A Missense De Novo Variant in the CASK-interactor KIRREL3 Gene Leading to Neurodevelopmental Disorder with Mild Cerebellar Hypoplasia.

KIRREL3 is a gene important for the central nervous system development-in particular for the process of neuronal migration, axonal fasciculation, and synaptogenesis-and colocalizes and cooperates in neurons with CASK gene. Alterations of KIRREL3 have been linked to neurodevelopmental disorders, ranging from developmental delay, to autism spectrum disorder, to attention deficit/hyperactivity disorder. The underlying mechanism is not yet fully understood, as it has been hypothesized a fully dominant effect, a risk factor role of KIRREL3 partially penetrating variants, and a recessive inheritance pattern. We report a novel and de novo KIRREL3 mutation in a child affected by severe neurodevelopmental disorder and with brain magnetic resonance imaging evidence of mega cisterna magna and mild cerebellar hypoplasia. This case strengthens the hypothesis that dominant KIRREL3 variants may lead to neurodevelopmental disruption; furthermore, given the strong interaction between KIRREL3 and CASK, we discuss as posterior fossa anomalies may also be part of the phenotype of KIRREL3-related syndrome.

Feasibility and Reliability Assessment of Video-Based Motion Analysis and Surface Electromyography in Children with Fragile X during Gait.

Fragile X Syndrome (FXS), the leading form of inherited intellectual disability and autism, is characterized by specific musculoskeletal conditions. We hypothesized that gait analysis in FXS could be relevant for the evaluation of motor control of gait, and help the understanding of a possible correlation between functional and intellectual abilities. Typical deficits in executive control and hyperactivity have hampered the use of standard gait analysis. The aim of our study was to quantitatively assess musculoskeletal alterations in FXS children in standard ambulatory conditions, in a friendly environment. Ten FXS children and sixteen controls, with typical neurodevelopment, were evaluated through four synchronized video cameras and surface electromyography; lower limb joints rotations, spatiotemporal parameters, duration of muscle contraction, activation timing and envelope peaks were determined. Reliability and repeatability of the video based kinematics analysis was assessed with respect to stereophotogrammetry. The Kruskal-Wallis Test (p < 0.05) or SPM1D were used to compare different groups of subjects. Results show a consistently altered gait pattern associated with abnormal muscle activity in FXS subjects: reduced knee and excessive hip and ankle flexion, and altered duration and activity onset on all the recorded muscles (Rectus/Biceps Femoris, Tibialis Anterior, Gastrocnemius Lateralis). Results of this study could help with planning personalized rehabilitations.

Frequency of Usher gene mutations in non-syndromic hearing loss: higher variability of the Usher phenotype.

Non-syndromic hearing loss (NSHL) is characterized by a vast genetic heterogeneity; some syndromic forms as Usher syndrome (USH) have onset as isolated deafness and then evolve later in life. We developed an NGS targeted gene-panel containing 59 genes and a customized bioinformatic pipeline for the analysis of DNA samples from clinically highly selected subjects with sensorineural hearing loss, previously resulted negative for GJB2 mutations/GJB6 deletions. Among the 217 tested subjects, 24 (11.1%) were found to carry mutations in genes involved both in NSHL and USH. For 6 out of 24 patients a diagnosis of USH was performed. Eleven subjects out of 24 had hearing loss without vestibular or ocular dysfunction and, due to their young age, it was not possible to establish whether their phenotype could be NSHL or USH. Seven subjects were diagnosed with NSHL, due to their age and phenotype. A total of 41 likely pathogenic/pathogenic mutations were identified, among which 17 novel ones. We report a high frequency of mutations in genes involved both in NSHL and in USH in a cohort of individuals tested for seemingly isolated deafness. Our data also highlight a wider than expected phenotypic variability in the USH phenotype.

Assessment of patient clinical descriptions and pathogenic variants from gene panel sequences in the CAGI-5 intellectual disability challenge.

The Critical Assessment of Genome Interpretation-5 intellectual disability challenge asked to use computational methods to predict patient clinical phenotypes and the causal variant(s) based on an analysis of their gene panel sequence data. Sequence data for 74 genes associated with intellectual disability (ID) and/or autism spectrum disorders (ASD) from a cohort of 150 patients with a range of neurodevelopmental manifestations (i.e. ID, autism, epilepsy, microcephaly, macrocephaly, hypotonia, ataxia) have been made available for this challenge. For each patient, predictors had to report the causative variants and which of the seven phenotypes were present. Since neurodevelopmental disorders are characterized by strong comorbidity, tested individuals often present more than one pathological condition. Considering the overall clinical manifestation of each patient, the correct phenotype has been predicted by at least one group for 93 individuals (62%). ID and ASD were the best predicted among the seven phenotypic traits. Also, causative or potentially pathogenic variants were predicted correctly by at least one group. However, the prediction of the correct causative variant seems to be insufficient to predict the correct phenotype. In some cases, the correct prediction has been supported by rare or common variants in genes different from the causative one.

Characterization of intellectual disability and autism comorbidity through gene panel sequencing.

Intellectual disability (ID) and autism spectrum disorder (ASD) are clinically and genetically heterogeneous diseases. Recent whole exome sequencing studies indicated that genes associated with different neurological diseases are shared across disorders and converge on common functional pathways. Using the Ion Torrent platform, we developed a low-cost next-generation sequencing gene panel that has been transferred into clinical practice, replacing single disease-gene analyses for the early diagnosis of individuals with ID/ASD. The gene panel was designed using an innovative in silico approach based on disease networks and mining data from public resources to score disease-gene associations. We analyzed 150 unrelated individuals with ID and/or ASD and a confident diagnosis has been reached in 26 cases (17%). Likely pathogenic mutations have been identified in another 15 patients, reaching a total diagnostic yield of 27%. Our data also support the pathogenic role of genes recently proposed to be involved in ASD. Although many of the identified variants need further investigation to be considered disease-causing, our results indicate the efficiency of the targeted gene panel on the identification of novel and rare variants in patients with ID and ASD.

Rhinencephalon changes in tuberous sclerosis complex.

PURPOSE: Despite complex olfactory bulb embryogenesis, its development abnormalities in tuberous sclerosis complex (TSC) have been poorly investigated. METHODS: Brain MRIs of 110 TSC patients (mean age 11.5 years; age range 0.5-38 years; 52 female; 26 TSC1, 68 TSC2, 8 without mutation identified in TSC1 or TSC2, 8 not tested) were retrospectively evaluated. Signal and morphological abnormalities consistent with olfactory bulb hypo/aplasia or with olfactory bulb hamartomas were recorded. Cortical tuber number was visually assessed and a neurological severity score was obtained. Patients with and without rhinencephalon abnormalities were compared using appropriate parametric and non-parametric tests. RESULTS: Eight of110 (7.2%) TSC patients presented rhinencephalon MRI changes encompassing olfactory bulb bilateral aplasia (2/110), bilateral hypoplasia (2/110), unilateral hypoplasia (1/110), unilateral hamartoma (2/110), and bilateral hamartomas (1/110); olfactory bulb hypo/aplasia always displayed ipsilateral olfactory sulcus hypoplasia, while no TSC patient harboring rhinencephalon hamartomas had concomitant forebrain sulcation abnormalities. None of the patients showed overt olfactory deficits or hypogonadism, though young age and poor compliance hampered a proper evaluation in most cases. TSC patients with rhinencephalon changes had more cortical tubers (47 ± 29.1 vs 26.2 ± 19.6; p = 0.006) but did not differ for clinical severity (p = 0.45) compared to the other patients of the sample. CONCLUSIONS: Olfactory bulb and/or forebrain changes are not rare among TSC subjects. Future studies investigating clinical consequences in older subjects (anosmia, gonadic development etc.) will define whether rhinencephalon changes are simply an imaging feature among the constellation of TSC-related brain changes or a feature to be searched for possible implications in the management of TSC subjects.

Pyridoxine-dependent epilepsies: an observational study on clinical, diagnostic, therapeutic and prognostic features in a pediatric cohort.

The aim of our study was to describe the clinical, electroencephalogram, molecular findings and the diagnostic and therapeutic flow-chart of children with pyridoxine-dependent epilepsies (PDEs). We performed a retrospective observational study on children with PDEs, diagnosed and followed-up in Italian Pediatric Departments. In each centre, the authors collected data from a cohort of children admitted for intractable seizures, responsive to pyridoxine administration and resistant to other anticonvulsant therapies. Data were retrospectively analysed from January 2016 to January 2017. Sixteen patients (13 males, and 3 females) were included. We found that 93.75% of patients underwent conventional anticonvulsant therapy before starting pyridoxine administration and 62.5% had ex-juvantibus diagnosis, as specific serum diagnostic tests had been performed in only 37.5% of patients by alpha-AASA and pipecolic acid blood and urine dosage. The most common type of seizure was generalized tonic-clonic in 7 patients and the most common EEG pattern was characterized by a "burst suppression" pattern. Before pyridoxine administration, other anticonvulsant drugs were used in 93.75% of patients, with consequent onset of drug-resistance. Phenobarbital was the most frequently used drug as first-line treatment. The importance of our study relies on the need of a deeper knowledge of PDEs in terms of early diagnosis, avoiding incorrect treatment and related adverse events, clinical and EEG pathognomonic features, and genetic aspects of the disease.

[Accurate compiling of the hospital discharge records according to clinicians' perception: critical issues and perspectives]. / L'accurata compilazione delle schede di dimissione ospedaliera secondo la percezione dei medici: criticità e prospettive.

OBJECTIVES: to explore clinicians vision on hospital discharge records in order to identify useful elements to foster a more accurate compiling. DESIGN: qualitative research with phenomenological approach. SETTING AND PARTICIPANTS: participants were selected through purposive sampling among clinicians of two hospitals located in Sardinia; the sample included 76 people (32 medical directors and 44 doctors in training). MAIN OUTCOME MEASURES: identified codes for themes under investigation: vision of accurate compiling, difficulties, and proposals. RESULTS: collected data highlighted two prevailing visions, respectively focused on the importance of an accurate compiling and on the burden of such activity. The accurate compiling is hindered by the lack of motivation and training, by the limits of the registration system and the information technology, by the distortions induced by the prominent role of the hospital discharge records in the evaluation processes. Training, timely updating of the information system accompanied by a proper cross-cultural validation process, improvement of the computer system, and activation of support services could promote more accurate compiling. CONCLUSIONS: the implementation of services, unconnected with evaluation and control processes, dedicated to training and support in the compiling of the hospital discharge records and in the conduction of related epidemiological studies would facilitate the compliance to the compilation. Such services will make tangible the benefits obtainable from this registration system, increasing skills, motivation, ownership, and facilitating greater accuracy in compiling.

Volumetric image-guided conformal radiotherapy for localized prostate cancer: Analysis of dosimetric and clinical factors affecting acute and late toxicity.

AIM: To identify factors influencing toxicity in patients affected by localized prostate cancer treated with conformal image-guided radiotherapy. BACKGROUND: Image guidance in combination with conformal techniques is the standard of care in localized prostate cancer, but factors affecting toxicity are still under investigation. MATERIALS AND METHODS: 294 patients were analyzed. Median age at diagnosis was 71 year. 76 Gy (38 × 2 Gy) were delivered to the target volume. We used the χ2 test to analyse associations between toxicity and dosimetric and clinical parameters. Multivariate analysis was performed using binary logistic regression. Kaplan-Meier method was used for survival analysis. RESULTS: Median follow-up was 62.9 months. Acute grade ≥2 gastro-intestinal toxicity (GI) was 12.1%. Acute genito-urinary (GU) toxicity of grade ≥2 was 33.9%. Actuarial 4 and 5 years late grade ≥2 GI was 3% and 4%, respectively. Four and 5-year late grade ≥2 GU toxicity was 6% and 10%. At multivariate analysis for acute toxicity rectal V70 was correlated with GI toxicity (p = 0.01, HR 2.73 CI 1.19-6.26), and smoking habit with GU toxicity (p < 0.01, HR 2.50 CI 1.51-4.14). For late toxicity, rectal V70 was correlated with gastro-intestinal toxicity (p = 0.04, HR 4.76 CI 1.07-21.13), and pre-radiotherapy urinary symptoms with genito-urinary toxicity (p = 0.01, HR 2.84 CI 1.29-6.22). DISCUSSION: Conformal image-guided radiotherapy shows low rates of toxicity. Smoking should be avoided during radiotherapy. Besides the evaluation of high doses received by the organs at risk, individual factors, such as co-morbidities and lifestyle choices, have an impact on normal-tissue complication risk.

Oligometastatic cancer: stereotactic ablative radiotherapy for patients affected by isolated body metastasis.

BACKGROUND: To evaluate the outcome of patients affected by a single isolated body metastasis treated with stereotactic body radiotherapy (SBRT). MATERIAL AND METHODS: Seven-eight patients were treated with SBRT for isolated body metastasis. The most frequent primary tumor was prostate cancer (28.2%), followed by colorectal cancer (23.1%) and lung cancer (20.5%). Median age at diagnosis of oligometastatic disease was 70 years (range 47-88). Median Karnofsky Performance Status (KPS) was 90 (range 70-100). The most common SBRT fractionation scheme was 5 × 7 Gy (total dose 35 Gy). Response to radiotherapy was determined according to RECIST criteria v1.1. Toxicity was registered according to Common Terminology Criteria for Adverse Events (CTCAE) v4.0. The survival analysis was performed with the Kaplan-Meier method. The correlation between time actuarial incidence and clinical parameters was studied, and the Kaplan-Meier method of log-rank test was applied. RESULTS: With a median follow-up of 22.68 months, local control was achieved in 89.7% of the cases. The two-year overall survival (OS) and progression-free survival (PFS) were 68% and 42%, respectively. On univariate analysis, KPS ≥80 is predictive for improved OS (p = .001) and PFS (p = .001). Acute toxicity of grade ≥2 occurred in eight (10.2%) patients and late grade ≥2 toxicity in five (6.4%) patients. CONCLUSIONS: Ablative radiotherapy in 'early oligometastatic state' is a safe, effective and minimally invasive treatment modality. A good performance status (KPS ≥80) seems to influence the clinical outcome.

Volumetric image-guided highly conformal radiotherapy of the prostate bed: Toxicity analysis.

AIM: To evaluate toxicity of high conformal image-guided radiotherapy of the prostate bed. BACKGROUND: Radiotherapy of the prostate bed has a pivotal role in the post-operative and salvage settings, but few clinical data are available on the use of daily image guidance in combination with highly conformal techniques, and data on long-term results are lacking. MATERIALS AND METHODS: We analyzed 118 patients irradiated on the prostate bed using conformal plans processed with a micro-multileaf collimator, and daily checking treatment set-up with a cone-beam CT system. Correlation between toxicity and clinical-dosimetric parameters was assessed by the Cox regression model and log-rank test. Survival analyses were performed with the Kaplan-Meier method. RESULTS: Median follow-up was 54.08 months. Late grade ≥2 gastro-intestinal (GI) and genito-urinary (GU) toxicity were 3.4% and 4.2%, respectively. Actuarial 4-year late grade ≥2 GI and GU toxicities were 4% and 6%, respectively. Four-year relapse-free survival was 87%. At log-rank test, acute grade ≥2 GI toxicity is associated with the use of antihypertensives (p = 0.03), and there is a trend toward significance between the use of anticoagulants and late grade ≥2 GI toxicity (p = 0.07). At Cox analysis, acute grade ≥2 GU toxicity is correlated with the percentage of bladder volume receiving more than 65 Gy (p = 0.02, HR 1.87 CI 1.25-2.8), and the maximal dose to the rectum is correlated to the development of late grade ≥2 GI toxicity (p = 0.03, HR 2.75 CI 1.10-6.9). CONCLUSIONS: Conformal volumetric image-guided radiotherapy of the prostate bed leads to low toxicity rates.

Functional characterization of CDK5 and CDK5R1 mutations identified in patients with non-syndromic intellectual disability.

Cyclin-dependent kinase 5 (CDK5) and cyclin-dependent kinase 5, regulatory subunit 1 (CDK5R1), encoding CDK5 activator p35, have a fundamental role in central nervous system (CNS) development and function, and are involved in the pathogenesis of several neurodegenerative disorders, thus constituting strong candidate genes for the onset of intellectual disability (ID). We carried out a mutation screening of CDK5 and CDK5R1 coding regions and CDK5R1 3'-UTR on a cohort of 360 patients with non-syndromic ID (NS-ID) using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. We found one novel silent mutation in CDK5 and one novel silent mutation in CDK5R1 coding regions, three novel intronic variations in CDK5, not causing any splicing defect, and four novel heterozygous variations in CDK5R1 3'-UTR. None of these variations was present in 450 healthy controls and single-nucleotide polymorphism (SNP) databases. The functional study of CDK5R1 p.A108V mutation evidenced an impaired p35 cleavage by the calcium-dependent protease calpain. Moreover, luciferase constructs containing the CDK5R1 3'-UTR mutations showed altered gene expression levels. Eight known polymorphisms were also identified displaying different frequencies in NS-ID patients compared with the controls. In particular, the minor allele of CDK5R1 3'-UTR rs735555 polymorphism was associated with increased risk for NS-ID. In conclusion, our data suggest that mutations and polymorphisms in CDK5 and CDK5R1 genes may contribute to the onset of the NS-ID phenotype.

Identification of four novel PCDH19 Mutations and prediction of their functional impact.

The PCDH19 gene encodes protocadherin-19, a transmembrane protein with six cadherin (EC) domains, containing adhesive interfaces likely to be involved in neuronal connection. Over a hundred mostly private mutations have been identified in girls with epilepsy, with or without intellectual disability (ID). Furthermore, transmitting hemizygous males are devoid of seizures or ID, making it difficult to establish the pathogenic nature of newly identified variants. Here, we describe an integrated approach to evaluate the pathogenicity of four novel PCDH19 mutations. Segregation analysis has been complemented with an in silico analysis of mutation effects at the protein level. Using sequence information, we compared different computational prediction methods. We used homology modeling to build structural models of two PCDH19 EC-domains, and compared wild-type and mutant models to identify differences in residue interactions or biochemical properties of the model surfaces. Our analysis suggests different molecular effects of the novel mutations in exerting their pathogenic role. Two of them interfere with or alter functional residues predicted to mediate ligand or protein binding, one alters the EC-domain folding stability; the frame-shift mutation produces a truncated protein lacking the intracellular domain. Interestingly, the girl carrying the putative loss of function mutation presents the most severe phenotype.

Acute and late toxicity after three-dimensional conformal image-guided radiotherapy for localized prostate cancer.

We evaluated the clinical impact of a high definition micro-multileaf collimator and a linac-integrated cone-beam computed tomography in 142 patients treated with conformal radiotherapy for localized prostate cancer to a total dose of 76 Gy. Details on treatment toxicity and tumour control were collected. The 3 years biochemical relapse-free survival was 90%. Acute and late gastrointestinal toxicities were low (3-year actuarial late toxicity of 11.2%). Acute genitourinary toxicity was relatively high, the 3-year actuarial genitourinary late toxicity was 12%. Conformal image-guided radiotherapy for localized prostate cancer leads to low rates of late toxicity with a high rate of tumor control.

Standard or hypofractionated radiotherapy in the postoperative treatment of breast cancer: a retrospective analysis of acute skin toxicity and dose inhomogeneities.

BACKGROUND: To identify predictive factors of radiation-induced skin toxicity in breast cancer patients by the analysis of dosimetric and clinical factors. METHODS: 339 patients treated between January 2007 and December 2010 are included in the present analysis. Whole breast irradiation was delivered with Conventional Fractionation (CF) (50 Gy, 2.0/day, 25 fractions) and moderate Hypofractionated Schedule (HS) (44 Gy, 2.75 Gy/day, 16 fractions) followed by tumour bed boost. The impact of patient clinical features, systemic treatments and, in particular, dose inhomogeneities on the occurrence of different levels of skin reaction has been retrospectively evaluated. RESULTS: G2 and G3 acute skin toxicity were 42% and 13% in CF patients and 30% and 7.5% in HS patients respectively. The retrieval and revaluation of 200 treatment plans showed a strong correlation between areas close to the skin surface, with inhomogeneities >107% of the prescribed dose, and the desquamation areas as described in the clinical records. CONCLUSIONS: In our experience dose inhomogeneity underneath G2 - G3 skin reactions seems to be the most important predictor for acute skin damage and in these patients more complex treatment techniques should be considered to avoid skin damage. Genetic polymorphisms too have to be investigated as possible promising candidates for predicting acute skin reactions.

Activation Ratio Correlates with IQ in Female Carriers of the FMR1 Premutation.

Carriers of the FMR1 premutation (PM) allele are at risk of one or more clinical conditions referred to as FX premutation-associated conditions (FXPAC). Since the FMR1 gene is on the X chromosome, the activation ratio (AR) may impact the risk, age of onset, progression, and severity of these conditions. The aim of this study was to evaluate the reliability of AR measured using different approaches and to investigate potential correlations with clinical outcomes. Molecular and clinical assessments were obtained for 30 PM female participants, and AR was assessed using both Southern blot analysis (AR-Sb) and methylation PCR (AR-mPCR). Higher ARs were associated with lower FMR1 transcript levels for any given repeat length. The higher AR-Sb was significantly associated with performance, verbal, and full-scale IQ scores, confirming previous reports. However, the AR-mPCR was not significantly associated (p > 0.05) with these measures. Similarly, the odds of depression and the number of medical conditions were correlated with higher AR-Sb but not correlated with a higher AR-mPCR. This study suggests that AR-Sb may be a more reliable measure of the AR in female carriers of PM alleles. However, further studies are warranted in a larger sample size to fully evaluate the methylation status in these participants and how it may affect the clinical phenotype.

Expanding the genetics and phenotypic spectrum of Lysine-specific demethylase 5C (KDM5C): a report of 13 novel variants.

Lysine-specific demethylase 5C (KDM5C) has been identified as an important chromatin remodeling gene, contributing to X-linked neurodevelopmental disorders (NDDs). The KDM5C gene, located in the Xp22 chromosomal region, encodes the H3K4me3-me2 eraser involved in neuronal plasticity and dendritic growth. Here we report 30 individuals carrying 13 novel and one previously identified KDM5C variants. Our cohort includes the first reported case of somatic mosaicism in a male carrying a KDM5C nucleotide substitution, and a dual molecular finding in a female carrying a homozygous truncating FUCA1 alteration together with a de novo KDM5C variant. With the use of next generation sequencing strategies, we detected 1 frameshift, 1 stop codon, 2 splice-site and 10 missense variants, which pathogenic role was carefully investigated by a thorough bioinformatic analysis. The pattern of X-chromosome inactivation was found to have an impact on KDM5C phenotypic expression in females of our cohort. The affected individuals of our case series manifested a neurodevelopmental condition characterized by psychomotor delay, intellectual disability with speech disorders, and behavioral features with particular disturbed sleep pattern; other observed clinical manifestations were short stature, obesity and hypertrichosis. Collectively, these findings expand the current knowledge about the pathogenic mechanisms leading to dysfunction of this important chromatin remodeling gene and contribute to a refinement of the KDM5C phenotypic spectrum.

CAGI6 ID-Challenge: Assessment of phenotype and variant predictions in 415 children with Neurodevelopmental Disorders (NDDs).

In the context of the Critical Assessment of the Genome Interpretation, 6th edition (CAGI6), the Genetics of Neurodevelopmental Disorders Lab in Padua proposed a new ID-challenge to give the opportunity of developing computational methods for predicting patient's phenotype and the causal variants. Eight research teams and 30 models had access to the phenotype details and real genetic data, based on the sequences of 74 genes (VCF format) in 415 pediatric patients affected by Neurodevelopmental Disorders (NDDs). NDDs are clinically and genetically heterogeneous conditions, with onset in infant age. In this study we evaluate the ability and accuracy of computational methods to predict comorbid phenotypes based on clinical features described in each patient and causal variants. Finally, we asked to develop a method to find new possible genetic causes for patients without a genetic diagnosis. As already done for the CAGI5, seven clinical features (ID, ASD, ataxia, epilepsy, microcephaly, macrocephaly, hypotonia), and variants (causative, putative pathogenic and contributing factors) were provided. Considering the overall clinical manifestation of our cohort, we give out the variant data and phenotypic traits of the 150 patients from CAGI5 ID-Challenge as training and validation for the prediction methods development.