Diamond-Blackfan syndrome: evidence against cell-mediated erythropoietic suppression

The profound anemia of Diamond-Blackfan syndrome (DBS) is due to marrow red cell failure, but the pathogenesis is not understood. Studies by others indicated cell-mediated erythropoietic suppression in this condition. To explore this mechanism further, Ficoll-Hypaque--separated peripheral blood lymphocytes (PBL) from four anemic untreated patients with DBS, or from normals were cocultured with control marrow in vitro and the growth of erythropoietin-responsive stem cell colonies (CFU-E) was dermined. CFU-E numbers obtained from cultures with added normal PBL were not significantly different from the number without PBL. Similarly, CFU-E from cultures with added DBS PBL were not significantly different from the number without PBL (215 versus 220, 229 versus 220 and 84 versus 60, 74 versus 94/10(5) cells, respectively). Mixing marrows from a control and one DBS patient in ratios of 2:1, 1:1, or 1:2 prior to culture failed to disclose a decrease of colony growth. We could not show cellular inhibition of erythropoiesis in these patients with DBS. The mechanism of anemia in this disorder remains an open question.     

Mutations in GAS8, a Gene Encoding a Nexin‐Dynein Regulatory Complex Subunit,Cause Primary Ciliary Dyskinesia with Axonemal Disorganization

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few—including CCDC39 and CCDC40—carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi‐allelic loss‐of‐function mutations in GAS8, a gene that encodes a subunit of the nexin‐dynein regulatory complex (N‐DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high‐speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients’ respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N‐DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N‐DRC integrity and in the proper alignment of axonemal microtubules in humans.     

p53蛋白的免疫亲和层析纯化

建立了ｐ５３单克隆抗体ｐＡｂ１８０１的免疫亲和层极法纯化ｐ５３蛋白，所纯化的ｐ５３蛋白经Ｗｅｓｔｅｒｎ Ｂｌｏｔ（ＥＣＬ法）检测证明：用此法从ｐ５３阳性的ＳＷ４８０细胞中分离到ｐ５３蛋白。银染显示ｐＨ２．０甘氨酸缓冲液比ｐＨ２．８的甘氨酸缓冲液洗脱效果好，这种方法的建立将为分离肿瘤细胞中引起ｐ５３蛋白功能失活和研究肿瘤发生机制提供一种有效途径。     

Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disease of motile cilia. Even though PCD is widely studied, North‐African patients have been rarely explored. In this study, we aim at confirming the clinical diagnosis and explore the genetic spectrum of PCD in a cohort of Tunisian patients. Forty clinically diagnosed patients with PCD belonging to 34 families were recruited from Tunisian pediatric departments. In each proband, targeted capture PCD panel sequencing of the 40 PCD genes was performed. PCD panel sequencing identified bi‐allelic mutations in 82% of the families in eight PCD genes. Remarkably, 23.5% of patients carried the same c.2190del CCDC39 mutation. Single nucleotide polymorphism profiling in six unrelated patients carrying this mutation has revealed a founder effect in North‐African patients. This mutation is estimated to date back at least 1,400–1,750 years ago. The identification of this major allele allowed us to suggest a cost‐effective genetic diagnostic strategy in North‐African patients with PCD.     

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin‐2 (PKP2) identified with microarray analysis and/or multiplex ligation‐dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.     

Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples

Frozen tissue is considered the gold standard if DNA is to be extracted for polymerase chain reaction (PCR) analysis. In molecular studies from paraffin-embedded material, only positive results are usually taken into account. Our goal was to evaluate both the sensitivity and the specificity of PCR techniques for immunoglobulin heavy chain (IgH) gene rearrangement according to various lengths and types of fixative before paraffin embedding. One set of studies compared IgH rearrangement in a case of mantle cell lymphoma tissue that had been fixed in 14 different ways before paraffin embedding and frozen tissue. Formalin fixation was found not to be deleterious for DNA, amplification being possible up to 15 days after fixation with good sensitivity. In contrast, the performance of PCR decreased for samples fixed in Bouin's liquid for longer than 6 hours or after 48 hours of incubation in a vacuum infiltration processor (in which Bouin's liquid-fixed and formalin-fixed samples are mixed). In addition, we undertook a retrospective study of 20 routinely processed B-cell lymphomas, with frozen formalin-fixed and Bouin's liquid-fixed tissues for each case. Of the 14 positive cases on frozen material, 13 were also clonal from paraffin-embedded tissues. Whatever the IgH locus analyzed, each time the adapted control was positive, results from paraffin-embedded material were identical to results obtained from frozen tissue. In this study, we showed that the use of paraffin-embedded tissue is efficient for the study of IgH gene rearrangement. Whenever adapted controls are used, it is even possible to assess negative results.     

Neurological symptoms in children with intussusception

Aim:  The classical combination of abdominal pain, vomiting, rectal blood loss and a palpable abdominal mass is only present in a minority of children with intussusception. Neurological signs and symptoms have been described, but are not a well understood phenomenon. We performed a retrospective study to ascertain the frequency and nature of these symptoms and to describe the characteristics of the patients presenting in this atypical way.
Methods:  The records of 58 children presenting with intussusception from 2003 to 2008 were reviewed for abdominal and neurological signs and symptoms, duration of symptoms and effectiveness of treatment.
Results:  In 10 out of 58 patients (17%), one or more neurological symptoms were recorded at presentation, with lethargy being the most frequent, followed by hypotonia and fluctuating consciousness. The patients with neurological abnormalities were significantly younger and presented with a shorter duration of symptoms. Therapy was more invasive, although not statistically significant, in this patient category.
Conclusion:  Intussusception should be considered in the differential diagnosis in young children presenting with lethargy, hypotonia and/or sudden alterations of consciousness even in the absence of the classical symptoms of intussusception.