Pino J. Poddighe

A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.

For amplification of in situ hybridization (ISH) signals, we describe a method using catalyzed reporter deposition (CARD). This amplification method is based on the deposition of biotinylated tyramine (BT) at the location of the DNA probe. The BT precipitate can then visualized with fluorochrome- or enzyme-labeled avidin. Both for bright-field ISH (BRISH) and for fluorescence ISH (FISH), the detection limit was highly increased. This method is especially suitable for visualization of very weak ISH signals, such as those obtained by ISH using locus-specific DNA probes. Furthermore, CARD amplification of ISH signals (CARD-ISH) is highly sensitive, rapid, flexible, and easy to implement. Successful application of CARD-ISH with locus-specific DNA probes on histological and cytological samples may improve the determination of structural chromosomal aberrations in archival material.

Array analysis and karyotyping: Workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands

Anomalies of chromosome number and structure are considered to be the most frequent cause of unexplained, non-syndromic developmental delay and mental retardation (DD/MR). High-resolution, genome-wide, array-based segmental aneusomy profiling has emerged as a highly sensitive technique for detecting pathogenic genomic imbalances. A review of 29 array-based studies of DD/MR patients showed that a yield of at least approximately 19% pathogenic aberrations is attainable in unselected, consecutive DD/MR referrals if array platforms with 30-70 kb median probe spacing are used as an initial genetic testing method. This corresponds to roughly twice the rate of classical cytogenetics. This raises the question whether chromosome banding studies, combined with targeted approaches, such as fluorescence in situ hybridisation for the detection of microdeletions, still hold substantial relevance for the clinical investigation of these patients. To address this question, we reviewed the outcome of cytogenetic studies in all 36,325 DD/MR referrals in the Netherlands during the period 1996-2005, a period before the advent of array-based genome investigation. We estimate that in a minimum of 0.78% of all referrals a balanced chromosomal rearrangement would have remained undetected by array-based investigation. These include familial rearrangements (0.48% of all referrals), de novo reciprocal translocations and inversions (0.23% of all referrals), de novo Robertsonian translocations (0.04% of all referrals), and 69,XXX triploidy (0.03% of all referrals). We conclude that karyotyping, following an initial array-based investigation, would give only a limited increase in the number of pathogenic abnormalities, i.e. 0.23% of all referrals with a de novo, apparently balanced, reciprocal translocation or inversion (assuming that all of these are pathogenic), and 0.03% of all referrals with 69,XXX triploidy. We propose that, because of its high diagnostic yield, high-resolution array-based genome investigation should be the first investigation performed in cases of DD/MR, detecting >99% of all pathogenic abnormalities. Performing both array investigation and karyotyping may not be a feasible option when laboratories are faced with a need to limit the number of genetic tests available for each patient. However, laboratories that supplant karyotyping by array-based investigation should be aware that, as shown here, a chromosomal abnormality, with possible pathogenic consequences for the patient or the family, will escape detection in about 0.78% of all DD/MR referrals.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome

Background: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. Aim: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. Results: We estimate the prevalence of the syndrome to be 1 in 16 000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729–41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10−5). Conclusion: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix

SUMMARY In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Changes in yearly birth prevalence rates of children with Down syndrome in the period 1986-2007 in the Netherlands

BACKGROUND The Netherlands are lacking reliable national empirical data in relation to the development of birth prevalence of Down syndrome. Our study aims at assessing valid national live birth prevalence rates for the period 1986-2007. METHOD On the basis of the annual child/adult ratio of Down syndrome diagnoses in five out of the eight Dutch cytogenetic centres, the national annual figures of the National Cytogenetic Network on total numbers of postnatal Down syndrome diagnoses were transformed into national figures on total numbers of postnatal Down syndrome diagnoses in newborn children only. In combination with the national annual data of the Working Group for Prenatal Diagnostics and Therapeutics on numbers of Down syndrome pregnancies not aborted after diagnosis, national figures on birth prevalence were constructed. RESULTS For the period 1986-2007, results based on the data of the cytogenetic centres are almost similar to the theory-based model data of de Graaf et al., with a small discrepancy of approximately 4%. Down syndrome birth prevalence in the Netherlands shows an upward trend from around 11 per 10,000 births in the early 1990s to around 14 per 10,000 births nowadays. CONCLUSION In spite of expansion of antenatal screening in the Netherlands, Down syndrome live birth prevalence has risen in the last two decades as a result of rising maternal age. This increase in Down syndrome birth prevalence is in contrast to studies from other European countries.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1–q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1–q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

5q11.2 deletion in a patient with tracheal agenesis

Tracheal agenesis (TA) is a rare congenital anomaly of the respiratory tract. Many patients have associated anomalies, suggesting a syndromal phenotype. In a cohort of 12 patients, we aimed to detect copy number variations. In addition to routine cytogenetic analysis, we applied oligonucleotide array comparative genomic hybridization. Our patient cohort showed various copy number variations, of which many were parentally inherited variants. One patient had, in addition to an inherited 16p12.1 deletion, a 3.6 Mb deletion on chromosomal locus 5q11.2. This patient had a syndromic phenotype, including vertebral, anal, cardiovascular and tracheo-oesophageal associated anomalies, and other foregut-related anomalies, such as cartilage rings in the oesophagus and an aberrant right bronchus. No common deletions or duplications are found in our cohort, suggesting that TA is a genetically heterogeneous disorder.

Impact of the Y-containing cell line on histological differentiation patterns in dysgenetic gonads

Objective  Gonadal karyotyping is considered a tool for increasing our knowledge of disturbed gonadal development in patients with gonadal dysgenesis and for estimating more accurately the risk for gonadoblastoma formation. The objective was to gain insight into the role of Y chromosome distribution in the histological heterogeneity of gonads of patients with gonadal dysgenesis.

Double-target in situ hybridization in brightfield microscopy.

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromosome imbalances.

Decreased expression of Ki-67 in atrophic cervical epithelium of post-menopausal women

Papanicolaou‐stained cervical smears (Pap smears) of post‐menopausal women often present difficulties in distinguishing atrophic cervical epithelium from high‐grade cervical intraepithelial neoplasia (CIN2–3). The aim of this study was to disclose differences in proliferative activity in normal cervical epithelium, cervical atrophy, and high‐grade CIN lesions, in order to develop specific and sensitive classifiers to discriminate between cervical atrophy and high‐grade CIN, both in cervical smears and in tissue sections. A case–control study was done on 83 patients. Proliferative activity was assessed in histological sections using the monoclonal antibody MIB1. An image analysis system was used to characterize different proliferation‐associated features. Preceding Pap smears were restained with MIB1 and proliferative activity was measured by a point‐counting procedure, carried out on a training set of 32 cases and a test set of 51 cases. In cervical atrophy, proliferative activity was significantly lower than in normal epithelium (p<0.001). Proliferative activity measured in both biopsies and cervical smears was considerably higher in high‐grade CIN than in normal epithelium (p<0.001). Discriminant analyses resulted in four classifiers, based on proliferation parameters, to discriminate between cervical atrophy and high‐grade CIN, and between CIN2 and CIN3, in biopsy specimens and cervical smears, respectively. The two classifiers for biopsy specimens resulted in 100% correct classification. Application of the classifier obtained from the training set of Pap smears resulted in 100% correct classification of the Pap smears in the test set. The classifier to discriminate between CIN2 and CIN3 in Pap smears, obtained from 36 patients, resulted in 87% and 90% correct classification, respectively. Copyright