Aneuploidy: the impact of chromosome imbalance on nuclear organization and overall genome expression.

The organization and dynamics of chromatin within the interphase nucleus as chromosome territories (CTs) and the relationship with transcriptional regulation are not fully understood. We studied a natural example of chromosomal disorganization: aneuploidy due to trisomies 13, 18 and 21. We hypothesized that the presence of an extra copy of one chromosome alters the CT distribution, which perturbs transcriptional activity. We used 3D-FISH to study the position of the chromosomes of interest (18 and 21) in cultured amniocytes and chorionic villus cells from pregnancies with a normal or aneuploid karyotype. We studied the volumes of nuclei and CTs in both conditions and performed a compared transcriptome analysis. We did not observe any differences between euploid and aneuploid cells in terms of the radial and relative CT positions, suggesting that the same rules govern nuclear organization in cases of trisomy. We observed lower volumes for CTs 18 and 21. Overall genome expression profiles highlighted changes in the expression of a subset of genes in trisomic chromosomes, while the majority of transcriptional changes concerned genes located on euploid chromosomes. Our results suggest that a dosage imbalance of the genes on trisomic chromosomes is associated with a disturbance of overall genomic expression.

Inverted duplication with deletion: first interstitial case suggesting a novel undescribed mechanism of formation.

Inverted duplications with terminal deletions are a well-defined family of complex rearrangements already observed for most of chromosome extremities. Several mechanisms have been suggested which could lead to their occurrence, either through non-homologous end joining, non-allelic homologous recombination, or more recently through an intrastrand fold-back mechanism. We describe here a patient with intellectual disability and pharmacoresistant epilepsy, for which array CGH analysis showed the first interstitial case of inverted duplication with deletion on chromosome 1p. Furthermore, SNP array analysis revealed an associated segmental isodisomy for the distal part of 1p, which led us to consider a replicative mechanism to explain this abnormality. This observation extends the range of this once telomeric rearrangement.

[Genetic mosaicism in Systemic Auto-Inflammatory Diseases: A review of the literature]. / Mosaïcisme génétique dans les maladies auto-inflammatoires : revue de la littérature.

Systemic auto-inflammatory diseases (SAIDs) are disorders associated with deregulation of innate immunity in which patients present classically with systemic inflammatory manifestations, in particular fever, skin-mucosal rashes, arthromyalgia and abdominal pain, with an increase in blood biomarkers of inflammation. At the time of their discovery, these diseases were associated with constitutional mutations in genes encoding proteins involved in innate immunity, and it was then considered that they had to begin in childhood. This dogma of constitutional mutations in SAIDs is no longer so unquestionable, since 2005 several cases of mosaicism have been reported in the literature, initially in cryopyrinopathies, but also in other SAIDs in patients with obvious clinical phenotypes and late onset of disease expression, in particular in the VEXAS syndrome (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic Syndrome) and very recently in MEVF gene. Next-generation sequencing techniques are more sensitive than Sanger for detecting mosaicisms. So, when a clinical diagnosis seems obvious but no constitutional mutation is found by low-depth genetic analysis, it is useful to discuss with expert geneticists whether to consider another genetic approach in a child or an adult. This modifies the situations in which clinicians can evoke these diseases. This review provides an update on mosaicism in SAIDs.

Mutations in the gene encoding mevalonate kinase cause hyper-IgD and periodic fever syndrome. International Hyper-IgD Study Group.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is a rare, apparently monogenic, autosomal recessive disorder characterized by recurrent episodes of fever accompanied with lymphadenopathy, abdominal distress, joint involvement and skin lesions. All patients have high serum IgD values (>100 U/ml) and HIDS 'attacks' are associated with an intense acute phase reaction whose exact pathophysiology remains obscure. Two other hereditary febrile disorders have been described. Familial Mediterranean fever (MIM 249100) is an autosomal recessive disorder affecting mostly populations from the Mediterranean basin and is caused by mutations in the gene MEFV (refs 5,6). Familial Hibernian fever (MIM 142680), also known as autosomal dominant familial recurrent fever, is caused by missense mutations in the gene encoding type I tumour necrosis factor receptor. Here we perform a genome-wide search to map the HIDS gene. Haplotype analysis placed the gene at 12q24 between D12S330 and D12S79. We identified the gene MVK, encoding mevalonate kinase (MK, ATP:mevalonate 5-phosphotransferase; EC 2.7.1.36), as a candidate gene. We characterized 3 missense mutations, a 92-bp loss stemming from a deletion or from exon skipping, and the absence of expression of one allele. Functional analysis demonstrated diminished MK activity in fibroblasts from HIDS patients. Our data establish MVK as the gene responsible for HIDS.

French protocol for the diagnosis and management of familial Mediterranean fever.

Familial Mediterranean fever is the most common monogenic auto-inflammatory disease in the world. It mainly affects people originating from the Mediterranean region. The mutated gene is MEFV, which codes for pyrin. Transmission is autosomal recessive. Patients present with recurrent attacks of fever since childhood associated with abdominal and/or thoracic pain lasting an average of 2-3days and a biological inflammatory syndrome. Other symptoms include arthralgia or arthritis in large joints such as the knees and ankles, myalgia in the lower limbs and pseudo-erysipelas in the ankles. The most serious complication is inflammatory amyloidosis, which can lead to kidney failure. Treatment is based on colchicine, which helps to prevent flares and the onset of renal amyloidosis. This paper proposes national guidelines for the diagnosis, management and follow-up of familial Mediterranean fever in France, where we estimate there are between 5000 and 10,000 patients with the disease at all stages of life. The diagnosis is suspected on the basis of clinical and anamnestic factors and confirmed by genetic analysis. These guidelines also suggest a "treat-to-target" approach to disease management, particularly in case of suspected colchicine resistance - a very rare situation that should remain a diagnosis of elimination, especially after colchicine compliance has been verified. Two special situations are also addressed in these guidelines: kidney failure and pregnancy.

Mutations in the autoinflammatory cryopyrin-associated periodic syndrome gene: epidemiological study and lessons from eight years of genetic analysis in France.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) consist of a continuum of autoinflammatory diseases caused by a defect in interleukin 1ß regulation. Although symptoms may vary widely, the discovery, in 2001, of the gene involved (NLRP3) has dramatically helped diagnosis. OBJECTIVES: To define the spectrum and prevalence of NLRP3 mutations in France and to delineate initial criteria before molecular analysis. METHODS: Retrospective review (2001-9) of genetic analysis data and request forms of patients living in France with an NLRP3 mutation since the set up of CAPS molecular diagnosis by the three French laboratories providing this test (GenMAI network). RESULTS: Over 800 analyses of this gene have been conducted, identifying 135 cases with an NLRP3 mutation (55 probands; 33 multiplex families); the estimated prevalence in France was equal to 1/360 000. A total of 21 different sequence variants were detected, among which four are common and nine are new mutations. CONCLUSIONS: Although the number of NLRP3 test requests has doubled over the past 5 years, genetic screening has not contributed to enhanced detection of new index cases each year. There are two possible reasons for this: (i) no clinical prerequisite for genetic diagnosis and (ii) few new large families are now identified (unlike the initial study based on a selection by linkage). A set of initial clinical criteria have been drawn up which it is recommended should be fulfilled before a patient is tested: at least three recurrent bouts, age at disease onset < 20 years and elevated levels of C-reactive protein, especially in individuals with urticaria and moderate fever.

New CIAS1 mutation and anakinra efficacy in overlapping of Muckle-Wells and familial cold autoinflammatory syndromes.

OBJECTIVES: Muckle-Wells syndrome (MWS) and familial cold autoinflammatory syndrome (FCAS) are rare periodic fevers associated with CIAS1 mutations. A third entity, the chronic infantile neurological, cutaneous, articular (CINCA) syndrome was also recently associated with mutation in the same gene. A phenotypic and genotypic continuum seems to exist from the most benign (FCAS) to the most severe forms (CINCA). Although a CIAS1 mutation can be associated with two different phenotypes. METHODS: We report a family of three patients exhibiting the MWS and FCAS phenotypes. These phenotypes were associated with a novel missense mutation in CIAS1. RESULTS: Anakinra controlled inflammatory flares in the three patients. CONCLUSIONS: FCAS, MWS and CINCA could be different phenotype expressions of the same disease.

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is an autosomal recessive inflammatory disorder characterised by recurrent episode of fever associated with lymphadenopathy, abdominal distress, joint involvement and skin lesions. We recently demonstrated that mutations in the mevalonate kinase gene (MVK) are associated with HIDS. Direct DNA sequencing was done to screen the entire coding region of MVK in 25 unrelated patients with HIDS. Mutations were detected in the coding region of the gene including 11 missense mutations, one deletion, the absence of expression of one allele, as well as three novel polymorphisms. Seven of these mutations are novel. The large majority of the patients were compound heterozygotes for two mutations. Of these, V377I (G-->A) is the most common mutation occurring in 20 unrelated patients and was found to be associated with I268T in six patients. Mutations were associated with a decrease of mevalonate kinase (MK) (ATP:mevalonate 5-phosphotransferase, EC 2.7.I.36) enzymatic activity but not as profound as in mevalonic aciduria, a syndrome also caused by a deficient activity of MK. In HIDS the mutations are located all along the protein which is different from mevalonic aciduria where MK mutations are mainly clustered to a same region of the protein. On the basis of this study, we propose that the diagnostic screen of MVK in HIDS should be first directed on V377I and I268T mutations. Three patients are also described to illustrate the genotypic and phenotypic overlap with mevalonic aciduria.

[Tumor necrosis factor receptor superfamily 1A-associated periodic syndrome (TRAPS)]. / Le syndrome de fièvre héréditaire lié à un dysfonctionnement du récepteur du TNF ou Traps.

PURPOSE: Tumor necrosis factor receptor superfamily 1A associated periodic syndrome (TRAPS) belongs to the group of hereditary fever syndromes, also called hereditary auto-inflammatory syndromes. CURRENT KNOWLEDGE AND KEY POINTS: The diagnosis of TRAPS should be evoked in presence of the following clinical signs, whatever the population of the affected patients. TRAPS acute inflammatory access, of 1 to 3 weeks' duration, is characterised by the presence of fever, abdominal pain, myalgias, various types of skin rash including erysepela-like erythema. Long term inflammatory response can lead to AA amyloidosis. Genetic testing will confirm the diagnosis when showing a mutation in the extracellular part of the TNFRSF1A receptor. Therapeutic management of TRAPS is not definitely established. Daily colchicine does not seem to prevent efficiently inflammatory attacks. Corticosteroids, in contrast can attenuate the intensity and diminish the duration of attacks. FUTURE PROSPECTS AND PROJECTS: The value of biological agents that inhibits TNF action is not yet completely determined in TRAPS. Mechanisms of the disease are not yet elucidated. In some families with specific mutations, a relative soluble TNF receptor deficiency has been found in the plasma. However this mechanism does not account for what is observed in other kindreds.

[Genetic basis of Prader-Willi and Angelman syndromes: implications for the biologic diagnosis]. / Bases génétiques des syndromes de Prader-Willi et d'Angelman: implications pour la conduite du diagnostic biologique.

Prader-Willi and Angelman syndromes are two genetic diseases whose clinical diagnosis is often impaired by a wide variability in some clinical findings. New insights in the genetic basis of these disorders allow the proposition of a biological approach to detect almost all Prader-Willi syndrome patients and over 80% of Angelman syndrome patients. Moreover, the results of these tests are indispensable for the evaluation of the recurrence risk.

[Hyperimmunoglobulinemia D and periodic fever syndrome]. / Le syndrome d'hyper-immunoglobulines D (HIGDS) avec fièvre périodique. À propos de deux observations.

We report the cases of two sisters born of parents who were first-degree cousins, who started recurrent fever with lymph node and digestive tract involvement at the age of 2 years. There was no mutation of the familial Mediterranean fever gene and a diagnosis of partial mevalonate kinase (MVK) deficiency was made. However, immunoglobulin (Ig) D and A levels were normal. Elevated mevalonic acid in the patients' urine during an episode and MVK gene analysis provided the diagnosis. Clinical remission was obtained under anti-TNF-alpha treatment with etanercept. These observations and those of several previously reported patients, particularly in French and Dutch series, illustrate the importance of considering the diagnosis in a child with early-onset auto-inflammatory syndrome even in the absence of hyper-IgD or -IgA.

Diagnostic value of serum immunoglobulinaemia D level in patients with a clinical suspicion of hyper IgD syndrome.

OBJECTIVE: The hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) was originally defined by the presence of a high serum level of immunoglobulin D associated with recurrent fever. Since the discovery of the mevalonate kinase gene (MVK) gene encoding the mevalonate kinase enzyme, most patients with a clinical diagnostic of HIDS are now found to have a mevalonate kinase deficiency based on metabolic and genetic data. We aimed to asses the value of a high IgD serum level for the diagnosis of HIDS in a cohort of patients with a phenotype of recurrent fever, and to characterize patients with a high IgD serum level without mevalonate kinase mutation. METHODS: Main clinical and biological data of 50 patients who presented with clinical signs compatible with HIDS have been prospectively registered on a standard form. Clinical data have been analysed according the IgD serum level and the presence of MVK mutation. RESULTS: The metabolic and genetic data establishing the diagnosis of HIDS correlated in all cases. In this series of 50 patients, the sensitivity of a high IgD value for the diagnosis of HIDS is 0.79. In five patients with MVK mutation, IgD levels were found to be in the normal range. Likelihood ratios indicate that IgD measurement is not relevant for the diagnostic of HIDS. Most patients with a high serum IgD level and no MVK mutation have no definite diagnosis. CONCLUSION: The clinical relevance of the IgD measurement for the diagnosis of MKD in our population appears as poor, as reflected by likelihood ratios which are both close to 1.

First-trimester enzymatic and molecular prenatal diagnosis of mevalonic aciduria.

Prenatal diagnosis was offered to a family at risk of mevalonic aciduria. A chorionic villus sample was obtained and both mevalonate kinase activity and mutation analysis were done. An affected fetus was diagnosed.

A protein phosphatase is involved in the inhibition of histone deacetylation by sodium butyrate.

Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases. Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate. These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases. Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.

Quantitative analysis of histone H1 degrees protein synthesis in HTC cells.

H1 degrees, a member of histone H1 family associated with cell growth arrest and differentiation, is barely expressed in most mammalian cells in culture. Depending on the cell type, serum deprivation or drugs, such as sodium butyrate, significantly increase H1 degrees mRNA level and H1 degrees protein accumulates. However, probably because of a lack of a simple quantitative procedure, little is known about the relationship between H1 degrees mRNA content and its effective translation rate. Using a rat hepatoma cell line and sodium butyrate as a model system, we attempted to evaluate this in different cellular conditions by measuring H1 degrees synthesis with a rapid quantitative procedure we described previously. We found that although the amount of H1 degrees mRNA rapidly increased and then stabilized under sodium butyrate treatment, its transcription was delayed and H1 degrees protein was synthesized in a progressive wave. Butyrate removal from cell culture confirmed that mRNA level and protein synthesis were independently regulated, and provided evidence that sodium butyrate would not directly target the translation apparatus. In contrast, during the S phase of the cell cycle, H1 degrees gene transcription and protein synthesis were concomitantly activated. Taken together these data provide evidence that H1 degrees accumulation results from an increase of its synthesis and that, depending on conditions, a cell exhibits a H1 degrees translation efficiency which may or may not reflect the mRNA level.

The effects of sodium butyrate on transcription are mediated through activation of a protein phosphatase.

In this study we have investigated the molecular mechanism by which sodium butyrate modulates gene expression when added to cultured cells. As a model system we used hepatoma tissue culture cells in which sodium butyrate treatment increases histone H1(0) mRNA level and decreases c-myc mRNA level. Because we observed that stimulation of histone H1(0) gene expression could take place in the absence of protein neosynthesis, we hypothesized that sodium butyrate induced a post-translational modification of a factor involved in the transcription process. Using different types of well known kinase and phosphatase inhibitors, we studied the implication of kinase or phosphatase activity in this pathway. Interestingly, cell treatment with potent serine-threonine-phosphatase inhibitors, calyculin A or okadaic acid, prevented the regulation of both histone H1(0) and c-myc gene expressions by sodium butyrate. On the other hand, the tyrosine phosphatase inhibitor, vanadate, or the protein kinase C inhibitor, staurosporine, did not significantly modify sodium butyrate effects. Using protein phosphatase 1 and 2A for in vitro assays, we found a 45% increase of phosphatase activity after cell treatment by sodium butyrate, possibly due to a protein phosphatase 1-type protein phosphatase. These data strongly suggest that signaling pathway(s) triggered by sodium butyrate to modulate gene expression involve(s) a serine-threonine-phosphatase activity.

Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney.

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.

Fetal phenotype of Prader-Willi syndrome due to maternal disomy for chromosome 15.

Prader-Willi syndrome (PWS) results from either paternal deletion of 15q11-q13, or maternal uniparental disomy (UPD) of chromosome 15 or imprinting center mutation. Prenatal diagnosis of PWS is currently indicated for chromosomal parental translocation involving chromosome 15 and for decreased fetal movements during the third trimester of gestation. Here we present the prenatal diagnosis of PWS during the first trimester of gestation and autopsy findings. Chorionic villus sampling (CVS) was performed for advanced maternal age at 13 weeks' gestation. CVS showed mosaicism including cells with a normal karyotype and cells with trisomy 15. Amniocentesis showed cells with a normal karyotype. Molecular analysis demonstrated that the fetus had a typical PWS abnormal methylation profile and maternal disomy for chromosome 15. Fetal ultrasound examination showed slightly enlarged lateral ventricles and hypoplasic male external genitalia without intra-uterine growth retardation. The autopsy showed a eutrophic male fetus with facial dysmorphy, hypoplasic genitalia, abnormal position of both feet and posterior hypoplasia of the corpus callosum. This report points out that in a karyotypically normal fetus with ambiguous male external genitalia and cerebral anomalies, extensive cytogenetic and molecular biology studies are strongly recommended because of risk of PWS.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.