Human inherited CCR2 deficiency underlies progressive polycystic lung disease.

We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.

Extensive genetic screening of Iranian Factor FVII-deficient individuals unraveled several novel mutations and postulated founder effects in some cases.

Background: As the most frequent congenital rare bleeding disorder that transmits in an autosomal recessive manner, factor VII (FVII) deficiency is a serious bleeding complication in populations with high rate of in-marriages. While diagnosis mainly relies on clinical and laboratory phenotypes, plasma FVII antigen and activity levels do not often correlate with symptoms' severity. Objectives: Genetic profiling of the affected individuals potentially improves our biological understanding of this complicated rare disorder. Methods: Conventional polymerase chain reaction-Sanger sequencing and whole-exome sequencing were applied for genetic profiling of F7 gene in 66 symptomatic FVII-deficient individuals from 62 independent pedigrees. Thirty-nine asymptomatic relatives of the patients were also studied. Results: Thirty different F7 pathogenic variations were identified in the studied cases of which 11 have not been reported before. The novel mutations include 5 missenses (c.715G>A, c.794T>C, c.1090C>G, c.1222C>A, c.1265T>C), 3 splicing (c.316+1G>T, c.682-2A>G, c.572-16C>G), 2 nonsenses (c.790delC, c.1248G>A), and 1 frameshift (c.1346delA). A founder effect is proposed for c.790delC that was detected in 8 independent pedigrees who were all from similar geographical regions and ethnic backgrounds. Homozygous c.790delC reduces plasma FVII activity to <1% and causes spontaneous intracranial hemorrhage in early infancy. Conclusion: From the 66 studied symptomatic FVII-deficient individuals, 58 were homozygous carriers of the identified variations. Identification of homozygotes clarifies the potential role of nucleotide variations in reducing FVII activity and their contributions to a certain phenotype. Some of those variations, such as c.1A>G, c.509G>A, c.634C>T, and c.1285G>A have only been previously reported as heterozygous.

Pro-haemostatic effect of DDAVP is partially derived through non-classical (CD14dim /CD16++ ) monocytes residing the spleen.

The splenic endothelial Weibel-palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin-specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro-haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin-treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co-cultured with non-classical (CD14dim /CD16++ ), but not other subtypes of monocytes or PBMCs. This in vitro study which resembles close proximity of endothelial cells of sinusoids to monocyte reservoir reside in parenchyma of subcapsular red pulp of the spleen sheds a light upon the role of this highly vascularized VWF-producing organ in driving indirect effect of desmopressin.

Familial Hemophagocytic Lymphohistiocytosis secondary to UNC13D mutation: a report of two cases.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by some clinical signs (e.g., non-remitting fever, hepatosplenomegaly) and laboratory findings (e.g., cytopenia, increased ferritin level, hypofibrinogenemia, lipid disorders, coagulopathy, and multiple organ failure). Depending on the etiology, HLH is divided into familial (i.e., primary) and acquired (i.e., secondary) forms. Familial HLH (FHL), an autosomal recessive condition, is classified into five subtypes based on underlying genetic defects. The PRF1, STX11, UNC13D, HPLH1, and STXBP2 are the most well-known genes of this type which are related to granule-mediated cytotoxic T and Natural killer (NK) cells. The treatment is based on the HLH-2004 protocol. CASE PRESENTATION: The current report presents two cases of HLH with presentations different from each other and previously reported cases. Case 1 was a 15-month-old boy with fever, skin rash, splenomegaly, and bicytopenia, raised triglyceride levels, AST (aspartate transaminase), and ALT (alanine aminotransferase), normal ferritin, and abundant hemophagocytic cell in bone marrow aspiration. He was diagnosed with HLH and received HLH protocol as treatment. The patient had a homozygous intronic mutation; NM_199242: c.2448-13G > A in UNC13D. The associated disease was Familial Hemophagocytic Lymphohistiocytosis 3 (FHL3). Case 2, a 37-day-old female presented with fever, a history of neonatal cholestasis, and huge hepatosplenomegaly. Her whole-exome sequencing report manifested that the patient had the same mutation as case 1. Unfortunately, both patients passed away. CONCLUSION: The sequencing of the entire UNC13D gene (coding and non-coding regions) is an applicable and valuable diagnostic procedure for the detection of deep intronic splicing variants and large inversions in patients with atypical manifestations of HLH (such as normal ferritin or triglyceride and cholesterol).

The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group.

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.

The taxonomic affiliation of Pseudomonas isolates is currently assessed by using the 16S rRNA gene, MultiLocus Sequence Analysis (MLSA), or whole genome sequencing. Therefore, microbiologists are facing an arduous choice, either using the universal marker, knowing that these affiliations could be inaccurate, or engaging in more laborious and costly approaches. The rpoD gene, like the 16S rRNA gene, is included in most MLSA procedures and has already been suggested for the rapid identification of certain groups of Pseudomonas. However, a comprehensive overview of the rpoD-based phylogenetic relationships within the Pseudomonas genus is lacking. In this study, we present the rpoD-based phylogeny of 217 type strains of Pseudomonas and defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level. To validate this approach, we sequenced the rpoD of 145 environmental isolates and complemented this analysis with whole genome sequencing. The rpoD sequence allowed us to accurately assign Pseudomonas isolates to 20 known species and represents an excellent first diagnostic tool to identify new Pseudomonas species. Finally, rpoD amplicon sequencing appears as a reliable and low-cost alternative, particularly in the case of large environmental studies with hundreds or thousands of isolates.

Protective effect of probiotic Lactobacillus acidophilus against the toxicity of beauvericin mycotoxin on the Caco-2 cell line.

The present study evaluated the protective effect of Lactobacillus acidophilus against the toxicity of the Beauvericin on the Caco-2 cell line. After culturing Caco-2 cells and applying different concentrations of Beauvericin and L. acidophilus individually and in combination, cell viability was assessed by enzyme-linked immunosorbent assay at different times. The results indicate the potential risk of Beauvericin to human health and the interventional role of L. acidophilus, which improved cell viability in the presence of Beauvericin.

Draft Genome Sequence of Cyclic Lipopeptide Producer Pseudomonas sp. Strain SWRI103, Isolated from Wheat Rhizosphere.

The draft genome sequence of wheat rhizosphere isolate Pseudomonas sp. strain SWRI103 is reported. This strain carries several gene clusters encoding nonribosomal peptide synthetases (NRPSs), including a system for cyclic lipopeptide (CLP) production, and genes for carotenoid biosynthesis.

Exome-first Approach Identified Novel Homozygous Dedicator of Cytokinesis 8 (DOCK8) Mutations in Three Unrelated Iranian Pedigrees Suspected with Hyper-IgE Syndrome.

The prevalence of primary immunodeficiency (PID) is rather high in Iran compared to the world average, mainly due to the high rate of consanguineous marriage. Despite that, little genetic information is available about primary immunodeficiencies in Iran. Autosomal recessive hyper IgE syndrome (AR-HIES) is a severe type of immunodeficiency, mainly caused by mutations in the dedicator of cytokinesis 8 (DOCK8). Rapid and precise diagnoses of patients suffering from AR-HIES can help to manage the patients and reach properly the treatment decision. However, in regions with low financial resources and limited expertise, deep phenotyping is uncommon. Therefore, an exome-first approach is helpful to make a genetic-based diagnosis. In the present study, whole-exome sequencing (WES) was applied to detect causative mutations in three unrelated primary immunodeficient patients with poor clinical information. One of the cases was a deceased patient with suspected hyper IgE syndrome (HIES) whose parents were subjected to WES. As a result, three novel pathogenic variants were detected in the DOCK8 gene, including two splicing sites (c.4241+1G>T and c.4886+1G>T) and one-stop-gain (c.4201G>T, p.Glu1401Ter) variants. Sanger sequencing confirmed the mutations' segregation in corresponding families. Further immunological investigations confirmed that HIES in the studied probands. The presence of frontal bossing and broad nose in one of the studied cases, in addition to the typical clinical presentation of DOCK8-AR-HIES, is notable. This work suggests that an exome-first approach can be a valuable alternative strategy for precise diagnosis of primary immunodeficiency patients.

A Novel Non-frameshift ADA Deletion Detected by Whole Exome Sequencing in an Iranian Family with Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of genetic disorders caused by early defects in the development and function of T cells. Other lymphocyte lineages (B and/or natural killer cells) are variably affected. With a worldwide frequency of approximately 1:50,000 live births, SCID may result from diverse mutations in over 16 genes. Whole-exome sequencing (WES) provides an opportunity for parallel screening of all those genes. This approach is also useful for genetic diagnosis in parents whose infant expired before genetic testing. Here, we describe a heterozygous novel non-frameshift deletion (c.587_598del p.196_199del) in the adenosine deaminase (ADA) gene identified by WES in healthy parents of an expired child with SCID. The mutation was subsequently confirmed to be homozygous in the deceased baby whose left-over blood sample volume was insufficient for direct WES analysis. In conclusion, we here describe a novel mutation in ADA, a well-known SCID gene.

Draft Genome Sequence of Pseudomonas aeruginosa Strain LMG 1272, an Atypical White Line Reaction Producer.

The draft genome sequence of Pseudomonas aeruginosa LMG 1272, isolated from mushroom, is reported here. This strain triggers formation of a precipitate ("white line") when cocultured with Pseudomonas tolaasii However, LMG 1272 lacks the capacity to produce a cyclic lipopeptide that is typically associated with white line formation, suggesting the involvement of a different diffusible factor.

A novel homozygous LRRK1 stop gain mutation in a patient suspected with osteosclerotic metaphyseal dysplasia.

Osteosclerotic metaphyseal dysplasia (OSMD) is a very rare autosomal-recessive disorder and a distinctive type of osteopetrosis, characterized mainly by skeletal fractures and deformity, osteosclerosis, and sometimes hypotonia, developmental delay, and seizures. Sequence variants in the leucine-rich repeat kinase 1 (LRRK1) gene underlying OSMD have been reported previously. In the present study, we investigated a 14-year-old girl suspected with OSMD in a consanguineous family of Iranian origin segregating the disease in an autosomal-recessive manner. The patient had severe short stature, multiple sclerotic lesions, sandwich vertebrae, Erlenmeyer flask deformity, and looser zones. The multifocal active bony pathology suggested multifocal bony inflammation or multiple looser fractures. Whole-exome sequencing followed by Sanger sequencing confirmation revealed a novel homozygous stop gain mutation (c.G2785T, p.E929X) in the LRRK1 gene. This is the first mutation in the LRRK1 gene, underlying OSMD, in the Iranian population and the third case worldwide. The mutation is located in the C terminal of the Roc domain, distinct from domains affected in the previous two LRRK1 mutations. Additionally, a new group of clinical indications different from the two previous cases is discussed.

Inherited IFNAR1 deficiency in otherwise healthy patients with adverse reaction to measles and yellow fever live vaccines.

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

GATA4 screening in Iranian patients of various ethnicities affected with congenital heart disease: Co-occurrence of a novel de novo translocation (5;7) and a likely pathogenic heterozygous GATA4 mutation in a family with autosomal dominant congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and a major health problem around the world. However, its exact etiology has remained unclear. Among various genetic contributing factors, GATA4 transcription factor plays a significant role in the CHD pathogenesis. In this study, GATA4 coding sequence was screened in Iranian patients of various ethnicities. METHODS: Sixty six individuals with familial CHD referred to our center were recruited in this study. After receiving written informed consent from each individual or their parents, chromosomal analyses and GATA4 variant screening were performed. Pathogenicity of the suspected variants was evaluated using available online software tools: CADD, Mutation Taster, SIFT, and PolyPhen-2. RESULTS: A total of twelve GATA4 variants were detected including five intronic, 2 exonic and 3 polymorphisms as well as 2 missense mutations, the c.1220C>A and c.1309G>A. Unlike the c.1220C>A, the likely pathogenic heterozygous c.1309G>A has not been previously associated with any phenotype. Here, we not only report, for the first time, a c.1309G>A-related CHD, but also report a novel de novo balanced translocation, 46,XY,t(5;7)(qter13;qter11), in the same patient which may have influenced the disease severity. CONCLUSION: From screening GATA4 sequence in 66 Iranian patients of various ethnicities, we conclude that cytogenetic analysis and PCR-direct sequencing of different candidate genes may not be the best approach for genetic diagnosis in CHD. Applying novel approaches such as next-generation sequencing (NGS) may provide a better understating of genetic contributing factors in CHD patients for whom conventional methods could not reveal any genetic causative factor.

Draft Genome Sequence of Micromonospora sp. Strain MW-13, a Bacterial Strain with Antibacterial Properties and Plant Growth Promotion Potential Isolated from the Rhizosphere of Wheat in Iran.

Strain MW13 exhibited broad-spectrum antibacterial activity toward Gram-positive and Gram-negative pathogens. The 7.1-Mb draft genome gives insight into the complete secondary metabolite production capacity and reveals genes putatively responsible for its antibacterial activity, as well as genes which contribute to plant growth promotion.

Whole exome sequencing identifies both nuclear and mitochondrial variations in an Iranian family with non-syndromic hearing loss.

Genetic contributing factors to non-syndromic hearing loss (NSHL) are remarkably diverse spanning over autosomal to X-linked to mitochondrial inheritance patterns. Facing a quite unconventional pedigree, here we report implementation of whole exome sequencing (WES) to uncover mitochondrial pathogenic variant in a six-generation Iranian family with four cases affected with hereditary NSHL of variable severity. As a result, heteroplasmic transition of A to G at position 1555 of MT-RNR1 gene was identified in all affected individuals co-existing with nuclear c.28G > T (p.A10S) variant in the TRMU gene, only in some patients. The reliability of WES to infer nuclear as well as mitochondrial variants in hearing loss were discussed.

Mosaic trisomy 22 in a 4-year-old boy with congenital heart disease and general hypotrophy: A case report.

BACKGROUND: Trisomy 22 mosaicism is a rare autosomal anomaly with survival compatibility. Recognition of the complete trisomy 22 which is incompatible with life from the mosaic form is critical for genetic counseling. Affected mosaic cases have prevalent clinical presentations such as webbed neck, developmental delay, abnormal ears, cardiac disorders, and microcephaly. Phenotype of these patients is milder than full chromosomal aneuploidy, and the severity of the phenotype depends on the count of trisomic cells. We describe a 4-year-old boy with mosaic trisomy 22 from healthy parents and no family history of any genetic disorders in the pedigree. METHOD AND RESULTS: The patient had determined dysmorphic clinical features including facial asymmetry, cleft palate, gastroenteritis, hydronephrosis, developmental delay, genital anomalies, dysplastic toenails, flattened nasal bridge, congenital heart defect, hearing loss, cryptorchidism, and hypotonic muscle. He is the first reported with hypothyroidism and larynx wall thickness in worldwide and the first with atrial septal defect (ASD) from Iran. Chromosomal analyses using G-banding indicated a de novo Mos 47,XY,+22(6)/46,XY(44) karyotype with no other chromosomal structural changes. CONCLUSIONS: Our observations confirm the importance of cytogenetic analyses for determining the cause of congenital anomalies and provide a useful genetic counseling. In addition, due to the fact that some of mosaic trisomy 22 features are unavoidable such as CHD and general hypotrophy, we suggest including echocardiography test for early diagnosis during the clinical assessment.

Assessing the impact of exact reads on reducing the error rate of read mapping.

BACKGROUND: Nowadays, according to valuable resources of high-quality genome sequences, reference-based assembly methods with high accuracy and efficiency are strongly required. Many different algorithms have been designed for mapping reads onto a genome sequence which try to enhance the accuracy of reconstructed genomes. In this problem, one of the challenges occurs when some reads are aligned to multiple locations due to repetitive regions in the genomes. RESULTS: In this paper, our goal is to decrease the error rate of rebuilt genomes by resolving multi-mapping reads. To achieve this purpose, we reduce the search space for the reads which can be aligned against the genome with mismatches, insertions or deletions to decrease the probability of incorrect read mapping. We propose a pipeline divided to three steps: ExactMapping, InExactMapping, and MergingContigs, where exact and inexact reads are aligned in two separate phases. We test our pipeline on some simulated and real data sets by applying some read mappers. The results show that the two-step mapping of reads onto the contigs generated by a mapper such as Bowtie2, BWA and Yara is effective in improving the contigs in terms of error rate. CONCLUSIONS: Assessment results of our pipeline suggest that reducing the error rate of read mapping, not only can improve the genomes reconstructed by reference-based assembly in a reasonable running time, but can also have an impact on improving the genomes generated by de novo assembly. In fact, our pipeline produces genomes comparable to those of a multi-mapping reads resolution tool, namely MMR by decreasing the number of multi-mapping reads. Consequently, we introduce EIM as a post-processing step to genomes reconstructed by mappers.