Cytogenetic analysis of spontaneously discharged products of conception by array-based comparative genomic hybridization.

BACKGROUND: Cytogenetic analysis of products of conception (POC) is essential for the management of recurrent pregnancy loss (RPL), but the currently-performed G-banding method is not necessarily applicable to spontaneously discharged POC because of poor quality for culture. We analyzed the karyotypes of 15 spontaneously discharged POC by array-based comparative genomic hybridization (array-CGH). RESULTS: All specimens were successfully analyzed and 10 cases had abnormal results: gain in copy number (n = 7) and loss in copy number (n = 3). Most of them were estimated to be whole chromosome aneuploidy, whereas one case was compatible with microdeletion. Two cases were suspected to be male diploid contaminated by maternal DNA or triploid because of the unsatisfactory signal patterns on X/Y chromosomes. Two of three cases with normal female DNA pattern were identified to be contaminated with maternal DNA by the additional analysis of short tandem repeats. CONCLUSIONS: Given the potential to analyze non-viable POC specimens, array-CGH is a feasible cytogenetic tool for women, in particular, with a history of RPL who desire non-surgical or expectant management of miscarriages and/or a thorough investigation on the cause for recurrent miscarriage, although it needs to take into account high incidence of maternal contamination in spontaneously discharged POC.

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies.

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

Multiplex PCR/liquid chromatography assay for screening of subtelomeric rearrangements.

In idiopathic or nonspecific mental retardation, the overall rate of cryptic subtelomeric rearrangements is estimated to be about 5%. Development of cost-effective screening for subtelomeric deletions would help clinical geneticists to make specific diagnoses in children with idiopathic mental retardation. Current screening modalities include fluorescence in situ hybridization (FISH) using subtelomeric probes and PCR-based quantitative analyses. Reductions in the cost and turnaround time will make the complete screening of subtelomeric rearrangements more widely used in clinical settings. Recently, a versatile method, called the multiplex PCR/liquid chromatography assay (MP/LC), was developed to assess copy numbers in this assay. Multiple genomic regions are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography. In the present study, we developed an MP/LC-based subtelomeric screening system that involves 21 multiple reactions and validated the protocol by analyzing 16 publicly available cell lines with known cytogenetic abnormalities involving at least one subtelomere per patient. To confirm the validity of the MP/LC method, we analyzed these cell lines concurrently with array-based comparative genomic hybridization (array-CGH), which gives higher resolution than the conventional G-banding technique. Among those 16 samples, the results from MP/LC and array-CGH agreed with each other perfectly. In 2 of the 16 samples, MP/LC correctly revealed subtelomeric duplications that were detected by array-CGH but were undetected by conventional cytogenetics, demonstrating the sensitivity of the MP/LC assay. This system is expected to be useful for making specific diagnoses and in genetic counseling for children with idiopathic mental retardation, a sizable fraction of whom have subtelomeric rearrangements.

Topical instillation of ciliary neurotrophic factor inhibits retinal degeneration in streptozotocin-induced diabetic rats.

Intraocular injections of ciliary neurotrophic factor (CNTF) or intraocular adenovirus-mediated CNTF gene transfer have been reported to inhibit retinal alterations in inherited retinal degeneration in mice strains. To investigate whether or not CNTF administered by eye drops prevents retinal degeneration in streptozotocin (STZ)-induced diabetic rats, recombinant CNTF was administered to eyes of diabetic rats (n=20) twice daily for 1 month after the onset of diabetes. The b-wave amplitude of electroretinogram in CNTF-administered diabetic rats was significantly larger than that of diabetic rats, and approached that of the controls. Atrophy of the inner plexiform layer, and cavity formation in the pigment epithelium, which were observed in diabetic rats, were prevented in CNTF-administered diabetic rats. These results indicate that CNTF administration by eye drops prevents retinal degeneration in STZ-induced diabetic rats.

Degeneration of retinal neuronal processes and pigment epithelium in the early stage of the streptozotocin-diabetic rats.

Early pathological and electro-physiological changes of the retina in the streptozotocin (STZ)-diabetic rats were investigated through optical and electron microscopy in two strains and electro-retinography in one strain. In Sprague-Dawley (SD) rats I month after the onset of diabetes, the thickness of the inner plexiform layer (IPL) and photoreceptor segment layer (PSL) was significantly reduced by 9.9% and 18.9%, respectively (P < 0.01, P < 0.05). In Brown-Norway (BN) rats STZ-diabetic for 1 month, the thickness of the IPL was also significantly reduced by 15.7% (P < 0.05). Cytochemical study using peanut agglutinin (PNA), a lectin binding selectively to the cone photoreceptor-associated domains of the inter-photoreceptor matrix, revealed a marked reduction in intensity, number and length of the PNA-binding cone photoreceptors. Electron microscopy showed deepened hollows in the basal infoldings of the retinal pigment epithelium (RPE) of STZ-rats diabetic for 1 month and large concavities into the cytoplasm in STZ-rats diabetic for 6 months. Blood vessels in the retina and choroid were unremarkable. Single-flash electro-retinogram revealed a reduction in the amplitudes of alpha- and beta-waves of electro-retinogram (ERG) of 1 month STZ BN rats (P < 0.05). These findings indicate that the degeneration of rods/cones in the PSL and RPE are the most prominent pathological alteration sites in the early stage of diabetic rats.

Alteration of midkine expression in the ischemic brain of humans.

Midkine (MK) is a heparin-binding growth factor that occurs as a product of the retinoic acid-inducible gene. Alteration of MK expression in ischemic brain lesions was examined in humans immunohistochemically in nine patients and in two control subjects without neurological disorders. Some neurons were MK-immunopositive, but no evident MK-immunoreactivity was observed in astrocytes in brains of control subjects. In the ischemic lesions, significant elevation of MK-immunoreactivity in the astrocytes and depletion of the reactivity in neurons were seen, especially in the early period, where edema and eosinophilic neurons were prominent. On the other hand, MK-immunoreactivity was not observed in hypertrophic and fibrillary astrocytes in the later period. These findings suggest that the MK in astrocytes play some role in the repair process in the early period of the ischemic brain lesions in humans.