Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

One-step NGS molecular analysis of the CFTR gene on newborn dried blood spots gives a higher diagnostic sensitivity in affected and carrier subjects: A pilot study.

BACKGROUND: Cystic fibrosis is the most common hereditary recessive disease with an incidence of about 1:2500/3000. It has long been known that the disease is caused by deleterious mutations in the CFTR gene. Conventionally, the disease is diagnosed in several phases. The analysis of all the possible disease-causing molecular alterations is time consuming and may not lead to a definitive diagnosis in several cases. Consequently, we propose, in this paper, a rapid sequencing method that, in a single procedural asset, reveals the presence of small mutations and also the copy number variants (CNVs) from the DNA extracted from the Guthrie Spot. MATERIALS AND METHODS: We first sequenced 30 blood spots, then we validated the method on 100 spots that underwent both traditional analyses and this complete NGS sequencing, and lastly, we tested the strategy on patients who normally do not reach the molecular sequencing step because of low level of Immune-Reactive Trypsinogen. RESULTS: Using this procedure, we identified 97 variants in the CFTR gene of our samples and 6 CNVs. Notably, the significant data were obtained in the group of patients with borderline or negative IRT who routinely would not undergo molecular testing. We also identified 6 carriers of "disease-causing" variants. CONCLUSION: This method is very robust. Indeed, there was a 100% concordance with Sanger sequencing validation, and 6 mutation carriers were identified who normally escaped molecular testing with actual conventional procedure. There were also 3 duplications of almost the entire gene in heterozygosity, which were not seen with traditional methods. Being quick and easy to perform, we suggest that complete sequencing of the CFTR gene, as in this study be considered for all newborns.

C-H Amination via Nitrene Transfer Catalyzed by Mononuclear Non-Heme Iron-Dependent Enzymes.

Expanding the reaction scope of natural metalloenzymes can provide new opportunities for biocatalysis. Mononuclear non-heme iron-dependent enzymes represent a large class of biological catalysts involved in the biosynthesis of natural products and catabolism of xenobiotics, among other processes. Here, we report that several members of this enzyme family, including Rieske dioxygenases as well as α-ketoglutarate-dependent dioxygenases and halogenases, are able to catalyze the intramolecular C-H amination of a sulfonyl azide substrate, thereby exhibiting a promiscuous nitrene transfer reactivity. One of these enzymes, naphthalene dioxygenase (NDO), was further engineered resulting in several active site variants that function as C-H aminases. Furthermore, this enzyme could be applied to execute this non-native transformation on a gram scale in a bioreactor, thus demonstrating its potential for synthetic applications. These studies highlight the functional versatility of non-heme iron-dependent enzymes and pave the way to their further investigation and development as promising biocatalysts for non-native metal-catalyzed transformations.

Endophytic biocatalysts with enoate reductase activity isolated from Mentha pulegium.

The biotransformation of (4R)-(-)-carvone by Mentha pulegium (pennyroyal) leaves and its endophytic bacteria was performed in order to search for novel biocatalysts with enoate reductase activity. The obtained results clearly indicated that endophytes play an important role in the biotransformation of (4R)-(-)-carvone with pennyroyal plant tissues. The best activity was associated to the endophytic bacteria Pseudomonas proteolytica FM18Mci1 and Bacillus sp. FM18civ1. Enoate reductase activity for the reduction of (4R)-(-)-carvone and (4S)-(+)-carvone as model substrates was evaluated for each strain. Finally, both isolated strains were evaluated for the kinetic resolution of racemic carvone. The two bacteria gave (1R, 4R) or (1R, 4S)-dihydrocarvone as major products. P. proteolytica FM18Mci1 had preference for the 4S-(-)-carvone, reaching a conversion 95% in 24 h. In contrast, Bacillus sp. FM18civ1 had preference for (4R)-(-)-carvone. The results obtained in the kinetic resolution of carvone indicated that the Bacillus strain could be useful for resolving a racemic mixture of carvone.

Twelve Novel Mutations in the SLC26A3 Gene in 17 Sporadic Cases of Congenital Chloride Diarrhea.

OBJECTIVES: We aimed to improve the knowledge of pathogenic mutations in sporadic cases of congenital chloride diarrhea (CCD) and emphasize the importance of functional studies to define the effect of novel mutations. METHODS: All member 3 of solute carrier family 26 (SLC26A3) coding regions were sequenced in 17 sporadic patients with CCD. Moreover, the minigene system was used to analyze the effect of 2 novel splicing mutations. RESULTS: We defined the SLC26A3 genotype of all 17 patients with CCD and identified 12 novel mutations. Using the minigene system, we confirmed the in silico prediction of a complete disruption of splicing pattern caused by 2 of these novel mutations: the c.971+3_971+4delAA and c.735+4_c.735+7delAGTA. Moreover, several prediction tools and a structure-function prediction defined the pathogenic role of 6 novel missense mutations. CONCLUSIONS: We confirm the molecular heterogeneity of sporadic CCD adding 12 novel mutations to the list of known pathogenic mutations. Moreover, we underline the importance, for laboratories that offer molecular diagnosis and genetic counseling, to perform fast functional analysis of novel mutations.

Identification, expression and characterization of an R-ω-transaminase from Capronia semiimmersa.

Chiral amines are essential precursors in the production of biologically active compounds, including several important drugs. Among the biocatalytic strategies that have been developed for their synthesis, the use of ω-transaminases (ω-TA) appears as an attractive alternative allowing the stereoselective amination of prochiral ketones. However, the problems associated with narrow substrate specificity, unfavourable reaction equilibrium and expensive amine donors still hamper its industrial application. The search for novel enzymes from nature can contribute to expand the catalytic repertoire of ω-TA and help to circumvent some of these problems. A genome mining approach, based on the work described by Höhne et al., was applied for selection of potential R-ω-TA. Additional criteria were used to select an enzyme that differs from previously described ones. A candidate R-ω-TA from Capronia semiimmersa was selected, cloned and expressed in Escherichia coli. Interestingly, alignment of this enzyme with previously reported TA sequences revealed the presence of two additional amino acid residues in a loop close to the active site. The impact of this change was analysed with a structural model based on crystallized R-ω-TAs. Analysis of the substrate specificity of R-ω-TA from C. semiimmersa indicates that it accepts a diversity of ketones as substrates yielding the corresponding amine with good yields and excellent enantioselectivity. The expressed enzyme accepts isopropylamine as amine donor what makes it suitable for industrial processes.

A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII).

Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL ß-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.

Saturation mutagenesis in selected amino acids to shift Pseudomonas sp. acidic lipase Lip I.3 substrate specificity and activity.

Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed. Substitution of residues Y29 and W310 by smaller amino acids provided increased activity on C18-substrates. Residues G152 and S154, modified to study their influence on interfacial activation, displayed a five and eleven fold increased activity.

An update on laboratory diagnosis of liver inherited diseases.

Liver inherited diseases are a group of genetically determined clinical entities that appear with an early chronic liver involvement. They include Wilson's disease (hepatolenticular degeneration), hereditary hemochromatosis, and alpha-1-antitrypsin deficiency. In addition, cystic fibrosis, although it is not specifically a liver disease, may cause a severe liver involvement in a significant percentage of cases. For all these pathologies, the disease gene is known, and molecular analysis may contribute to the unequivocal diagnosis. This approach could avoid the patient invasive procedures and limit complications associated with a delay in diagnosis. We review liver inherited diseases on the basis of the genetic defect, focusing on the contribution of molecular analysis in the multistep diagnostic workup.

Omics in laboratory medicine.

In the last years, molecular diagnosis of human genetic diseases has greatly improved thanks to the knowledge of more than 7,000 disease genes (genomics). However, the study of such diseases revealed the very complex relationships between the phenotype of each disease and the molecular alterations responsible. The analysis of proteins (proteomics) revealed that most proteins are subjected to post-translational changes or to alternative splicing; the study of gene expression identified a series of mechanisms that modulate gene expression (epigenomics) which include microRNA regulation, histone acetylation and gene methylation. The alteration of all these mechanisms may contribute to the pathogenesis or to the phenotypic expression of most human genetic diseases. Molecular analysis became more and more complex, but "omics" studies revealed that each single individual is "unique".

Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis?

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3' untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

Molecular and functional analysis of the large 5' promoter region of CFTR gene revealed pathogenic mutations in CF and CFTR-related disorders.

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

Three novel CFTR polymorphic repeats improve segregation analysis for cystic fibrosis.

BACKGROUND: Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. METHODS: We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. RESULTS: The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. CONCLUSIONS: The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.