Whole Genome Sequencing Revealed Inherited Rare Oligogenic Variants Contributing to Schizophrenia and Major Depressive Disorder in Two Families.

Schizophrenia and affective disorder are two major complex mental disorders with high heritability. Evidence shows that rare variants with significant clinical impacts contribute to the genetic liability of these two disorders. Also, rare variants associated with schizophrenia and affective disorders are highly personalized; each patient may carry different variants. We used whole genome sequencing analysis to study the genetic basis of two families with schizophrenia and major depressive disorder. We did not detect de novo, autosomal dominant, or recessive pathogenic or likely pathogenic variants associated with psychiatric disorders in these two families. Nevertheless, we identified multiple rare inherited variants with unknown significance in the probands. In family 1, with singleton schizophrenia, we detected four rare variants in genes implicated in schizophrenia, including p.Arg1627Trp of LAMA2, p.Pro1338Ser of CSMD1, p.Arg691Gly of TLR4, and Arg182X of AGTR2. The p.Arg691Gly of TLR4 was inherited from the father, while the other three were inherited from the mother. In family 2, with two affected sisters diagnosed with major depressive disorder, we detected three rare variants shared by the two sisters in three genes implicated in affective disorders, including p.Ala4551Gly of FAT1, p.Val231Leu of HOMER3, and p.Ile185Met of GPM6B. These three rare variants were assumed to be inherited from their parents. Prompted by these findings, we suggest that these rare inherited variants may interact with each other and lead to psychiatric conditions in these two families. Our observations support the conclusion that inherited rare variants may contribute to the heritability of psychiatric disorders.

Molecular Modeling Unveils the Effective Interaction of B-RAF Inhibitors with Rare B-RAF Insertion Variants.

The Food and Drug Administration (FDA) has approved MAPK inhibitors as a treatment for melanoma patients carrying a mutation in codon V600 of the BRAF gene exclusively. However, BRAF mutations outside the V600 codon may occur in a small percentage of melanomas. Although these rare variants may cause B-RAF activation, their predictive response to B-RAF inhibitor treatments is still poorly understood. We exploited an integrated approach for mutation detection, tumor evolution tracking, and assessment of response to treatment in a metastatic melanoma patient carrying the rare p.T599dup B-RAF mutation. He was addressed to Dabrafenib/Trametinib targeted therapy, showing an initial dramatic response. In parallel, in-silico ligand-based homology modeling was set up and performed on this and an additional B-RAF rare variant (p.A598_T599insV) to unveil and justify the success of the B-RAF inhibitory activity of Dabrafenib, showing that it could adeptly bind both these variants in a similar manner to how it binds and inhibits the V600E mutant. These findings open up the possibility of broadening the spectrum of BRAF inhibitor-sensitive mutations beyond mutations at codon V600, suggesting that B-RAF V600 WT melanomas should undergo more specific investigations before ruling out the possibility of targeted therapy.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.

[Cystic fibrosis - ETI and type 2 N-of-1 trials : the next step]. / Mucoviscidose : valider un traitement révolutionnaire par de brefs essais thérapeutiques individuels bien supervisés.

In evidence-based medicine, N-of-1 trials are increasingly attractive for rare and heterogeneous conditions. A recent French study illustrates this convincingly in the field of cystic fibrosis. A highly effective triple therapy (ETI) is currently available in Europe, which will eventually help the 85 % of Belgian patients carrying at least one copy of the F508del mutation. Most other 2.000 or so putative mutations of this gene are poorly characterised and very rare or private. To predict the efficacy of ETI at the individual level in currently ineligible patients, sophisticated tools are advocated, but they are expensive, not widely available, often partially standardised and there still remains a «grey area¼ concerning their reliability in this context. With-out using them, the French study suggests that more than half of these patients show clinically meaningful responses to a 4-6 weeks trial of ETI. What makes this pragmatic, cost-effective, non-invasive and simplified approach possible (type 2 N-of-1 trials) is the dramatic and rapid efficacy of a life-saving treatment without alternative and the fact that it can be assessed using simple and robust clinical and paraclinical outcomes. Here, we describe one such trial and discuss the value and limitations of this approach.

Dans la médecine basée sur les preuves, les essais de taille 1 suscitent un intérêt croissant dans les affections rares et hétérogènes. Une récente étude française l'illustre de manière convaincante dans la mucoviscidose. Une trithérapie extrêmement efficace (ETI) est actuellement disponible en Europe, concernant à terme en Belgique les 85 % de patients porteurs d'au moins une copie de la mutation F508del. La majorité des quelque 2.000 autres mutations putatives de ce gène sont mal caractérisées et rarissimes. Des techniques sophistiquées sont évoquées pour prédire, à l'échelle individuelle, l'efficacité d'ETI chez les patients actuellement non éligibles, mais elles sont peu disponibles, coûteuses, souvent imparfaitement standardisées et leur interprétabilité conserve une «zone grise¼. Sans y recourir, l'étude française montre que plus de la moitié de ces patients répondent d'une manière évidente à un essai d'ETI pendant quelques semaines seulement. Ce qui permet cette approche pragmatique, économique, non invasive et simplifiée (essai de taille 1, de type 2), c'est l'efficacité spectaculaire et rapide d'un traitement salvateur sans alternative et le fait qu'elle puisse être appréhendée à partir de critères cliniques et paracliniques simples et robustes. Nous rapportons ici un essai de ce type et discutons l'intérêt et les limites de cette approche.

Rare and New Mutations of B-Globin in Azari Population of Iran, a Considerable Diversity.

Background: Thalassemia, as the most common single-gene genetic disorder, is related to a defect in the synthesis of one or more hemoglobin chains. More than 200 mutations have been identified in the ß-globin gene. Globally, every susceptible racial group has its own specific spectrum of the common mutations that are well-known to a particular geographic region. On the other hand, varying numbers of diverse rare mutations may occur. Materials and Methods: The subjects of the study included 2113 heterozygote or homozygote ß-thalassemia cases selected among couples who participated in the Iranian national thalassemia screening program from January 2011 to November 2019. Molecular characterization of the ß-thalassemia mutation was initially carried out by the amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) technique for common mutations, followed by sequencing, Gap PCR, and Multiple ligation-dependent probe amplification (MLPA) methods - in cases not detected by the ARMS-PCR. Results: The existence of 39 rare and new point mutations and 4 large deletions were described in our cohort. Sicilian (-13,337bp) deletion, CD36/37 (-T), and CD15 TGG>TGA were encountered more often than the others in a decreasing order, in terms of frequency. The least frequent mutations/deletions were deletion from HBD exon 1 to HBB promoter, 619 bp deletion, Deletion from up HBBP1-Exon3 HBBP1 and up HBB-0.5Kb down HBB, CAP+8 C>A, CD37 (G>A), CD6 (-A), IVSI-2 (T>C), IVSII-705 T>G, and IVSII-772 (G>A). Each occurred once. Five mutations/variants were also determined which have not been reported previously in Iran. Conclusion: According to the findings of the study, the Northwestern Iranian population displayed a wide variety of thalassemia allelic distributions. Identification of rare and new mutations in the ß-thalassemia in the national population is beneficial for screening programs, genetic counseling, and prenatal diagnosis.

KATK: Fast genotyping of rare variants directly from unmapped sequencing reads.

KATK is a fast and accurate software tool for calling variants directly from raw next-generation sequencing reads. It uses predefined k-mers to retrieve only the reads of interest from the FASTQ file and calls genotypes by aligning retrieved reads locally. KATK does not use data about known polymorphisms and has NC (no call) as the default genotype. The reference or variant allele is called only if there is sufficient evidence for their presence in data. Thus it is not biased against rare variants or de-novo mutations. With simulated datasets, we achieved a false-negative rate of 0.23% (sensitivity 99.77%) and a false discovery rate of 0.19%. Calling all human exonic regions with KATK requires 1-2 h, depending on sequencing coverage.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies.

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.

Rare mutations in the autophagy-regulating gene AMBRA1 contribute to human neural tube defects.

Neural tube defects (NTDs) are severe congenital malformations caused by failed neural tube closure. Recently, autophagy is revealed to play a vital role in neuroepithelium development and neurulation. Autophagy and beclin 1 regulator 1 (Ambra1) is a crucial regulator of autophagy initiation, and its deficiency in mice leads to exencephaly and/or spina bifida. However, the genetic contribution of AMBRA1 to the etiology of human NTDs remains unknown. In this study, we identified five rare missense mutations of AMBRA1 in 352 NTDs cases, which were absent in 224 matched controls. Western blotting and fluorescence puncta counting for MAP1LC3A/LC3 in HEK293T cells suggested that four of the mutations (AMBRA1 p.Thr80Met, p.Leu274Phe, p.Ser743Phe, and p.Met884Val) affected autophagy initiation to various extents. Furthermore, these four mutations also displayed loss-of-function effects compared with wild-type AMBRA1 when we injected messenger RNA (mRNA) to overexpress or rescue ambra1a-morpholino oligos (MO) knockdown in zebrafish. It is intriguing that trehalose, a natural disaccharide, could rescue ambra1a-MO knockdown in a dose-dependent manner independently or together with AMBRA1 mRNA. Taken together, our findings suggest that rare mutations of the autophagy regulator gene AMBRA1 may contribute to the etiology of human neural tube defects, and trehalose is a promising treatment for a subset of NTDs caused by autophagy impairment.

Genetics and mechanisms leading to human cortical malformations.

Cerebral cortical development involves a complex series of highly regulated steps to generate the laminated structure of the adult neocortex. Neuronal migration is a key part of this process. We provide here a detailed review of cortical malformations thought to be linked to abnormal neuronal migration. We have focused on providing updated views related to perturbed mechanisms based on the wealth of genetic information currently available, as well as the study of mutant genes in animal models. We discuss mainly type 1 lissencephaly, periventricular heterotopia, type II lissencephaly and polymicrogyria. We also discuss functional classifications such as the tubulinopathies, and emphasize how modern genetics is revealing genes mutated in atypical cases, as well as unexpected genes for classical cases. A role in neuronal migration is revealed for many mutant genes, although progenitor abnormalities also predominate, depending on the disorder. We finish by describing the advantages of human in vitro cell culture models, to examine human-specific cells and transcripts, and further mention non-genetic mechanisms leading to cortical malformations.

Enrichment of rare genetic variants in astrocyte gene enriched co-expression modules altered in postmortem brain samples of schizophrenia.

The transcriptome profiles of the cingulate gyrus region from the postmortem brain tissues of a set of well-characterized patients with schizophrenia (SCZ) and matched controls were investigated using an integrated approach that analyzed both the alterations in transcription expression pattern and rare genetic variants in expressed genes. We demonstrated increased expression of astrocyte-related genes using spatiotemporal co-expression modules that have previously been established for developing human brain, and showed these results are independent of medication dosage. The relationship between genetic variants and expression pattern in the context of neurodevelopment was further investigated, and we identified an enrichment of rare genetic variants in a set of signature genes that were specific to astrocytes and up-regulated in the patients with SCZ. Our result suggested the involvement of astrocyte malfunction in SCZ pathophysiology. In addition, our approach indicated a novel strategy of narrowing down genetic variants that might contribute to the pathophysiology in the patients with SCZ to a subset of genes that are highly expressed in an affected brain region.

First Report on the Coinheritance of α-Thalassemia and a Rare ß-Thalassemia Compound Heterozygosity for the IVS-I-I(G>A)/IVS-II-705(T>G) Mutations in a Syrian Family.

We describe a proband originating from Al-Quneitra Province, Syria. His hematology data was as follows: Hb A 24.7%, Hb F 71.1%, Hb A2 4.2%. Molecular analysis, based on DNA sequencing of the ß-globin gene mutation, showed for the first time a compound heterozygous IVS-I-1 (G>A) (HBB: c.92+1G>A)/IVS-II-705 (T>G) (HBB: c.316-146T>G) on the ß-globin gene. A reverse hybridization technique revealed that the proband was also an α-thalassemia (α-thal) -α3.7 (rightward) deletion carrier. Haplotypes analysis for the seven polymorphic restriction sites showed that the compound heterozygous mutations, IVS-I-1/IVS-II-705, were linked to two haplotypes: I [+ - - - - + +] and VI [- + + - - - +], respectively. Our results showed, for the first time, the presence of rare ß-thalassemia (ß-thal) IVS-II-705 (T>G) mutation associated with IVS-I-1 (G>A). Our findings suggest the presence of these mutations resulted from past migrations.

Heterogeneous Responses to Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKIs) in Patients with Uncommon EGFR Mutations: New Insights and Future Perspectives in this Complex Clinical Scenario.

Uncommon Epidermal Growth Factor Receptor (EGFR) mutations represent a distinct and highly heterogeneous subgroup of Non-Small Cell Lung Cancers (NSCLCs), that accounts for approximately 10% of all EGFR-mutated patients. The incidence of uncommon EGFR mutations is growing, due to the wider adoption of next-generation sequencing (NGS) for diagnostic purposes, which enables the identification of rare variants, usually missed with available commercial kits that only detect a limited number of EGFR mutations. However, the sensitivity of uncommon mutations to first- and second-generation EGFR Tyrosine Kinase Inhibitors (TKIs) is widely heterogeneous and less well known, compared with classic mutations (i.e., exon 19 deletions and exon 21 L858R point mutation), since most of the pivotal studies with EGFR TKIs in the first line, with few exceptions, excluded patients with rare and/or complex variants. Recently, the third generation EGFR TKI osimertinib further revolutionized the therapeutic algorithm of EGFR-mutated NSCLC, but its role in patients harboring EGFR mutations besides exon 19 deletions and/or L858R is largely unknown. Therefore, a better knowledge of the sensitivity of uncommon mutations to currently available EGFR TKIs is critical to guiding treatment decisions in clinical practice. The aim of this paper is to provide a comprehensive overview of the treatment of NSCLC patients harboring uncommon EGFR mutations with currently approved therapies and to discuss the emerging therapeutic opportunities in this peculiar subgroup of patients, including chemo-immunotherapy combinations, next-generation EGFR TKIs, and novel targeted agents.

Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing.

We present a genome-wide comparative and comprehensive analysis of three different sequencing methods (conventional next generation sequencing (NGS), tag-based single strand sequencing (e.g., SSCS), and Duplex Sequencing for investigating mitochondrial mutations in human breast epithelial cells. Duplex Sequencing produces a single strand consensus sequence (SSCS) and a duplex consensus sequence (DCS) analysis, respectively. Our study validates that although high-frequency mutations are detectable by all the three sequencing methods with the similar accuracy and reproducibility, rare (low-frequency) mutations are not accurately detectable by NGS and SSCS. Even with conservative bioinformatical modification to overcome the high error rate of NGS, the NGS frequency of rare mutations is 7.0 × 10-4. The frequency is reduced to 1.3 × 10-4 with SSCS and is further reduced to 1.0 × 10-5 using DCS. Rare mutation context spectra obtained from NGS significantly vary across independent experiments, and it is not possible to identify a dominant mutation context. In contrast, rare mutation context spectra are consistently similar in all independent DCS experiments. We have systematically identified heat-induced artifactual variants and corrected the artifacts using Duplex Sequencing. Specific sequence contexts were analyzed to examine the effects of neighboring bases on the accumulation of heat-induced artifactual variants. All of these artifacts are stochastically occurring rare mutations. C > A/G > T, a signature of oxidative damage, is the most increased (170-fold) heat-induced artifactual mutation type. Our results strongly support the claim that Duplex Sequencing accurately detects low-frequency mutations and identifies and corrects artifactual mutations introduced by heating during DNA preparation.

MIB1 mutations reduce Notch signaling activation and contribute to congenital heart disease.

Congenital heart disease (CHD) is one of the most common birth defects in humans, but its genetic etiology remains largely unknown despite decades of research. The Notch signaling pathway plays critical roles in embryonic cardiogenesis. Mind bomb 1 (Mib1) is a vital protein that activates the Notch signaling pathway through promoting ubiquitination, endocytosis and subsequent activation of Notch ligands. Previous studies show that Mib1 knockout in mice completely abolishes Notch signaling, leading to cardiac deformity. However, the function of MIB1 and its potential disease-causing mutations are poorly studied in human CHD. In this research, we identified four novel non-synonymous heterozygous rare mutations of MIB1 from 417 Han Chinese CHD patients. The following biochemical analyses revealed that mutations p.T312K fs*55 and p.W271G significantly deplete MIB1's function, resulting in a lower level of JAGGED1 (JAG1) ubiquitination and Notch signaling induction. Our results suggest that pathologic variants in MIB1 may contribute to CHD occurrence, shedding new light on the genetic mechanism of CHD in the context of the Notch signaling pathway.

Rare FMR1 gene mutations causing fragile X syndrome: A review.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, typically due to CGG-repeat expansions in the FMR1 gene leading to lack of expression. We identified a rare FMR1 gene mutation (c.413G>A), previously reported in a single patient and reviewed the literature for other rare FMR1 mutations. Our patient at 10 years of age presented with the classical findings of FXS including intellectual disability, autism, craniofacial findings, hyperextensibility, fleshy hands, flat feet, unsteady gait, and seizures but without the typical CGG-repeat expansion. He had more features of FXS than the previously reported patient with the same mutation. Twenty individuals reported previously with rare missense or nonsense mutations or other coding disturbances of the FMR1 gene ranged in age from infancy to 50 years; most were verbal with limited speech, had autism and hyperactivity, and all had intellectual disability. Four of the 20 individuals had a mutation within exon 15, three within exon 5, and two within exon 2. The FMR1 missense mutation (c.413G>A) is the same as in a previously reported male where it was shown that there was preservation of the post-synaptic function of the fragile X mental retardation protein (FMRP), the encoded protein of the FMR1 gene was preserved. Both patients with this missense mutation had physical, cognitive, and behavioral features similarly seen in FXS.

One novel and two uncommon MEFV mutations in Japanese patients with familial Mediterranean fever: a clinicogenetic study.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in the MEFV gene and characterized by recurrent episodes of fever and polyserositis. To date, over 317 MEFV mutations have been reported, only nine of which account for almost all Japanese patients with FMF. Therefore, the prevalence of rare MEFV variants and their clinical characteristics remains unclear. This study identified MEFV mutations previously unreported in the Japanese population and described their clinical features. We performed MEFV genetic testing in 488 Japanese patients with clinically suspected FMF. Of these patients, we retrospectively analyzed three patients with novel or very uncommon MEFV mutations. In all patients, the clinical diagnosis of FMF was made according to Tel-Hashomer's criteria. One novel missense mutation (N679H) and two rare mutations (T681I and R410H) were identified in the MEFV gene. These mutations were found in compound heterozygous or complex genotypes with other known mutations in exons 1 or 2. According to clinical images, all three patients exhibited typical FMF symptoms. A number of patients with FMF caused by novel or uncommon MEFV variants might exist in the Japanese population; therefore, careful genetic testing is required for accurate diagnosis of this curable genetic disorder.

Description of a Rare ß-Globin Gene Mutation: -86 (C>G) (HBB: c.-136C>G) Observed in a Syrian Family.

We present the description of a ß-thalassemia (ß-thal) -86 (C>G) (HBB: c.-136C>G) mutation in a Syrian family from Damascus, As-Suwayda Province, Syria, who was referred to the laboratory for prenatal diagnosis (PND). The mutation was found in the mother in a homozygous state, while it was in the father and in the amniotic fluid sample in a heterozygous state. This mutation is located at -86 within the proximal CACCC box in the promoter of the ß-globin gene and is possibly linked with a phenotype of ß+-thal. Polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) analysis indicated that the -86 mutation was linked with haplotype I [+ - - - - + +]. We propose that Lebanon may be the origin of this mutation. To the best of our knowledge, this is the first report describing this mutation in As-Suwayda Province. These findings provide novel information on the region-specificity of this mutation in southwestern Syria.

mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis.

Rare ß-Globin Gene Mutations in Pakistan.

The aim of this study was to analyze the rare ß-thalassemia (ß-thal) mutations in the Pakistani population. A total of 8716 unrelated Pakistani individuals having children with transfusion-dependent thalassemia were investigated by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) for the previously reported common and rare ß-thal mutations. Genomic sequencing of the ß-globin gene and its immediate 5' and 3' flanking regions was done where no known mutation was found. Out of the 8716 individuals studied, 88 (1.0%) were not characterized by ARMS-PCR. Genomic sequencing revealed that 67 (0.82%) individuals had 19 different ß-thal mutations including one novel mutation (HBB: c.136delT). The remaining 21 (0.26%) individuals did not show any mutation on the ß-globin gene and its immediate flanking regions. The characterized alleles included seven (0.09%) in the 5' untranslated region (5'UTR), 29 (0.35%) in the coding regions, and 31 (0.38%) in the splice junction regions. HBB: c.92+1G>A and HBB: c.113G>A were the most frequently seen rare mutations. The spectrum of ß-thal mutations in the Pakistani population is very diverse. In addition to the already reported mutations, another 19 different types of mutations were found. Interestingly, 21 individuals who had children with transfusion-dependent thalassemia and one known ß-thal mutation, did not show any mutation on the ß-globin gene. HBB: c.92+1G>A and HBB: c.113G>A are the most frequently seen rare mutations in Pakistan.