A Witteveen-Kolk Syndrome Patient with Reflux Disease and a de novo Deletion of the SIN3A Gene.

Introduction: The Witteveen-Kolk syndrome (WITKOS) (OMIM: 613406) is a heterogeneous emerging disorder caused by pathogenic variants or microdeletions encompassing the SIN3A gene (SIN3 Transcription Regulator Family Member A). It is characterized by distinctive facial features, developmental delay, intellectual disability, microcephaly, short stature, and subtle anomalies on brain magnetic resonance imaging (MRI). To date, about 50 patients have been reported in the medical literature. Patient Presentation: In this article, we reported a patient with classic findings of WITKOS including global developmental delay, microcephaly, hypotonia, vomiting, malnutrition, autistic and dysmorphic facial features, and cardiac abnormalities. Also, a barium esophagogram suggested severe motility disorder and gastroesophageal reflux disease. Affymetrix CytoScan 750K microarray showed a de novo 1.6-Mb deletion at 15q24.1q24.2, including the whole SIN3A gene. We have also summarized the clinical features of WITKOS patients in the medical literature and cardiac abnormalities detected in 4 out of 10 patients in studies that clearly state that cardiac examination was performed in the patients. Conclusion: Our findings showed that cardiac defects are not uncommon findings in WITKOS. Physicians should also be aware of reflux disease and motility disorder in patients with feeding difficulty together with early cardiac examination in terms of an improved quality of life in WITKOS patients.

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function.

TRAPPC9-Related Intellectual Disability: Report of Two New Cases and Review of the Literature.

Introduction: Hereditary forms of intellectual disability (ID), an estimated prevalence ranging between 1% and 3% in the general population, are among the most important problems in health care. Especially, autosomal-recessive ID has a very heterogeneous molecular basis and a lack of specific phenotypic features. Methods: Here, we report on two unrelated patients with autosomal-recessive ID, microcephaly, and autistic features and review the patients with TRAPPC9-related ID. Whole-exome sequencing and array CGH were performed for molecular diagnosis of the patients. Results: The first case has a microdeletion on chromosome 8q24.23-q24.3 region which is 1.7 Mb in length and includes the last 5 exons of TRAPPC9, and c.3435delG [p.Thr1146Profs*8] deletion. The second case has a homozygous missense c.623A>C (p.His208Pro) variant in TRAPPC9 which is detected by means of whole-exome sequencing study of the proband. We also reviewed the clinical findings and mutation spectrum of all patients with TRAPPC9-related ID reported so far. Conclusions: Our study showed that the most consistent clinical findings for TRAPPC9-related ID are ID, microcephaly, and some structural brain MRI abnormalities. The mutations in the TRAPPC9 are scattered throughout all exons of TRAPPC9 indicating there is no hot spot mutation region in this gene.

Chromosome 1p status in neuroblastoma correlates with higher expression levels of miRNAs targeting neuronal differentiation pathway.

Neuroblastoma (NB) is characterized by acquired segmental and numerical chromosome aberrations. Although deletions of distal 1p and 11q are frequent alterations, no candidate tumor suppressor gene residing in these chromosomal sites could be identified so far. In the present study, we detected the genomic imbalances of six neuroblastoma cell lines using the multiplex ligation-dependent probe amplification (MLPA) technique and the microRNA (miRNA) expression profiles of the cell lines by a microarray study. According to MLPA results, we aimed to assess the miRNA expression profiles of the cell lines harboring 11q and 1p deletions. The cell lines with 1p deletions revealed statistically significant higher levels of expression for 29 miRNAs in contrast to the cell lines without 1p deletion in microarray study. We also performed GO enrichment analysis for predicted targets of the differentially expressed miRNAs. According to GO enrichment analysis, miRNAs that showed the high change in expression was associated with neuronal differentiation. We showed that hsa-miR-494, hsa-miR-495, and hsa-miR-543 target most of mRNAs in neuronal differentiation pathway. Although limited to the cell lines, our results highly suggest that NBs with different segmental chromosome abnormalities may have different dysregulated miRNA expression signatures that target the genes involved in neuronal differentiation.

KCNB1 frameshift variant caused inherited intellectual disability, developmental delay, and seizure.

Potassium voltage-gated channel subfamily B member 1 (KCNB1) encodes Kv2.1 potassium channel. KCNB1 mutations are known to cause global developmental delay, behavioral disorders, and various epilepsies. Most variants occur de novo and are rarely inherited. Here, we report a 14-year-old male patient who was admitted to our clinic with seizures, developmental delay history, and intellectual disability. Brain magnetic resonance image (MRI) was normal and electroencephalogram (EEG) showed spike and sharp-wave complexes emerging in the left hemisphere parietooccipital areas, which were paroxysmally generalized. We performed whole exome sequence analysis (WES) and identified a heterozygous frameshift mutation c.522delA in exon 1 of KCNB1 (NM_004975.4) predicting a premature stop codon p.Lys174Asnfs*20 in the proband. Sanger sequencing confirmed the heterozygous c.522delA mutation in the proband and his mother who also had epilepsy and learning difficulties. His 45 year old mother had used antiepileptic drugs for 9 years after a seizure episode at 12 years old. Also, his mother's uncle's son is nonverbal and has developmental delay and epilepsy. Our study shows that frameshift mutation cytoplasmic domain of KCNB1 gene can cause intrafamilial phenotypic variability and relatively mild clinical findings in these patients.

Rapid identification of Helicobacter pylori and assessment of clarithromycin susceptibility from clinical specimens using FISH.

Helicobacter pylori remains one of the most common bacterial infections worldwide. Clarithromycin resistance is the most important cause of H. pylori eradication failures. Effective antibiotic therapies in H. pylori infection must be rapidly adapted to local resistance patterns. We investigated the prevalence of clarithromycin resistance due to mutations in positions 2142 and 2143 of 23SrRNA gene of H. pylori by fluorescence in situ hybridisation (FISH), and compared with culture and antimicrobial susceptibility testing in 234 adult patients with dyspepsia who were enrolled. Antrum and corpus biopsy specimens were obtained for rapid urease test, histopathology and culture. Epsilometer test was used to assess clarithromycin susceptibility. H. pylori presence and clarithromycin susceptibility were determined by FISH in paraffin-embedded biopsy specimens. We found that 164 (70.1%) patients were positive for H. pylori based on clinical criteria, 114 (69.5% CI 62.5-76.6%) were culture positive, and 137 (83.5% CI 77.8-89.2%) were FISH positive. Thus the sensitivity of FISH was significantly superior to that of culture. However specificity was not significantly different (91.4 versus 100.0%, respectively). The resistance rate to clarithromycin for both antrum and corpus was detected in H. pylori-positive patients; 20.2% by FISH and 28.0% by E-test.The concordance between E-test and FISH was only 89.5% due to the presence of point mutations different from A2143G, A2142G or A2142C. We conclude that FISH is significantly more sensitive than culture and the E-test for the detection of H. pylori and for rapid determinination of claritromycin susceptibility. The superior hybridisation efficiency of FISH is becoming an emerging molecular tool as a reliable, rapid and sensitive method for the detection and visualisation of H. pylori, especially when the management of H. pylori eradication therapy is necessary. This is particularly important for the treatment of patients with H. pylori eradication failure.

The Detection of Genetic Parameters for Prognostic Stratification of Neuroblastoma Using Multiplex Ligation-Dependent Probe Amplification Technique.

BACKGROUND: Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system and the most frequent extra cranial solid tumor of early childhood. These tumors display a wide range of clinical behavior and are characterized by complex chromosomal changes, some of which are associated with distinct clinical phenotypes. We investigated the contribution of genetic variables to staging and histology by logistic regression analyses. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to detect segmental genomic imbalances and gene copy number changes in 202 primary NBs. RESULTS: Cases with NB were categorized into four distinct groups based on the genomic changes. Group 1 (48 cases, 23.7%) contained tumors with a 1p deletion and/or MYCN gene amplification (MNA). Group 2 included 46 cases (22.8%) with 3p and/or 11q deletions without 1p deletion and MYCN gene amplification. Tumors harboring at least two commonly observed deletions with or without MNA were classified as Group 3 (25 cases, 12.4%). Tumors with chromosomal imbalance other than MYCN gene amplification and 1p, 3p, and 11q deletions were in Group 4 (83 cases, 41.1%). MYCN gene amplification and 17q gain were significant predisposing factors for unfavorable histology. Significant correlations were detected between 1p deletion and MYCN gene amplification; 3p and 11q deletions; and 11q deletion and 17q gain. CONCLUSION: MLPA can be used effectively to simultaneously detect multiple genomic imbalances and these changes can be utilized to classify neuroblastomas by prognostic subtypes. The genetic changes detected in NB in this study and their associations with clinical characteristics are in line with previously published reports.

Copy number status and mutation analyses of anaplastic lymphoma kinase (ALK) gene in 90 sporadic neuroblastoma tumors.

Somatic and germline mutations of the anaplastic lymphoma kinase (ALK) gene were recently described in neuroblastoma (NB). In this study, we investigated the association of ALK copy number alterations with copy number status 2p24.1 amplicon harboring DEAD box polypeptide 1 (DDX1), MYCN and neuroblastoma-amplified (NAG) genes in 90 primary tumors of sporadic NB cases by multiplex ligation-dependent probe amplification (MLPA). We also performed mutation analysis of ALK gene by directly sequencing the exons 20-28 which cover the region that encodes juxtamembrane and kinase domains. A total of 39 (43.3%) NB cases revealed copy numbers alterations of ALK gene. There was highly significant association of ALK copy number gains with gains of one or more of the genes at 2p24.1 (DDX1, MYCN or NAG) in MYCN unamplified tumors (P<0.000). In addition, 15 of 17 MYCN amplified cases (88.2%) had aberrant ALK status. Solitary gain of ALK with normal copy number status of all other genes was observed only in one case. DNA sequencing of exons 20-28 of ALK revealed two different nucleotide changes in three cases leading to amino acid substitutions of F1245V and R1275Q in tyrosine kinase domain. In conclusion, the frequency of ALK mutations in NB is low and solitary copy number change of it is rarely observed.

Association of MYCN amplification and 1p deletion in neuroblastomas with high tumor vascularity.

The biologic behavior of neuroblastoma (NB) is extremely variable; therefore, the clinical behavior may be reliably predicted based on the analysis of a panel of prognostic parameters. High vascular density has been correlated with aggressive tumor progression in many types of cancers. The goal of this study was to correlate the tumor vascularity in NB with status of MYCN and the short arm of chromosome 1 (1p) to address the association between angiogenesis and genetic markers of prognostic significance. The study population consisted of 33 patients with histologically proven diagnosis of primary NB and no history of previous chemotherapy. Histologic quantitation of tumor angiogenesis was performed using 3 different methods: microvessel density, vascular grading, and Chalkley counting. MYCN amplification and 1p deletion were determined by using fluorescence in situ hybridization technique. The differentiation and mitosis-karyorrhexis index of tumor cells were also assessed using the Shimada System. MYCN amplification was present in 12 cases (36.3%), and 1p deletion in 16 (48.5%). Both genetic changes significantly correlated with increased tumor vascularity. In addition, tumor vascularity was significantly increased in tumors with high mitosis-karyorrhexis index or of undifferentiated histology. We conclude that angiogenesis shows close association with histologic and genetic prognosticators in NB. Our data support the validity of recent applications of antiangiogenic agents which interfere or block NB progression.

Detection of Helicobacter pylori and determination of clarithromycin susceptibility using formalin-fixed, paraffin-embedded gastric biopsy specimens by fluorescence in situ hybridization.

BACKGROUND: Clarithromycin resistance and poor compliance to therapy are often responsible for Helicobacter pylori eradication therapy failure. AIM: To evaluate fluorescence in situ hybridization (FISH) as a nonculture method to simultaneously detect H. pylori and to identify clarithromycin resistance. METHODS: Fifty-four patients with dyspepsia (17 male, 37 female subjects; mean age, 46.5; range, 21-78 years) were studied. Two antrum and corpus biopsies were taken from each patient. Positive rapid urease test (RUT) and histopathologic examinations defined H. pylori positivity. A total of 108 formalin-fixed paraffin-embedded gastric mucosal biopsies were examined retrospectively by the FISH (seaFAST H. pylori Combi-Kit) method. RESULTS: Forty-five patients (83.3%) were H. pylori positive and 43 (95.5%) were also positive by FISH. There were two false-positive FISH results. Fourteen patients (31.1%) had clarithromycin-susceptible strains, 4 (8.9%) resistant strains, and 27 (60%) both susceptible and resistant strains. CONCLUSION: FISH results correlated well with H. pylori infection and were able to identify clarithromycin-susceptible and -resistant strains. This technique will be helpful in determining the bacterial density and the success of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Correlation of modified Shimada classification with MYCN and 1p36 status detected by fluorescence in situ hybridization in neuroblastoma.

Neuroblastoma (NB) is a childhood cancer derived from neural crest cells, with a highly variable clinical course and biologic behavior. NB cells harbor complex genetic changes. Also, MYCN amplification is a well-known molecular marker for aggressive progression, and deletion of the short arm of chromosome 1 is frequently observed in NB. The aim of this study was to investigate the correlation between genetic markers and prognostic morphological parameters to address the biology and underlying the clinical complexity of NB. Therefore, we performed fluorescence in situ hybridization analyses of chromosome band 1p36 and MYCN in a series of tumors from 43 cases classified according to the recommendation of International Neuroblastoma Pathology Committee (modification of Shimada classification). The correlations of MYCN amplification status and two distinct types of 1p36 alterations (deletion and imbalance) with Shimada classification and histologic prognostic factors were statistically analyzed. Amplification of MYCN and 1p36 deletion was present in 14 (32.6%) and 18 (41.9%) cases, respectively. Sixteen cases (37.2%) displayed a favorable histology, while 27 (62.8%) had an unfavorable histology. The 1p36 deletion was found to be an independent predictor of unfavorable histology by multivariate analysis (logistic regression test, P = 0.03), but the 1p36 imbalance did not show any significance. Both 1p36 deletion and MYCN amplification showed significant correlation with undifferentiated tumors (chi-square test, P = 0.002 and 0.03, respectively). Highly significant correlation was found between the higher mitotic karyorrhectic index (MKI) and MYCN amplification (chi-square test, P < 0.001), whereas neither 1p36 deletion nor 1p36 imbalance significantly correlated with a higher MKI (chi-square test, P > 0.05). We conclude that 1p36 deletion may be a reliable parameter in determining unfavorable histology and predicting prognosis in NB. Further studies with prognostic data are needed to highlight its clinical significance.