X-chromosome inactivation in female patients with Fabry disease.

Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females.

Primary adhalinopathy: a common cause of autosomal recessive muscular dystrophy of variable severity.

Marked deficiency of muscle adhalin, a 50 kDa sarcolemmal dystrophin-associated glycoprotein, has been reported in severe childhood autosomal recessive muscular dystrophy (SCARMD). This is a Duchenne-like disease affecting both males and females first described in Tunisian families. Adhalin deficiency has been found in SCARMD patients from North Africa Europe, Brazil, Japan and North America (SLR & KPC, unpublished data). The disease was initially linked to an unidentified gene on chromosome 13 in families from North Africa, and to the adhalin gene itself on chromosome 17q in one French family in which missense mutations were identified. Thus there are two kinds of myopathies with adhalin deficiency: one with a primary defect of adhalin (primary adhalinopathies), and one in which absence of adhalin is secondary to a separate gene defect on chromosome 13. We have examined the importance of primary adhalinopathies among myopathies with adhalin deficiency, and describe several additional mutations (null and missense) in the adhalin gene in 10 new families from Europe and North Africa. Disease severity varies in age of onset and rate of progression, and patients with null mutations are the most severely affected.

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.

Peripheral selection of V delta 1+ cells with restricted T cell receptor delta gene junctional repertoire in the peripheral blood of healthy donors.

To characterize the T cell receptor (TCR) repertoire expressed by the V delta 1+ gamma/delta T cell population, we have studied the V delta 1-J delta 1 junctional sequences from peripheral blood samples of healthy donors. We show that, surprisingly, this repertoire is restricted in most healthy adults, with a donor-specific and relatively stable pattern, whereas this repertoire remains unrestricted in infants, and is similar to that of thymocytes. These data contrast with the general assumption that the junctional repertoire of V delta 1+ gamma/delta T cells is extensive, and strongly suggest that peripheral recruitment of V delta 1+ cells bearing particular TCR occurs in humans during the postnatal stage.

Refinement of cortical dysgeneses spectrum associated with TUBA1A mutations.

OBJECTIVE: We have recently shown that de novo mutations in the TUBA1A gene are responsible for a wide spectrum of neuronal migration disorders. To better define the range of these abnormalities, we searched for additional mutations in a cohort of 100 patients with lissencephaly spectrum for whom no mutation was identified in DCX, LIS1 and ARX genes and compared these data to five previously described patients with TUBA1A mutations. RESULTS: We detected de novo TUBA1A mutations in six patients and highlight the existence of a prominent form of TUBA1A related lissencephaly. In four patients, the mutations identified, c.1190T>C (p.L397P), c.1265G>A (p.R422H), c.1264C>T (p.R422C), c.1306G>T (p.G436R), have not been reported before and in two others, the mutation corresponds to a recurrent missense mutation, c.790C>T (p.R264C), likely to be a hot spot of mutation. All together, it emerges that the TUBA1A related lissencephaly spectrum ranges from perisylvian pachygyria, in the less severe form, to posteriorly predominant pachygyria in the most severe, associated with dysgenesis of the anterior limb of the internal capsule and mild to severe cerebellar hypoplasia. When compared with a large series of lissencephaly of other origins (ILS17, ILSX or unknown origin), these features appear to be specific to TUBA1A related lissencephaly. In addition, TUBA1A mutated patients share a common clinical phenotype that consists of congenital microcephaly, mental retardation and diplegia/tetraplegia. CONCLUSIONS: Our data highlight the presence of consistent and specific abnormalities that should allow the differentiation of TUBA1A related lissencephalies from those related to LIS1, DCX and ARX genes.

[Epileptogenic brain malformations: radiological and clinical presentation and indications for genetic testing]. / Malformations cérébrales et épilepsie: présentations radiocliniques et implications pour le diagnostic génétique.

Malformations of cortical development (MCD) represent a major cause of developmental disabilities and severe epilepsy. Advances in imaging and genetics have improved the diagnosis and classification of these conditions. Up to now, eight genes have been involved in different types of MCD. Lissencephaly-pachygyria and subcortical band heterotopia (SBH) represent a malformative spectrum resulting from mutations of either LIS1 or DCX genes. LIS1 mutations cause a more severe malformation in the posterior brain regions. DCX mutations usually cause anteriorly predominant lissencephaly in males and SBH in female patients. Additional forms are X-linked lissencephaly with corpus callosum agenesis and ambiguous genitalia associated with mutations of the ARX gene. Lissencephaly with cerebellar hypoplasia (LCH) encompass heterogeneous disorders named LCH type a to d. LCHa are related with mutation in LIS1 or DCX, LCHb with mutation of RELN gene, and LCHd could be related with TUBA1A gene. Polymicrogyria encompass a wide range of clinical, aetiological and histological findings. Among several syndromes, recessive bilateral fronto-parietal polymicrogyria has been associated with mutations of the GPR56 gene. Bilateral perisylvian polymicrogyria showed a linkage to chromosome Xq28 in some pedigrees, and mutations in SRPX2 gene in others conditions. X-linked bilateral periventricular nodular heterotopia (BPNH) consists of BPNH with focal epilepsy in females and prenatal lethality in males. Filamin A (FLNA) mutations have been reported in some families and in sporadic patients. It is possible to infer the most likely causative gene by brain imaging studies and other clinical findings. Based on this experience, a detailed phenotype analysis is needed to develop the most efficient research on MCD in the future.

Prenatally diagnosed periventricular nodular heterotopia: Further delineation of the imaging phenotype and outcome.

OBJECTIVES: Periventricular nodular heterotopia (PNH) is a malformation of cortical development which presents with heterogeneous imaging, neurological phenotype and outcome. There is a paucity of comprehensive description detailing the prenatal diagnosis of PNH. The aim of this study is to report neuroimaging features and correlated outcomes in order to delineate the spectrum of prenatally diagnosed PNH. METHODS: It was a retrospective study over 15 years in five tertiary centers. All fetuses with prenatally diagnosed PNH were collected. Fetal ultrasound and MRI were reviewed and genetic screening collected. Prenatal findings were analyzed in correlation to fetopathological analyses and post-natal follow up. RESULTS: Thirty fetuses (22 females and 8 males) with PNH were identified. The two major ultrasound signs were ventriculomegaly associated with dysmorphic frontal horns (60%) and posterior fossa anomalies (73.3%). On MRI, two groups of PNH were identified: the contiguous and diffuse PNH (n = 15, 50%), often associated with megacisterna magna, and the non-diffuse, either anterior, posterior or unilateral PNH. FLNA mutations were found in 6/11 cases with diffuse PNH. Additional cortical malformations were exclusively observed in non diffuse PNH (9/15; 60%). Twenty-four pregnancies (80%) were terminated. Six children aged 6 months to 5 years are alive. Five have normal neurodevelopment (all had diffuse PNH) whereas one case with non diffuse PNH has developmental delay and epilepsy. CONCLUSION: PNH is heterogeneous but patients with diffuse PNH are a common subgroup with specific findings on prenatal imaging and implications for prenatal counseling.

Biological and clinical significance of concurrent p53 gene alterations, MDR1 gene expression, and S-phase fraction analyses in breast cancer patients treated with primary chemotherapy or radiotherapy.

We investigated the interrelationship between p53 gene alterations, MDR1 gene expression, and S-phase fraction (SPF) in breast carcinomas treated primarily with chemotherapy or radiotherapy and correlated the results with patient outcome to determine the potential clinical significance of these factors. In a consecutive series of 64 fine-needle samplings of breast cancer patients who underwent either neoadjuvant chemotherapy (n = 53) or radiotherapy (n = 11), p53 (exons 5-9) gene alterations by denaturating gradient gel electrophoresis and subsequent direct sequencing, MDR1 gene expression by semiquantitative reverse transcription-PCR, and SPF by DNA flow cytometry were determined. Our results show that p53 mutations (n = 20) were significantly associated (P = 0.01) with high SPF but not with de novo MDR1 gene expression. Most patients with wild-type p53 tumors were found to be resistant to neoadjuvant chemotherapy. No correlation was observed between p53 mutations and the induction of MDR1 gene expression during treatment. Although a significant correlation between shorter distant disease-free survival and high (>/=5%) SPF (P = 0.016) was found, no correlation between distant disease-free survival and p53 status or intrinsic MDR1 gene expression was found. Poor overall survival was observed in patients with tumors with high SPF (P < 0.0004) or lacking MDR1 gene expression (P = 0.03) before treatment, but not with p53 alterations. These data suggest that SPF remains the most relevant biological factor for breast cancer patients treated by primary chemotherapy or radiotherapy and that p53 and MDR1 status may identify a small subset of patients that may resist therapy or pursue an aggressive course.

Five novel frameshift mutations in exon 3 and 4 of the MECP2 gene identified in Rett patients: Consequences for the molecular diagnosis strategy.

Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in 70-85% of RTT cases. We report here five novel frameshift mutations (named 345delC, 895del202, 989ins18del8, 996insAG and 1124del53) in exon 3 and 4 of the MECP2 gene. To avoid the missing of few small deletions in RTT patients using classical mutation screening approaches, we suggest that screening of the mutations in the MECP2 gene in RTT girls should include at least a large PCR to amplify exon 4 entirely.

The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects.

To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.

No mutations in the coding region of the Rett syndrome gene MECP2 in 59 autistic patients.

Autistic disorder is a pervasive developmental disorder considered to have a multigenic origin. Mental retardation is present in 75% of autistic patients. Autistic features are found in Rett syndrome, a neurological disorder affecting girls and associated with severe mental retardation. Recently, the gene responsible for the Rett syndrome, methyl CpG-binding protein (MECP2) gene, was identified on the X chromosome by a candidate gene strategy. Mutations in this gene were also observed in some mentally retarded males. In this study we tested MECP2 as a candidate gene in autistic disorder by a DGGE analysis of its coding region and intron-exon boundaries. Among 59 autistic patients, 42 males and 17 females, mentally retarded or not, no mutations or polymorphisms were present in the MECP2 gene. Taking into account the size of our sample, we conclude that MECP2 coding sequence mutations are not an important factor (less than 5% of cases) in the aetiology of autistic disorder.

Parental origin of de novo MECP2 mutations in Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females as sporadic cases. Recently, DNA mutations in the MECP2 gene have been detected in approximately 70% of patients with RTT. To explain the sex-limited expression of RTT, it has been suggested that de novo X-linked mutations occur exclusively in male germ cells resulting therefore only in affected daughters. To test this hypothesis, we have analysed 19 families with RTT syndrome due to MECP2 molecular defects. In seven informative families we have found by DHPLC a nucleotide variant which could be used to differentiate between the maternal and the paternal allele. In each subject investigated from these families, we have amplified specifically each allele and sequenced allele-specific PCR products to identify the allele bearing the mutation as well as the parental origin of each X chromosome. This approach allowed us to determine the parental origin of de novo mutations in all informative families. In five cases, the de novo MECP2 mutations have a paternal origin and in the two other cases a maternal origin. In all transitions at CpG, the de novo mutation observed was of paternal origin. The high frequency of male germ-line transmission of the mutation (71% of RTT informative cases) is consistent with a predominant occurrence of the disease in females.

Analysis of alternative splicing patterns in the cystic fibrosis transmembrane conductance regulator gene using mRNA derived from lymphoblastoid cells of cystic fibrosis patients.

Using in vitro amplification of cDNA by the polymerase chain reaction, we analyzed alternatively spliced events of cystic fibrosis transmembrane conductance regulator gene in lymphoblastoid cells. Ten alternatively spliced transcripts were identified using analysis of 6 overlapping segments of amplified cDNA, 4 of which have not been described previously. These include transcripts lacking exon 16, 17b, 22 and a transcript resulting from the use of a cryptic acceptor and donor splice sites. Moreover, in 2 cystic fibrosis (CF) patients bearing nonsense mutations E60X or W1282X, we observed that nonsense mutations are associated with an alteration of splice site selection in vivo resulting in exon skipping of constitutive exons or in the use of cryptic splice sites. In addition, even though lymphoblastoid cells are not the relevant tissue to address the question of the relationship between clinical respiratory phenotype and genotype, our results concerning adult CF patients (delta F508/ delta F508) suggest that individual-specific RNA splicing patterns could influence the severity of the CF pulmonary disease. If this phenomenon of alternative splicing events proves to be significant in CF and to be a common feature of disease genes, the study of RNA splicing could become an important tool for the analysis of the genotype-phenotype relationship in many inherited disorders.

Somatic mosaicism for partial paternal isodisomy in Wiedemann-Beckwith syndrome: a post-fertilization event.

Genomic imprinting has been implicated in the aetiology of an overgrowth cancer-prone syndrome, the Wiedemann-Beck-with syndrome (WBS). We have demonstrated uniparental disomy (UPD) for paternal chromosome 11p markers in 5 out of 25 sporadic cases (20%). Delineation of the extent of the disomy region may help in understanding the mechanism and the stage, meiotic or mitotic, of disomy formation in this disease and in associated tumours. Our current studies in WBS patients with seventeen 11p and one 11q markers reveal paternal isodisomy, not heterodisomy, in the five cases. For one case we demonstrate unambiguously that partial isodisomy for 11p and somatic mosaicism for UPD resulted from a post-fertilization event. The restriction of isodisomy to part of 11p in another case, and somatic mosaicism for UPD in three other cases, suggest a mitotic recombinational event that must have occurred after fertilization. Mosaic phenotypes reflect the timing of their origin and the fate of the cells involved, as well as the cell-specific pattern of imprinting. Somatic mosaicism for UPD in four cases may thus explain the incomplete forms of WBS. The association of hemihypertrophy in sporadic WBS and even some cases of isolated hemihypertrophy. This is in agreement with a recent report of paternal isodisomy for 11p markers in a patient with hemihypertrophy, Wilms' tumour and adrenocortical carcinoma. Moreover, the risk of developing a tumour seems higher (50%) for patients with paternal 11p UPD than for WBS patients in general (7.5%).

Mapping of the X-breakpoint involved in a balanced X;12 translocation in a female with mild mental retardation.

Balanced chromosomal abnormalities such as translocations and inversions have been identified in many genetic diseases. Cloning of the breakpoints involved in these abnormalities has led to the identification of the disease-related genes. Recent reports suggest the presence of a mental retardation locus at Xq11-12. We have identified a female patient with a balanced translocation t (X;12) (q11;q15) associated with mild mental retardation. We identified a yeast artificial chromosome spanning the X-chromosome breakpoint by using fluorescent in situ hybridization techniques. A cosmid library of this YAC has been constructed and the search for candidate genes is in progress.

Detection of polymorphism in the Trypanosoma cruzi TcP2 beta gene family by single strand conformational analysis (SSCA).

Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2 beta gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5' end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4 degrees C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4 degrees C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project.

Cloning and characterization of the human V3 pituitary vasopressin receptor.

Arginine-vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G-protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (Kd = 0.55 +/- 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT-PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a approximately 4.8 kb messenger in human corticotropic pituitary adenomas.

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes.

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

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.