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Congenital malformations the human hindbrain, including the cerebellum, are poorly understood largely because their recognition is a relatively recent advance for imaging diagnostics. Cerebellar malformations are the most obvious and best characterized hindbrain malformations due to their relative ease to view by MRI and the recent identification of several causative genes1. Malformations of the pons and medulla have also been described both in isolation and in association with cerebellar malformations2. Although little is understood regarding the specific developmental pathologies underlying hindbrain malformations in humans, much is known regarding the mechanisms and genes driving hindbrain development in vertebrate model organisms. Thus, studies in vertebrate models provide a developmental framework in which to categorize human hindbrain malformations and serve to inform our thinking regarding disrupted developmental processes and candidate genes. Here we survey the basic principles of vertebrate hindbrain development and integrate our current knowledge of human hindbrain malformations into this framework.
The hindbrain controls numerous important physiological processes including respiration, circulation, arousal and motor coordination. Understanding the pathology of human hindbrain malformations, and devising strategies to diagnose and treat them ultimately relies on identifying the causative genes and elucidating the molecular and cellular mechanisms underling the early events that form the nervous system. Studies from vertebrate model systems have already revealed much of the genetic control that underlies the establishment of hindbrain structures and circuits controlling these functions. Given the high degree of neuroanatomical and functional conservation across vertebrates, insights from the analyses of brain development in model organisms are essential to decipher the developmental and genetic bases of human hindbrain malformations.
The vertebrate central nervous system (CNS) derives from the neural plate, an epithelial sheet that arises from the dorsal ectoderm of the gastrula-stage embryo. Subsequently, the neural plate closes to form the neural tube. The neural tube is then patterned by secreted molecules from signaling centers, which regulate a hierarchy of localized transcription factors to establish a Cartesian-like coordinate system of positional information along the anterior/posterior (A/P) and dorsal/ventral (DV) axes of the CNS.
The vertebrate embryonic neuroepithelium is characterized morphologically by a series of swellings that appear transiently around neural tube closure. Ultimately the anterior portions give rise to the forebrain, midbrain and hindbrain, while the narrow posterior epithelium transforms into the spinal cord. These early morphological features are positioned and foreshadowed by localized expression of developmental control genes that orchestrate the regional plan of the CNS and indicate future functional specializations3,4. As development proceeds, neurogenesis further leads to the formation of distinct neuronal groups that can be identified by morphology, molecular markers, axonal trajectory and neurotransmitter type. As neurons mature, they migrate from their birthplace to their final destination, where they form synaptic connections and integrate into functional circuits (reviewed in5). All of these features of nervous system development are largely conserved across vertebrates.
The hindbrain is the most evolutionarily ancient part of the vertebrate brain4,6, and is fated to give rise to the medulla oblongata, the pons, and the cerebellum. As the hindbrain becomes distinguished from the midbrain and the spinal cord, around the time of neural tube closure, it is further subdivided into a series of seven A/P segments, or rhombomeres (r1-r7) (Fig. 1A,B). Rhombomeres are transient structures first distinguished by the expression of molecular markers and later as morphological segments. Recent fate mapping studies in mice, together with older chick/quail studies, show that dorsal r1 gives rise to the cerebellum and ventral r1 to the anterior pons, while more posterior rhombomeres form the remainder of the pons and the medulla7.
The hindbrain begins to show segmental gene expression patterns at the end of gastrulation, before the morphological appearance of rhombomere boundaries. The initial establishment of the rhombomeric pattern does not follow a strict rostro-caudal order8,9, but rather boundaries form in stereotypical sequence. The presumptive r4 territory, in the center of the hindbrain is the first to be generated and it in turn acts as a local organizing center, signaling to adjacent territories and initiating a molecular cascade leading to the further partitioning of the r3, r5, and r6 segments10,11. As the rhombomeres emerge, distinct physical boundaries form to separate the molecularly and neuroanatomically distinct segments12,13. The process of compartmentalization first involves formation of an interface between adjacent segments, and this is followed by the induction of a specialized population of boundary cells at the interface. The initial formation of a clear interface between rhombomeres is concomitant with sharpening of initially indistinct segmental gene expression domains, such as those of hox genes and krox-2014,15.
Transplantation studies in chick have demonstrated that the process of boundary formation is regulated by a cell sorting mechanism16,17, which requires Ca2+dependent adhesion molecules18. Studies in zebrafish have shown that this cell sorting mechanism is controlled by the repulsive interaction between Eph receptors and Ephrin ligands in alternating segments, and possibly by differential adhesion within the same segment14,15,19–21 to ultimately prevent intermingling of cells across boundaries (Fig. 1C). Embryological studies in chick have also indicated that following the establishment of sharpened segmental gene expression, the interactions between adjacent rhombomeres induce the formation of specialized boundary cells at the segment interface16,22. Recent work in zebrafish showed that Notch activation in boundary cells regulates their formation23; boundary expression of the Notch target gene Hes1 in the mouse suggests that Notch may play a conserved role in rhombomere boundary establishment24. In addition, in zebrafish the boundaries also function as sources of secreted Wnts, which help to pattern adjacent rhombomeres25,26. Although Wnt expression has not been detected in boundaries of other vertebrates, another secreted signal, Fgf3, is expressed in chick rhombomere boundaries27. In summary, the formation of rhombomere boundaries is required not only for prevention of cell mixing between adjacent segments, but also to establish local signaling centers that regulate hindbrain neurogenesis and thus to establish segmental neuronal organization of the hindbrain.
Interestingly, unlike the segmented nature of the hindbrain, the posterior part of the neural tube remains unsegmented during embryonic development. Recent work in zebrafish has identified Cdx transcription factors as key determinants of the unsegmented spinal cord region, demonstrating that repression of the hindbrain developmental program is required for the specification of the vertebrate spinal cord28,29. This regulatory mechanism of establishing the boundary between hindbrain and spinal cord is most likely conserved across vertebrates, based on the similarity between patterns of Cdx gene expression during nervous system development of various vertebrate species28.
At the same developmental stages that the hindbrain is undergoing morphological segmentation it is also become regionalized via segment-specific gene expression. In particular, the segmentally expressed Hox genes play a critical part in conferral of rhombomere identity. Hox genes code for a family of Antennapedia class homeodomain transcription factors that are well known for their conserved role in regionalization of the body plan. A defining feature of Hox genes is a clustered organization, with all vertebrates possessing multiple Hox clusters as a consequence of ancient genome duplication events30. The Hox genes are further arranged in 13 paralog groups, with no one cluster retaining all 13 paralogs. The genes demonstrate an interesting property termed colinear expression, whereby the expression domains along the embryonic A/P axis mirror the locations of the genes along the chromosome. It is the most 3’ located genes, members of paralog groups 1–4, that are expressed most anteriorly, within the developing hindbrain. Each rhombomere expresses a specific combination of Hox genes (with r1 being devoid of Hox expression), thus providing a mechanism to specify unique segment identities (Fig. 1A). Establishment of these segment-specific Hox expression domains is dependent on retinoic acid signaling31,32.
The roles of several Hox genes in hindbrain patterning have been studied in the mouse via generation of knock-out alleles, and in chick and zebrafish via gain-of-function and knockdown approaches (reviewed in4,33). Classic studies in Drosophila have established that Hox loss-of-function tends to cause anteriorizing homeotic transformations, in which posterior segments take on the identity of more anterior segments, whereas gain-of-function tends to cause posteriorizing homeotic transformations. Consistent with this model, when the r4 specific Hoxb1 gene is misexpressed r2 takes on the identity of r4, and when Hoxb1 function is disrupted r4 takes on the identity of r234–36. Interestingly, knock-out of the paralogous gene Hoxa1, which is the earliest expressed of the hindbrain Hox genes, causes an alteration not in segmental identity, but rather in the segmentation process itself37–39, such that r5 is missing. Mutations in both of these Hox genes have been correlated with human malformations.
The mouse Hoxb1 knock-out causes loss of the r4-derived VIIth (facial) motor nerve35. This is turn causes paralysis of the muscles of facial expression, similar to the pathology of Bell’s Palsy or Moebius Syndrome. While this disease has not been directly correlated with alterations in human HOXB1, changes in HOXA1 have been linked with Bosley-Salih-Alorainy (BSAS) syndrome. Linkage analysis and positional cloning in BSAS patients has indicated homozygous truncating mutations in the HOXA1 gene40. These patients exhibit horizontal gaze abnormalities, deafness, facial weakness, hypoventilation, vascular malformations of the internal carotid arteries and cardiac outflow tract and mental retardation. Interestingly, it was this spectrum of defects that suggested a potential HOX mutation, due to the similarities between the BSAS pathology and the phenotype of Hoxa1 mutant mice37–39, which show variable abnormalities of hindbrain segmentation, with a nearly complete loss of r5, partial loss of r4, and alterations to r3, 6, and 7. These mice also show reduced numbers of cell bodies and exiting cranial nerves of the abducens and facial nuclei; absence of the superior olive, and variable defects in cranial autonomic and sensory ganglia, the skull and the external and inner ears. Interestingly, the phenotypes of patients with BSAS also show that loss of HOXA1 function segregates with cognitive impairment and suggest that brainstem dysgenesis may lead to higher cortical dysfunction40.
Other human hindbrain malformations have also been suggested to arise through alterations in the segmentation process. For example, brain stem disconnection syndrome (BSDS) has been proposed to also be a segmentation disorder. In this very rare and very severe malformation, the junction of the midbrain and pons or the pons and the medulla are completely discontinuous41,42, a feature not observed in model organisms. In all experimental situations in model vertebrates where hindbrain segments are missing, the remaining transformed hindbrain segments always remain contiguous with each other. This difference may suggest that an early catastrophic vascular accident is the cause of BSDS rather than a disruption in hindbrain segmentation.
A recent study in human patients has reported a reduced size of the basis pontiis and an associated abnormally long and thick medulla with rounded ventral surfaces resembling the pons more than the normal medulla2. These rare cases may represent true examples of abnormal human hindbrain segmentation, reflecting the hypothesis that they arose via posterior to anterior rhombomere transformations during early segmentation. Given the limited resolution of brain imaging studies it is likely that more subtle segmentation abnormalities exist but cannot be recognized as on MRI analysis as is the case with BSDS. It has been proposed that developmental mispatterning of neurons that regulate the specification of respiration control centers in r3 and r4 may underlie some forms of congenital respiratory failure43. To fully prove that any human hindbrain malformation is the result of abnormal hindbrain segmentation, gene identification is required together with extensive neuropathological analysis using markers specific for the affected neurons and circuits, together with comparisons with appropriate animal models.
The cerebellum is the most prominent derivative and best studied feature of the hindbrain. Thus cerebellar malformations are recognized with relative ease in both mice44 and humans45,46. However, confusion regarding the classification of human hindbrain and particularly human cerebellar malformations has been a major obstacle to progress in defining their causes. Despite this confusion, it is worth noting that many distinct malformations have been delineated based on very specific imaging criteria45,46 (Figure 2). For example, the relatively rare Joubert and related disorders (JSRD) and are defined by cerebellar vermis hypoplasia (CVH) and a complex midbrain clefting and cerebellar peduncle malformation that has a “molar tooth” appearance in axial brain images. CVH and Dandy-Walker malformation (DWM) are the most common human cerebellar phenotypes46,47. Isolated CVH consists of vermis hypoplasia affecting the posterior more than the anterior vermis with normal vermis position and posterior fossa size. Classic DMW includes CVH, upward rotation of the vermis producing cystic enlargement of the 4th ventricle, and enlarged posterior fossa. While the “molar tooth” feature distinguishes JRSD, the lack of similarly unique features and the overlap in appearance between CVH and DWM creates a significant source of diagnostic confusion.
Further complicating diagnosis is the presentation of DWM in a mild form. Mild DWM is similar to classic DWM, except that the vermis is mildly rotated so that the inferior roof of the 4th ventricle is parallel to the brainstem (rather than being rotated up), and the posterior fossa is only mildly enlarged. Mega cisterna magna (MCM) is another phenotype that resembles aspects of both CVH and DWM. MCM as a true malformation includes CVH, normal position of the vermis with no rotation, and enlarged posterior fossa1,45,46. An increased understanding of the developmental mechanisms regulating cerebellar development and the recent identification of a number of causative genes for several of these malformations is beginning to illuminate the previously confusing literature. There is also growing recognition that cerebellar development does not occur in isolation, rather it accompanies the development of the rest of the brain. Thus, many recognized cerebellar malformations do not exclusively affect the cerebellum, but also involve developmentally related structures, including the midbrain and pons.
The cerebellum arises exclusively from dorsal r1 adjacent to the 4th ventricle. The developmental boundary at the junction of the midbrain and hindbrain does not form analogously to the boundaries between the other hindbrain rhombomeres. Instead, r1 segregates from the midbrain via formation of the mid/hindbrain boundary, which becomes the isthmic organizer (IsO) (Fig. 1A). The, IsO is an important signaling center which secretes Wnt and Fgf molecules that promote proliferation and identity of the adjacent midbrain and hindbrain regions. The generation of the mid/hindbrain junction is the earliest segmentation event of the entire developing CNS, occurring at early neural plate stages. Its formation is coordinated through the activation of an extensive genetic network acting to juxtapose the expression of two transcription factors, Otx2 and Gbx2, respectively located anterior and posterior to the mid-hindbrain junction, by signals from the underlying anterior mesoderm and dorsal non-neural ectoderm. These transcription factors together establish Wnt and Fgf8 expression in the resulting IsO signaling center48,49.
Loss of either Wnt1 or Fgf8 results in complete loss of posterior midbrain and anterior hindbrain tissue causing an abnormal fusion of the tectum with the posterior cerebellum. This results in neonatal lethality in mice. An equivalent phenotype has never been reported in humans and is presumed to be incompatible with life. Alterations in the positioning of the IsO cause less severe phenotypes. For example, in mice, ectopic expression of Otx2 in the presumptive anterior hindbrain results in nearly complete loss of the anterior cerebellar vermis and enlargement of the inferior colliculus, a posterior midbrain derivative50. Potential IsO positioning phenotypes have recently been described in humans. For example, one patient with a substantially enlarged tectum accompanied by severe hypoplasia or aplasia of the cerebellar vermis suggests posterior displacement of the mid-hindbrain junction. The same authors also described a contrasting phenotype of a patient with a small tectum and an abnormally large anterior cerebellum2. Confirmation of a developmental mispatterning of the IsO awaits identification of the molecular lesions in these patients. Notably, all of these patients were originally identified as DWM patients. However, awareness of the neurodevelopmental literature for model organism and careful evaluation of the imaging studies enabled reinterpretations of these particular images to recognize the nuanced specific features in these phenotypes. These examples illustrate that a clear understanding of the principles of developmental neurobiology in model organisms provides valuable paradigms with which to assess human hindbrain malformations and generate new hypotheses about their etiology.
Cerebellar neurons arise from one of two germinal zones within the embryonic cerebellar anlage – the ventricular zone and the rhombic lip (Figure 3 top). The ventricular zone, expressing the bHLH factor Ptf1a, gives rise to the GABAergic neurons of the cerebellum51, including Purkinje cells. Post-mitotic neurons leave the VZ and migrate radially within the anlage. The rhombic lip, expressing bHLH factor Atoh1 (Math1), is induced by Bmp-signals from the adjacent dorsal midline roof plate52 and gives rise to all cerebellar glutamategic neurons in addition to several neuronal populations of the brain stem including the pontine nuclei7,53–55. Neuronal progenitors leaving the rhombic lip continue to proliferate as they migrate anteriorly over the surface of the developing cerebellar anlage to form the external granule layer (EGL). Granule neuron progenitors within the EGL proliferate extensively, driven by Shh secreted by the underlying Purkinje cells56–58. Once differentiated, inward radial migration of EGL granule neurons establishes the internal granule layer (IGL) underneath the monolayer of Purkinje cells to achieve the final laminar and folial structure of the mature cerebellum (Figure 3 bottom).
Malformations caused by abnormal cerebellar neurogenesis have been described. For example, loss of PTF1A function in humans and mouse causes a failure in the initial generation of all cerebellar GABAergic neurons. This secondarily causes loss of newly born cerebellar glutamateric cells since their trophic support and targets are now missing, resulting in complete cerebellar atrophy or agenesis at birth51,59. We have recently demonstrated that defects in rhombic lip neurogenesis likely underlie some forms of human CVH. This was based on the observation that most human cerebellar malformations preferentially affect the posterior cerebellar vermis45. Only one mouse mutant has been shown to model this specific phenotype of posterior CVH. These mice harbor mutations in the gene encoding the transcription factor Lmx1a60. Specifically, Lmx1a is required to define posteriorly fated vermis granule cell progenitors within the early rhombic lip61. Loss of Lmx1a causes these cells to adopt more anterior fates and early regression of the rhombic lip so that posterior vermis progenitors are completely lost. This is the first developmental insight into the cause of specific posterior cerebellar vermis hypoplasia. Though it is unlikely that human LMX1A mutations are compatible with life due to the other roles of this important transcription factor throughout the developing embryo, this data raise the possibility that disruption of rhombic lip neurogenesis is central to the pathology of some forms of human cerebellar malformations.
Not all cerebellar hypoplasia is caused by rhombic lip abnormalities. For example, the cerebellar vermis is profoundly hypoplastic in JSRD. Currently, nine genetic loci have been mapped for the various subtypes of Joubert syndrome and most genes have been identified. JSRD genes include: AHI1, NPHP1, CEP290, TMEM67, RPGRIP1L, ARL13B and CC2D2A1,62. Although the function of JSRD proteins remains largely unknown, several of the JSRD proteins localize to the basal body or cilium, and recent evidence suggests roles in either mediating the assembly/stability of cilia or mediating cargo transport within cilia. Analysis of cerebellar development in mice mutant for homologues of these specific genes has not been published. However, in mouse models that lack functional cilia in the developing brain, a primary defect in cerebellar granule cell progenitor proliferation is observed once granule cell progenitors leave the rhombic lip63,64. Failure to proliferate precludes the dramatic expansion of cerebellar size. Instead, the newly born progenitors differentiate early and form a small IGL. Proliferative failure is caused the inability of granule cell progenitors to respond to the Purkinje-cell secreted Shh mitogenic signal, since the Shh receptor, smoothened, must be located on cilia to transmit the signal65.
Severe CVH is also observed in an X-linked mental retardation (XLMR) syndrome that is caused by loss of function of the OPHN1 gene66. OPHN1 is a rhoGAP protein expressed in neurons and gila of the developing and adult brain67. In vitro experiments have demonstrated a role for Ophn1 in the dendritic spine morphogenesis of hippocampal neurons in mice and rats, though no obvious cerebellar morphological abnormalities were observed in Ophn1 mutant mice68,69. Thus, OPHN1 impacts cerebellar morphology through a currently unknown mechanism.
Many cerebellar malformations also involve the pons2. This is not particularly surprising given the developmental relationship of cerebellar granule cells and pontine nuclei which are both derived from the cerebellar rhombic lip. Any disruption of rhombic lip neurogenesis is likely to disrupt the neurogenesis of pontine neurons. Developmental degenerations of the cerebellum and pons also occur. Pontocerebellar hypoplasias (PCH) are rare, heterogeneous disorders characterized primarily by hypoplasia of the cerebellum and ventral pons. Presently, four genes, RARS2, TSEN2, TSEN34 and TSEN54, encoding components of tRNA charging or splicing machinery have been identified for subtypes of PCH using linkage or candidate gene approaches70,71. In the human fetus, TMEM54 is strongly expressed in the neurons of the pons, cerebellar dentate and olivary nuclei70. However, the PCH genes have not been studied in vertebrate model organisms, thus their mechanisms of action within the hindbrain are unknown.
An XLMR PCH phenotype that includes microcephaly is caused by CASK loss of function66. CASK encodes a multidomain calcium/calmodulin-dependent serine protein kinase that is targeted to neuronal synapses by anchoring to membrane-associated proteins such as neurexin72. In vitro experiments demonstrate a role for Cask in presynaptic neurotransmitter release, while hypomorphic Cask mutant mice exhibit cerebellar hypoplasia in addition to partially penetrant cleft palate73,74. Interestingly, Tarpey et al. (2009) recently identified four missense variants in CASK by sequencing X-chromosome coding exons in families with XLMR. These variants were associated with mild to moderate mental retardation and nystagmus, which is not usually associated with XLMR75 and imaging studies for these patients were not published. These recent findings serve to emphasize that the full spectrum of genotype-phenotype associations are not known. Rarely have developmental outcomes been rigorously correlated with specific structural malformations and in most instances, extensive phenotype-genotype correlations have not been conducted since the numbers of patients with identified molecular lesions remains low.
Once born in the rhombic lip, pontine nuclei neurons must undergo extensive migration around and through the tegmentum of the brain stem to arrive at their final ventral position. In mice, deciphering the developmental cues that specifically regulate this precise migration is in its infancy76,77, but it is likely that many mechanisms controlling these migrations are similar to mechanisms that control neuronal migration throughout the brain. It is also very likely that these mechanisms are conserved in humans. For example, in mice, loss of the Large glycosyltransferase gene causes absence of the pons due to abnormal migration of pontine progenitors78. Pontine hypoplasia and CVH are also features of congenital disorders of glycoslyation in humans79.
Pontine deficiency may not only be caused by cell migration abnormalities. It is important to note that model organism studies have demonstrated that many of the same molecules that regulate neuronal migration also regulate axon guidance. These molecules include ephrins, slits, netrins, semaphorins and Wnts80. Axonal tract malformations of both the pons and medulla have been described both with cerebellar malformations or in isolation2,81, though few causative genes have been identified. Model organisms provide interesting candidate genes and also can be used to test specific disease-relevant hypotheses. For example, in JSRD, a variety of major tract abnormalities are evident by MRI. Since JSRD are now identified as ciliopathies65, it will be interesting learn whether cilia are required for long range cell migration and axon guidance in the developing brain stem in cilia deficient mouse models.
In the last few years, identification of genes for JSRD and PCH has significantly clarified the developmental basis of these important, yet rare hindbrain malformations. There has also been major progress towards gene identification for DWM, the most common human cerebellar malformation. The first DWM causative genes, ZIC1 and ZIC4, were chosen as strong candidate genes within the first DWM locus on chromosome 3q24 due to their clear expression within the mouse cerebellum during development. Mice with heterozygous deletion of the homologues of these two linked transcription factors further display a phenotype that recapitulates human DWM82. Thus, the first paradigm for identifying DWM-causative genes held that the aberrant genes must normally be expressed in the cerebellum in order for their alteration to impact cerebellar development.
We recently characterized a second DWM locus on chromosome 6p25.383. Within this locus no clear DWM-candidate genes emerged based on strong cerebellar expression. However, mice with null mutations in Foxc1 displayed a striking, early embryonic cerebellar defect. Foxc1 is a forkhead box transcription factor that is never expressed with the developing brain. Instead, Foxc1 is expressed in the adjacent head mesenchyme and is required for normal skull development84. We demonstrated that Foxc1 in the early posterior fossa mesenchyme is also required for the expression of growth factors, including Tfgb1 and several Bmps. Without these growth factors, the adjacent cerebellum does not maintain cerebellar Atoh1 expression which is required for normal EGL development83. Thus Foxc1 regulates both posterior fossa development and cerebellar development, two defining features of abnormal DWM pathology. Based on these studies, we are now reexamining our Zic1/4 mutant mouse models to determine whether the Zic genes have additional roles in posterior fossa mesenchyme development.
The complementary analyses between human and model organisms not only recognized a new role for FOXC1 in cerebellar development, but also highlighted a novel mechanism for early cerebellum development through neuroepithelium-mesenchyme interactions. These reciprocal analyses also informed our understanding of human cerebellar phenotypes and led us to revise our paradigm for selecting future candidate genes. Interestingly, patients with deletions or duplications encompassing FOXC1 exhibit a wide range of cerebellar phenotypes, including classic DWM, mild DWM, MCM and isolated CVH (listed in order of severity), suggesting a single spectrum with shared etiology in these patients83. In contrast, mutations in genes such as OPHN1 and CASK are associated with CVH or diffuse cerebellar hypoplasia, but not MCM or DWM66,74. Together these observations lead us now to propose that genes with expression in the developing cerebellum will be associated with CVH or diffuse cerebellar hypoplasia, while genes with posterior fossa mesenchymal expression will be associated with the complete CVH-MCM-DWM spectrum of malformations.
Hindbrain developmental neurobiology has been well described in vertebrate model organisms, though it is not completely understood. Easy manipulation of model organisms further provides a conduit for unraveling the specific genes and mechanisms underlying hindbrain development. In turn, these hindbrain developmental programs provide valuable insights for interpreting the origin of human hindbrain phenotypes and suggesting functionally relevant candidate genes. Future synergistic analyses of human and model organism hindbrain phenotypes are the keys to improvements in diagnoses and potential treatments for these disorders.
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