Whole-genome sequencing reveals a novel pathogenic GRIN2B variant in a patient with neurodevelopmental disorder and an inv(6)(p24p11.2)pat.

INTRODUCTION: Neurodevelopmental disorders (NDDs) are diverse and can be explained by either genomic aberrations or single nucleotide variants (SNVs). Most likely due to methodological approaches and/or disadvantages, the concurrence of both genetic events in a single patient has hardly been reported and even more rarely the pathogenic variant has been regarded as the cause of the phenotype when a chromosomal alteration is initially identified. CASE PRESENTATION: Here, we describe a NDD patient with a 6p non-pathogenic paracentric inversion paternally transmitted and a de novo pathogenic variant in the GRIN2B gene. Molecular-cytogenetic studies characterized the familial 6p inversion and revealed a paternal 9q inversion not transmitted to the patient. Subsequent whole-genome sequencing (WGS) in the patient-father dyad corroborated the previous findings, discarded inversions-related cryptic genomic rearrangements as causative of the patient's phenotype, and unveiled a novel heterozygous GRIN2B variant (p.(Ser570Pro)) only in the proband. In addition, Sanger sequencing ruled out such a variant in her mother and thereby confirmed its de novo origin. Due to predicted disturbances in the local secondary structure, this variant may alter the ion channel function of the M1 transmembrane domain. Other pathogenic variants in GRIN2B have been related to the autosomal dominant neurodevelopmental disorder MRD6 (Intellectual developmental disorder, autosomal dominant 6, with or without seizures), which presents with a high variability ranging from mild intellectual disability (ID) without seizures to a more severe encephalopathy. In comparison, our patient's clinical manifestations include, among others, mild ID and brain anomalies previously documented in subjects with MRD6. CONCLUSION: Occasionally, gross chromosomal abnormalities can be coincidental findings rather than a prime cause of a clinical phenotype (even though they appear to be the causal agent). In brief, this case underscores the importance of comprehensive genomic analysis in unraveling the wide-ranging genetic causes of NDDs and may bring new insights into the MRD6 variability.

Genomic Characterization of a Rare, de Novo Unbalanced ins(3;1)(p25.3;q21.3q23.3) in a Female Child with Multiple Congenital Anomalies.

"Simple" 1-way interchromosomal insertions involving an interstitial 1q segment are rare, and therefore, their characterization at the base pair level remains understudied. Here, we describe the genomic characterization of a previously unreported de novo interchromosomal insertion (3;1) entailing an about 12-Mb pure gain of 1q21.3q23.3 that causes typical (microcephaly, developmental delay, and facial dysmorphism) and atypical (interauricular communication, small feet with bilateral deep plantar creases, syndactyly of II-IV toes, and mild pachyonychia of all toes) clinical manifestations associated with this region. Based on our analyses, we hypothesize that the duplication of a subset of morbid genes (including LMNA, USF1, VANGL2, LOR, and POGZ) could account for most clinical findings in our patient. Furthermore, the apparent disruption of a promoter region (between CPNE9 and BRPF1) and a topologically associated domain also suggests likely pathogenic reconfiguration/position effects to contribute to the patient's phenotype. In addition to further expanding the clinical spectrum of proximal 1q duplications and evidencing the phenotypical heterogeneity among similar carriers, our genomic findings and observations suggest that randomness - rather than lethality issues - may account for the paucity of "simple" interchromosomal insertions involving the 1q21.3q23.3 region as genomic donor and distal 3p25.3 as receptor. Moreover, the microhomology sequence found at the insertion breakpoint is consistent with a simple nonhomologous end-joining mechanism, in contrast to a chromothripsis-like event, which has previously been seen in other nonrecurrent insertions. Taken together, the data gathered in this study allowed us to inform this family about the low recurrence risk but not to predict the reproductive prognosis for hypothetical carriers. We highlight that genomic-level assessment is a powerful tool that allows the visualization of the full landscape of sporadic chromosomal injuries and can be used to improve genetic counseling.

Familial 3-Way Balanced Translocation Causes 1q43→qter Loss and 10q25.2→qter Gain in a Severely Affected Male Toddler.

Constitutional complex chromosomal rearrangements (CCRs) are rare events that typically involve 2 or more chromosomes with at least 3 breakpoints and can result in normal or abnormal phenotypes depending on whether they disturb the euchromatic neighborhood. Here, we report an unusual balanced CCR involving chromosomes 1, 9, and 10 that causes an unbalanced karyotype in a severely affected toddler. The CCR was initially reported as a maternal 2-way translocation but was reclassified as a 3-way translocation after a microarray analysis of the propositus revealed the involvement of another chromosome not identified by G-banding in his phenotypically normal mother. FISH assays on maternal metaphase cells confirmed that the 1qter region of der(1) was translocated to der(10), whereas the 10qter segment was translocated to der(9), which in turn donated a segment to der(1). Subsequently, this CCR was also identified in her phenotypically normal father (the patient's grandfather). Thus, the patient inherited the previously unreported pathogenic combination of der(1) with a loss of 1q43→qter (including AKT3, ZBTB18, HNRNPU, and SMYD3) and der(9) with a gain of 10q25.2→qter (including FGFR2), leading to a compound phenotype with key features of the 1q43→qter deletion and distal 10q trisomy syndromes. Our observations suggest that the loss of SMYD3 accounts for cardiac defects in a subset of patients. Moreover, due to recurrent miscarriages in this family, our findings allowed improved genetic counseling.

A de novo t(10;19)(q22.3;q13.33) leads to ZMIZ1/PRR12 reciprocal fusion transcripts in a girl with intellectual disability and neuropsychiatric alterations.

We report a girl with intellectual disability (ID), neuropsychiatric alterations, and a de novo balanced t(10;19)(q22.3;q13.33) translocation. After chromosome sorting, fine mapping of breakpoints by array painting disclosed disruptions of the zinc finger, MIZ-type containing 1 (ZMIZ1) (on chr10) and proline-rich 12 (PRR12) (on chr19) genes. cDNA analyses revealed that the translocation resulted in gene fusions. The resulting hybrid transcripts predict mRNA decay or, if translated, formation of truncated proteins, both due to frameshifts that introduced premature stop codons. Though other molecular mechanisms may be operating, these results suggest that haploinsufficiency of one or both genes accounts for the patient's phenotype. ZMIZ1 is highly expressed in the brain, and its protein product appears to interact with neuron-specific chromatin remodeling complex (nBAF) and activator protein 1 (AP-1) complexes which play a role regulating the activity of genes essential for normal synapse and dendrite growth/behavior. Strikingly, the patient's phenotype overlaps with phenotypes caused by mutations in SMARCA4 (BRG1), an nBAF subunit presumably interacting with ZMIZ1 in brain cells as suggested by our results of coimmunoprecipitation in the mouse brain. PRR12 is also expressed in the brain, and its protein product possesses domains and residues thought to be related in formation of large protein complexes and chromatin remodeling. Our observation from E15 mouse brain cells that a Prr12 isoform was confined to nucleus suggests a role as a transcription nuclear cofactor likely involved in neuronal development. Moreover, a pilot transcriptome analysis from t(10;19) lymphoblastoid cell line suggests dysregulation of genes linked to neurodevelopment processes/neuronal communication (e.g., NRCAM) most likely induced by altered PRR12. This case represents the first constitutional balanced translocation disrupting and fusing both genes and provides clues for the potential function and effects of these in the central nervous system.

Downregulation of inducible nitric oxide synthase (iNOS) expression is implicated in the antiviral activity of acetylsalicylic acid in HCV-expressing cells.

Previously, we described that acetylsalicylic acid (ASA) decreases HCV expression, but the mechanisms involved have not been clearly established. We evaluated the participation of inducible nitric oxide synthase (iNOS) in the regulation of HCV-RNA induced by ASA. Huh7 cells expressing non-structural HCV proteins were exposed to 4 mM ASA and incubated at the same times we reported HCV downregulation (24-72 h), and iNOS mRNA and protein levels were then measured by real-time PCR and Western blot, respectively. Nitric oxide levels were measured at the same time. Inhibition of iNOS mRNA by small interfering RNAs (siRNA) and activation of the iNOS gene promoter by ASA treatment were evaluated. In Huh7 replicon cells treated with ASA, we found decreased levels of iNOS mRNA, iNOS protein and nitrosylated protein levels at 48-72 h. ASA exposure also reduced the transactivation of the iNOS promoter in HCV replicon cells at 48 h, and this was partly due to the decrease in the affinity of transcription factor C/EBP-ß for its binding site in the iNOS promoter. siRNA silencing of iNOS decreased HCV-RNA expression (65 %) and potentiated the antiviral effect (80 %) of ASA compared with control cells. ASA reduces iNOS expression by downregulating promoter activity, mRNA and protein levels at the same time that it decreases HCV expression. These findings suggest that the antiviral activity of ASA is mediated partially through the modulation of iNOS.

Review of the Impact of Biofilm Formation on Recurrent Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) may recur in approximately 10-30% of patients, and the risk of recurrence increases with each successive recurrence, reaching up to 65%. C. difficile can form biofilm with approximately 20% of the bacterial genome expressed differently between biofilm and planktonic cells. Biofilm plays several roles that may favor recurrence; for example, it may act as a reservoir of spores, protect the vegetative cells from the activity of antibiotics, and favor the formation of persistent cells. Moreover, the expression of several virulence genes, including TcdA and TcdB toxins, has been associated with recurrence. Several systems and structures associated with adhesion and biofilm formation have been studied in C. difficile, including cell-wall proteins, quorum sensing (including LuxS and Agr), Cyclic di-GMP, type IV pili, and flagella. Most antibiotics recommended for the treatment of CDI do not have activity on spores and do not eliminate biofilm. Therapeutic failure in R-CDI has been associated with the inadequate concentration of drugs in the intestinal tract and the antibiotic resistance of a biofilm. This makes it challenging to eradicate C. difficile in the intestine, complicating antibacterial therapies and allowing non-eliminated spores to remain in the biofilm, increasing the risk of recurrence. In this review, we examine the role of biofilm on recurrence and the challenges of treating CDI when the bacteria form a biofilm.

Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence.

The transcriptomic profile in a biofilm model of ribotypes (RT) 001 and 027 associated with recurrent Clostridioides difficile infection (R-CDI) and not associated with recurrent (NR)-CDI was analyzed to identify genes that may favor the recurrence. Twenty strains were selected, 10 RT001 and 10 RT027. From each ribotype, 5 were R-CDI and 5 NR-CDI. Biofilm and nonadherent cells were prepared from each clinical isolate, and the RNA was extracted. RNA samples were pooled in 8 combinations implying ribotype, recurrence, and biofilm formation. Each pool was separately labeled with Cy3 dye and hybridized on a microarray designed for this study. Slides were scanned, analyzed, and gene expression was compared between unique and grouped pools using the Student's t-test with Benjamini-Hochberg correction when appropriate. Validation was carried out by qRT-PCR for selected genes. Results: After comparisons of differentially expressed genes from both ribotypes of R-CDI strains (nonadherent cells vs. biofilm) and both ribotypes in biofilm (R-CDI vs. NR-CDI), we found 3 genes over-expressed and 1 under-expressed in common (adj. p ≤ 0.05). Overexpressed genes were CAJ70148 (a putative dehydrogenase), CAJ68100 (a secretion type II system protein from the GspH (pseudopilins) family), and CAJ69725 (a putative membrane protein); under-expressed was CAJ68151 (a segregation and condensation protein A). Because CAJ70148, CAJ68100, CAJ69725 and CAJ68151 were differentially expressed in biofilm in strains associated with R-CDI, they may support the biofilm favoring the recurrence of CDI. However, further studies will be needed for poorly studied genes.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

Carbapenemase-Encoding Genes and Colistin Resistance in Gram-Negative Bacteria During the COVID-19 Pandemic in Mexico: Results from the Invifar Network.

In this study, we report the carbapenemase-encoding genes and colistin resistance in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa in the second year of the COVID-19 pandemic. Clinical isolates included carbapenem-resistant K. pneumoniae, carbapenem-resistant E. coli, carbapenem-resistant A. baumannii, and carbapenem-resistant P. aeruginosa. Carbapenemase-encoding genes were detected by PCR. Carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates were analyzed using the Rapid Polymyxin NP assay. mcr genes were screened by PCR. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on representative isolates. A total of 80 carbapenem-resistant E. coli, 103 carbapenem-resistant K. pneumoniae, 284 carbapenem-resistant A. baumannii, and 129 carbapenem-resistant P. aeruginosa isolates were recovered. All carbapenem-resistant E. coli and carbapenem-resistant K. pneumoniae isolates were included for further analysis. A selection of carbapenem-resistant A. baumannii and carbapenem-resistant P. aeruginosa strains was further analyzed (86 carbapenem-resistant A. baumannii and 82 carbapenem-resistant P. aeruginosa). Among carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates, the most frequent gene was blaNDM (86/103 [83.5%] and 72/80 [90%], respectively). For carbapenem-resistant A. baumannii, the most frequently detected gene was blaOXA-40 (52/86, 60.5%), and for carbapenem-resistant P. aeruginosa, was blaVIM (19/82, 23.2%). For carbapenem-resistant A. baumannii, five indistinguishable pulsotypes were detected. Circulation of K. pneumoniae New Delhi metallo-ß-lactamase (NDM) and E. coli NDM was detected in Mexico. High virulence sequence types (STs), such as K. pneumoniae ST307, E. coli ST167, P. aeruginosa ST111, and A. baumannii ST2, were detected. Among K. pneumoniae isolates, 18/101 (17.8%) were positive for the Polymyxin NP test (two, 11.0% positive for the mcr-1 gene, and one, 5.6% with disruption of the mgrB gene). All E. coli isolates were negative for the Polymyxin NP test. In conclusion, K. pneumoniae NDM and E. coli NDM were detected in Mexico, with the circulation of highly virulent STs. These results are relevant in clinical practice to guide antibiotic therapies considering the molecular mechanisms of resistance to carbapenems.

Whole genome analysis of Gram-negative bacteria using the EPISEQ CS application and other bioinformatic platforms.

OBJECTIVES: To determine genomic characteristics and molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa from medical centres of Mexico using whole genome sequencing data analysed with the EPISEQⓇ CS application and other bioinformatic platforms. METHODS: Clinical isolates collected from 28 centres in Mexico included carbapenem-non-susceptible K. pneumoniae (n = 22), E. coli (n = 24), A. baumannii (n = 16), and P. aeruginosa (n = 13). Isolates were subjected to whole genome sequencing using the Illumina (MiSeq) platform. FASTQ files were uploaded to the EPISEQⓇ CS application for analysis. Additionally, the tools Kleborate v2.0.4 and Pathogenwatch were used as comparators for Klebsiella genomes, and the bacterial whole genome sequence typing database was used for E. coli and A. baumannii. RESULTS: For K. pneumoniae, both bioinformatic approaches detected multiple genes encoding aminoglycoside, quinolone, and phenicol resistance, and the presence of blaNDM-1 explained carbapenem non-susceptibility in 18 strains and blaKPC-3 in four strains. Regarding E. coli, both EPISEQⓇ CS and bacterial whole genome sequence typing database analyses detected multiple virulence and resistance genes: 20 of 24 (83.3%) strains carried blaNDM, 3 of 24 (12.4%) carried blaOXA-232, and 1 carried blaOXA-181. Genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, phenicols, trimethoprim, and macrolides were also detected by both platforms. Regarding A. baumannii, the most frequent carbapenemase-encoding gene detected by both platforms was blaOXA-72, followed by blaOXA-66. Both approaches detected similar genes for aminoglycosides, carbapenems, tetracyclines, phenicols, and sulfonamides. Regarding P. aeruginosa, blaVIM, blaIMP, and blaGES were the more frequently detected. Multiple virulence genes were detected in all strains. CONCLUSION: Compared to the other available platforms, EPISEQⓇ CS enabled a comprehensive resistance and virulence analysis, providing a reliable method for bacterial strain typing and characterization of the virulome and resistome.

A chromoanagenesis-driven ultra-complex t(5;7;21)dn truncates neurodevelopmental genes in a disabled boy as revealed by whole-genome sequencing.

Germline or constitutional chromoanagenesis-related complex chromosomal rearrangements (CCRs) are rare, apparently "all-at-once", catastrophic events that occur in a single cell cycle, exhibit an unexpected complexity, and sometimes correlate with a severe abnormal phenotype. The term chromoanagenesis encompasses three distinct phenomena, namely chromothripsis, chromoanasynthesis, and chromoplexy. Herein, we found hallmarks of chromothripsis and chromoplexy in an ultra-complex t(5;7;21)dn involving several disordered breakpoint junctions (BPJs) accompanied by some microdeletions and the disruption of neurodevelopmental genes in a patient with a phenotype resembling autosomal dominant MRD44 (OMIM 617061). G-banded chromosomes and FISH showed that the CCR implied the translocation of the 5p15.2→pter segment onto 7q11.23; in turn, the fragment 7q11.23→qter of der(7) separated into two pieces: the segment q11.23→q32 translocated onto 5p15.2 and fused to 21q22.1→ter in the der(5) while the distal 7q32→qter segment translocated onto der(21) at q22.1. Subsequent whole-genome sequencing unveiled that CCT5, CMBL, RETREG1, MYO10, and TRIO from der(5), IMMP2L, TES, VPS37D, DUS4L, TYW1B, and FEZF1-AS1 from der(7), and TIAM1 and SOD1 from der(21), were disrupted by BPJs, whereas some other genes (predicted to be haplosufficient or inconsequential) were completely deleted. Although remarkably CCT5, TRIO, TES, MYO10, and TIAM1 (and even VPS37D) cooperate in key biological processes for normal neuronal development such as cell adhesion, migration, growth, and/or cytoskeleton formation, the disruption of TRIO most likely caused the patient's MRD44-like phenotype, including intellectual disability, microcephaly, finger anomalies, and facial dysmorphia. Our observation represents the first truncation of TRIO related to a chromoanagenesis event and therefore expands the mutational spectrum of this crucial gene. Moreover, our findings indicate that more than one mechanism is involved in modeling the architecture of ultra-complex rearrangements.

Clinical Exome Sequencing Enables Congenital Sialidosis Type II Diagnosis in Two Siblings Presenting with Unreported Clinical Features from a Rare Homozygous Sequence Variant p.(Tyr370Cys) in NEU1.

Sialidosis is a rare autosomal recessive disease that presents with progressive lysosomal storage of sialylated glycopeptides and oligosaccharides caused by homozygous or compound heterozygous sequence variants in the neuraminidase 1 (NEU1) gene. These sequence variants can lead to sialidosis type I and II; the latter is the most severe and presents prenatally or at early age. However, sialidosis diagnosis is challenging, especially in those health systems with limited resources of developing countries. Consequently, it is necessary to dip into high-throughput molecular diagnostic tools to allow for an accurate diagnosis with better cost-effectiveness and turnaround time. We report a 4-member pedigree segregating an ultrarare missense variant, c.1109A>G; p.Tyr370Cys, in NEU1 as detected by whole-exome sequencing. Two short-lived siblings, who presented with previously unreported clinical features from such a homozygous sequence variant, were diagnosed with sialidosis type II. Additionally, we present a novel molecular model exhibiting the consequences of the variant in the sialidase-1 tridimensional structure. This study allowed us to provide a definitive diagnosis for our patients, increase our understanding of this pathogenic variant, and improve genetic counseling.

Complete Genome Sequences of Mycobacterium tuberculosis Isolates Subjected to 200 Continuous Passages.

We report 6 draft genome sequences corresponding to Mycobacterium tuberculosis H37Rv and M. tuberculosis DR689, a Beijing isolate, plus their counterparts subjected to 200 continuous passages in Middlebrook 7H9 broth, either alone or with ox bile.

Whole-exome sequencing in three children with sporadic Blau syndrome, one of them co-presenting with recurrent polyserositis.

Blau syndrome (BS) is a rare, chronic autoinflammatory disease with onset before age 4 and mainly characterised by granulomatous arthritis, recurrent uveitis, and skin rash. Sporadic (also known as early-onset sarcoidosis) or familial BS is caused by gain-of-function mutations in the NOD2 gene, which encodes for a multi-task protein that plays a crucial role in the innate immune defense. We report on three Mexican patients clinically diagnosed with BS who exhibited a likely pathogenic variant in NOD2 as revealed by whole-exome sequencing (WES) and Sanger sequencing: two variants (c.1000 C > T/p.Arg334Trp and c.1538 T > C/p.Met513Thr) lie in the ATP/Mg2+ binding site, whereas the other (c.3019dupC/p.Leu1007ProfsTer2) introduces a premature stop codon disrupting the last LRR domain (LRR9) formation; all three variants are consistent with gain-of-function changes. Interestingly, all these patients presented concomitant likely pathogenic variants in other inflammatory disease-related genes, i.e. TLR10, PRR12, MEFV and/or SLC22A5. Although the clinical presentation in these patients included the BS diagnostic triad, overall it was rather heterogeneous. It is plausible that this clinical variability depends partly on the patients' genetic background as suggested by our WES results. After this molecular diagnosis and given the absence of NOD2 mutations (demonstrated in two trios) and related symptoms in the respective parents (confirmed in all trios), patients 1 and 2 were considered to have sporadic BS, while patient 3, a sporadic BS-recurrent polyserositis compound phenotype. Altogether, our observations and findings underscore the overlapping among inflammatory diseases and the importance of determining the underlying genetic cause by high-throughput methods. Likewise, this study further reinforces a pathogenic link between the here found NOD2 variants and BS and envisages potential additive effects from other loci in these, and probably other patients.

Williams-Beuren syndrome in Mexican patients confirmed by FISH and assessed by aCGH.

Williams-Beuren syndrome (WBS) has a prevalence of 1/7500-20000 live births and results principally from a de novo deletion in 7q11.23 with a length of 1.5 Mb or 1.8 Mb. This study aimed to determine the frequency of 7q11.23 deletion, size of the segment lost, and involved genes in 47 patients with a clinical diagnosis of WBS and analysed by fluorescence in situ hybridization (FISH); among them, 31 had the expected deletion. Micro-array comparative genomic hybridization (aCGH) confirmed the loss in all 18 positive-patients tested: 14 patients had a 1.5 Mb deletion with the same breakpoints at 7q11.23 (hg19: 72726578-74139390) and comprising 24 coding genes from TRIM50 to GTF2I. Four patients showed an atypical deletion: two had a 1.6 Mb loss encompassing 27 coding genes, from NSUN5 to GTF2IRD2; another had a 1.7 Mb deletion involving 27 coding genes, from POM121 to GTF2I; the remaining patient presented a deletion of 1.2 Mb that included 21 coding genes from POM121 to LIMK1. aCGH confirmed the lack of deletion in 5/16 negative-patients by FISH. All 47 patients had the characteristic facial phenotype of WBS and 45 of 47 had the typical behavioural and developmental abnormalities. Our observations further confirm that patients with a classical deletion present a typical WBS phenotype, whereas those with a high (criteria of the American Association of Pediatrics, APP) clinical score but lacking the expected deletion may harbour an ELN point mutation. Overall, the concomitant CNVs appeared to be incidental findings.

Complete Genome Sequence of Houston Virus, a Newly Discovered Mosquito-Specific Virus Isolated from Culex quinquefasciatus in Mexico.

We fully sequenced the genome of Houston virus, a recently discovered mosquito-associated virus belonging to the newly established family Mesoniviridae. The isolate was recovered from Culex quinquefasciatus in southern Mexico, which shows that the geographic range of Houston virus is not restricted to the United States in North America.

Uncommon runs of homozygosity disclose homozygous missense mutations in two ciliopathy-related genes (SPAG17 and WDR35) in a patient with multiple brain and skeletal anomalies.

We describe a patient severely affected with multiple congenital anomalies, including brain malformations and skeletal dysplasia suggestive of cranioectodermal dysplasia (CED) ciliopathy, who unusually carries several homozygosity tracts involving homozygous missense mutations in SPAG17 (exon 8; c.1069G > C; p.Asp357His) and WDR35 (exon 13; c.1415G > A; p.Arg472Gln) as revealed by homozygosity mapping and next generation sequencing. SPAG17 is essential for the function and structure of motile cilia, while WDR35 belongs to the same intraflagellar transport (IFT) gene family whose protein products are part of functional IFT A and B complexes. Formerly, SPAG17 was related - through polymorphic variants - to an influence on individuals' height; more recently, Spag17-/- mice models were reported to present skeletal and bone defects, reduced mucociliary clearance, respiratory distress, and cerebral ventricular enlargement. Homozygous or compound heterozygous mutations in WDR35 have mainly been related to CED2 or short-rib thoracic dysplasia 7, with only three cases showing some brain anomalies. Given that our patient presents these clinical features and the close functional relationship between SPAG17 and WDR35, it is feasible that the combined effects from both mutations contribute to his phenotype. To our knowledge, this patient is the first to harbor a likely pathogenic homozygous mutation in both genes at the same time. Thus, the resulting complex phenotype of this patient illustrates the heterogeneity associated with ciliopathies and further expands the clinical and mutational spectrum of these diseases. Finally, we highlight the combined use of high-throughput tools to diagnose and support the proper handling of this and other patients.

Two girls with a de novo Xq rearrangement of paternal origin: t(X;9)(q24;q12) or rea(X)dup q.

OBJECTIVE: We report on two rare Xq rearrangements, namely a t(X;9)(q24;q12) found in a mildly-affected girl (Patient 1) and a rea(X)dup q concomitant with a rob(14;21)mat in a Down syndrome girl (Patient 2). CASE REPORT: Both rearrangements were characterized by banding techniques [Giemsa (G), constitutive heterochromatin (C), and bromodeoxyuridine (BrdU) pulse], fluorescence in situ hybridization (FISH) assays, human androgen receptor (HUMAR) assays, and microarray analyses. Patient 1 had a t(X;9)(q24;q12)dn. Patient 2 had a de novo rea(X)(qter→q23 or q24::p11.2→qter) concomitant with an unbalanced rob(14;21)mat. X-Inactivation studies in metaphases and DNA revealed a fully skewed inactivation: the normal homolog was silenced in Patient 1 and the rea(X) in Patient 2. Both rearranged X chromosomes were of paternal descent. Microarray analyses revealed no imbalances in Patient 1 whereas loss of Xp (∼52 Mb) and duplication of Xq (∼44 Mb) and 21q were confirmed in Patient 2. CONCLUSION: Our observations further document the cytogenetic heterogeneity and predominant paternal origin of certain de novo X-chromosome rearrangements.