Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR.

Genetic testing for cystic fibrosis and CFTR-related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next-generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

α-Thalassemia syndromes in the United Arab Emirates.

α-Thalassemia (α-thal) is usually due to deletions within the α-globin gene cluster, leading to loss of function of one or both α-globin genes. α-Thalassemia is prevalent in the Arabian Peninsula, particularly in the United Arab Emirates (UAE) and Saudi Arabia. There are no large-scale reports regarding the prevalence of α-thal in the Arabian populations apart from sporadic surveys in the mid-1980s on red cell indices from Saudi Arabia and a more recent study from Kuwait. Several studies were conducted in an attempt to elucidate the frequency of α-thal in the UAE. Cord blood samples were collected from 419 consecutive newborns of UAE national mothers. The study involved polymerase chain reaction (PCR)-based analysis of the α-globin genes and sequencing using an ABI Genetic Analyser 3130. The findings demonstrated that 49% of the neonates had α-thal, one of the highest in the world. The incidence of α-thal, particularly the -α(3.7) deletion, was extremely high. The distribution of mutations was extensive, ranging from the simple -α(3.7) genotype to severe nondeletional type α-thalassemias such as the polyadenylation signal (polyA1) [α(PA-1) (AATAAA>AATAAG)], polyA2 [α(PA-2) (AATAAA>AATGAA)], Hb Constant Spring [Hb CS, α142 (α(CS)α/α(CS)α) TAA>CAA (α2)] and pentanucleotide deletion [α(-5 nt) (GAGGTGAGG>GAGG)]. The nondeletional mutations, denoted α(T)α or αα(T), are markedly frequent in the UAE and are well characterized. The report here describes the analysis of 84 chromosomes having deletional and nondeletional types of α-thal. Of the 84 chromosomes, 47.4% had the polyA1 mutation (α(PA-1)α), 28.2% had the small deletion -α(3.7), 11.5% had Hb CS (α(CS)α), and 5% were positive for the pentanucleotide deletion (α(-5 nt)α). These findings show that nondeletional α-thal has a significant impact on the genotype/phenotype correlation in the UAE. It is important to note that the polyA1 mutation accounted for almost 50% of all the α-thal alleles, making it one of the most common mutations in the Gulf Region.

Molecular basis of ß-thalassemia in the United Arab Emirates.

In an attempt to define the prevalence of ß-thalassemia (ß-thal) in the United Arab Emirates (UAE), we have conducted molecular studies on nearly 2000 randomly-selected adult UAE nationals. The results demonstrated that the prevalence of ß-globin gene defects in the UAE was 8.5%. Among these anomalies were ß-thal mutations, abnormal hemoglobin (Hb) variants, e.g., Hb S, Hb D-Punjab, Hb O-Arab, Hb C and Hb E. The sickle gene (ß(S) or Hb S) contributed significantly to the molecular epidemiology of the hemoglobinopathies in the UAE. In this article, Hb S and other abnormal Hbs are excluded as they are comprehensively described by other contributors in this current issue. The molecular characterization and mutational analyses of all ß-thal patients were carried out using current molecular techniques including amplification refractory mutation system (ARMS), restriction enzyme analysis (REA), dot-blot hybridization, ß-strip hybridization, allele-specific oligonucleotide (ASO), polymerase chain reaction (PCR), gap-PCR and DNA Sequencing. Most of these techniques are now virtually obsolete. Almost all molecular characterizations are currently performed through PCR followed by DNA sequencing using a fully automated ABI PRISM™ 3130 Genetic Analyzer. Our molecular studies showed that the majority of the ß-thal mutations in the UAE are very severe; the most common allele was the IVS-I-5 (G>C). Although this allele is a ß(+)-thal, its phenotype is very severe. All other mutations are also severe ß(0)-thal. High frequency of moderate or severe ß-thal mutations have implications in the wide spectrum of clinical manifestations seen in patients whose phenotypes vary from ß-thal intermedia (ß-TI) to severe transfusion-dependent ß-thal major (ß-TM). The molecular pathology of the ß-thal patients demonstrated that a vast majority were homozygous. The most frequent homozygous mutation was the IVS-I-5(G>C)/IVS-I-5(G>C) (53.0%) followed by -25 bp del/-25 bp del (6.8%), codons 8/9(+G)/codons 8/9(+G) (2.8%) and codon 39(C>T)/codon 39(C>T) (2.4%). Four mutations accounted for 65.0% of the homozygous patient population. Remarkably, the two most prevalent mutations, IVS-I-5 and Hb S, accounted for 77% of all the homozygous ß-thal patients from the UAE. We showed 13 discrete homozygosities in the UAE national patients in contrast to 23 homozygosities in the expatriate population. Since the number of homozygous mutations has a direct correlation with the degree of consanguinity, the data shown here corroborate the social tendency towards family planning. In fact, in the UAE, more than 50% of all marriages are between relatives and more than half of these are between first cousins.

Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.

BACKGROUND: alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. METHODS: We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). RESULTS: Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. CONCLUSIONS: The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.

The cypriot and Iranian National Mutation Frequency Databases.

The National Mutation Frequency Databases are continuously updated mutation depositories, which contain extensive information over the described genetic heterogeneity of an ethnic group or population. Here, we report the construction of the Cypriot (http://www.goldenhelix.org/cypriot) and Iranian National Mutation Frequency Databases (http://www.goldenhelix.org/iranian), both derived from an academic effort to provide high quality and up-to-date information on the underlying genetic heterogeneity of inherited disorders in the Cypriot and Iranian populations, respectively. Both databases have been built and maintained online using ETHNOS platform, a specialized software, which provides the means for national mutation database construction and curation. Each database contains brief summaries of the various genetic disorders studied for each population, and an easy-to-use query interface provides, both to specialist as well as to non-specialist users (i.e. patients and their families), instant access to the list and frequencies of the different mutations responsible for the inherited disorders in these populations. Furthermore, numerous links to the respective Online Mendelian Inheritance in Man (OMIM) entries and, when available, to the locus-specific databases fruitfully integrate the databases content into a single Web site. Both databases can serve as valuable online tools for molecular genetic testing of inherited disorders in these populations and could potentially motivate further investigations of yet unknown genetic diseases in the Cypriot and Iranian populations.