the people from the hospital’s Center of Human Genetics, especially Catherine Rydlewski, Laurence Desmyter and Cindy Badoer for sharing information and answering my numerous emails. I thank all the referring geneticists and neuro-pediatricians for their collaboration, and for sharing their patients’ clinical information. I especially thank Alain Verloes and his team for sharing with us their patients’ exome data for our mutation burden test cohort.

I thank all my friends for the good times we spend together, that makes my life sunnier. I thank all my family, my parents, my brother, my grandparents, my step-family, for their support, their confidence, their useful advice and so much more. I thank Ward for his unconditional trust that everything will be okay, and our two wonderful children for showing us day after day what matters the most.

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Abstract

Primary Microcephalies (PMs) are characterized by a small head since birth, resulting from an insufficient production of mature neurons during neurogenesis. PMs carry a heavy burden of intellectual deficiency, and serve as model diseases for brain volumic development. Known PM genes are ascribed to several cellular pathways such as the centriole duplication pathway, the control of cell-cycle checkpoints and the general control of DNA replication licensing, although the exact mechanisms remain unclear. Genes causing monogenic forms of PM can be identified in fewer than 50% of patients. Digenic inheritance has recently been described in the mouse for PM caused by Aspm and Wdr62, but it is not known whether this applies to humans. The genetic dissection of PM will provide interesting clues about the cellular mechanisms involved.

In this study, we used two different holistic, in vivo approaches: high throughput DNA sequencing of multiple PM genes in human PM patients, and genome-edited zebrafish modeling of selected PM genes.

Analysis of a large mostly outbred PM cohort revealed 15 novel mutations in known PM genes, ASPM and WDR62 being the most frequently mutated. Mutations in AP4M1 and TRAPPC9 were for the first time associated with PM in two families. A mutation in MCPH1 truncating the last BRCT domain was surprisingly not associated with PM. The analysis of the exome cohort also revealed variants in four candidate genes that are not yet associated with human pathologies. Furthermore, exomes of PM patients showed a significant excess of variants in 75 PM genes, that persisted after removing monogenic causes of PM. A PM gene panel showed that the burden was carried by six centrosomal genes. In zebrafish, non-centrosomal gene casc5 biallelic invalidation produced a severe PM phenotype, that was not modified by centrosomal genes aspm or wdr62 invalidation. A digenic, quadriallelic PM phenotype was produced by aspm and wdr62. Digenic analysis of the PM cohort revealed candidates for digenic inheritance, among which heterozygous mutations in CEP135 and WDR62 in one PM patient.

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Table of illustrations

Figure 1. The different models of digenic inheritance. ... 10

Figure 2. Molecular mechanisms of digenic disorders – nonallelic noncomplementation. ... 11

Figure 3. Molecular mechanisms of digenic disorders – noninteracting noncomplementation. ... 12

Figure 4. Mendelian versus oligogenic inheritance. ... 14

Figure 5. Exome data filtering parameters. ... 15

Figure 6. Microcephaly. ... 17

Figure 7. Division modes of neural progenitors, and their effect on cortical expansion. ... 18

Figure 8. Cortical neurogenesis during embryological development. ... 19

Figure 9. Transcription activator-like effectors nucleases (TALENs). ... 34

Figure 10. Study workflow and conclusive causes in a PM patients’ cohort. ... 48

Figure 11. OFC evolution. ... 51

Figure 12. C*** variant. ... 100

Figure 13. IGF2BP3 variant and igf2bp3 mutant fishes. ... 103

Figure 14. DNAH2 variants. ... 105

Figure 15. Pictures of the two aspm +/+ casc5 -/- larvae from Figure S2B. ... 138

Figure 16. Relative reductions of head area and body length in zebrafish larvae. ... 139

Figure 17. A suspicion of digenic inheritance in a PM proband. ... 142

Table 1. Overview of MCPH genes. ... 32

Table 2. Primers used for amplification and Sanger sequencing of human DNA. ... 40

Table 3. Novel mutations identified in our cohort. ... 49

Table 4. PM patients with a molecular diagnosis and clinical information. ... 54

Table 5. Clinical features in reported cases of AP4 defects and in the present proband. ... 82

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List of abbreviations

AP4: Adaptor Protein Complex-4

CADD: Combined Annotation Dependent Depletion Cas: CRISPR-associated protein

CNV: Copy Number Variant

CRISPR: Clustered regularly interspaced short palindromic repeat HAT: Half-A-TPR

MCPH: MicroCephaly Primary Hereditary MRI: magnetic resonance imaging OFC: Occipito frontal circumference PCR: Polymerase Chain reaction PM: Primary microcephaly SD: standard deviation

SNP: Single Nucleotide Polymorphism

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Introduction

Digenic inheritance

The identification of genes and mutations causing human diseases is a challenge in human genetics. It allows for a more precise prognosis, precision therapies, and better genetic counselling in patients and families. It also provides better understanding of the pathology and the biological pathways involved. Apparently Mendelian disorders are traditionally explained by a monogenic model, the variants at one locus being associated with the phenotype. After finding a causal variant at one locus, we usually stop looking at other variants, introducing a bias in discovering more precise causes or modifiers of genetic diseases. Nevertheless, evidence accumulates showing that apparently Mendelian disorders can sometimes be better explained by the genotypic variants at more than one locus [1].

Definition of digenic inheritance

Digenic inheritance refers to a form of inheritance where the genotypes at two different, genetically unlinked loci explain the phenotypes of the patients better than the genotype at one locus alone [2]. In the case of an on/off effect (true digenic), variants at both loci equally determine the occurrence of the disease. In the case of a severity effect, the major variant causes a phenotype, and the modifier variant changes the severity of the phenotype, or modifies the age of onset [2], [3].

A digenic model may provide a better correlation between genotype and phenotype than the monogenic model, especially in the presence of incomplete penetrance, variable expressivity and/or locus heterogeneity [2], [3]. Incomplete penetrance refers to a situation where all the patients carrying a particular mutation do not present the phenotype. Variable expressivity is present when the patients carrying a particular mutation have different phenotypes in term of severity, age of onset or variability of the features presented. Locus heterogeneity refers to the implication of different loci in a same Mendelian, or complex pathology.

The different models of digenic inheritance

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number of mutant alleles that are required to induce a pathology [5]. The different models of digenic inheritance are represented in Figure 1.

Molecular mechanisms of digenic disorders

Different molecular mechanisms explaining digenic disorders are described in [1], Oligogenic disease chapter.

Nonallelic noncomplementation describes a situation where two mutant alleles at two different loci act together to cause a same phenotype. This situation can be explained by a dosage model or a poison model. In the dosage model, a decrease in two interacting proteins is necessary to cause the phenotype. If there is only one mutant protein, the interaction between the two proteins is still possible, but if both proteins are mutated, the interaction is not possible anymore (Figure 2A). Similarly, in a complex, if there is only one mutant protein, the other proteins are still able to form the complex, but two mutant proteins prevent the formation of the complex, or prevent the complex from performing its normal biological function (Figure 2B). In the poison model, one mutant protein poisons the complex by blocking the binding of another protein, but there are still enough functional complexes to prevent the phenotype. A second mutant protein results in very few functional complexes and the phenotype becomes apparent (Figure 2C) [1].

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Noninteracting noncomplementation describes a situation where the two mutant proteins are not interacting but are in the same pathway or have a redundant functionality. In the same pathway model, a hypomorph mutation in one protein reduces the signal but this signal is still sufficient to prevent the phenotype, while two mutant proteins result in the absence of signal (Figure 3A). In the redundant functionality model, mutations in two redundant proteins are required to lower the signal under a critical threshold and induce a disease (Figure 3B) [1].

Protein-protein interactions are thus an important type of evidence for digenic inheritance. Mutations in two interacting proteins could in theory be compensatory, but are most of the time a double hit [2]. In most of the digenic pairs described there is a relationship between the two genes: direct interaction between the two proteins, indirect interaction via one intermediate protein, belonging to the same pathway, co-expression or similar function [4].

A better knowledge of digenic interactions thus facilitates the elucidation of the biological pathways involved in the diseases, improves the clinical predictions associated with particular mutations and can also help to develop novel drug targets, e.g. by mimicking the effects of modifiers that affect the penetrance [6].

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Digenic inheritance and human pathologies

Several examples of digenic inheritance in human pathologies have been described. Fascioscapulohumeral muscular dystrophy type 2 was first thought to follow an autosomal dominant mode of inheritance with incomplete penetrance. It was then shown to result from a digenic interaction between DUX4 and SMCHD1 loci, the molecular basis for this digenic interaction being a protein-DNA interaction [7]. Some cases of midline craniosynostosis were shown to result from the combined effect of a rare mutation in SMAD6 and a common variant in BMP2, both genes being involved in bone formation [8]. One form of retinitis pigmentosa is another example of digenic inheritance with variants at two loci encoding two interacting proteins [9]. Long QT syndrome is another pathology that was first considered autosomal dominant with incomplete penetrance, but that seem in fact to be better explained by a digenic inheritance (double heterozygosity between two genes that were already known in this pathology) [10]–[12].

One debated case of digenic inheritance in human pathology is Biedl syndrome. The Bardet-Biedl phenotype is variable between and within families, and includes pigmentary retinal dystrophy, polydactyly, obesity, developmental delay and renal defects. Bardet-Biedl syndrome is usually considered as autosomal recessive, with clinical and genetic heterogeneity. Twelve BBS loci have been identified. Two studies proposed that digenic inheritance, triallelic model, could better explain inheritance of the the phenotype than a Mendelian model in some selected families [13], [14]. However, this observation was not replicated in six latter studies [15]–[20].

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The Digenic diseases Database (DIDA) provides detailed information on genes and variants involved in digenic diseases [4]. The current version of DIDA includes digenic gene pairs involved in 54 different pathologies.

From digenic to oligogenic and complex inheritance

Digenic inheritance is the simplest form of oligogenic inheritance. Oligogenic and polygenic inheritance describe diseases caused or modulated by mutations in multiple genes [1]. Oligogenic diseases involve a few genes, while polygenic diseases involve a lot of genes, the limit between the two being vague. Complex disease is a synonym for polygenic disease. Multifactorial diseases are attributed to the concomitant action of multiple genetic variants and environmental factors. Actually, all disorders should be considered multifactorial because there is always in some way an influence of genetic factors and of environmental factors. If some diseases are considered Mendelian, it is because most of their phenotype is recapitulated in a monogenic cause [1]. For example, cystic fibrosis is attributed to CFTR mutations, but the phenotypic variability is wide. Allelic heterogeneity, environmental factors and genetic modifiers are involved in this variability [1].

As mentioned above, a high phenotypic variability in a disease considered as monogenic, or a poor genotype-phenotype correlation, should lead to suspect multiple genes being involved in the pathology. Indeed, an oligogenic model could provide a better correlation between genotype and phenotype. In multifactorial diseases, a distinction between genetic variation and environmental factors needs to be done by determining the heritability of the phenotype. The heritability is the proportion of phenotypic variation that can be attributed to genetic variation. It can be assessed with twin studies comparing the concordance of phenotypes between monozygotic and dizygotic twins, adoption studies, or by comparing interfamilial and intrafamilial variability [1], [6], [14].

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Similarly to what is observed in digenic inheritance, the genes involved in monogenic forms of a disease are often good candidates modifier genes, or candidates for oligogenic inheritance; and the genes involved in oligogenic inheritance are likely involved in the same biological pathway. Several features suggest the presence of oligogenic inheritance: unclear genotype-phenotype correlations, different phenotypes in animal models depending on the genetic background, transmission of a disease trait other than Mendelian, or linkage to more than one locus in the same family [1].

Evidence of digenic inheritance

The discovery of digenic diseases is highly challenging because of the difficulty to demonstrate the pathogenicity of a pair of variants. Proof of digenic inheritance is given by evidence of protein-protein or protein-DNA interaction, segregation of the phenotype in the family and/or functional studies like animal models showing a combined effect of the variants [2]. It is fundamental to have detailed patients’ clinical information and also longitudinal information to be able to compare the phenotypes of the patients [6].

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The contribution of new technologies

The era of high throughput DNA sequencing should accelerate the discoveries of digenic inheritance since it makes it possible to sequence many genes simultaneously [2]. It does not give the proof of digenic inheritance, but at least it enables the quick identification of variants in multiple candidate genes.

Most high throughput DNA sequencing studies target the protein-coding region of the genome, called the exome, since 85% of known disease-causing variants reside in exons [22]. For whole exome sequencing, the exome is targeted by hybridization. The sequencing step is performed and millions of short sequence copies are read. The reads are then aligned to the reference genome, and the aligned sequence is inspected for positions differing from the human reference sequence. These positions are identified as Single Nucleotide Variants (SNVs). 15000 to 20000 variants are usually observed in an exome, and a measure of quality is given for each variant. The BAM file format is used for the raw sequencing data, while the Variant Call format is used to report the variants [23]. The first challenge in analyzing high throughput sequencing data is to distinguish the true variants from the sequencing errors. The variants are first filtered based on quality criteria (e.g. the number of independent reads harboring the variant). Second, the variants occurring outside the coding regions and the synonymous variants are filtered out, keeping only the variants altering the protein sequence. Third, only the rare variants are kept (e.g. allelic frequency lower than 1% in public databases), the cut-off allelic frequency being adapted to the frequency of the studied disease. The remaining variants are prioritized according to the assumed mode of inheritance of the disease, the available information on gene function in relation to the phenotype and the impact of variants on proteins structure or function calculated by several prediction programs [24]. An example of exome data filtering parameters in one patient is given in Figure 5.

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Several study designs for the discovery of digenic inheritance are described that can benefit from the technology of high throughput DNA sequencing [2].

The candidate gene design consists of

- identifying a set of genes usually mutated in the monogenic forms of the disease - sequencing these genes in a set of patients and relatives

- identifying patients with mutations in two different genes

- performing additional experiments to show how the two proteins interact or reproduce the digenic inheritance in an animal model [2].

The high throughput sequencing design consists of

- sequencing the whole exome of a set of patients and relatives

- identify some pairs of genes that are recurrently mutated in patients, without a preselection of candidate genes

- compare the sequences of these genes in patients and relatives, and perform functional experiments [2].

The protein-protein interaction design is a candidate gene design where the gene set is chosen according to its interaction with a particular gene involved in the monogenic forms of the disease [2]. Similarly, the genes included in a same biological pathway could also be screened [6]. Interactomes can be useful tools in order to establish a set of candidate genes for digenic inheritance. An interactome is a network that integrates all protein physical interaction within a cell, e.g. protein-protein, protein-lipids and protein-DNA/RNA interactions [25]. Disease-associated proteins seem to interact with each other, forming what is called a disease module. Disease modules can only been discovered if enough disease-associated genes are known [25].

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In a study, 37 probands with Charcot-Marie-Tooth disease, a clinically and genetically heterogeneous peripheral neuropathy had their exome sequenced [27]. In less than half of the patients, a monogenic cause was found. A mutation burden test showed an excess of rare variants in 58 known neuropathy-associated genes in the patients’ cohort in comparison to control individuals. The burden remained significant after removing the highly penetrant Mendelian variants. The test was successfully replicated in an independent Turkish cohort. Morpholinos co-injection of several gene pairs in zebrafish showed an exacerbation of the phenotype, suggesting that the combined effect of rare variants in neuropathy genes could contribute to disease penetrance and variable expressivity [27].

Primary microcephaly

Definition of Primary Microcephaly

Microcephaly is a clinical condition characterized by a small head. It is assessed by measuring the Occipito frontal circumference (OFC) [28]. It reflects a small brain, mostly a small neocortex (Figure 6).

Brain size being distributed on a roughly Gaussian curve, about 2% of the population have a brain size smaller than 2 standard deviations (SD) below the mean, and most have a normal intellect. Using 3 SD below the mean as a cut-off for microcephaly, most subjects will have intellectual deficiency, and some will have additional neurological deficits like epilepsy or weakness.

Primary microcephaly (PM), or congenital microcephaly, consists of a prenatal defect of brain volumic development. Secondary microcephaly, or progressive microcephaly, refers to a progressive atrophy of an initially normal brain, usually starting after birth [29].

PMs are usually transmitted as autosomal recessive traits, and have a global incidence of 1/10,000 to 1/100,000 births depending on ethnicity and rate of consanguinity [30]. More importantly, PM is a

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model disease for defects of brain volumic development. The understanding of the genetic basis of PM and the identification of the numerous genes involved in PM provide substantial insights into human brain evolution and development of the cerebral cortex. Indeed, the genes that underwent a positive selection during evolution contributed to human brain development. Invalidating mutations of the same genes could hence result in a PM phenotype in human.

Development of the human cerebral cortex

The cerebral cortex is a complex six-layered structure, that underwent a considerable increase in relative size and complexity during vertebrate evolution. Cortical surface area was increased by a factor 1000, and cortical thickness by a factor 2 from mouse to human [31], [32]. This increase in cortical size results from an increase in the number of neurons. The neurons are generated in a process called neurogenesis, consisting of a proliferative phase of neural progenitors’ multiplication, followed by a neurogenic phase of neurons production. Schematically, an increased number of proliferative divisions increases the cortical surface, while an increase in neurogenic divisions increases the cortical thickness (Figure 7) [31], [33].

Several types of cortical neurons exist, among which the pyramidal neurons. These neurons derive from neural progenitors called neuroepithelial cells, generated during gestation at the surface of the cerebral ventricle, where they form a monolayered neuroepithelium, the ventricular zone. The neuroepithelium looks pseudostratified because of the migration of nuclei during cell cycle. Neuroepithelial cells are polarized along the apico-basal axis, with an apical process contacting the ventricular lumen, and a basal process extending to the basal lamina. They undergo symmetric

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proliferative divisions generating two neuroepithelial cells and increasing exponentially the pool of neural progenitors (Figure 8) [31], [33], [34].

As neurogenesis progress, neuroepithelial cells turn into neural progenitors with a more restricted potential, the radial glia cells, and that marks the transition from the proliferative to the neurogenic phase of neurogenesis. The radial glia cells are characterized by a long radial process extending to the pial surface and guiding neuronal migration. Radial glia cells undergo symmetric proliferative divisions generating two radial glia cells, asymmetric neurogenic divisions generating one radial glia cell and one neuron, and asymmetric differentiating divisions generating one radial glia cell and one intermediate progenitor cell [33]–[35]. Intermediate progenitor cells have a rond morphology, localize in the subventricular zone, and divide symmetrically to generate either two neurons, either two intermediate progenitor cells (Figure 8) [33].

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Radial glia cells thus generate the neurons of the different cortical layers, either directly, or indirectly via the intermediate progenitors. The postmitotic neurons migrate radially through the cortex to form the different cortical layers. The neurons generated first form the deepest layers and the neurons generated later form the more superficial layers (inside-out generation of the cortical neurons) (Figure 8) [35].

As mentioned before, the duration of the proliferative phase of neurogenesis determines the area of cortical surface, and the timing from symmetric to asymmetric divisions is thus a crucial determinant of brain growth. The symmetric division of a neuroepithelial cell requires exquisite precision of the cleavage plane positioning, in order to bisect the apical plasma membrane. The poles of the mitotic spindle have to be positioned perfectly, with a vertical metaphase plane, and a horizontal spindle axis, that has to be maintained horizontal. Finally, the cleavage furrow moving from the basal to the apical membrane, the fusion of plasma membrane on completion of cytokinesis has to result in inheritance of a part of the apical plasma membrane by both daughter cells. A subtle change, e.g. in spindle pole orientation, would result in bypassing in place of bisecting the apical plasma membrane, and would lead to an asymmetric division [33], [34]. More recent studies propose another model for asymmetric divisions, involving primary cilia, key cell sensory organelles anchored in the plasma membrane through the mother centriole. Primary cilia were first taught to disassemble prior to mitosis. It was recently shown that they become very small, and that the part of plasma membrane attached to the mother centriole, including the ciliary remnant, is endocytosed at the onset of mitosis. This ciliary remnant was shown to persist at one of the spindle poles and to be inherited by one of the daughter cells, that retain the stem cell character, resulting in an asymmetric division [36].

The centrosome is the major microtubule organizing center in mammals. It has the capacity to serve as basal body and anchor for cilia, or for spindle pole during cell division [37]. A large number of genes are already known to encode centrosomal-associated proteins involved in the processes mentioned above, and several mutations in those genes were shown to be associated with a PM phenotype in humans.

The many causes of small brain

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syndromic or non-syndromic, and be associated with an abnormal or a normal brain architecture. Causes of syndromic PM include 16p11.2 chromosomal duplication syndrome, Seckel syndrome, Bloom syndrome, Wolcott-Rallison syndrome. Examples of non-syndromic PM with an abnormal brain architecture are holoprosencephaly, lissencephaly, schizencephaly. The subgroup where microcephaly is primary and non-syndromic and the brain architecture is normal used to be referred to as Microcephalia Vera. An important autosomal recessive subgroup thereof is referred to as MicroCephaly Primary Hereditary (MCPH) [38]. These cases have a high recurrence risk in siblings, especially if parents are consanguineous, indicating a large contribution of autosomal recessive causes [39]. The MCPH phenotype is typically mild, usually consisting of mild to moderate mental retardation only [30].

The genetics of PM

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Seminars in Cell & Developmental Biology 76 (2018) 76–85

Contents lists available atScienceDirect

Seminars in Cell & Developmental Biology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

The genetics of congenitally small brains

Sarah Duerinckxa,∗_{, Marc Abramowicz}a,b

a_{IRIBHM, Université Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium}

b_{Department of Medical Genetics, Hôpital Erasme, Université Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium}

a r t i c l e i n f o Article history:

Received 26 July 2017

Received in revised form 5 September 2017 Accepted 8 September 2017

Available online 12 September 2017 Keywords: Intellectual disability Congenital malformation Brain development Centriole Complex inheritance a b s t r a c t

Primary microcephaly (PM) refers to a congenitally small brain, resulting from insufﬁcient prenatal pro-duction of neurons, and serves as a model disease for brain volumic development. Known PM genes delineate several cellular pathways, among which the centriole duplication pathway, which provide interesting clues about the cellular mechanisms involved. The general interest of the genetic dissection of PM is illustrated by the convergence of Zika virus infection and PM gene mutations on congenital microcephaly, with CENPJ/CPAP emerging as a key target. Physical (protein-protein) and genetic (digenic inheritance) interactions of Wdr62 and Aspm have been demonstrated in mice, and should now be sought in humans using high throughput parallel sequencing of multiple PM genes in PM patients and control subjects, in order to categorize mutually interacting genes, hence delineating functional pathways in vivo in humans.

Contents

1. Deﬁnition of primary microcephaly . . . 77

1.1. Microcephaly, primary (PM) and secondary . . . 77

1.2. PM, a model disease for brain volumic development . . . 77

2. The many causes of small brains . . . 77

3. MCPH, the non syndromic, autosomal recessive PM . . . 77

4. Syndromes with PM . . . 79

4.1. PM syndromes with dysostoses and/or short stature . . . 79

4.2. PM syndromes with radiosensitivity . . . 79

4.3. PM syndromes with diabetes . . . 79

5. Zika virus infection and PM genes converge on congenital microcephaly . . . 79

6. A common ﬁnal pathway in primary microcephaly? . . . 80

6.1. Centriole duplication and the centrosomal cycle . . . 80

6.2. Mitotic checkpoint activation . . . 80

6.3. Regulation of mRNA translation . . . 80

7. The Zebraﬁsh as a model organism . . . 80

8. Beyond monogenic inheritance: dissecting the more complex inheritance of primary microcephaly . . . 81

9. Conclusion . . . 82

Acknowledgements . . . 82 References . . . 82

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S. Duerinckx, M. Abramowicz / Seminars in Cell & Developmental Biology 76 (2018) 76–85 77 1. Deﬁnition of primary microcephaly

1.1. Microcephaly, primary (PM) and secondary

Microcephaly refers to a small head. It is a clinical condition, readily assessed by measuring the Occipito frontal circumference

[1]. Microcephaly almost always reﬂects a small brain, mostly a

small neocortex. The occipito-frontal circumference is hence a sur-rogate for brain volume.

If we admit that brain size is distributed on a roughly Gaussian curve, a little over 2% of the general population will have brain sizes smaller than 2 SD below the mean, and most of this group will have normal intellect. Using 3 SD below the mean as a cut-off, most subjects have intellectual deﬁciency (ID), and some have additional neurological deﬁcits like epilepsy or weakness.

Primary microcephaly (PM), i.e. congenital microcephaly, once referred to as microcephalia vera, consists of a prenatal defect of brain volumic development, and secondary microcephaly, i.e. pro-gressive microcephaly, refers to propro-gressive atrophy of an initially

normal brain, usually starting after birth[2].

1.2. PM, a model disease for brain volumic development

Primary genetic microcephalies are a group of conditions, usually transmitted as autosomal recessive traits, with a global incidence of 1/10 000–1/100 000 births depending on ethnicity

and rate of consanguinity[3]. PM is a model disease for defects of

brain volumic development, and the numerous genes already iden-tiﬁed as causes of PM provide insights into the pathways involved. This review will aim at a synthesis of current data and emerging patterns.

2. The many causes of small brains

Recognized causes of microcephaly are vastly heterogeneous. Hundreds of syndromes have been described that feature micro-cephaly. As an illustration, a query of the Online Mendelian

Inheritance in Man[4]retrieved 666 entries with microcephaly in

a clinical synopsis section, i.e. indexed because of a speciﬁc phe-notype. These can be categorized into detailed nosological entities

using both clinical and genetic data[5].

More generally, environmental and genetic microcephaly can be

classiﬁed as inTable 1. A subgroup emerges where microcephaly

is primary and non-syndromic. Such cases have a high recurrence risk in siblings, especially if parents are consanguineous,

indicat-ing a large contribution of autosomal recessive causes[6]. They are

referred to as MicroCephaly Primary Hereditary (MCPH). The MCPH brain is small but its architecture is conserved, and the phenotype is typically mild, usually consisting of mild to moderate mental

retardation only[3].

The known functions of genes involved in syndromic and non-syndromic microcephaly, point to the centriole as a major, but not sole player, as will be overviewed below.

3. MCPH, the non syndromic, autosomal recessive PM

Even in this well circumscribed nosological entity, genetic het-erogeneity is striking, with 17 genes reported at the time of this

writing. An overview of MCPH genes is given inTable 2. Many of

these genes’ products however are localized at the centrosome dur-ing interphase, or spindle pole durdur-ing mitosis. The centrosome is the major microtubule organizing center in mammals. It has the capacity to serve as basal body and anchor for cilia, or for spindle

pole during cell division[7].

Microcephalin (BRCT-repeat inhibitor of hTERT expression, BRIT1, MCPH1) was the ﬁrst gene linked with MCPH. MCPH1 is localized at the centrosome and plays roles in mitotic spindle alignment and checkpoint, and also in DNA damage repair. MCPH1 is required for Chk1 (Checkpoint kinase 1) localization to centrosomes, where it inhibits Cdc25 (Cell division cycle 25) phosphorylation. Prema-ture Cdc25 phosphorylation and Cdk1 (Cyclin-dependent kinase 1) activation in MCPH1 deﬁciency causes a premature entry into mitosis, uncoupling mitosis from the centrosome cycle and leading

to abnormal spindles and chromosome misalignment[8]. MCPH1

deﬁciency is associated with a cytogenetic phenotype of premature

chromosome condensation (PCC)[9–11]. The N-terminal BRCT1

domain (BRCA1 C-terminal domain) of Mcph1 is necessary for cen-trosomal localization, while C-terminal BRCT2 and BRCT3 domains are required for ionizing radiation-induced nuclear foci (IRIF)

for-mation and response to DNA damage[12]. BRCT2 and BRCT3 bind

E2F1 (E2F transcription factor 1) to form a complex able to

transac-tivate BRCA1 and CHK1[13], and also interact with Cdc27, a subunit

of the anaphase-promoting complex[14], and H2AX (H2A histone

family, member X)[15].

WDR62 (WD40-repeat protein 62, MCPH2) is after ASPM a preva-lent cause of MCPH. WDR62 localizes at the spindle poles during

mitosis, and in the nucleus during interphase [16]. It plays a

role in stabilizing the spindle pole after bipolar spindle formation

[17]. Depletion of wdr62 in mice resulted in modest microcephaly

caused by reduced proliferation of neuroprogenitors, abnormal-ities in asymmetric centrosome inheritance leading to neuronal migration delays and cell death, and altered neuronal

differen-tiation[18]. Some patients with WDR62 defects have associated

phenotype abnormalities like agyria, pachygyria, hypoplasia of the

corpus callosum or other brain malformations[19], indicating a

role of WDR62 in neuronal migration. In mice, Wdr62 recruits Jnk1 (c-Jun N-terminal kinase) to spindle poles, and hence

pro-motes neurogenesis[20]. Pericentriolar matrix protein Aurora A

phosphorylates WDR62 which allows for spindle organization and

metaphase chromosome alignment[21]. Wdr62 stabilizes

inter-phase microtubules, which in turn recruit Plk1 (Polo like kinase 1)

to the centrosome[22].

CDK5RAP2 (CDK5 regulatory subunit associated protein 2, MCPH3) is expressed in neuroprogenitor cells during neurogenesis,

and localizes at the centrosome[23]. CDK5RAP2 depletion causes

a delocalization of gamma-tubulin from centrosomes and thus

the absence of centrosomal microtubules formation[24]. Mutant

mice were modestly microcephalic with reduced superﬁcial

corti-Table 1

A general classiﬁcation of causes of microcephaly with selected examples.

• Environmental: prenatal infection by Zika virus or cytomegalovirus; prenatal exposure to alcohol or other teratogens; perinatal hypoxia; hypoglycemia • Genetic: 666 phenotypic entries in OMIM

-Postnatal (secondary): inborn errors of intermediate metabolism, others -Congenital (primary)

• Syndromic: 16p11.2 chromosomal duplication syndrome, Seckel syndrome, Bloom syndrome, Wolcott-Rallison syndrome • non-syndromic

-with abnormal brain architecture: holoprosencephaly, lissencephaly, schizencephaly -with normal brain architecture: « microcephalia vera »

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78 S. Duerinckx, M. Abramowicz / Seminars in Cell & Developmental Biology 76 (2018) 76–85

Table 2

Overview of MCPH genes.

Locus Gene name Localization Prevalence of human

mutations

MCPH1 MCPH1, microcephalin 1 centrosome

MCPH2 WDR62, WD repeat domain 62 spindle poles during mitosis, nucleus during interphase

prevalent cause of MCPH after ASPM

MCPH3 CDK5RAP2, CDK5 regulatory subunit associated protein 2 centrosome MCPH4 CASC5, cancer susceptibility candidate 5, KNL1, kinetochore

scaffold 1

kinetochore

MCPH5 ASPM, abnormal spindle microtubule assembly spindle poles during mitosis, nucleus during interphase

most common cause of MCPH (25–50% in consanguineous patients)

MCPH6 CENPJ, centromere protein J centrosome allelic to Seckel syndrome

MCPH7 STIL, SCL/TAL1 interrupting locus centriole

MCPH8 CEP135, centrosomal protein 135 centrosome

MCPH9 CEP152, centrosomal protein 152 pericentriolar matrix allelic to Seckel syndrome MCPH10 ZNF335, zinc ﬁnger protein 335 component of H3K4 methyltransferase

complex (chromatin remodeling)

severe neurological phenotype MCPH11 PHC1, polyhomeotic homolog 1 component of polycomb (chromatin

remodeling)

MCPH12 CDK6, cyclin dependent kinase 6 centrosome during mitosis, cytosol and nucleus during interphase

MCPH13 CENPE, centromere protein E centromere

MCPH14 SASS6, SAS-6 centriolar assembly protein centriole

MCPH15 MFSD2A, major facilitator superfamily domain containing 2A endothelium of the blood-brain-barrier MCPH16 ANKLE2, ankyrin repeat and LEM domain containing 2 NA

MCPH17 CIT, citron rho-interacting serine/threonine kinase cleavage furrow and midbody

cal layers. They showed a disrupted architecture of centrosomes, an altered centriole replication cycle resulting in centriole ampli-ﬁcation and multipolar spindles[25]. They also showed reduced vertical cleavage planes, increased neuronal differentiation and decreased proliferation of neuroprogenitors, premature cell cycle

exit and increased apoptosis of neuroprogenitors[26,27].

Pericen-triolar matrix component (PMC) PCNT interacts with Cdk5rap2 and

recruits it to the centrosome[26].

CASC5 (cancer susceptibility candidate 5, KNL1, kinetochore scaffold 1, MCPH4) is localized at the kinetochore, and is required for the attachment of chromatin to the mitotic apparatus. The CASC5 locus maps to the same chromosome arm as CEP152 but is clearly

distinct from it[28]. CASC5 binds BUBR1 to control the spindle

assembly checkpoint[29]. Patient-derived mutated cells showed

CASC5 localization at the metaphase plane but also in the cyto-plasm, chromosome misalignment, increase in mitotic cells due to a probable mitotic delay, increased apoptosis, fragmented nuclei and additional micronuclei which may be the consequence of unre-paired DNA breaks, and a greater DNA damage load signing an

impaired DNA damage response[30].

ASPM (abnormal spindle microtubule assembly, MCPH5) is the most common cause of MCPH, accounting for 25–50% of the cases

in consanguineous patients[31–33]. ASPM is localized in

neuroep-ithelial cells at the spindle pole during mitosis, and in the nucleus during interphase. It is down-regulated as neuroepithelial cells

switch from proliferative to neurogenic divisions[34,35]. Aspm

deﬁciency in mice induced a lengthening of the cell cycle upstream of the G1 restriction point, mediated by the Cdk2/Cyclin E complex,

causing down-regulation of symmetric stem cell division[36].

CENPJ (centromere protein J, SAS-4, CPAP, MCPH6) is localized at the centrosome and plays a role in the elongation step of cen-triole duplication. It interacts with STIL, CEP135 and CEP152, as

well as WDR62 and ASPM, and binds microtubules (MTs)[37–39].

CENPJ mutant cells presented aberrant ciliogenesis with long cilia, retarded cilium disassembly and retarded cell-cycle re-entry. Retarded cilium disassembly caused premature neural progenitor

cells (NPCs) differentiation[40].

STIL (SCL/TAL1 interrupting locus, MCPH7) mutations in human

required for the correct loading of SASS6 and CENPJ to the base of

the procentriole to initiate procentriole assembly[38,43,44].

CEP135 (centrosomal protein 135, MCPH8) is localized at the

centrosome with PCNT[45]. CEP135 is required for centriole

dupli-cation and mitotic spindle assembly. It interacts directly with SASS6, forms a complex with SASS6 and CENPJ, and it also directly

binds to MTs[46].

CEP152 (centrosomal protein 152, MCPH9) is a relatively rare cause of MCPH but is the most common cause of Seckel

syn-drome[38]. Cep152 interacts directly with Cep63 and localizes

to the pericentriolar matrix in a mutually dependent manner, and promote centriole duplication via recruitment of SASS6

[47]. CEP152-deﬁcient cells display an early anaphase block, an

increased chromosome instability, with increased activation of ATM (Ataxia-Telangiectasia Mutated) signaling pathways, and an

increased formation of hydroxyurea-induced␥H2AX foci[48].

ZNF335 (zinc ﬁnger protein 335, MCPH10) mutations cause a severe phenotype of extreme microcephaly with neuronal

disor-ganization, small size and neonatal death[49]. Its qualiﬁcation as

a MCPH gene is hence questionable. ZNF335 is a component of the H3K4 methyltransferase complex involved in chromatin remodel-ing, is expressed in neural progenitors, and prevents premature cell-cycle exit. Mutant mice showed early embryonic lethality, while conditional knock out mice presented with severely reduced cortical size, premature neuronal fate determination, and impaired

neuronal morphogenesis[49].

PHC1 (polyhomeotic homolog 1, MCPH11) encodes a component of polycomb, involved in chromatin remodeling. It plays a role in

cell-cycle control[38]. Patient-derived cells showed a decreased

H2A ubiquitination and a subsequent defect in DNA repair[50].

CDK6 (cyclin dependent kinase 6, MCPH12) is a member of the cyclin-dependent kinase family, crucial for cell cycle progression of G1 to S phase. It is localized in the cytosol and the nucleus in

interphase, and at the centrosome during mitosis[51].

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S. Duerinckx, M. Abramowicz / Seminars in Cell & Developmental Biology 76 (2018) 76–85 79 SASS6 (SAS-6 centriolar assembly protein, MCPH14) interacts

directly with CEP152, CEP63 and CEP135. CEP135 interaction is

critical for centriole duplication[46,53].

MFSD2A (major facilitator superfamily domain containing 2A, MCPH15) is expressed in the endothelium of the blood-brain-barrier and transports docosahexaenoic acid (DHA) and other fatty acids in the form of lysophosphatidylcholine across the blood-brain

barrier, in a sodium-dependent manner[54–56].

ANKLE2 (ankyrin repeat and LEM domain containing 2, MCPH16) depletion in drosophila causes a severe reduction in neuroblasts number, reduced cell proliferation and increased apoptosis. Asymmetric division or centriole number seemed

unaf-fected[57].

CIT (citron rho-interacting serine/threonine kinase, MCPH17) has two isoforms, K and N which lacks the kinase domain.

CIT-K localizes to the cleavage furrow and midbody[58]. Loss of CIT-K

resulted in cytokinesis failure and apoptosis speciﬁcally in neural

progenitors and in male germ cells[59]. Mutant mice showed an

excess of DNA damage (␥H2AX foci) due to increased DNA

double-strand breaks[60].

4. Syndromes with PM

Numerous PM syndromes feature either dysostoses/short stature, or radiosensitivity/chromosome breakage, or diabetes.

4.1. PM syndromes with dysostoses and/or short stature

Seckel syndrome is characterized by proportionate short stature, severe microcephaly with intellectual disability, and a typi-cal ‘bird-head’ facial appearance (receding forehead and chin, large

beaked nose, and large or bulging eyes)[61]. Known genes include

ATR (SCKL1), RBBP8/CTIP (SCKL2), ATRIP, CEP63 (SCKL6), NIN (SCKL7), DNA2 (SCKL8), TRAIP (SCKL9), NSMCE2 (SCKL10), as well as CENPJ (SCKL4) and CEP152 (SCKL5) which in some families cause MCPH only. MCPH and Seckel are now considered part of a phenotypic

continuum[62]. ATR plays a role in homologous recombination

DNA repair[63]. ATR has also a role in cilia signaling[64]. ATRIP is a

direct interactor of ATR involved in DNA repair[65]. Dna2 promotes

DNA replication and chromosome detachment after replication

fork stalling[66]. TRAIP, NSMCE2 and RBBP8 play a role in DNA

damage response[67–69].

Microcephalic osteodysplastic primordial dwarﬁsm type II (MOPDII) associates severe growth retardation and marked micro-cephaly, with characteristic facies, skeletal dysplasia, abnormal dentition, and an increased risk for cerebrovascular disease and insulin resistance. It is caused by mutations in PCNT (pericentrin)

[70]. PCNT is required for removal of CDK5RAP2 from centrosomes

during late mitosis, which promotes centriole disengagement and

separation[71]. PCNT also plays a role in DNA damage-dependent

signaling[72,73].

Meier-Gorlin syndrome is an association of proportionate short stature, microcephaly, microtia, and patellar aplasia or hypoplasia. This syndrome is caused by mutations in components of the DNA

pre-replication complex ORC1, ORC4, ORC6, CDT1, or CDC6[74]. A

defect in any of those genes is presumed to impair the S phase of the mitotic cycle, and to impede cell proliferation. ORC1 might also

play a role in centrosome duplication[75].

Defects in DONSON are associated with marked microcephaly, reduced cortical size, decreased gyral folding and short stature with minor skeletal abnormalities including hypoplastic patellae. DON-SON is a component of the replisome. It maintains replication fork stability and genome integrity, preventing spontaneous DNA

dam-4.2. PM syndromes with radiosensitivity

LIG4 mutations cause a syndrome of microcephaly, short stature, bone marrow failure, immunodeﬁciency, and radiation

hypersensi-tivity[77]. Mutated patient cells have increased sensitivity to DNA

double strand breaks induced by ionizing radiation[78]. LIG4 plays

a role in the ﬁnal step of non-homologous end joining, by ligating

the DNA strands together[79].

Bloom syndrome (BS) and Nijmegen Breakage syndrome (NBS) are characterized by microcephaly, growth failure, increased cancer

incidence and immunodeﬁciency[80]. NBS is caused by mutations

in NBN (NBS1) gene. NBN plays a role in the cellular response to DNA injury, being a major component of the MRE11/RAD50/NBN

double strand breaks (DSBs) repair complex[80]. BS is caused by

mutations in BLM/RECQL3, a DNA helicase that functions in DNA

double-strand-break repair[81]. It plays a role in maintaining the

stability of DNA through the replication processes[80].

BUBR1 mutations are associated with severe growth retardation, microcephaly, increased cancer incidence, premature chromatid separation of all chromosomes, and mosaicism for various tri-somies and monotri-somies referred to as mosaic variegate aneuploidy

(MVA)[82–84]. BUBR1 localizes to the kinetochore[82,83], and is

crucial for the spindle checkpoint. 4.3. PM syndromes with diabetes

Wolcott Ralisson syndrome associates neonatal/early-onset non-autoimmune insulin-requiring diabetes with skeletal dyspla-sia and growth retardation. It is caused by mutations in the gene encoding the endoplasmic reticulum (ER) transmembrane protein EIF2AK3, a stress sensor that plays a key role in translation

con-trol during the unfolded protein response[85]. In mice, Eif2ak3

deﬁciency caused accumulation of misfolded proteins leading to

apoptosis in beta cells[86].

IER3IP1 is associated with a syndrome of neonatal diabetes, primary microcephaly with simpliﬁed gyral pattern and severe

infantile epileptic encephalopathy[87]. It is also associated with

skeletal changes like scoliosis and osteopenia[88]. IER3IP1

local-izes to the ER[89], and is thought to be involved in the ER stress

response. Increased apoptosis in cerebral cortex and in pancreas were observed in a patient autopsy. Patient-derived cells presented

an increased apoptotic cell death under stress conditions[90].

TRMT10A mutations cause a syndrome of young onset diabetes, short stature and microcephaly. TRMT10A is a tRNA methyl-transferase enriched in brain and pancreas, and localizes to the nucleolus, where tRNA modiﬁcations occur. TRMT10A silencing in

beta cells resulted in ER stress-induced apoptosis[91].

5. Zika virus infection and PM genes converge on congenital microcephaly

In the past few years, an outbreak of fetal Zika virus (ZIKV) infection in Brazil was paralleled with a surge in congenital

micro-cephaly incidence [92]. ZIKV infection of neuroprogenitor cells

(NPCs) derived from human induced pluripotent stem cells (hiPSCs) showed a decreased proliferation and an increased differentiation of NPCs. Increased cell death was also observed. Centrosomes in interphase were structurally abnormal. Infected brain organoids had a limited growth. A vertically oriented division plane, leading to neurogenic divisions, was observed in a higher proportion of

apical progenitors[93,94].

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80 S. Duerinckx, M. Abramowicz / Seminars in Cell & Developmental Biology 76 (2018) 76–85 RNAseq and real time PCR data showed that several

micro-cephaly associated genes are downregulated following ZIKV

infection (ASPM, CASC5, CENPF, MCPH1, RBBP8, STIL, TBR2)[96,97].

Experimental ZIKV infection of brain organoids derived from human cells was found to target apical progenitors, causing prema-ture neurogenesis, mediated by a direct interaction of a viral protein

with CEP63[93]. Subtle changes in cilium structure were observed

that mimicked ciliary changes observed in organoids derived from

patients with a CPAP gene mutation[40]. In another line of study,

Zika virus mRNA was shown to interfere with Musashi-1 (MSI1), a RNA binding protein expressed in neural progenitors. This dis-rupted the normal interaction of MSI1 with MCPH1 mRNA, required for efﬁcient translation of MCPH1. Signiﬁcantly, the very rare patients with biallelic mutations of MSI1 presented with congen-ital microcephaly and their lymphocytes displayed the premature chromosome condensation phenotype typical of MCPH1 gene

loss-of-function mutations[98].

6. A common ﬁnal pathway in primary microcephaly? 6.1. Centriole duplication and the centrosomal cycle

A majority of MCPH-causing gene products are localized at the

centrosome during interphase or spindle pole during mitosis[38],

suggesting a general centrosomal mechanism for all forms of PM

[99]. It has been hypothesized that subtle changes in mitotic

spin-dle orientation would commit apical neural progenitors to either symmetric or asymmetric cell division, by respectively bisecting or bypassing the portion of the apical membrane that conveys apical progenitor speciﬁcity. As symmetric divisions increase the pool of neural progenitors, a spindle orientation mechanism would

pro-vide an elegant explanation hypothesis for PM[35]. While spindle

alignment has not been validated as the cause, inheritance of a progenitor-specifying portion of the cell membrane tightly associ-ated with the primary cilium is now recognized as an important mechanism in the regulation of neural progenitor proliferation [100,101].

Crucial to volumic development of the brain is the timing of the transition from proliferative to postmitotic neuron-producing cell divisions. A premature transition, in particular from apical to basal progenitors, will cause a reduced ﬁnal number of neurons and

pri-mary microcephaly ([102]and references therein). When mitosis

was experimentally prolonged in radial glia neural progenitors in mice, either by a speciﬁc genetic mutation, or pharmacologically, altered fate speciﬁcation was observed in the cell progeny, with an increased neuron/progenitor balance, and with an increased rate

of apoptotic daughter cells[102]. This observation is reminiscent

of the prolonged prophase and PCC phenotype in patients with

MCPH1 mutations and PM[9–11]. Of note, prolonged mitosis of

outer radial glia progenitors was also observed in hiPSC-derived brain organoids from patients with Miller-Dieker lissencephaly, a

brain developmental disorder with PM as a feature[103].

MCPH proteins are recruited stepwise to the centrosome in C.

Elegans and in the mouse[104,105]. Cep63 and Cep152 bind the

mother centriole to initiate its duplication[106]. Cep63 recruits

Wdr62 and Aspm, which physically interact and in turn recruit Cpap/Cenpj. The latter protein may be the ﬁnal common target of centrosome-associated PM genes that is eventually required for

centriole duplication[39,107]. Interestingly, the physical

interac-tion of Wdr62 and Aspm is paralleled by their genetic interacinterac-tion

in a digenic manner: Aspm−/− mice display a modest imbalance

in the ratio of apical to basal progenitors and a modest reduction in

Wdr62−/− and Aspm −/− genotypes. Wdr62 and Aspm are thus

required in a dose-dependent manner for centriole duplication in

mice[39]. Of note, loss of Wdr62 and/or Aspm caused

disorganiza-tion of apical epithelial structure, independently of their centriolar effect, which resulted in untimely neurogenesis by inducing early

loss of apical progenitor speciﬁcity[39].

Evidence from many MCPH genes thus identiﬁes PM as a

centro-somal disease[99], and more speciﬁcally as a centriolopathy[39],

with partial redundancy, at least in mice, of some genes like Aspm and Wdr62. The centriolar pathway however fails to unify data from other important PM-causing molecules, which expose at least two other possible pathways: the activation of mitotic checkpoints, and

the control of protein translation (seeFig. 1).

6.2. Mitotic checkpoint activation

Dysfunction of several checkpoints of mitosis progression, e.g. the intrinsic S-phase checkpoint, the G2/M checkpoint, or the spin-dle assembly checkpoint, have been reported in PM caused by various genes, e.g. ORC1, DONSON, and CASC5 respectively (see above). Loss of checkpoint activity will hamper cell proliferation

and initiate cellular apoptosis[108], providing a possible

expla-nation for the depletion in the final number of neurons. While specific checkpoints are directly dependent on PM genes, e.g. CASC5 or BUBR1 for the spindle assembly checkpoint, mitotic checkpoint activity is also dependent on DNA replication stress, defined as the slowing or stalling of replication fork progression and/or DNA

synthesis[109]. Deﬁcient licensing of DNA replication, deﬁcient

chromosome condensation, or deﬁcient DNA damage repair, will all lead to replication stress.

MCPH1 has been involved at several steps. In mice, Mcph1 is localized at the centrosome where it recruits Chk1 which in turn activates Cdc25. Mcph1 loss results in premature Cdk1 acti-vation by Cdc25, which uncouples the centrosome cycle with the

mitotic cycle, causing early mitotic entry[8]. Furthermore, MCPH1

is associated with premature chromosome condensation[9], via its

interaction with condensin components[110].

6.3. Regulation of mRNA translation

Syndromes of PM with diabetes have been associated with

apoptosis via the endoplasmic reticulum stress response[86,91].

More generally, a group of PM genes emerged that regulate mRNA

translation including EIF2AK3[85], IER3IP1[90], and TRMT10A[91].

While apoptosis has so far been demonstrated mainly in pancreatic beta cells, providing a likely explanation for the diabetes feature of the phenotypes, apoptosis in neural progenitors is also speculated to be the cause of the PM feature.

7. The Zebraﬁsh as a model organism

In spite of the conspicuously human nature of a large neocor-tex, the Zebraﬁsh (ZF) is now a recognized organism to model PM, starting with aspm-targeting morpholinos in 2011. Injected embryos had reduced head and eye sizes in comparison with

control embryos [111]. Stil, aspm and wdr62 silencing caused a

reduction in head size and in retinal cell number, with an increase in

the fraction of mitotic cells and high rates of apoptosis[112].

Anti-atr morpholino caused an underdeveloped head, smaller eyes and a

smaller body[64]. ZF modeling proved able to dissect the

molecu-lar cause of microcephaly within a genetic segment encompassing

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S. Duerinckx, M. Abramowicz / Seminars in Cell & Developmental Biology 76 (2018) 76–85 81

Fig. 1. Pathways in Primary Microcephaly.

A selection of PM-causing genes involved in speciﬁc mechanisms (dotted boxes) are shown that converge on three pathways (solid boxes). Possible cellular mechanisms that mediate reduced proliferation are shown in italics. MCPH1 couples the centriole duplication pathway and the mitotic checkpoint pathway via Cdc25 and Cdk1 (dotted arrow), and is involved in chromosome condensation and DNA damage repair (see text).

a gene cause microcephaly, like the K78E missense mutation, as

opposed to other genetic variants, of the RPL10 gene[114].

Finally, the ZF has been used to modelize digenic inheritance. As an example, suboptimal silencing of various combinations of two genes out of six genes causing microcephaly and local-ized to human chromosomal segment 17p13.1 showed genetic interactions unique for each combination, suggesting additive, multiplicative or absent effect on microcephaly depending on the

combination[115].

8. Beyond monogenic inheritance: dissecting the more complex inheritance of primary microcephaly

Apparently Mendelian disorders may in fact result from muta-tions in two distinct loci, which is referred to as digenic inheritance [116]. The simplest model in digenic inheritance consists of het-erozygous mutations at two loci (double heterozygosity). A more complex model consists of homozygosity/biallelic mutations at one locus and heterozygosity at a second locus (triallelic inheritance). This latter model is of special interest in phenotypes with appar-ent autosomal recessive inheritance, like PM, assuming that a third

mutation might be necessary to cause the phenotype[117]. Digenic

inheritance indicates a functional relationship between two loci, including protein-protein interaction, protein-DNA interaction, or

a shared pathway[118].

Interestingly, digenic, triallelic inheritance has been described

for PM caused by Wdr62 and Aspm in the mouse[39]. Whether this

applies to humans remains to be determined, and it should be noted that ﬁnding digenic inheritance of PM in humans would be of con-siderable interest. First, it would pave the way to dissecting more complex, oligogenic and multigenic inheritance of microcephaly, probably pinpointing lighter phenotypes and endophenotypes along the process, and eventually helping to solve clinical

conun-variable expressivity. Second, it would categorize mutually

inter-acting genes[39,118], hence delineating functional pathways using

a holistic, in vivo method.

With the advent of high throughput, parallel DNA sequencing of multiple PM-causing genes in panels or exomes, such a holistic approach becomes feasible in human patients. Exome sequencing data from over a hundred of PM and control subjects are amenable to a mutation burden test, in order to quantify the absolute num-ber of genetic variants in a pre-deﬁned subset of several dozen PM-causing genes in the PM cases compared with control sub-jects. The cut-off for minor allele frequency should be low, since almost all variants described in digenic inheritance are unknown

from public databases or rare (<1%)[119]. The subset of PM genes

could be extracted from the literature, and protein interaction

databases like ConsensusPathDB[120]could be used to reﬁne the

list. Control tests should include permutation testing with sub-sets of non-PM-associated, housekeeping genes. Furthermore, in order to rule out the trivial hypothesis that an increased muta-tion burden would merely reflect the presence in patients of the biallelic mutations of the major PM gene as expected under a monogenic model, i.e. the diagnostic mutations, the analysis should be performed after removing such biallelic mutations form the PM patients’ data. A mutation burden test specifically showing an excess of variants of the PM-related genes in the PM patients will indicate digenic, or more complex inheritance of PM. If positive, the data should be re-analyzed to identify which pairs of genes drove the result to statistical significance. This would indicate functional pathways in vivo, and the genetic interactions could then be val-idated by modeling digenic inheritance of the orthologues in ZF using genome-editing methods based on Transcription

activator-like effectors nucleases (TALENs) [121] or Clustered regularly

interspaced short palindromic repeat (CRISPR)/CRISPR-associated

Monogenic and Digenic Inheritance of Primary Microcephaly

Monogenic and Digenic Inheritance

of Primary Microcephaly

Thesis submitted by Sarah DUERINCKX

in fulfilment of the requirements of the PhD Degree in medical science

(“Docteur en médecine”)

Academic year 2018-2019

Supervisor: Professor Marc ABRAMOWICZ

Co-supervisors: Professors Tom LENAERTS and Isabelle PIRSON

Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire

(IRIBHM)

Thesis jury:

Monogenic and Digenic Inheritance

of Primary Microcephaly

Thesis submitted by Sarah DUERINCKX

in fulfilment of the requirements of the PhD Degree in medical science

(“Docteur en médecine”)

Academic year 2018-2019

Acknowledgements

Abstract

Table of contents

Table of illustrations

List of abbreviations

Introduction

Digenic inheritance

Primary microcephaly

Seminars in Cell & Developmental Biology

The genetics of congenitally small brains