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MRX87 family with Aristaless Xdup24bp mutation and implication for polyAlanine expansions
© Laperuta et al; licensee BioMed Central Ltd. 2007
Received: 22 November 2006
Accepted: 04 May 2007
Published: 04 May 2007
Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect.
We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing.
MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified.
Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.
X-linked mental retardation (XLMR) is a heterogeneous genetic condition characterized by variable cognitive handicap with IQ below 70. To date more than 50 XLMR genes have been recognized [1–3]. Each of them accounts for a very small proportion of the affected families with the exception of FMR1, whose loss of function mutation causes the Fragile X syndrome, and the Aristaless X (ARX) gene mutated in several syndromic and non syndromic mentally retarded patients [4–9].
The ARX gene (OMIM #300382) was identified as the causative gene in several allelic brain diseases with MR such as i) XLAG or X-linked lissencephaly with abnormal genitalia (OMIM #300215) ; ii) Proud syndrome or mental retardation with agenesis of the corpus callosum, microcephaly, limb contractures, scoliosis, coarse faces, tapered digits and urogenital abnormalities (OMIM #30004) ; iii) myoclonic epilepsy syndrome (OMIM #300432) ; iv) West syndrome or X-linked infantile spasm syndrome with hypsarrhythmia and mental retardation (OMIM #308350) ; v) Partington dystonic syndrome (OMIM #309510) ; vi) non syndromic X-linked mental retardation (OMIM #300382) .
ARX encodes the Aristaless-related protein, a bi-functional homeobox transcription factor essential for cerebral patterning and for the maintenance of specific neuronal subtypes in the cerebral cortex . It belongs to the Q 50 Paired-like (Prd-like) class genes, an ancient family of transcription factors with a key role in the early evolution of the animal head and development of the central nervous system . The ARX protein contains a number of conserved domains, including the two DNA binding domains (Homeobox and Aristaless), and four distinct hydrophobic polyalanine tracts (polyA_I, II, III and IV) with a hypothetical role as transcriptional suppressor [17, 18].
The Arx knockout mouse is characterized by a small brain with aberrant migration and differentiation of GABAergic interneuron progenitors and altered testes, a complex phenotype similar to the human XLAG syndrome [19, 20]. Murine expression studies showed that Arx is widespread throughout telencephalic structures implicated in the pathophysiology of learning formation [13, 14, 20].
ARX gene represents a hot spot for mutations in families with cognition disorders because its mutations account for 9.5% of X-linked MR families . The most frequent mutation is c.428_451dup24, also known as ARXdup24, a 24 bp duplication in exon 2 resulting in elongation of the second polyalanine tract (polyA12_II), that alone might account for 6.6% of all XLMR and 41% of families with mutations in ARX gene [4–9]. The c.428_451dup24 mutation has never been found in association with severe brain malformations (i.e. XLAG or Proud syndromes). However, variable phenotypic expression is often observed within the same family with c.428_451dup24 [21, 22] reinforcing the notion that ARX is a pleiotropic gene that, in a diverse genetic context and/or under the influence of modifier genes, controls different aspects of human brain morphogenesis and function.
Here we present the molecular and clinical characterization of a new XLMR family (MRX87) linked to the Xp21 region in which we found the segregation of the c.428_451dup24 associated to intra-familial clinical variability. Our study aims to enrich the clinical and genetic description of mental defects due to polyalanine expansions in Aristaless protein.
Ascertainment of family members
Genomic DNA was isolated from the nucleated peripheral blood leukocytes using the Salting out procedure. A standard set of microsatellite markers on the X chromosome, evenly spaced every 10 cM (ABI PRISM Linkage Mapping Sets vs2, Applied Biosystems) was PCR amplified using conditions already described . Thirteen individuals of the family were genotyped (Figure 1) and PCR products were analysed on automatic sequencer (ABI PRISM 3100, Applied Biosystem). Extra fluorescently labelled primers were synthesized for seven additional polymorphic markers chosen in public databases. Two-point linkage analysis was performed by the MLINK program version 5.1, from the LINKAGE software package . The approved gene symbol MRX87 (Mental Retardation X-Linked 87) was assigned according to the HUGO (Human Genome Organization) nomenclature. X-inactivation status was tested by HUMARA (Human Androgen Receptor Gene) fluorescent assay according to Fimiani et al . XCI patterns were classified as random (XCI ≤ 70%), non random (70% ≤ XCI ≤ 80%) or skewed (XCI ≥ 90%).
All coding exons and the flanking intronic sequences of the ARX gene were amplified using DNA from affected and non-affected members of the MRX87 family. Eight primer pairs were used, namely: 1F 5'-CCA ACA CAC ACC CAT CCA T-3' and 1R 5'-CCG AAC ACC AAA CAT CCA A-3' for exon 1; 2aF 5'-CAA GGC GTC GAA GTC TGG TG-3' and 2aR 5'-GTA CGA CTT GCT GCG GCT GA-3', 2bF 5'-CTC CTT CAG GGT GCG GCA GC-3' and 2bR 5'-CCA GCA GCT CCT CCT CGT CG-3', 2cF 5'-CGT CAC GCA CCC GGA GGA GC-3' and 2cR 5'-AGC CCG CTG TCC CTC CCT GG-3' for exon 2; 3F 5'-TGG AGT AGG CCT GCC ATA GA-3' and 3R 5'-CCA ACC CAT CTC TCT CTC TCC-3' for exon 3; 4aF 5'-GCC AAG GGA AGG GAC GGG TA-3' and 4aR 5'-GGT AGG GGC TGA GCG GGT GG-3', 4bF 5'-GAG AAG GCA GGC GCG CAG AC-3' and 4bR 5'-ACT CCT GCC TCC TCC CTG CC-3' for exon 4; 5F 5'-CCT CGG GGA ATA TCT GGA CT-3' and 5R 5'-TTG AGT GGT GCT GAG TGA GG-3' for exon 5. The PCR fragments were sequenced in both directions with the ABI PRISM 3100 DNA sequencer (Applied Biosystem). The 24 base pair duplication in exon 2 was also visualized in patients and carrier women by separating the PCR fragments of amplicon 2a (472 bp) and 2b (409 bp) on a 3% agarose gel.
Neurological and physical examination
A diagnosis of MR associated with minor anomalies was made after examination of the patients (II:5, III:5, III:10, IV:13 and IV:14; Figure 1) at the Neurological Center (CARSIC, Venafro, Italy). Careful examination of the patients' phenotypes was performed and the intelligence quotient was assessed by Wechsler Adult intelligence Scale (WAIS). All affected men were born at term after normal pregnancy; no statural growth deficiency was observed. No adverse prenatal events of interest were reported and an extensive metabolic work-up yielded negative results. None of them had convulsion or hand dystonia. All had normal vision and no gonad malformations were recorded.
Synopsis of MRX87 male patients
Bilateral Babinski sign;
Deficit of the VII cranial nerve;
Cerebellar tonsils below the level of the foramen magnum;
Wide subarachnoid spaces
Patient II:5 and patient IV:13 had severe urinary incontinence, a clinical sign often observed in association with MR . Only in patient IV:14, we diagnosed a moderate intellectual handicap associated with a language deficit. Three out of four probands showed a flatfoot deformity (III:10, IV:13 and IV:14), a defect that was not evident in the unaffected men of the family. The obligate carrier women are of normal intelligence and clinically indistinguishable from their non carrier sisters. No carrier mothers recalled serious abnormalities in pregnancy.
MRX87 was linked to Xp22-Xp21 and is due to a dup24 mutation in the Aristaless related homeobox X-chromosome linked gene
Two-point LOD scores analysis across the markers DXS7140 and DXS1214 linking MRX87 family to Xp22-p21 interval
X inactivation analysis (XCI) was performed in the leukocytes of four MRX87 carrier women (II:3, II:4, II:6 and III:7). We analysed the methylation status of the CpG islands of the AR gene, using the human Androgen Receptor gene fluorescent assay (HUMARA) and excluded the presence of skewed XCI in carrier women (Figure 1). This finding is not completely unexpected because most of the mutations that impair neurocognitive functioning do not confer a selective advantage in leukocytes, as in the case of individuals with Rett syndrome , or with Incontinentia Pigmenti . Moreover, X-inactivation was measured in cells of an unaffected tissue (blood) but XCI may be different in the brain or at a critical time during brain development.
Summary of clinical data observed in other ARXdup24 families
Moderate to profound
Long face, thin lips, large ears, epilepsy
Language deficit, hyperkinesia
Moderate to severe
Moderate to severe
Obesity, large head, epilepsy
Depressive and psychotic features
General developmental delays, dystonic hand movements
Severe language development delay
Long chin and deep-set eyes, strabismus, neonatal hypotonia
Learning and walking difficulties
Hypertelorism, broad nasal root, cleft upper lip, growth hormone deficiency
Transsphenoidal encephalocele and agenesis of corpus callosum (ACC) and hypopituitarism
The ARXdup24 underlies only a part of the complex phenotypic spectrum of ARX mutations. We can distinguish three groups of ARX mutations with different outcomes (Figure 3) [10–14, 19, 27, 30–33]: 1. severe mutations causing severe brain patterning malformations due to alterations of the DNA binding domains (HD and Aristaless); 2. expansion in the polyA_I motif causing familial ISSX phenotypes; 3. expansion in the polyA_II motif (c.428_451dup24) causing a spectrum of XLMR conditions with huge inter- and intra-familial heterogeneity. With the exception of the severe ARX alterations classified as "loss of function", we cannot establish the functional effect of the polyA expansion mutations. Indeed, in vitro data, obtained for only the polyA_I motif, are still controversial [34, 35] and no transgenic mice has been produced for each polyA mutation. On the other hand because ARX expansions in both motifs cause varying degree of MR in humans, the functions of the polyA tracts in the ARX protein could be related to the complexity of brain functions such as those controlling memory and learning.
In conclusion, the identification of a new MRX family linked to Xp22 and carrying the c.428_451dup24 (ARXdup24) underlines the high contribution of ARX to X linked mental retardation. Furthermore, the clinical findings of the affected members of the MRX87 family enhance the striking phenotypic variability associated with polyA_II expansion.
The authors are greatly indebted to the patients and their parents for contributing blood samples and medical records. They would like to thank C. Carbone for technical assistance. Work on XLMR in our laboratories was supported by FIRB grant from MIUR-Italy. The research conducted in this study complies with current Italian laws. Written consent was obtained from their relative for publication of the patient's details and their photographs.
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