Additional lesions identified by genomic microarrays are associated with an inferior outcome in low-risk chronic lymphocytic leukaemia patients.

We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low-risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL-IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time-to-first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time-to-first treatment in the subset of patients with wild-type TP53 and mutated IGHV (p = 0.025). In CLL patients with low-risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk-adapted follow-up and for early treatment including targeted agents within clinical trials.

Protection against Ischemic Heart Disease: A Joint Role for eNOS and the KATP Channel.

Genetic susceptibility may influence ischemic heart disease (IHD) predisposition and affect coronary blood flow (CBF) regulation mechanisms. The aim of this study was to investigate the association among single nucleotide polymorphisms (SNPs) of genes encoding for proteins involved in CBF regulation and IHD. A total of 468 consecutive patients were enrolled and divided into three groups according to coronary angiography and intracoronary functional tests results: G1, patients with coronary artery disease (CAD); G2, patients with coronary microvascular dysfunction (CMD); and G3, patients with angiographic and functionally normal coronary arteries. A genetic analysis of the SNPs rs5215 of the potassium inwardly rectifying channel subfamily J member 11 (KCNJ11) gene and rs1799983 of the nitric oxide synthase 3 (NOS3) gene, respectively encoding for the Kir6.2 subunit of ATP sensitive potassium (KATP) channels and nitric oxide synthase (eNOS), was performed on peripheral whole blood samples. A significant association of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 genes was detected in healthy controls compared with CAD and CMD patients. Based on univariable and multivariable analyses, the co-presence of rs5215_G/G of KCNJ11 and rs1799983_T/T of NOS3 may represent an independent protective factor against IHD, regardless of cardiovascular risk factors. This study supports the hypothesis that SNP association may influence the crosstalk between eNOS and the KATP channel that provides a potential protective effect against IHD.

KDM6A missense variants hamper H3 histone demethylation in lung squamous cell carcinoma.

KDM6A is the disease causative gene of type 2 Kabuki Syndrome, a rare multisystem disease; it is also a known cancer driver gene, with multiple somatic mutations found in a few cancer types. In this study, we looked at eleven missense variants in lung squamous cell carcinoma, one of the most common lung cancer subtypes, to see how they affect the KDM6A catalytic mechanisms. We found that they influence the interaction with histone H3 and the exposure of the trimethylated Lys27, which is critical for wild-type physiological function to varying degrees, by altering the conformational transition.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

Small Bowel Obstruction After Laparoscopic Roux-en-Y Gastric Bypass Caused by Hemobezoar: A Case Series and Review of Literature.

BACKGROUND: The development of an intraluminal blood clot (hemobezoar), represents a rare cause (0.05% to 1.9%) of small bowel obstruction (SBO) after laparoscopic Roux-en-Y gastric bypass (LRYGB). We present a case series of 6 cases of SBO caused by hemobezoar. METHODS: A retrospective analysis of a prospective database including patients who underwent LRYGB from January 2010 to December 2019 has been performed. All the patients who underwent reoperation because of an SBO caused by an intraluminal blood clot were included in the present study. RESULTS: Six of 843 LRYGB patients developed an hemobezoar (0.71%). Primary LRYGB was uneventful in all cases. SBO symptoms developed after a mean interval of 26.6 hours from the primary procedure. All reoperations were performed with the laparoscopic approach. In all cases, the hemobezoar was located at the level of the jejunojejunal anastomosis (JJA) and was removed through an enterotomy performed at the distal end of the biliopancreatic stump. Three postoperative complications occurred: 1 ab-ingestis pneumonia, 1 leak of the JJA requiring further reoperation, and 1 pelvic abscess treated with radiologic drainage. The mean hospital stay was 11 days. DISCUSSION: SBO due to hemobezoar is a rare but worrisome early complication after LRYGB. It almost always affects the JJA and requires a prompt diagnosis to avoid dreadful sequelae. In the absence of anastomotic leak or stenosis, surgical management may consist of the removal of the blood clot without refashioning the anastomosis and it may be accomplished with the laparoscopic approach.

GDF5 mutation case report and a systematic review of molecular and clinical spectrum: Expanding current knowledge on genotype-phenotype correlations.

INTRODUCTION: Brachydactyly is a bone development abnormality presenting with variable phenotypes and different transmission patterns. Mutations in GDF5 (Growth and Differentiation Factor 5, MIM *601146) account for a significant amount of cases. Here, we report on a three-generation family, where the proband and the grandfather have an isolated brachydactyly with features of both type A1 (MIM #112500) and type C (MIM #113100), while the mother shows only subtle hand phenotype signs. MATERIALS AND METHODS: Whole Exome Sequencing (WES) was performed on the two affected individuals. An in-depth analysis of GDF5 genotype-phenotype correlations was performed through literature reviewing and retrieving information from several databases to elucidate GDF5-related molecular pathogenic mechanisms. RESULTS: WES analysis disclosed a pathogenic variant in GDF5 (NM_000557.5:c.157dup; NP_000548.2:p.Leu53Profs*41; rs778834209), segregating with the phenotype. The frameshift variant was previously associated with Brachydactyly type C (MIM #113100), in heterozygosity, and with the severe Grebe type chondrodysplasia (MIM #200700), in homozygosity. In-depth analysis of literature and databases allowed to retrieve GDF5 mutations and correlations to phenotypes. We disclosed the association of 49 GDF5 pathogenic mutations with eight phenotypes, with both autosomal dominant and recessive transmission patterns. Clinical presentations ranged from severe defects of limb morphogenesis to mild redundant ossification. We suggest that such clinical gradient can be linked to a continuum of GDF5-activity variation, with loss of GDF5 activity underlying bone development defects, and gain of function causing disorders with excessive bone formation. CONCLUSIONS: Our analysis of GDF5 pathogenicity mechanisms furtherly supports that mutation and zygosity backgrounds resulting in the same level of GDF5 activity may lead to similar phenotypes. This information can aid in interpreting the potential pathogenic effect of new variants and in supporting an appropriate genetic counseling.

Prenatal whole exome sequencing detects a new homozygous fukutin (FKTN) mutation in a fetus with an ultrasound suspicion of familial Dandy-Walker malformation.

BACKGROUND: Posterior fossa malformations are among the most diagnosed central nervous system (CNS) anomalies detected by ultrasound (US) in prenatal age. We identified the pathogenic gene mutation in a male fetus of 17 weeks of gestation with US suspicion of familial Dandy-Walker spectrum malformation, using Next Generation Sequencing approach in prenatal diagnosis. METHODS: Whole exome sequencing (WES) approach has been performed on fetal genomic DNA. After reads preprocessing, mapping, variant calling, and annotation, a filtering strategy based on allelic frequency, recessive inheritance, and phenotypic ontologies has been applied. A fetal magnetic resonance imaging (MRI) at 18 weeks of gestation has been performed. An in silico analysis of a potential causative missense variant in the fukutin protein has been carried out through a structural modeling approach. RESULTS: We identified a new homozygous missense mutation in fukutin gene (FKTN, NM_006731.2: c.898G>A; NP_006722.2: p.Gly300Arg). Fetal MRI supported molecular findings. Structural modeling analyses indicated a potential pathogenetic mechanism of the variant, through a reduced activation of the sugar moieties, which in turn impairs transfer to dystroglycan and thus its glycosylation. These findings pointed to a redefinition of the US suspicion of recurrence of Dandy-Walker malformation (DWM) to a muscular dystrophy-dystroglycanopathy type A4. CONCLUSIONS: The present case confirmed WES as a reliable tool for the prenatal identification of the molecular bases of early-detected CNS malformations.

Intussusception After Roux-en-Y Gastric Bypass: Laparoscopic Management.

INTRODUCTION: Intussusception represents an uncommon cause of intestinal obstruction after Rouxen-Y gastric bypass. Symptoms are not specific and clinical presentation may vary from acute intestinal obstruction with or without bowel necrosis to intermittent or chronic pain. CT scan is the diagnostic test of choice. MATERIALS AND METHODS: A 38-year-old woman who had undergone RYGBP 5 months prior was admitted to our Emergency Department with acute abdominal pain, alimentary and bilious vomiting, and fever. A CT scan revealed an intussusception after the anastomosis and dilatation of the biliopancreatic limb and the gastric remnant. An emergency laparoscopic exploration was performed. RESULTS: The patient undergoes an explorative laparoscopy. A bowel intussusception starting distally at the jejunojejunostomy and involving the latter is discovered. The common channel is divided first, and after that, the alimentary limb is resected. The biliary limb is identified, marked, and finally divided. A side-to-side jejunojejunal anastomosis is created between the alimentary limb and the common limb. Finally, the anastomosis between the common limb and the biliopancreatic limb is fashioned about 30 cm distally from the latter anastomosis. The total operative time was 130 min. Postoperative course was uneventful, and the patient was discharged on the fifth postoperative day. CONCLUSION: Although rare, intussusception after RYGBP must be considered as a possible cause of intestinal obstruction. In case of a small bowel intussusception, a surgical resection is recommended. A laparoscopic approach to treat bowel intussusception after RYGBP is safe and feasible.

Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome.

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies.

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

The emerging role of MicroRNA in schizophrenia.

MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that control gene expression post-transcriptionally by base pairing to mRNAs. MiRNAs are predicted to target ~50% of all protein-coding genes and functional studies indicate that they participate in the regulation of almost every cellular process. They also play a key role in pathogenetic mechanisms underlying several diseases, e.g. cancer, cardiovascular diseases, autoimmune diseases, and neurodegenerative diseases. Several miRNAs are expressed in the human brain where they contribute to equilibrium between maintenance and differentiation of neural stem cells. MiRNAs specific mechanisms of action and their roles in brain development and synaptic plasticity resulted in a great interest in the analysis of their potential role in the pathogenesis and pathophysiology of neuropsychiatric disorders. Currently, schizophrenia is one of the fields in psychiatry where miRNAs have been most widely investigated. The understanding of miRNAs role in schizophrenia has been achieved through association, functional and expression profiling studies on post mortem brain and peripheral tissues. Several studies identified association between neuropsychiatric disorders and variants in miRNAs including variations in miRNA/primary-/precursor-miRNAs sequences, in miRNAs biogenesis machinery genes, in the 3'UTR of target genes and in miRNAs expression. In summary, there is growing evidence that miRNAs exert a crucial role in gene expression regulation in the central nervous system and are altered in the development, presentation and response to treatment of psychiatric disorders. In this review we summarize the most significant results of experimental studies aimed at highlighting the involvement of human miRNAs in schizophrenia.

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor ß and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.

Ago1 and Ago2 differentially affect cell proliferation, motility and apoptosis when overexpressed in SH-SY5Y neuroblastoma cells.

Argonaute are a conserved class of proteins central to the microRNA pathway. We have highlighted a novel and non-redundant function of Ago1 versus Ago2; the two core factors of the miRNA-associated RISC complex. Stable overexpression of Ago1 in neuroblastoma cells causes the cell cycle to slow down, a decrease in cellular motility and a stronger apoptotic response upon UV irradiation. These effects, together with a significant increase in p53 levels, suggest that Ago1 may act as a tumor-suppressor factor, a function also supported by GEO Profiles microarrays that inversely correlate Ago1 expression levels with cell proliferation rates.

Heterozygous germline mutations in the CBL tumor-suppressor gene cause a Noonan syndrome-like phenotype.

RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies.

Quantification of small non-coding RNAs allows an accurate comparison of miRNA expression profiles.

MicroRNAs (miRNAs) are highly conserved approximately 22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3'-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family.

Brody disease is a rare muscle disorder characterized by exercise-induced impairment in muscle relaxation, due to a markedly reduced influx of calcium ions in the sarcoplasmic reticulum. A subset of autosomal recessive families harbour mutations in the ATP2A1 gene, encoding the fast-twitch skeletal muscle sarcoplasmic reticulum Ca(2+) ATPase (SERCA1). Rare autosomal dominant families have been described, in which ATP2A1 was excluded as the causative gene, further supporting genetic heterogeneity. We report four individuals from a three-generation Italian family with a clinical phenotype of Brody disease, in which linkage analysis excluded ATP2A1 as the responsible gene. The disease cosegregates in an autosomal dominant fashion with an apparently balanced constitutional chromosome translocation (2;7)(p11.2;p12.1), suggesting a causal relationship between the rearrangement and the phenotype. FISH analysis using YAC and PAC clones as probes refined the breakpoint regions to genomic segments of about 164 and 120 kb, respectively, providing a possible clue to pinpoint the location of a novel gene responsible for this rare muscle disorder.

Hereditary early-onset Parkinson's disease caused by mutations in PINK1.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.