Sharon L. Wenger

Molecular and phenotypic characterization of ring chromosome 22.

We performed a phenotype study of 35 individuals (19 males, 16 females) with ring chromosome 22 or r(22) with a mean age of 10 years. In common with other studies, a phenotype of moderate‐to‐profound learning difficulties and delay or absence of speech affected all individuals with the exception of the case with the smallest deletion. Autistic traits were significantly associated with r(22), as shown by an autism screening questionnaire. Mild and variable dysmorphic features, predominantly craniofacial and distal limb, were observed. Internal organ involvement was uncommon. Even though ring chromosomes are reportedly associated with growth abnormalities, only 2 out of 24 individuals showed evidence of growth failure, while 2 showed accelerated growth. Chromosome 22 long arm deletions, as determined by hemizygosity for informative microsatellite markers, varied from <67 kb to 10.2 Mb in size (or <0.15 to 21% of total chromosome length), with no significant differences in the parental origin of the ring chromosome. Few phenotypic features correlated with deletion size suggesting a critical gene, or genes, of major effect lies close to the telomere. Loss of the SHANK3/PROSAP2 gene has been proposed to be responsible for the main neurological developmental deficits observed in 22q13 monosomies. This study supports this candidate gene by identifying a phenotypically normal r(22) individual whose ring chromosome does not disrupt SHANK3. All other r(22) individuals were hemizygous for SHANK3, and we propose it to be a candidate gene for autism or abnormal brain development.

Bone marrow stromal cells regulate caspase 3 activity in leukemic cells during chemotherapy

The interaction between leukemic cells and stromal cells of the bone marrow microenvironment has been shown to enhance leukemic cell survival during exposure to chemotherapeutic agents. In the current study we investigated whether association of B lineage acute lymphoblastic leukemic cells with human bone marrow stromal cells altered caspase activation during chemotherapy treatment. Following treatment with Ara-C or VP-16 in vitro, caspase 3 activity in leukemic cells was consistently reduced by co-culture of leukemic cells with human bone marrow stromal cell layers. These observations suggest that the protective effect of the bone marrow microenvironment on leukemic cells may be due, in part, to regulation of caspase 3 activity.

Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of jacobsen syndrome

The 11q terminal deletion disorder or Jacobsen syndrome is a contiguous gene disorder. It is characterized by psychomotor retardation, cardiac defects, blood dyscrasias (Paris‐Trousseau syndrome) and craniofacial anomalies. We report on a female patient with an approximately 10 Mb interstitial deletion with many of the features of Jacobsen syndrome: A congenital heart defect, dysmorphic features, developmental delay, and Paris‐Trousseau syndrome. The karyotype of the patient is 46,XX,del(11)(q24.1q24.3). The interstitial deletion was confirmed using FISH probes for distal 11q, and the breakpoints were characterized by microarray analysis. This is the first molecularly characterized interstitial deletion in a patient with the clinical features of Jacobsen syndrome. The deletion includes FLI‐1, but not JAM‐3, which will help to determine the critical genes involved in this syndrome.

Tissue Culture of Human Renal Epithelial Cells Using a Defined Serum-Free Growth Formulation

Background: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory’s cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. Methods: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms’ tumors. Results: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms’ tumors. Conclusions: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.

BRCA1 contributes to cell cycle arrest and chemoresistance in response to the anticancer agent irofulven

Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. We demonstrated previously that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair, and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials and the activation and foci formation of γ-H2AX, BRCA1, and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or -proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G2/M cell cycle arrest and is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.

Human Telomere Length Correlates to the Size of the Associated Chromosome Arm

The majority of human telomere length studies have focused on the overall length of telomeres within a cell. In fact, very few studies have examined telomere length for individual chromosome arms. The objective of this study was to examine the relationship between chromosome arm size and the relative length of the associated telomere. Quantitative Fluorescence In Situ Hybridization (Q-FISH) was used to measure the relative telomere length of each chromosome arm in metaphases from cultured lymphocytes of 17 individuals. A statistically significant positive correlation (r = 0.6) was found between telomere length and the size of the associated chromosome arm, which was estimated based on megabase pair measurements from http://www.ncbi.nlm.nih.gov/projects/mapview/.

Use of the Glycophorin A Somatic Mutation Assay for Rapid, Unambiguous Identification of Fanconi Anemia Homozygotes Regardless of GPA Genotype

A 7‐year‐old girl was hospitalized with pancytopenia requiring blood transfusion. She and an older brother with suspicious symptoms were referred for laboratory testing to confirm a clinical diagnosis of Fanconi anemia (FA). Blood samples from these two children and one parent were examined with the GPA somatic mutation assay. The patients total GPA somatic mutation frequency of 1.4 × 10−4 was determined despite the confounding effects of her recent transfusion, and was greater than 10‐fold higher than that of a population of pediatric controls, consistent with the known FA phenotype. Her brother was not informative for the standard GPA assay, which requires heterozygosity for the MN blood group, but was analyzed with a modified assay that measured only allele loss mutation. His mutation frequency, 6.8 × 10−4 was also supportive of a diagnosis of FA. Both analyses also showed evidence of ongoing mutation through terminal erythroblast differentiation, a characteristic of patients with DNA repair syndromes which further confirmed the diagnoses. These conclusions were confirmed with traditional DEB‐induced chromosome breakage studies. The quantitative and qualitative aspects of the GPA assay relevant for applying this test for FA diagnosis, and perhaps for carrier detection, are discussed.

Del(5q) Is Associated With Clinical and Histological Parameters in Small Cell Neuroendocrine Lung Carcinoma

To elucidate the relationship between del(5q) and the clinical and histological features of small cell neuroendocrine lung carcinoma, 33 tissue samples from patients with this tumor were evaluated. By using fluorescence in situ hybridization, del(5q) was identified in almost 50% of cases (15/33, 45%). Clinically, patients with tumors showing del(5q) were older (mean age = 71 years) with a correspondingly greater pack-year smoking history (mean = 61) than patients with tumors (mean age = 59 years, mean pack-years = 44) without del(5q). Histologically, tumors with del(5q) had a greater frequency of spindle cell morphology (11/14 [79%] vs 6/16 [38%], P < .025) than those without del(5q). This is the first study to find an association between del(5q) and tumor histology in small cell neuroendocrine lung carcinoma.

Mosaic ring 12p and total anomalous pulmonary venous return

An infant born with total anomalous pulmonary venous return (TAPVR) was found to have an extra chromosome present as a small ring. Spectral karyotyping and FISH analysis identified the material as a duplication involving the short arm of chromosome 12. Previous cases describing a variety of cytogenetic abnormalities that have been associated with TAPVR are reviewed along with prior cases of duplication 12p with their associated findings. We believe ours is the first case to report the occurrence of mosaic ring 12p and its association with TAPVR.

Prenatal diagnosis of tetrasomy 9p

Dhandha et al. [2002] report three cases of tetrasomy 9p andprovide a useful and interesting case review from the literature. Tetrasomy 9p is a rare clinical syndrome with only 32 cases reported to date, with reports of both mosaic and non-mosaic cases. The phenotypic differences of tetrasomy 9p seem to be the result of the degree of mosaicism. We recently identified a patient with tetrasomy 9p diagnosed prenatally and report our findings here. The mother was a 22-year-old gravida 1 para 0 white woman.During the pregnancy,multiple fetal anomalies were identified by repeated ultrasounds, as early as 22 weeks of gestation. These fetal anomalies included a strawberry shaped skull, mildly enlarged fetal ventricles, bilateral cleft lip and palate, a small-sized stomach, a possible horseshoe kidney, and rockerbottom deformityof the footand/or clubfeet.Alsonotedwaspersistent abnormal posturing of the fetal fists with overlapping of the fetal digits. Amniocentesis was performed at 24weeks of gestation,which showed47,XX,þi(9)(p10) in 20 cells. The isochromosome was confirmed as chromosome 9 by FISH. The patient was born by induced labor at 3557-week gestation due to premature rupture of membranes. The infant was a female weighing 1,730 g, 41 cm long, with occipital frontal circumference (OFC) of 28.5 cmat birth. Apgar scores were 1 and 6 at 1 and 5 min, respectively. Physical examination at birth revealed strawberryshaped head, downslanting palpebral fissures, large anterior fontanel, hypertelorism, bilateral small low-set ears, micrognathia, beaked nose, bilateral cleft lip and cleft palate, brachydactyly with hypoplastic thumb, and bilateral foot deformities with eversion. The patient was also found to have patent foramen ovale, bicuspid aortic valve, and patent ductus arteriosus. At 33 days of age, the patient died of aspiration pneumonitis with respiratory distress. Ultrasound and MRI of the head were performed postnatally, and diagnoses of hydrocephalus, ventriculomegaly with left ventricle being larger in size than the right one, agenesis of the corpus callosum, and Dandy– Walker variantweremade. The above physical findings, along with ventriculomegaly, Dandy–Walker variant, and horseshoe kidney detected by ultrasound, are consistent with the emerging phenotype for tetrasomy 9p, summarized by Dhandha et al. [2002]. Karyotyping from amniocentesis confirmed our case as tetrasomy 9p. Chromosomal analysis was also performed on peripheral blood lymphocytes and cord fibroblasts of the infant at birth.All but one of the 20 cells from lymphocytes, and all but three of the 20 cells from fibroblasts, showed identicalkaryotypeasheramniocytes, 47,XX,þi(9)(p10). The one cell from lymphocytes and three cells from fibroblasts analyzed showed anormal female karyotype, suggesting low level of mosaicism. In our patient, we were able to analyze three different tissues, and found low level of mosaicism in two of the three cell types. No early deaths are reported for mosaic cases of tetrasomy 9p. The level of mosaicism seen in our case at birth was very low. Considering that all of her amniocytes analyzed had an abnormal karyotype, this could represent a non-mosaic case of tetrasomy 9p in the fetus, and thus account for the early death in our patient.