X-linked Lissencephaly With Abnormal Genitalia Research Articles

Analysis of ARX gene variant in a child with X-linked lissencephaly with abnormal genitalia

To explore the clinical characteristics and genetic basis for a child with X-linked lissencephaly with abnormal genitalia (XLAG). A child with XLAG who had presented at the Third Affiliated Hospital of Zhengzhou University in May 2021 was selected as the study subject. Peripheral blood samples of the child and his parents were collected and subjected to high-throughput sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the result was analyzed by using bioinformatic software. The child was found to have harbored a hemizygous c.945_948del variant in exon 2 of the ARX gene, which as a frameshifting variant has resulted in a truncated protein. His mother was found to be heterozygous for the variant, whilst his father was of wild type. The variant was unreported previously. The hemizygous c.945_948del variant of the ARX gene probably underlay the XLAG in this patient. Above finding has provided a basis for the diagnosis and genetic counseling for this family.

Deregulation of microtubule organization and RNA metabolism in Arx models for lissencephaly and developmental epileptic encephalopathy.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein–protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

INTERNEURONOPATHIES AND THEIR ROLE IN EARLY LIFE EPILEPSIES AND NEURODEVELOPMENTAL DISORDERS.

Summary GABAergic interneurons control the neural circuitry and network activity in the brain. The advances in genetics have identified genes that control the development, maturation, and integration of GABAergic interneurons and implicate them in the pathogenesis of epileptic encephalopathies and neurodevelopmental disorders. For example, mutations of the aristaless‐related homeobox X‐linked gene (ARX) may result in defective GABAergic interneuronal migration in infants with epileptic encephalopathies like West syndrome (WS), Ohtahara syndrome, or X‐linked lissencephaly with abnormal genitalia (XLAG). The concept of “interneuronopathy,” that is, impaired development, migration, or function of interneurons, has emerged as a possible etiopathogenic mechanism for epileptic encephalopathies. Treatments that enhance γ‐aminobutyric acid (GABA) levels may help seizure control but do not necessarily show disease modifying effect. On the other hand, interneuronopathies can be seen in other conditions in which epilepsy may not be the primary manifestation, such as autism. In this review, we plan to outline briefly the current state of knowledge on the origin, development, and migration and integration of GABAergic interneurons, present neurodevelopmental conditions, with or without epilepsy, that have been associated with interneuronopathies, and discuss the evidence linking certain types of interneuronal dysfunction with epilepsy and/or cognitive or behavioral deficits.

An Emerging Female Phenotype with Loss-of-Function Mutations in the Aristaless-Related Homeodomain Transcription Factor ARX.

The devastating clinical presentation of X-linked lissencephalywith abnormal genitalia (XLAG) is invariably caused by loss-of-function mutations in the Aristaless-related homeobox (ARX) gene. Mutations in this X-chromosome gene contribute to intellectual disability (ID) with co-morbidities including seizures and movement disorders such as dystonia in affected males. The detection of affected females with mutations in ARX is increasing. We present a family with multiple affected individuals, including two females. Two male siblings presenting with XLAG were deceased prior to full-term gestation or within the first few weeks of life. Of the two female siblings, one presented with behavioral disturbances, mild ID, a seizure disorder, and complete agenesis of the corpus callosum (ACC), similar to the mother's phenotype. A novel insertion mutation in Exon 2 of ARX was identified, c.982delCinsTTT predicted to cause a frameshift at p.(Q328Ffs* 37). Our finding is consistent with loss-of-function mutations in ARX causing XLAG in hemizygous males and extends the findings of ID and seizures in heterozygous females. We review the reported phenotypes of females with mutations in ARX and highlight the importance of screening ARX in male and female patients with ID, seizures, and in particular with complete ACC.

O-010 Comparative Neuropathology Of Lissencephaly With Arx Mutation: Consideration Of Neocortical Interneuron Distribution

Background X-linked lissencephaly with abnormal genitalia (XLAG) is established as one disease entity. XLAG, showing severe neonatal seizure and developmental delay, is a rare disorder caused by mutations in the aristaless-related homeobox ( ARX ) gene, located in Xp22.13. Arx -null mice for human XLAG model showed loss of tangential migration of GABAergic interneurons. Objectives We investigated subpopulation of GABAergic interneurons in the brain of an infant with XLAG, who had a nonsense mutation of the ARX gene, compared with those of age-matched normal control, Miller-Dieker syndrome (MDS) as a type I lissencephaly, and polymicrogyria of Fukuyama type congenital muscular dystrophy (FCMD) as a type II lissencephaly. Methods We used paraffin-embeded brain tissues of two XLAG, three MDS and four FCMD, with an informed consent of their parents. We performed immunocytochemistry for interneuron and migration markers. Results Glutamic acid decarboxylase (GAD) and calretinin (CR) containing (+) cells were significantly very few in the neocortex and located in the white matter and neocortical subventricular zone. In the neocortical subventricular region, the GAD+ and CR+ cells had Mash -1 protein, like a radial migration marker, and nestin protein. On the contrary, MDS showed relative low concentration of GAD+ cells. FCMD revealed random distribution of these marked cells. Conclusions ARX controls not only tangential migration of GABAergic interneurons from the ganglionic eminence, but also may serve to induce radial migration from the neocortical subventricular zone. MDS and FCMD also demonstrated abnormal distribution of neocortical interneurons, but those severities are different in each type of lissencephaly.

Mutational screening of ARX gene in Iranian families with X-linked intellectual disability.

Mutations in the human aristaless-related homeobox (ARX) gene are amongst the major causes of developmental and neurological disorders. They are responsible for a wide spectrum of phenotypes, including nonsyndromic X-linked intellectual disability (NS-XLID), and syndromic (XLIDS) forms such as X-linked lissencephaly with abnormal genitalia (XLAG), Partington syndrome (PRTS), and X-linked infantile spasm syndrome (ISSX). The recurrent 24 bp duplication mutation, c.428_451dup(24 bp), is the most frequent ARX mutation, which accounts for ~40% of all cases reported to date. We have screened the entire coding sequences of the ARX gene in 65 Iranian families with intellectual disabilities in order to obtain the relative prevalence of ARX mutations. At first these families were screened for the most recurrent mutation, the c.428_451dup(24 bp). For samples with negative results, single strand conformation polymorphism (SSCP) analysis was performed. We identified one family with the c.428_451dup(24 bp) duplication. Three shifts (one shift in exon 5 and two shifts in exon 4) were also identified among the total families. According to the results of the sequencing analysis, two shifts were not associated with any mutation and the other one was a c.1347C>T (p.G449G) substitution in exon 4. Hence, we suggest that molecular analysis of ARX mutations as a second cause of XLID should be considered as routine diagnostic procedure in any male who presents with either NS-XLID or XLIDS.

Asymmetric polymicrogyria and periventricular nodular heterotopia due to mutation in ARX

Mutations in the ARX gene, at Xp22.3, cause several disorders, including infantile spasms, X-linked lissencephaly with abnormal genitalia (XLAG), callosal agenesis and isolated intellectual disability. Genotype/phenotype studies suggested that polyalanine tract expansion is associated with non-malformative phenotypes, while missense and nonsense mutations cause cerebral malformations, however, patients with structural normal brain and missense mutations have been reported. We report on a male patient born with cleft lip and palate who presented with infantile spasms and hemiplegia. MRI showed agenesis of corpus callosum (ACC), an interhemispheric cyst, periventricular nodular heterotopia (PVNH), and extensive left frontal polymicrogyria (PMG). Sequencing of the ARX gene in the patient identified a six basepair insertion (c.335ins6, exon 2). The insertion leads to a two-residue expansion of the first polyalanine tract and was described previously in a family with non-syndromic X-linked mental retardation. To our knowledge, ARX mutation causing PMG and PVNH is unique, but the spasms and ACC are common in ARX mutations. Clinicians should be aware of the broad clinical range of ARX mutations, and further studies are necessary to investigate the association with PMG and PVNH and to identify possible modifying factors.

Neocortical Layer Formation of Human Developing Brains and Lissencephalies: Consideration of Layer-Specific Marker Expression

To investigate layer-specific molecule expression in human developing neocortices, we performed immunohistochemistry of the layer-specific markers (TBR1, FOXP1, SATB2, OTX1, CUTL1, and CTIP2), using frontal neocortices of the dorsolateral precentral gyri of 16 normal controls, aged 19 gestational weeks to 1 year old, lissencephalies of 3 Miller-Dieker syndrome (MDS) cases, 2 X-linked lissencephaly with abnormal genitalia (XLAG) cases, and 4 Fukuyama-type congenital muscular dystrophy (FCMD) cases. In the fetal period, we observed SATB2+ cells in layers II-IV, CUTL1+ cells in layers II-V, FOXP1+ cells in layer V, OTX1+ cells in layers II or V, and CTIP2+ and TBR1+ cells in layers V and VI. SATB2+ and CUTL1+ cells appeared until 3 months of age, but the other markers disappeared after birth. Neocortices of MDS and XLAG infants revealed SATB2+, CUTL1+, FOXP1+, and TBR1+ cells diffusely located in the upper layers. In fetal FCMD neocortex, neurons labeled with the layer-specific markers located over the glia limitans. The present study provided new knowledge indicating that the expression pattern of these markers in the developing human neocortex was similar to those in mice. Various lissencephalies revealed abnormal layer formation by random migration.

Partial loss of pancreas endocrine and exocrine cells of human ARX-null mutation: Consideration of pancreas differentiation

Aristaless-related homeobox gene ( ARX) mutation leads to several neurological disorders including X-linked lissencephaly with abnormal genitalia (XLAG), West syndrome and Partington syndrome, with XLAG being the most severe form. Although some of the brain pathologies of XLAG have already been described, the crucial extra-brain symptoms are severe growth retardation, transient hyperglycemia and intractable diarrhea. Since ARX expresses in the islets of Langerhans during the embryonic stage, these visceral phenotypes may be related to a loss of ARX function, which develops endocrine cells in the pancreas. We investigated the abnormal pancreatic development of XLAG patients with ARX-null mutation. We performed immunohistochemistry of XLAG pancreases, using the antibodies against glucagon, insulin, somatostatin, pancreatic polypeptide, ghrelin, Brn4, Nkx2.2, Mash1, amylase and pancreatic lipase. As the results, the glucagon- and pancreatic polypeptide-producing cells were found to be completely deficient in the islets of Langerhans. We also discovered marked interstitial fibrosis, small exocrine cells with loss of amylase-producing cells and an enlargement of the central lumen of the glandular acini. These pathological findings indicate that ARX contributes not only to endocrine development, but also to exocrine development of the human pancreas, and its deficiency may lead to the severe phenotypes of XLAG patients.

Genetic malformations of the human frontal lobe.

Interest in genetic malformations of the frontal lobe has grown from the recognition that certain brain malformations have a predilection for the frontal lobes or are more severe in the anterior brain. These malformations can be deleterious, as the frontal lobes in humans are particularly large in comparison with those of other species and play an important role in cognitive developmental functions.

Analysis of the hypothalamus in a case of X-linked lissencephaly with abnormal genitalia (XLAG)

X-linked lissencephaly with abnormal genitalia (XLAG) is characterized by lissencephaly, absent corpus callosum and ambiguous genitalia. We examined hypothalamic dysfunctions in a XLAG case with a novel mutation of the ARX gene, and performed immunohistochemical evaluation of the diencephalons in autopsy brain. A 1-year-old boy showed intractable epilepsy, persistent diarrhea and disturbed temperature regulation. This case had abnormalities in circadian rhythms and pituitary hormone reserve test. He died of pneumonia. The globus pallidus and subthalamic nucleus was not identified, and the putamen and thalamus were dysplasic. The suprachiasmatic nucleus was absent. A few neurons immunoreactive for vasopressin seemed to form the ectopic supraoptic-like nucleus. The diencephalons were disturbed differently in each sub-region, and the changes may be related to various hypothalamic dysfunctions.

Aristaless-related homeobox gene disruption leads to abnormal distribution of GABAergic interneurons in human neocortex: evidence based on a case of X-linked lissencephaly with abnormal genitalia (XLAG)

X-linked lissencephaly with abnormal genitalia (XLAG) is a rare disorder caused by mutations in the aristaless-related homeobox (ARX) gene, located on Xp22.13. Arx-null mice show loss of tangential migration of GABAergic interneurons, presumably being related to caudal ganglionic eminence tangential migration. In the present study, we investigated a subpopulation of GABAergic interneurons in the brain of an infant with XLAG, who had a novel nonsense mutation of the ARX gene, compared with those of age-matched normal controls and Miller-Dieker syndrome. We performed immunocytochemistry for interneuron and migration markers. We found that glutamic acid decarboxylase (GAD)- and calretinin (CR)-containing cells were significantly reduced in the neocortex and located in the white matter and neocortical subventricular zone, while neuropeptide Y- or cholecystokinin-containing cells were normally distributed. Moreover, in the neocortical subventricular region, the GAD- and CR-containing cells expressed the radial migration marker Mash-1 as well as nestin. Our findings suggest that ARX protein controls not only the tangential migration of GABAergic interneurons from the ganglionic eminence, but also may serve to induce radial migration from the neocortical subventricular zone.

Screening of ARX in mental retardation families: consequences for the strategy of molecular diagnosis

Mutations in the human ARX gene have been shown to cause nonsyndromic X-linked mental retardation (MRX) as well as syndromic forms such as X-linked lissencephaly with abnormal genitalia (XLAG), Partington syndrome and X-linked infantile spasm. The most common causative mutation, a duplication of 24 bp, was found in families with a variety of phenotypes, but not in the more severe XLAG phenotypes. The aim of the study was to access the frequency of ARX mutations in families with established or putative X-linked mental retardation (XLMR) collected by the European XLMR Consortium. We screened the entire coding region of ARX for mutations in 197 novel XLMR families by denaturing high-performance liquid chromatography, and we identified eight mutations (six c.428_451dup24, one insertion and one novel missense mutation p.P38S). To better define the prevalence of ARX mutations, we included previously reported results of 157 XLMR families. Together, these data showed the relatively high rate (9.5%) of ARX mutations in X-linked MR families and an expectedly low rate in families with affected brother pairs (2.2%). This study confirms that the frequency of ARX mutations is high in XLMR, and the analysis of ARX in MRX should not be limited to duplication.

Familial West syndrome and dystonia caused by an Aristaless related homeobox gene mutation

Boys with unexplained West syndrome should be examined for a mutation in the Aristaless related homeobox gene, especially, when the family history is positive for mental retardation and epilepsy. X-linked West syndrome is very rare. We report on two brothers with West syndrome and dystonia with polyalanine expansion of the Aristaless related homeobox gene (ARX). The index patient (Fig. 1a; III-2) was the second child of non-consanguineous parents, born in 2001 at term, after an uneventful pregnancy and delivery (birth weight 3659 g, 75th percentile; length 51 cm, 75th percentile; head circumference 35 cm, 50th percentile). At the age of 3 months he developed infantile spasms and a hypsarrhythmic EEG pattern. He promptly responded to vigabatrin therapy. At 5 months a generalised dystonia, i.e. increased muscle tone with dystonic posturing of limbs, was evident. At the age of 3 years, he is able to walk a few steps with help and grasping objects is very difficult. He has no expressive speech. The elder brother of the index patient (Fig. 1a; III-1) was born in 1996 by caesarean section because of neonatal macrosomia (birth weight 4200 g, 90th percentile; length 53 cm, 90th percentile; head circumference 38 cm, >90th percentile). At the age of 3 months he showed dystonic movements and marked truncal hypotonia. One month later he developed infantile spasms and a hypsarrhythmic EEG pattern. He did not respond to vigabatrin, but to ACTH. At the age of 8 years he is wheel-chair bound, not talking, grasping objects is not possible. In both children metabolic tests and neuroimaging (MRI) were normal. Actually, both brothers suffer from a severe dystonia, mental impairment and rare generalised tonic-clonic seizures (the older brother), treated with valproic acid. The family history was remarkable. In the maternal uncle (Fig. 1a; II-3, now 37 years old), spastic tetraplegia, mental retardation and epilepsy have been present since early infancy. In retrospect, the epilepsy syndrome could not be classified. MRI was not performed in this uncle, the mother or the grandmother. From the pedigree and the clinical findings we suspected a mutation in the ARX gene. Following informed consent, a sequence analysis of the coding region and flanking intronic sequences of the ARX gene was performed. The male proband (index patient; Fig. 1a; III-2) as well as his brother and uncle were found to be hemizygous for a 21 bp GCG repeat expansion in exon 2 of the ARX gene c.333_334ins(GCG)7, which expands the first of four alanine tracts from normally 16 to 23 alanine residues (Fig. 1b,c). Both the mother (Fig. 11; II-2) and the maternal grandmother (Fig. 1a; I-2) were identified as heterozygous mutation carriers using an optimised fluorescence-based PCR assay. Mutations in the ARX gene have been found in a broad spectrum of disorders including X-linked infantile spasms (ISSX)/West syndrome, mental retardation [2], ataxia and dystonia (Partington syndrome), syndromic and non-syndromic forms of mental retardation, myoclonic epilepsy and X-linked lissencephaly with abnormal genitalia (XLAG) [5, 6,8]. The mutation found in our Swiss family, which is not related to previously reported families, has been described before in boys with infantile spasms and normal MRI, severe mental and motor retardation [7]. In addition to Partington syndrome, dystonia was described in a few unpublished Australian and Norwegian cases [6], but G. Wohlrab (&) AE B. Schmitt AE E. Boltshauser Department of Neuropaediatrics and Neurophysiology, University Children’s Hospital, Steinwiesstrasse 75, 8032 Zurich, Switzerland E-mail: gabriele.wohlrab@kispi.unizh.ch Tel.: +41-1-2667701 Fax: +41-1-2667165

X-linked lissencephaly with abnormal genitalia (XLAG) associated with renal phosphate wasting and exocrine pancreatic insufficiency

X-linked lissencephaly with abnormal genitalia (XLAG) is caused by loss-of-function mutations of the aristaless-related homeo-box (ARX) gene. The clinical picture is imperfectly delineated due to the small number of reported patients and death in 7 of 10 cases during the first 3 months. We report on a XLAG-patient with a 1bp-deletion (790delC) of the ARX-gene who also suffered from renal phosphate wasting and exocrine pancreatic insufficiency. This has not been described previously. An MRI of his clinically healthy mother being heterozygous for the mutation revealed agenesis of the corpus callosum. The boy displayed the characteristic clinical symptoms (micropenis and cryptorchism, secondary microcephaly, intractable epilepsy, chronic diarrhoea, temperature instability) and typical neuroradiological findings previously described in XLAG. A low serum phosphate was noticed since age 6 months and tubular phosphate reabsorption was found to be markedly reduced subsequently (48–55%). An adequate therapy was impossible due to episodes of chronic diarrhoea associated with exocrine pancreatic insufficieny as expressed by markedly reduced pancreatic elastase in stool. Demineralisation of bones progressed and rib fractures occurred. The boy died at age 18 months. We conclude that the clinical picture of XLAG may be broader than hitherto known and that it may include renal phosphate wasting and exocrine pancreatic insufficiency.

Mutations of ARX are associated with striking pleiotropy and consistent genotype-phenotype correlation.

We recently identified mutations of ARX in nine genotypic males with X-linked lissencephaly with abnormal genitalia (XLAG), and in several female relatives with isolated agenesis of the corpus callosum (ACC). We now report 13 novel and two recurrent mutations of ARX, and one nucleotide change of uncertain significance in 20 genotypic males from 16 families. Most had XLAG, but two had hydranencephaly and abnormal genitalia, and three males from one family had Proud syndrome or ACC with abnormal genitalia. We obtained detailed clinical information on all 29 affected males, including the nine previously reported subjects. Premature termination mutations consisting of large deletions, frameshifts, nonsense mutations, and splice site mutations in exons 1 to 4 caused XLAG or hydranencephaly with abnormal genitalia. Nonconservative missense mutations within the homeobox caused less severe XLAG, while conservative substitution in the homeodomain caused Proud syndrome. A nonconservative missense mutation near the C-terminal aristaless domain caused unusually severe XLAG with microcephaly and mild cerebellar hypoplasia. In addition, several less severe phenotypes without malformations have been reported, including mental retardation with cryptogenic infantile spasms (West syndrome), other seizure types, dystonia or autism, and nonsyndromic mental retardation. The ARX mutations associated with these phenotypes have included polyalanine expansions or duplications, missense mutations, and one deletion of exon 5. Together, the group of phenotypes associated with ARX mutations demonstrates remarkable pleiotropy, but also comprises a nearly continuous series of developmental disorders that begins with hydranencephaly, lissencephaly, and agenesis of the corpus callosum, and ends with a series of overlapping syndromes with apparently normal brain structure.

X-Linked Lissencephaly with ARX Mutations, Abnormal Genitalia, and Corpus Callosum Agenesis

The clinical and genetic findings of two X-linked lissencephaly with abnormal genitalia (XLAG) pedigrees with ARX mutations are reported from the University of Regensburg, Germany.