Relevance of Extending FGFR3 Gene Analysis in Osteochondrodysplasia to Non-Coding Sequences: A Case Report.

Skeletal dysplasia, also called osteochondrodysplasia, is a category of disorders affecting bone development and children's growth. Up to 552 genes, including fibroblast growth factor receptor 3 (FGFR3), have been implicated by pathogenic variations in its genesis. Frequently identified causal mutations in osteochondrodysplasia arise in the coding sequences of the FGFR3 gene: c.1138G>A and c.1138G>C in achondroplasia and c.1620C>A and c.1620C>G in hypochondroplasia. However, in some cases, the diagnostic investigations undertaken thus far have failed to identify the causal anomaly, which strengthens the relevance of the diagnostic strategies being further refined. We observed a Caucasian adult with clinical and radiographic features of achondroplasia, with no common pathogenic variant. Exome sequencing detected an FGFR3(NM_000142.4):c.1075+95C>G heterozygous intronic variation. In vitro studies showed that this variant results in the aberrant exonization of a 90-nucleotide 5' segment of intron 8, resulting in the substitution of the alanine (Ala359) for a glycine (Gly) and the in-frame insertion of 30 amino acids. This change may alter FGFR3's function. Our report provides the first clinical description of an adult carrying this variant, which completes the phenotype description previously provided in children and confirms the recurrence, the autosomal-dominant pathogenicity, and the diagnostic relevance of this FGFR3 intronic variant. We support its inclusion in routinely used diagnostic tests for osteochondrodysplasia. This may increase the detection rate of causal variants and therefore could have a positive impact on patient management. Finally, FGFR3 alteration via non-coding sequence exonization should be considered a recurrent disease mechanism to be taken into account for new drug design and clinical trial strategies.

A phase-II study based on dose adjustment according to UGT1A1 polymorphism: is irinotecan underdosed in first-line FOLFIRI regimen for mCRC?

PURPOSE: Irinotecan has considerable importance in the treatment of metastatic colorectal cancer (mCRC). UDP-glucoronyltransferase (UGT) 1A1 is responsible for the inactivation of SN-38, a metabolite of irinotecan. Depending on UGT1A1 polymorphism, the activity of the UGT enzyme can be reduced leading to more frequent occurrence of adverse events related to irinotecan. The present study aimed to assess the safety and efficacy of different doses of irinotecan adjusted according to UGT1A1 polymorphism. METHODS: Thirty-four patients treated with FOLFIRI as first-line treatment for mCRC were included in this study. The irinotecan dosage was adapted on the basis of UGT1A1 polymorphisms: *1/*1 (370 mg/m2); *1/*28 (310 mg/m2), and *28/*28 (180 mg/m2). The incidence of grades 3 and 4 toxicities (neutropenia, febrile neutropenia, and diarrhoea) was recorded. Response was assessed according to the RECIST 1.1 criteria. RESULTS: On the basis of UGT1A1 genotyping, 20 patients were *1/*1 (58.8%), 12 were *1/*28 (35.3%) and 2 were *28/*28 (5.9%). Seven patients experienced at least one severe toxicity, i.e., 21% of the population, amounting to eleven adverse events. Concerning the response rate, 15 patients (44%) had partial or complete response. CONCLUSION: This study demonstrates that mCRC patients treated with FOLFIRI can tolerate a higher dose of irinotecan than the standard dose, i.e., > 180 mg/m2, on the basis of their UGT1A1 genotype, without increased toxicities. TRIAL REGISTRATION: NCT01963182 (registered on 16/10/2013, Clermont-Ferrand, France).

Paracetamol and Pain Modulation by TRPV1, UGT2B15, SULT1A1 Genotypes: A Randomized Clinical Trial in Healthy Volunteers.

BACKGROUND: The influence of the genetic polymorphism of enzymes and receptors involved in paracetamol metabolism and mechanism of action has not been investigated. This trial in healthy volunteers investigated the link between paracetamol pain relief and the genetic polymorphism of 23 enzymes and receptors. DESIGN: This randomized double-blind crossover controlled pilot study took place in the Clinical Pharmacology Department, University Hospital, Clermont-Ferrand, France. Forty-seven Caucasian volunteers were recruited. The trial consisted of two randomized sessions one week apart with oral paracetamol or placebo, and pain changes were evaluated with mechanical pain stimuli. The genetic polymorphism of 23 enzymes and receptors was studied, and correlations were made with pain relief. All tests are two-sided with a type I error at 0.05. RESULTS: Paracetamol was antinociceptive compared with placebo (222 ± 482 kPaxmin vs 23 ± 431 kPaxmin; P = 0.0047), and the study showed 30 paracetamol responders and 17 paracetamol nonresponders. Responders were characterized by TRPV1rs224534 A allele, UGT2B15rs1902023 TT genotype, and SULT1A1rs9282861 GG genotype (P < 0.05 for all). These findings confirm for the first time the involvement of a specific TRPV1 rs224534 variant in paracetamol antinociception. They also reveal a new antinociceptive role for specific variants of hepatic phase II enzymes associated with paracetamol metabolism. CONCLUSIONS: The study warrants larger clinical trials on these potential genomic markers of paracetamol analgesia in patients. Confirmation of the present findings would open the way to effective individualized pain treatment with paracetamol, the most commonly used analgesic worldwide.

Pathological Implications of Receptor for Advanced Glycation End-Product (AGER) Gene Polymorphism.

The receptor for advanced glycation end-products (RAGE) is a cell surface transmembrane multiligand receptor, encoded by the AGER gene. RAGE presents many transcripts, is expressed mainly in the lung, and involves multiple pathways (such as NFκB, Akt, p38, and MAP kinases) that initiate and perpetuate an unfavorable proinflammatory state. Due to these numerous functional activities, RAGE is implicated in multiple diseases. AGER is a highly polymorphic gene, with polymorphisms or SNP (single-nucleotide polymorphism) that could be responsible or co-responsible for disease development. This review was designed to shed light on the pathological implications of AGER polymorphisms. Five polymorphisms are described: rs2070600, rs1800624, rs1800625, rs184003, and a 63 bp deletion. The rs2070600 SNP may be associated with the development of human autoimmune disease, diabetes complications, cancer, and lung diseases such as chronic obstructive pulmonary disease and acute respiratory distress syndrome. The rs1800624 SNP involves AGER gene regulation and may be related to reduced risk of heart disease, cancer, Crohn's disease, and type 1 diabetes complications. The rs1800625 SNP may be associated with the development of diabetic retinopathy, cancer, and lupus but may be protective against cardiovascular risk. The rs184003 SNP seems related to coronary artery disease, breast cancer, and diabetes. The 63 bp deletion may be associated with reduced survival from heart diseases during diabetic nephropathy. Here, these potential associations between AGER polymorphisms and the development of diseases are discussed, as there have been conflicting findings on the pathological impact of AGER SNPs in the literature. These contradictory results might be explained by distinct AGER SNP frequencies depending on ethnicity.

Genetic Analysis of Mu and Kappa Opioid Receptor and COMT Enzyme in Cancer Pain Tunisian Patients Under Opioid Treatment.

BACKGROUND: Pain and its opioid treatments are complex measurable traits. Responses to morphine in terms of pain control is likely to be determined by many factors, including the underlying pain sensitivity of the patient, along with nature and extent of the painful process, concomitant medications, genetic and other clinical and environmental factors. This study investigated genetic polymorphisms implicated in the inter-individual pain response variability to opioid treatment in the Tunisian population. METHODS: This prospective association study investigated seven variations in the OPRM1, OPRK1 and COMT gene, which encode Mu and KAPPA opioid receptors, and Catechol-O-methyltransferase enzyme respectively, in a cohort of 129 Tunisian cancer pain patients under oral morphine treatment. Genotyping was performed by simple probe probes on Light Cyler for rs17174629, rs1799972, rs1799971, rs1051659, rs1051660 and rs4680 and by PCR assay for the indel in the promoter region of OPRK1 (rs35566036). A statistical associations study between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated using linear multiple regressions and logistic regressions respectively adjusted on metastases and pain covariates in the R software. RESULTS: We detected significant association of the rs1051660 adjusted on metastasis and pain (P=0.02), no other association has been detected between the 7 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This can be explained by the strong genetic heterogeneity in the cosmopolitan areas where our patients were recruited for this study, compared to more homegenous population recruited in other studies.

Association of the OPRM1 and COMT genes' polymorphisms with the efficacy of morphine in Tunisian cancer patients: Impact of the high genetic heterogeneity in Tunisia?

BACKGROUND: Genetic causes for inter-individual variability response to opioids are clinical difficulties for treatment efficiency. The aim of the present study was to investigate the possible association of opioid treatment outcome with single nucleotide polymorphisms (SNPs) in the mµ opioid receptor (OPRM1) and catechol-o-methyltransferase (COMT) genes, in Tunisian cancer pain patients. METHODS: We genotyped one hundred and twenty-nine cancer patients treated with different doses of morphine for 3 SNPs in OPRM1 gene (rs17174629, rs1799972 and rs1799971) and one in the COMT gene (rs4680). Associations between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated. RESULTS: Unlike other studies on Caucasian and Chinese populations, no significant association were found between the 4 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This result can be explained by the genetic heterogeneity and cosmopolitan areas of our Tunisian patients compared to the others homogenous population.

NOD2 gene mutations associate weakly with ulcerative colitis but not with Crohn's disease in Indian patients with inflammatory bowel disease.

BACKGROUND: Three mutations (two missense and one frameshift) in the NOD2 gene are associated with Crohn's disease (CD) in a proportion of patients with Crohn's disease in North America, Europe and Australia. These three mutations are not found in Indian patients with CD. We undertook new studies to identify polymorphisms in the NOD2 gene in the Indian population and to detect whether any of these were associated with inflammatory bowel disease (IBD) in this population. METHODS: Individual exons of the NOD2 gene were amplified by PCR and subjected to denaturing high performance liquid chromatography (DHPLC) to detect heteroduplex formation. All 12 exons of the NOD2 gene were amplified and Sanger-sequenced to detect polymorphisms in the NOD2 gene. 310 patients with CD, 318 patients with ulcerative colitis (UC) and 442 healthy controls (HC) were recruited for association studies. DNA from these participants was evaluated for the identified eight polymorphisms by Sequenom analysis. RESULTS: Heteroduplex formation was noted by DHPLC in exons 2 and 4 of the NOD2 gene. Sequencing of the entire NOD2 gene data revealed eight polymorphisms - rs2067085, rs2066842, rs2066843, rs1861759, rs2111235, rs5743266, rs2076753, and rs5743291 - of which the latter four were described for the first time in Indians. None of these polymorphisms was associated with CD. The SNPs rs2066842 and rs2066843 were in significant linkage disequilibrium. Both SNPs showed a significant association with UC (P=0.03 and 0.04 respectively; odds ratio 1.44 and 1.41 respectively). CONCLUSION: Four NOD2 polymorphisms were identified for the first time in the Indian population. Of 8 NOD2 polymorphisms, none were associated with CD but two were weakly associated with UC. NOD2 polymorphisms do not play a major role in CD genesis in India.

Longest form of CCTG microsatellite repeat in the promoter of the CD2BP1/PSTPIP1 gene is associated with aseptic abscesses and with Crohn disease in French patients.

PURPOSE: Aseptic abscesses syndrome (AA) is an inflammatory disease in which non-infectious deep abscesses develop; these respond quickly to corticosteroids. AA is associated with Crohn disease (CD) in 57% of cases and with neutrophilic dermatosis (ND) in 20%. Pyoderma gangrenosum is usually a sporadic ND. A hereditary autosomal dominant syndromic kind of pyoderma gangrenosum, the PAPA syndrome, is linked to mutations in the CD2BP1/PSTPIP1 gene. We systematically screened this gene in French AA patients. RESULTS: One microsatellite (CCTG)n with 3 alleles was identified in the promoter. The longest form (CCTG)7 was significantly more frequent in AA patients than in French controls (P = 0.0154). We also found an association of the (CCTG)7 allele with CD in French patients (P = 0.0351). This association was not found in a sample of Indian patients. CONCLUSIONS: The CCTG repeat in the PSTPIP1 promoter may play a role in the pathogenesis of AA and of CD. Further investigations are required to demonstrate the possible modulation of gene expression by the (CCTG)n motif.

Intra-familial phenotypic heterogeneity in adult onset vanishing white matter disease.

Vanishing white matter (VWM) disease, also known as childhood ataxia with central nervous system hypomyelination (CACH) syndrome, is an autosomal recessive transmitted leukodystrophy. Classically characterised by early childhood onset, adult onset formed with slower progression have been recently recognized. The course of neurological impairment is usually progressive with possible occasional episodes of acute deterioration following febrile illnesses or head trauma. Neurological features are dominated by cerebellar ataxia and spasticity with relatively preserved mental abilities. Brain MRI shows diffuse abnormal signal of the cerebral white matter and cystic degeneration. Mutations in one of the genes coding for the five subunits of the translation factor eukaryotic initiation factor 2B (eIF2B) have been identified. We report here on two sisters affected by adult onset VWM with variable phenotypic expression. The proband is remarkable by the very late age of the disease onset (age of 42). A homozygous p.Arg113His mutation in the eIF2Bvarepsilon gene was identified. This mutation had been recurrently associated with adult onset VWM establishing phenotype-genotype correlations. We will show an important intra-familial phenotypic variability and discuss it in the light of recent molecular progresses. External precipitating factors are contributing for some of the differences observed.

Outcome of intracytoplasmic sperm injection for a couple in which the man is carrier of CFTR p.[R74W;V201M;D1270N] and p.P841R mutations and his spouse a heterozygous carrier of p.F508del mutation of the cystic fibrosis transmembrane conductance regulator gene.

OBJECTIVE: To document the phenotype associated with the p.[R74W;V201M;D1270N] and p.P841R mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. DESIGN: Case report. SETTING: Biology and medicine of reproduction in a university hospital. PATIENT(S): A couple in which the man is carrier of the triple mutant p.[R74W;V201M;D1270N] allele in trans to p.P841R mutation and his spouse a heterozygous carrier for the severe p.F508del mutation of the CFTR gene, who became pregnant after intracytoplasmic sperm injection (ICSI) with twins. INTERVENTION(S): Genetic counseling; CFTR gene sequencing; ICSI; children's follow-up. MAIN OUTCOME MEASURE(S): First report of a male phenotype associated with the p.P841R mutation. RESULT(S): The triple mutant p.[R74W;V201M;D1270N] allele associated with the unknown p.P841R mutations were detected in this man with congenital bilateral absence of the vas deferens, which may presume p.P841R as a severe mutation. After genetic counseling, the couple preferred prenatal diagnosis after ICSI than preimplantation genetic diagnosis, which revealed that the boys were both carriers of p.[R74W;V201M;D1270N] and p.F508del mutations. They are now 4 years old and show normal growth without nutritional deficiency. CONCLUSION(S): This case report documents for the first time a male phenotype associated with the p.P841R mutation and underlines the difficulties in counseling a man with congenital bilateral absence of the vas deferens carrying uncommon mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene before ICSI.

Analysis of the NOD2/CARD15 gene in patients affected with the aseptic abscesses syndrome with or without inflammatory bowel disease.

OBJECTIVES: NOD2/CARD15 is a susceptibility gene for Crohn's disease (CD). It is also involved, via different mutations, in the Blau syndrome. The syndrome of aseptic abscesses (AA) is characterized by visceral sterile collections of mature neutrophils that do not respond to antibiotics but regress quickly with corticosteroids. It is associated in two cases out of three with inflammatory bowel disease (IBD), and in particular with CD. We wanted to assess if changes on gene NOD2/CARD15 could contribute to the development of AA in patients with and without IBD. METHODS: Seventeen unrelated patients with AA from the French national register were genotyped for c.802C>T (p.Pro268Ser) and the three main CD associated variants, c.2104C>T (p.Arg702Trp), c.2722G>C (p.Gly908Arg) and c.3019_3020insC (p.Leu1007fsX1008), and 16 were screened for the 11 coding exons of NOD2/CARD15. RESULTS: The main variants associated with CD were found at a similar frequency in patients free of IBD and in those with CD. There was no significant difference in the main variants between patients with CD and those without IBD in our study and patients with CD and controls, respectively, from a large study of an ethnically similar population. No rare variant was found. A significant association between carriers of the silent variant c.1377 C>T and markers of severity of AA was observed. CONCLUSIONS: These results suggest that the emergence of AA is not closely related to gene NOD2/CARD15. NOD2/CARD15 and other susceptibility genes might enhance the expression of AA as the result of a combination of polymorphisms.

A potential genomic biomarker for the detection of polycyclic aromatic hydrocarbon pollutants: multidrug resistance gene 49 in Drosophila melanogaster.

Polycyclic aromatic hydrocarbons (PAHs) are a major source of air, water, and soil pollution. The multidrug resistance (mdr)/permeability glycoprotein (P-gp) complex is implicated in the multidrug resistance pattern developed against various drugs and xenobiotics, including polycyclic aromatic hydrocarbons. In order to develop a genomic biomarker, we investigated the response of the mdr49 gene (mdr49) of Drosophila melanogaster to PAHs. Structural analysis of mdr49-PA, which is the putative protein expressed from Drosophila mdr49 gene, demonstrated that this transmembrane protein indeed belongs to the adenosine triphosphate-binding cassette transporter superfamily. Polymerase chain reaction (PCR) and real-time PCR analysis revealed that the mdr49 gene is expressed continuously at all the stages of fly development, including embryos, pupae, larvae, and adults, as well as in embryonic Drosophila S12 cells. In the adult fly, the mdr49 gene was expressed in all the analyzed segments (head, thorax, and abdomen) and organs (olfactory and sexual organs). The quantification of mdr49 transcripts by real-time PCR in adult flies exposed to benzo[a]pyrene over time or in presence of increasing concentrations of this pollutant showed a clear dose-dependent response. Similarly, mdr49 gene expression increased after adult flies were exposed to structurally varied PAHs. The detection of tested PAHs by Drosophila P-gp efflux pump was checked by flow cytometry.

Mouse SCO-spondin, a gene of the thrombospondin type 1 repeat (TSR) superfamily expressed in the brain.

SCO-spondin is specifically expressed in the subcommissural organ (SCO), a secretory ependymal differentiation lining the roof of the third ventricular cavity of the brain. When released into the cerebro-spinal fluid (CSF), SCO-spondin aggregates and forms Reissner's fiber (RF), a structure present in the central canal of the spinal cord. SCO-spondin belongs to the superfamily of proteins exhibiting conserved motifs called TSRs for 'thrombospondin type 1 repeats' and involved in axonal pathfinding during development. The mouse SCO-spondin coding sequence was searched by alignement of the coding bovine SCO-spondin sequence with the mouse whole genome shotgun (WGS) supercontig (NW 000250). Compared to the bovine, mouse SCO-spondin shows 66.8% identity of amino acids. This extracellular matrix glycoprotein has a modular arrangement of several conserved domains including 25 TSRs, 10 low-density lipoprotein receptor (LDLr) type A repeats and cystein-rich regions in the -NH2 and -COOH ends. The spatio-temporal expression of SCO-spondin was analyzed using specific antisera and an homospecific SCO-spondin riboprobe. In the adult, the patterns obtained by in situ hybridization (ISH) and immunohistochemistry correlated well in the SCO, while Reissner's fiber and the ampulla caudalis were immunoreactive only. In the fetus, both the immuno and ISH reactions appeared between 14 and 15 days post coïtum (dpc) in the SCO anlage. In addition, the mouse SCO-spondin gene was located at chromosome 6, between marker D6Mit352 and D6Mit119, in a conserved syntenic region.

Detection of trisomy 21 by quantitative fluorescent-polymerase chain reaction in uncultured amniocytes.

Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.