A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation.

We demonstrate here the importance of interleukin signalling pathways in cognitive function and the normal physiology of the CNS. Thorough investigation of an MRX critical region in Xp22.1-21.3 enabled us to identify a new gene expressed in brain that is responsible for a non-specific form of X-linked mental retardation. This gene encodes a 696 amino acid protein that has homology to IL-1 receptor accessory proteins. Non-overlapping deletions and a nonsense mutation in this gene were identified in patients with cognitive impairment only. Its high level of expression in post-natal brain structures involved in the hippocampal memory system suggests a specialized role for this new gene in the physiological processes underlying memory and learning abilities.

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Gene for nonspecific X-linked mental retardation (MRX 47) is located in Xq22.3-q24.

We describe a large family with nonspecific X-linked mental retardation (MRX 47). An X-linked recessive transmission is suggested by the inheritance from the mothers in two generations of a moderate to severe form of mental retardation in six males, without any specific clinical findings. Two point linkage analysis demonstrated significant linkage between the disorder and two markers in Xq23 (Zmax = 3.75, theta = 0). Multipoint linkage analyses confirmed the significant linkage with a maximum lod score (Z = 3.96, theta = 0) at DXS1059. Recombination events observed with the flanking markers DXS1105 and DXS8067 delineate a 17 cM interval. This interval overlaps with several loci of XLMR disorders previously localized in Xq23-q24, which are reviewed herein.

X-linked neurodegenerative syndrome with congenital ataxia, late-onset progressive myoclonic encephalopathy and selective macular degeneration, linked to Xp22.33-pter.

Linkage analysis was performed in a previously described family segregating for an X-linked progressive neurological disorder [Bertini et al., 1992]. In three generations, the disease was inherited from the mothers in seven affected males (Fig. 1). Five had severe congenital hypotonia and died during the first year of life. Two other boys (maternal cousins) were found to have severe congenital ataxia, late-onset progressive myoclonic encephalopathy, and selective macular degeneration; brain CT-scan showed moderate cerebellar vermis hypoplasia. Linkage analysis was carried out in 12 informative relatives using 35 microsatellite markers (Généthon) evenly distributed on the X chromosome. A multipoint analysis showed a significant linkage (Z > 2) between the disease and three markers in the Xp22.33 region: DYS403 (Z = 2.37, theta = 0) which maps in the pseudoautosomal region, DXS7099 (Z = 2.45, theta = 0), and DXS7100 (Z = 2.48, theta = 0). Further linkage analysis with more telomeric markers will refine the location of this severe X-linked encephalopathy.

Human amniotic fluid alpha-glucosidase.

Amniotic fluid in midpregnancy contains significant alpha-glucosidase activity. This enzyme is distinguishable from the lysosomal acid alpha-glucosidase, deficiency of which is associated with Pompe's disease. The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic alpha-glucosidase is also different from the classical neutral form. Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that alpha-glucosidase in amniotic fluid is linked to lipids.

Amniotic fluid protease activity and the prenatal detection of cystic fibrosis.

Changes in the protease activity in amniotic fluid has been proposed as a valid method for the prenatal detection of cystic fibrosis (CF). We have studied by quantitative and qualitative procedures, sixty four amniotic fluids: two of them from CF-affected fetuses. Interpretation of the benzoyl arginine ethyl ester (BAEE)-staining patterns after isoelectric focusing was often difficult, and repeated experiments gave variable results. In order to improve gel discrimination, we performed amniotic fluid electrofocusing in the presence of detergents: 0.1 per cent Triton X-100, 0.1 per cent DOC, or 0.1 per cent SDS. In these conditions, the pattern revealed by BAEE was modified, but no differences were observed between CF and normal amniotic fluids.

Autosomal dominant polycystic kidney disease and alpha -4.2 thalassemia in a Caucasian family.

We describe the first known association between autosomal dominant polycystic kidney disease (ADPKD) and alpha-4.2 thalassemia in a Caucasian family. Linkage studies have been carried out using two probes (3'HVR and 24-1) linked to ADPKD on locus PKD1 and two probes (alpha 1-PstI and BamH-I/EcoRI-zeta 2 fragment) allowing detection of alpha-thalassemia with either a 3.7-kb deletion or a 4.2-kb deletion. Our results show that to avoid misinterpretation it is important to investigate the occurrence of an alpha-gene deletion when polymorphisms situated in the alpha-globin locus are used for linkage studies on ADPKD. The studied family is one of the rare cases of leftward deletional thalassemia described in a non-Asian population.

Antenatal diagnosis in three pregnancies at risk for mannosidosis.

Three pregnancies were monitored in two families at risk for mannosidosis, alpha mannosidase and alpha fucosidase were determined on extracts from white blood cells of several members of these families, on fibroblast cell lines from the index cases and on uncultured and cultured amniotic cells of the foetuses. Two foetuses were diagnosed as affected and one as being free from the disease. The conclusions were confirmed on the affected foetuses after interruption of the pregnancy. The noticeable residual activity of alpha mannosidase found in some fibroblasts and cultured amniotic cells required complementary investigations (heat lability and dependency of substrate concentration) to reach a reliable diagnosis. Invesitgation of uncultured amniotic cells may give useful additional information.

Linkage study of a large family with autosomal dominant polycystic kidney disease with reduced expression. Absence of linkage to the PKD 1 locus.

We describe a large three generation family with autosomal dominant polycystic kidney disease (PKD). Ultrasonographic screening of 60 family members revealed 20 individuals, whose age ranged from ten to eighty years, with one or several cysts in only one kidney and 7 individuals with cysts in both kidneys. Transmission of unilateral cysts seems to be autosomal dominant, although there are some generation gaps. Linkage studies with several markers of the PKD1 locus on the short arm of chromosome 16 showed no linkage with the disease. Lod scores for linkage between the disease and the most informative marker 3'HVR were computed using different penetrance models and several hypotheses concerning the clinical status of individuals with unilateral renal cysts. Results varied from Z = 1.31 to Z = -21.47 (theta = 0). Smith's test of heterogeneity gave a conditional probability of non-linkage between 0.9 and 1.0. We conclude that this family presents a form of autosomal dominant PKD with reduced penetrance and no linkage to the PKD1 locus on the short arm of chromosome 16. Other hypotheses, such as the existence of two distinct hereditary diseases in this large family, or neomutation in one branch of the family associated with a high frequency of isolated renal cysts, are also considered.

Linkage analysis of families with severe childhood autosomal recessive muscular dystrophy in Morocco indicates genetic homogeneity of the disease in north Africa.

It has been previously shown in Tunisian and Algerian families that the locus for SCARMD maps to the proximal part of 13q, and in Algerian families that the disease is associated with deficiency of the 50 kDa dystrophin associated glycoprotein (50DAG). We have tested this linkage in six families from Morocco where this disease is also prevalent. In one family the 50DAG was tested and found to be negative in a muscle biopsy. Our results showed similar linkage in this country, with statistical tests indicating genetic homogeneity between the three Maghreb countries.

A gene for dominant nonspecific X-linked mental retardation is located in Xq28.

A large family (MRX48) with a nonspecific X-linked mental retardation condition is described. An X-linked semidominant inheritance is suggested by the segregation in three generations of a moderate to severe mental retardation in seven males and by a milder intellectual impairment in two females, without any specific clinical, radiological, or biological feature. Two-point linkage analysis demonstrated significant linkage between the disorder and several markers in Xq28 (maximum LOD score [Zmax] = 2.71 at recombination fraction [theta] = 0); multipoint linkage analyses confirmed the significant linkage with a Zmax of 3.3 at theta = 0, at DXS1684. A recombination event observed with the flanking marker DXS8011 delineates a locus between this marker and the telomere. The approximate length of this locus is 8-9 cM, corresponding to 5.5-6 Mb. In an attempt to explain the variable intellectual impairment in females, we examined X-chromosome inactivation in all females of the family. Inactivation patterns in lymphocytes were random or moderately skewed, and no correlation between the phenotypic status and a specific inactivation pattern was observed. The interval of assignment noted in this family overlaps with five MRX loci previously reported in Xq28.

Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system.

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.

Inherited microdeletion in Xp21.3-22.1 involved in non-specific mental retardation.

X-linked mental retardation (XLMR) is a genetically and clinically heterogeneous common disorder. A cumulative frequency of about 1/600 male births was estimated by different authors, including the fragile X syndrome, which affects 1/4000 males. Given this very high cumulative frequency, identification of genes and molecular mechanisms involved in other XLMRs, represents a challenging task of considerable medical importance. In this report we describe clinical and molecular investigations in the family of a mentally retarded boy for whom a microdeletion in Xp21.3-22.1 was detected within the frame of a previously reported systematic search for deletion using STS-PCR screening. Thorough clinical investigation of the sibling showed that two affected brothers exhibit a moderate non-specific mental retardation without any additional neurological impairment, statural growth deficiency or characteristic dysmorphy. Molecular analysis revealed that the microdeletion observed in this family is an inherited defect which cosegregates with mental retardation as an X-linked recessive condition, since both non-deleted boys and transmitting mother are normal. These results and the inherited microdeletion detected within the same region associated with non-specific MR, reported by Raeymaekers et al., suggest that Xp21.3 MR locus is prone to deletions. Therefore, search for microdeletions in the eight families assigned by linkage analysis to this region might allow a better definition of the critical region and an identification of the gene involved in this X-linked mental retardation.

Identification by STS PCR screening of a microdeletion in Xp21.3-22.1 associated with non-specific mental retardation.

X-linked non-specific mental retardation (MRX) is a heterogeneous condition in which mental retardation (MR) appears to be the only consistent manifestation. The genetic and phenotypic heterogeneity exclude any possibility of pooling families and, therefore, of fine-mapping the related disease genes. In order to identify genomic critical regions involved in the MRX condition assigned to Xp21.3-22.1 region, we have implemented the PCR screening of non fragile X MR patients for the presence of deletions in this region. The amplification by PCR of 12 markers located between POLA and DXS704 using genomic DNA from 192 MR males led to the identification, in a 9 year old mentally retarded boy, of a microdeletion which extends from DXS1202 to DXS1065. None of the known genes, POLA, MAGE genes cluster, DAX1, GK and DMD, that map in the Xp21.3-22.1 region is affected by this deletion. This approach, which could easily be applied to several other MRX loci, allowed not only a confirmation of the presence of a potential locus in Xp21.3-22.1 involved in non-specific mental retardation, but also a better definition of the genomic critical region corresponding to this locus.

MECP2 mutations account for most cases of typical forms of Rett syndrome.

Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females, with an estimated prevalence of approximately one in 10 000-15 000 female births. Most cases are sporadic, but several reports about familial recurrence support X-linked dominant inheritance with male lethality. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in <25% of RTT cases in this first report. To characterize the spectrum of mutations in the MECP2 gene in RTT patients, we selected 46 typical RTT patients and performed mutation screening by denaturing gradient gel electrophoresis combined with direct sequencing. We identified 30 mutations, accounting for 65% of RTT patients. They include 12 novel mutations (11 located in exon 3 and one in exon 2). Mutations, such as R270X and frameshift deletions in a (CCACC) (n) rich region, have been found with multiple recurrences. Most of the mutations were de novo, except in one family where the non-affected transmitter mother exhibited a bias of X inactivation. Although this study showed that MECP2 mutations account for most cases of typical forms of RTT (65%) and mutations in non-coding regions cannot be excluded for the remaining cases, an alternative hypothesis that takes into account the homogeneous phenotype and exclusive involvement of females, could be the implication in RTT of a putative second X-linked gene.

Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia.

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome.

X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.

Dominant X linked subcortical laminar heterotopia and lissencephaly syndrome (XSCLH/LIS): evidence for the occurrence of mutation in males and mapping of a potential locus in Xq22.

X linked subcortical laminar heterotopia and lissencephaly syndrome (XSCLH/ LIS) is an intriguing disorder of cortical development, which causes classical lissencephaly with severe mental retardation and epilepsy in hemizygous males, and subcortical laminar heterotopia (SCLH) associated with milder mental retardation and epilepsy in heterozygous females. Here we report an exclusion mapping study carried out in three unrelated previously described families in which males are affected with lissencephaly and females with SCLH, using 38 microsatellite markers evenly distributed on the X chromosome. Most of the X chromosome was excluded and potential intervals of assignment in Xq22.3-q23 or in Xq27 are reported. Although the number of informative meioses did not allow a decision between these two loci, it is worth noting that the former interval is compatible with the mapping of a breakpoint involved in a de novo X;autosomal balanced translocation 46,XX,t(X;2)(q22;p25) previously described in a female with classical lissencephaly. In addition, haplotype inheritance in two families showed a grandpaternal origin of the mutation and suggested in one family the presence of mosaicism in germline cells of normal transmitting males.

Non-specific X-linked semidominant mental retardation by mutations in a Rab GDP-dissociation inhibitor.

Non-specific X-linked mental retardation (MRX) is a very common disorder which affects approximately 1 in 600 males. Despite this high frequency, little is known about the molecular defects underlying this disorder, mainly because of the clinical and genetic heterogeneity which is evident from linkage studies. Recently, a collaborative study using the candidate gene approach demonstrated the presence of mutations in GDIalpha, a Rab GDP-dissociation inhibitor encoded by a gene localized in Xq28, associated with non-specific mental retardation. GDIalpha is mainly a brain-specific protein that plays a critical role in the recycling of Rab GTPases involved in membrane vesicular transport. The study presented here was designed to assess the prevalence of mutations in the GDIalpha in mentally retarded patients and to discuss the clinical phenotypes observed in affected individuals. Mutation screening of the whole coding region of the GDIalpha gene, using a combination of denaturing gradient gel electrophoresis and direct sequencing, was carried out in 164 patients found negative for expansions across the FRAXA GCC repeat. In addition to the nonsense mutation recently reported in MRX48, we have identified a novel missense mutation in exon 11 of the GDIalpha gene in one familial form of non-specific mental retardation. In this family (family R), all affected males show moderate to severe mental retardation, and the X-linked semidominant inheritance is strongly suggested by the severe phenotypes in males with respect to mildly affected females or unaffected obligatory carriers. This study showed that the prevalence of GDIalpha mutations in non-specific mental retardation could be estimated to be 0.5-1%, and molecular diagnosis and genetic counselling in some cases of non-specific mental handicap can now be provided.