Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males.

BACKGROUND: Mental retardation (MR) affects 2-3% of the human population and some of these cases are genetically determined. Although several genes responsible for MR have been identified, many cases have still not been explained. METHODS: We have identified a pericentric inversion of the X chromosome inv(X)(p22.3;q13.2) segregating in a family where two male carriers have severe MR while female carriers are not affected. RESULTS: The molecular characterisation of this inversion led us to identify two new genes which are disrupted by the breakpoints: KIAA2022 in Xq13.2 and P2RY8 in Xp22.3. These genes were not previously fully characterised in humans. KIAA2022 encodes a protein which lacks significant homology to any other known protein and is highly expressed in the brain. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family. It is located in the pseudo-autosomal region of the X chromosome and is not expressed in brain. CONCLUSIONS: Because the haploinsufficiency of P2RY8 in carrier mothers does not have a phenotypic consequence, we propose that the severe MR of the affected males in this family is due to the absence of the KIAA2022 gene product. However, screening 20 probands from X linked MR families did not reveal mutations in KIAA2022. Nonetheless, the high expression of this gene in fetal brain and in the adult cerebral cortex could be consistent with a role in brain development and/or cognitive function.

Abnormal expression of the KLF8 (ZNF741) gene in a female patient with an X;autosome translocation t(X;21)(p11.2;q22.3) and non-syndromic mental retardation.

Non-syndromic X linked mental retardation (MRX) is a heterogeneous group of conditions in which all patients have mental retardation as the only constant phenotypic feature. We have identified a female patient with mental retardation and a balanced translocation involving chromosomes X and 21, t(X;21)(p11.2;q22.3). Physical mapping of the translocation breakpoint on the human X chromosome was performed using fluorescence in situ hybridisation. We have mapped the X chromosome breakpoint to a 21 kb DNA fragment upstream of the first exon of the KLF8 (ZNF741) gene in Xp11.21. We have subsequently shown that the KLF8 transcript is no longer detected in cells from the patient, although KLF8 expression is otherwise normally present in control lymphoblasts. Mutation screening of probands from 20 unrelated XLMR families linked to the proximal short arm of the human X chromosome failed to show any mutation in the coding region of the KLF8 gene.

Two unrelated patients with inversions of the X chromosome and non-specific mental retardation: physical and transcriptional mapping of their common breakpoint region in Xq13.1.

Two unrelated mildly retarded males with inversions of the X chromosome and non-specific mental retardation (MRX) are described. Case 1 has a pericentric inversion 46,Y,inv(X) (p11.1q13.1) and case 2 a paracentric inversion 46,Y,inv(X) (q13.1q28). Both male patients have severe learning difficulties. The same chromosomal abnormalities were found in their mothers who are intellectually normal. Fluorescence in situ hybridisation mapping showed a common area of breakage of each of the inverted chromosomes in Xq13.1 near DXS131 and DXS162. A detailed long range restriction map of the breakpoint region was constructed using YAC, PAC, and cosmid clones. We show that the two inverted chromosomes break within a short 250 kb region. Moreover, a group of ESTs corresponding to an as yet uncharacterised gene was mapped to the same critical interval. We hypothesise that the common inversion breakpoint region of the two cases in Xq13.1 may contain a new MRX gene.

Transcript map of the human chromosome Xq11-Xq21 region: localization of 33 novel genes and one pseudogene.

The human Xq11-Xq21.3 region has been implicated in several inherited disorders including dystonia-parkinsonism (DYT3), sideroblastic anemia and several specific and non-specific forms of mental retardation (MR) syndromes. As part of a positional cloning effort to identify MR genes, we have generated a YAC-based transcript map. We first constructed a YAC/STS framework by extending previously published contigs. This framework map consists of a minimal set of 119 clones, covering approximately 20 Megabases (Mb) and allowing the precise ordering of 71 STSs between DXS136 and DXS472. This YAC contig was then used to define the positions of genes and expressed sequence tags (ESTs) assigned to the Xcen-Xq21.3 region. In addition to the genes previously localized to this part of the X chromosome, 18 transcription units corresponding to additional known genes or gene family members, one pseudogene and 15 novel transcripts were mapped. This transcriptional map incorporates 51 transcription units and provides a useful resource of candidate genes for some of the disorders assigned to this region of the X chromosome.

Evaluation of a mutation screening strategy for sporadic cases of ATR-X syndrome.

We report on the evaluation of a strategy for screening for XNP/ATR-X mutations in males with mental retardation and associated dysmorphology. Because nearly half of the mutations in this gene reported to date fall into a short 300 bp region of the transcript, we decided to focus in this region and to extend the mutation analysis to cases with a negative family history. This study includes 21 mentally retarded male patients selected because they had severe mental retardation and a typical facial appearance. The presence of haemoglobin H or urogenital abnormalities was not considered critical for inclusion in this study. We have identified six mutations which represents a mutation detection rate of 28%. This figure is high enough for us to propose this strategy as a valid first level of screening in a selected subset of males with mental retardation. This approach is simple, does not require RNA preparation, does not involve time consuming mutation detection methods, and can thus be applied to a large number of patients at a low cost in any given laboratory.

Determination of the genomic structure of the XNP/ATRX gene encoding a potential zinc finger helicase.

The XNP/ATR-X gene is involved in several X-linked mental retardation phenotypes: the ATR-X syndrome, the Juberg-Marsidi syndrome, and some severe mental retardation phenotypes without alpha-thalassemia. Using a vectorette strategy, we have identified and sequenced the intron/exon boundaries of this gene. The gene is composed of 35 exons. It encodes a potential protein of 2492 amino acids. A search of the databases identified three zinc finger motifs within the 5' end of the gene. Expression analysis in different tissues indicated that an alternative splicing event that involves exon 6 is occurring. One of these alternatively spliced transcripts is predominantly expressed in embryonic tissues. These data led us to search for mutations in the 5' region in ATRX patients without other mutations in the 3' region. In one patient a mutation was found in which part of exon 7 was removed from the XNP transcript, as a result of a mutation creating a novel splice site that is substituted for the natural splice site. This new splicing event removed one zinc finger motif. This is the first example of a mutation in XNP within the 5' coding region. It suggests that mutations will be predominantly found in the helicase region as well as in the zinc finger regions and leads us to propose a large screening of additional patients.

Splicing mutation in the ATR-X gene can lead to a dysmorphic mental retardation phenotype without alpha-thalassemia.

We have previously reported the isolation of a gene from Xq13 that codes for a putative regulator of transcription (XNP) and has now been shown to be the gene involved in the X-linked alpha-thalassemia with mental retardation (ATR-X) syndrome. The widespread expression and numerous domains present in the putative protein suggest that this gene could be involved in other phenotypes. The predominant expression of the gene in the developing brain, as well as its association with neuron differentiation, indicates that mutations of this gene might result in a mental retardation (MR) phenotype. In this paper we present a family with a splice junction mutation in XNP that results in the skipping of an exon and in the introduction of a stop codon in the middle of the XNP-coding sequence. Only the abnormal transcript is expressed in two first cousins presenting the classic ATR-X phenotype (with alpha-thalassemia and HbH inclusions). In a distant cousin presenting a similar dysmorphic MR phenotype but not having thalassemia, approximately 30% of the XNP transcripts are normal. These data demonstrate that the mode of action of the XNP gene product on globin expression is distinct from its mode of action in brain development and facial morphogenesis and suggest that other dysmorphic mental retardation phenotypes, such as Juberg-Marsidi or some sporadic cases of Coffin-Lowry, could be due to mutations in XNP.

Construction of a YAC contig spanning the Xq13.3 subband.

The loci involved in several X-linked mental retardation syndromes have been linked to the pericentromeric region of the X chromosome long arm (Xq12-q21). To isolate candidate genes for these diseases, we set up the construction of YAC contigs spanning this region. Two of these syndromes (the Juberg-Marsidi syndrome and the alpha-thalessemia mental retardation syndrome) have been recently linked, with high lod scores, to polymorphic probes previously assigned to Xq13.3. We therefore constructed a first YAC contig, encompassing this band, from DXS441 to PGK1. The physical map, deduced from the isolated clones, extends over 2.1 Mb of genomic DNA. Restriction analysis of the YAC contig allowed us to map precisely the loci previously assigned to that chromosomal region and to define their relative order. The validity of this physical map has been checked by comparing Sfi I digests of the YACs to genomic fragments obtained with the same enzyme. A cDNA selection approach, already performed with a previous partial contig, has been extended to cover the whole region.

Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction.

To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).

Physical and transcriptional mapping of DXS56-PGK1 1 Mb region: identification of three new transcripts.

Several new techniques for isolation expressed sequences have been recently described considerably speeding up the identification of unknown genes. Here we present a transcriptional map of the 1 Mb DXS56-PGK1 region in Xq13.3. Rare cutter restriction site mapping, direct cDNA selection on membrane discs and probing of Northern blots with total YAC DNA, were the methods explored in order to achieve this goal. In addition to three known genes from this region which have been recloned, two new cDNA clones corresponding to two new genes were isolated, mapped and characterized. Moreover one more transcript, highly expressed in placenta, has been detected in the region with a total YAC as a probe. In summary there are at least six genes known to reside in the DXS56-PGK1 region. As several human disease gene loci (i.e. SCID, CMTX1, WWS, MRX, XDP, ASB) were tightly linked to the markers from the region (PGK, CA repeats), the three new transcripts may be considered as their potential candidate genes.

The mRNA transcripts from a mutant beta-globin gene derived from splicing at preferential cryptic sites.

We have analyzed mRNA transcripts from beta-globin genes carrying a homozygous point mutation at the 5' splicing site of the first intron, using a method allowing in vivo analysis of mRNA transcripts. As expected, this mutation decreases normal splicing of mRNA when cryptic splicing are utilized. We have observed that, in reticulocytes, most mature mRNA transcribed from beta-globin genes derives from specific sites of abnormal splicing. Our results differ from those previously obtained using mutant beta-globin genes introduced in cultured cells and indicate a preferential processing of the abnormal globin mRNA species in red cell precursors.