Catherine Costa

Real-Time PCR Coupled with Automated DNA Extraction and Detection of Galactomannan Antigen in Serum by Enzyme-Linked Immunosorbent Assay for Diagnosis of Invasive Aspergillosis

ABSTRACT To improve the diagnosis of invasive aspergillosis (IA), we developed a LightCycler PCR assay targeted to Aspergillus fumigatus and A. flavus mitochondrial DNA. To avoid contamination, fully automated nucleic acid extraction with the MagNA Pure LC apparatus was used. The linearity of the results was achieved over a 6-log range of input A. fumigatus DNA, from 0.3 ng to 3 fg/10 μl of water. We retrospectively compared the LightCycler PCR and an enzyme-linked immunosorbent assay for the detection of galactomannan (GM) in serum from 14 patients with IA. The GM assay was more frequently positive (57 of 109; 52%) than the PCR assay (49 of 109; 45%). The LightCycler PCR assay, combined with automated DNA extraction, could be used in association with the GM assay to improve the reliability of IA diagnosis.

Analysis of microsatellite markers of Candida albicans used for rapid typing.

ABSTRACT To obtain a rapid genotyping method of Candida albicans, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, calledCDC3, EF3, and HIS3, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3,EF3, and HIS3, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans.

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.

Mutation spectrum in the French cohort of galactosemic patients and structural simulation of 27 novel missense variations

BACKGROUND Classic galactosemia refers to galactose-1-phosphate uridyltransferase (GALT) deficiency and is characterized by long-term complications of unknown mechanism and high allelic heterogeneity of GALT gene. AIM To report molecular characterization of GALT variations in 210 French families, to analyze the structural effects of novel missense variations and to assess informativity of structural data in predicting outcome. METHODS Sequencing of exons and intron-exon junctions of GALT gene was completed in unsolved cases by analysis of a long range PCR product. Structural consequences of novel missense variations were predicted using a homology model of GALT protein and a semi-automated analysis which integrates simulation of variations, structural analyses and two web servers dedicated to identify mutation-induced change of protein stability. RESULTS Forty four novel variations were identified, among them 27 nucleotide substitutions. In silico modeling of these missense variations showed that 12 variations are predicted to impair subunit interactions and/or active site conformation and that 23 variations modify H-bond or salt-bridge networks. Twenty variations decrease the global stability of the protein. Five variations had apparently no structural effect. CONCLUSION Our results expand the mutation spectrum in GALT gene and the list of GALT variations analyzed at the structural level, providing new data to assess the pathophysiology of galactosemia.

Hereditary fructose intolerance: Frequency and spectrum mutations of the aldolase B gene in a large patients cohort from France—Identification of eight new mutations

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 160 patients from 92 families by means of a PCR-based mutation screening strategy, consisting of restriction enzyme digestion and direct sequencing. Sixteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in previous studies, p.A150P (64%), p.A175D (16%) and p.N335K (5%) were the most common mutated alleles, followed by p.R60X, p.A338V, c.360_363delCAAA (p.N120KfsX30), c.324G>A (p.K108K) and c.625-1G>A. Eight novel mutations were also identified in 10 families with HFI: a one-base deletion (c.146delT (p.V49GfsX27)), a small deletion (c.953del42bp), a small insertion (c.689ins TGCTAA (p.K230MfsX136)), one splice site mutation (c.112+1G>A), one nonsense mutation (c.444G>A (p.W148X)), and three missense mutations (c.170G>C (p.R57P), c.839C>A (p.A280P) and c.932T>C (p.L311P)). Our strategy allows to diagnose 75% of HFI patients using restriction enzymatic analysis and to enlarge the diagnosis to 97% of HFI patients when associated with direct sequencing.

Mutational spectrum and DNA-based prenatal diagnosis in carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69-70insTGTGC, and 609-1g>a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

A One-Step Real-Time PCR Assay for Rapid Prenatal Diagnosis of Sickle Cell Disease and Detection of Maternal Contamination

AbstractIntroduction: Mutations at the codon 6 of the β-globin gene (hemoglobin [Hb] S and HbC) can be routinely identified by various methods and prenatal diagnosis has been available to affected families for several years. However, the presence of maternal cells in fetal samples constitutes a serious potential source of prenatal misdiagnosis and most methods currently used to detect maternal contamination are based on the analysis of highly polymorphic loci. In addition, these methods are labor intensive and time consuming and risk carry-over contamination. Method: We describe here a one-step method for mutation detection that uses fluorescent hybridization probes with melting curve analysis for both simultaneously prenatal diagnosis of sickle cell disease and potential maternal contamination. Results: Retrospective and prospective prenatal diagnosis studies (conducted in 20 and 50 cases, respectively), using both the regular procedure and real-time PCR assay show perfect concordant results. We show in addition, that as little as 5% maternal contamination can be detected and that genotype determinations are unambiguous.

Mosaicism in men in hemophilia: is it exceptional? Impact on genetic counselling

Haemophilia A is an X-linked bleeding disorder caused by a wide range of mutations in the factor VIII (F8) gene [1]. About one third of cases are due to a de novo mutation. The majority are thought to occur in a single germ cell but some, occurring during early embryogenesis, produce a germline and/or somatic mosaic. In haemophilia, somatic mosaicism has been generally observed in women and seems to represent a fairly common event [2]. We report here a case of exceptional mosaicism in the asymptomatic maternal grandfather of a haemophilia A patient. The proband has severe haemophila A with factor (F)VIIIc levels <1% and no previous family history of the disorder. Gene mutation studies were performed in order to identify the deleterious mutation and offer genetic counselling to the mother and the family. The mutation p.Arg336X in exon 8 was identified in the proband by direct sequencing and subsequently searched for in the mother and maternal grandmother. It was found only in the mother, suggesting a de novo germline mutation in one of the grandparents or a de novo somatic mutation early during embryogenesis in the mother. The maternal aunt, who had not been tested, was at first reassured as being probably not a carrier. Several years later, when undergoing medically assisted procreation because of the infertility of her partner, a genetic test was performed. Unexpectedly, the mutation p.Arg336X was identified, leading to a modification of her status as being a carrier of severe haemophilia A. The presence of the mutation in the two sisters thus first suggested the grandmother was a carrier with a somatic mosaicism. The absence of the mutation in her peripheral blood as well as in her buccal and uroepithelial cells, which have different embryological origins, then raised the question of the mechanism of occurrence of this mutation. Linkage analysis, using intragenic and extragenic markers linked to the F8 gene, actually showed that the deleterious allele originated from the asymptomatic maternal grandfather whose FVIIIc was normal FVIIIc=96% (Fig1). Figure 1 A-Family pedigree, haplotypes and mutation studies. B-Electropherogram obtained by direct sequencing. C-Denaturing High Liquid Chromatography (DHPLC) elution profiles of carrier, control and grandfather: in female carriers, heteroduplexes, which contain ... Somatic mosaicism in the grandfather was then hypothesized. However, it is well known that somatic mosaicism may be difficult to detect with conventional methods such as direct sequencing. Mutation-enrichment procedures, not used during routine test analyses, are often required [2]. Nowadays, due to technology progress, methods presenting higher sensitivity are available. One of them, denaturing-high-liquid-pressure-chromatography (DHPLC) was used in this family. DHPLC is well known for its efficiency to detect heteroduplexes that are DNA molecules containing mismatched base pairs and created during amplification reaction (PCR) when a mutation is present in heterozygosity. Under partial denaturation, heteroduplexes are eluted from the column by an acetonitrile gradient flow before homoduplexes [3]. Analysis of the grandfather’s leucocytes, buccal and uroepithelial cells showed the presence of the mutated allele with a proportion estimated between 15–20% (fig 1). Karyotype analysis showed a normal 46, XY karyotype, ruling out Klinefelter syndrome. The presence of the mutation in all tested grandpaternal tissues and in his two daughters suggested that the mutation had arisen very early during embryonic development. The distinction between isolated or sporadic cases is of major importance in genetic counselling. A case may appear to be isolated because family size is small; DNA testing may help for carrier diagnosis but negative results will not rule out the possibility of an occult mosaic. In a recent study only a small number (11%) of maternal grandmothers of isolated cases had the mutation in their white blood cells, while 85% of mothers were carriers, which favours the hypothesis that isolated cases may have originated as a de novo germline mutation in one of the grandparents or a de novo somatic mutation early during embryogenesis in the proband’s mother [2, 4]. Somatic mosaicism has been found in around 10% of mothers of isolated cases [2] and in 13% of patients’ mothers and grandmothers in a study which used mutation enrichement procedures [5]. These results indicate that mosaicism is a fairly common event in haemophilia, but is still underestimated due to the limited sensitivities of the methods for detection of mosaicism and probably also because the distinction between carrier and 50% mosaicism is difficult. It is of note that most of the time, mosaicism has been reported in families with point mutations while only once in an isolated case with intron 22 inversion. [2, 5, 6]. Somatic mosaicism in families with apparent de novo mutations is however rarely explored in women, and grandfathers are usually not considered. In this present case we have been questioned because the proband’s mother and aunt were carriers while the grandmother was not. In the literature only three cases of mosaicism in men have been reported, all of them in grandfathers in families with point mutations |7–9]. Our case underlies that somatic mosaicism in men is probably underestimated because of the difficulties of obtaining blood sample from grandfathers, and points to the need for testing men in such apparent isolated cases. In these situations and even if the mutation is characterized, linkage analysis remains a precious help to identify the origin of the deleterious allele. Assessment of mosaicism in mothers of apparent isolated cases is now part of genetic counselling. It also seems important now to take into account the risk of mosaicism in grandfathers as well as grandmothers with a view to the genetic counselling of all their daughters.

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.

Multiplex Allele-Specific Fluorescent PCR for Haplotyping the IVS8 (TG)m(T)n Locus in the CFTR Gene

BACKGROUND Precise genotyping of the intron 8 poly(TG) and poly(T) tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is of clinical relevance in CFTR pathology. The (TG)(m) locus influences the penetrance of the (T)(5) allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens (CBAVD) or other CFTR-related disorders (CFTR-RD), in particular in the context of (TG)(12) and (TG)(13). Simple and accurate genotyping of both loci should thus be routinely offered in laboratories. METHODS We designed a new single test method relying on multiplex allele-specific fluorescent PCR: (T)(5)-, (T)(7)-, and (T)(9)-specific primers, labeled with different fluorophores, in combination with a common primer. Each fluorescent PCR product was identified on a capillary sequencer by its fluorescence color, specific for (T)(n), and size, indicative of the (TG) length. We first validated the assay in 2 different laboratories on 52 DNA samples with already known genotypes. We then evaluated the method prospectively, compared with sequencing, on 62 samples from healthy individuals and 108 samples from patients with CBAVD or other CFTR-RDs. RESULTS We observed a 100% match in both validation steps. Results found in CBAVD and CFTR-RD patients are in keeping with data in the literature. CONCLUSIONS The assay proved to be simple, rapid, and accurate for single-test (TG)(m)(T)(n) genotyping and suited for analysis in clinical laboratories.