Thue Bryndorf

Comparative genomic hybridization reveals a recurrent pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and in advanced‐stage cervical carcinoma

We analyzed 17 cases of dysplasia/carcinoma in situ (CIS) of the cervix and 29 advanced‐stage cervical squamous cell carcinomas by comparative genomic hybridization (CGH). A comparable recurrent pattern of aberrations was detected in both preinvasive and invasive cases, although the total number of aberrations was much higher in the latter category. The most consistent chromosomal gain was mapped to chromosome arm 3q in 35% of preinvasive cases and in 72% of invasive cases. Chromosome aberrations were detected in 13/17 preinvasive cases with a total of 61 involved chromosome arms. In the invasive cases, frequent gains also occurred on 1q (45%), 8q (41%), 15q (41%), 5p (34%), and Xq (34%), and frequent losses were mapped to chromosome arms 3p (52%), 11q (48%), 13q (38%), 6q (38%), and 4p (34%). A recurrent pattern of aberrations has not previously been described in preinvasive lesions of the cervix. Our finding is surprising considering that only few preinvasive lesions are expected to progress to invasive cancer. Genes Chromosomes Cancer 24:144–150, 1999.

Investigation of patients with mental retardation and dysmorphic features using comparative genomic hybridization and subtelomeric multiplex ligation dependent probe amplication

For a number of years we have used high resolution metaphase comparative genomic hybridization (CGH, referred to as HR-CGH in our prior papers) for detection of cryptic chromosomal imbalances in patients with mental retardation and dysmorphic features. Although this technique detects imbalances in as many as 12% of patients, additional diagnoses can be obtained if CGH is combined with subtelomeric FISH analysis [Kirchhoff et al., 2004]. In this study, we set out to test the diagnostic yield of subtelomeric multiplex ligation-dependent probe amplification (MLPA) in patients already investigated by CGH. In this study, we present the CGH and subtelomeric MLPA results of 258 patients with mental retardation and dysmorphic features. Introduction of a newmethod in a diagnostic routine setting requires careful consideration regarding evaluation of results. Thus, the diagnostic criteria for the MLPA analysis and the strategy used for exclusion of putative polymorphisms are discussed. The MLPA assay was performed using the SALSA P019, P020, and P036 human subtelomeric probe sets (MRC-Holland, Amsterdam, The Netherlands). Samples of DNA from 20 normal individuals were used to create reference values for eachMLPAprobe set. The references for P019, P020, and P036 werebased on86, 81, and92analyses respectively.Henceforth, P019 and P020 aremerged and referred to as a single probe set as combination of these two sets interrogate all subtelomeric regions (except the acrocentric p-arms) as does the P036 probe set. Following capillary electrophoresis on an ABI Prism 3100, peak areas were normalized in relation to the mean of neighboring peaks. The ratio of each peak and the correspondingmean peak area of the reference were computed, and it was measured in SDs how far each peak area was from the corresponding mean area of the reference. The ‘‘area distance in SDs’’ reflects the individual probe reliability, because a peak ratio indicating an imbalance get a low area distance in SDs, if the probe shows extensive variation (i.e., high SD value) in the reference data set; hereby false positives may be spotted (for normally distributed data the probability for observing an area outside 3 SD is less than 1%) [Gerdes et al., 2005; Gerdes et al., in press] (details of data analysis and downloads of the software are available on www.chromosomelab.dk). Thirteen cases with 20 known aberrations in subtelomeric regions (12 deletions and eight duplications) were used to set up a diagnostic threshold, which was based on both peak area ratios and the area distance in SDs. The average ratio of the deletion probes to the reference was 0.50 with a range of 0.40– 0.60. The average area distance in SDs was 7.4 with a range of 4.1–11.8. The average ratio of the duplication probes to the referencewas 1.46with a range of 1.35–1.64. The average area distance in SDs was 7.7 with a range of 4.4–10.2. On the basis of these values, a diagnostic threshold was set: a peak was consideredabnormalwhen the peakareawasmore than4.0SDs from the reference mean and the ratio was 1.35. The diagnostic threshold was tested on samples from 258 consecutive patients with mental retardation and dysmorphic features who were referred for CGH analysis. The results are summarized in Table I. Abnormalities were detected in 29 (11.2%) patients by subtelomericMLPA. In 14 of those 29 cases (5.4% of 258), the imbalances were detected by both probe sets (in cases 8, 11, and 29 one of the ratio values or one of the area distances in SDs values were close to, but did not reach, the diagnostic threshold) and in remaining 15 cases (5.8% of 258) the imbalances were detected by one probe set. In two cases, the P019/P020 probe set showed a deletion of 3pter. Sequencing analysis showed that these patients had aG instead of an A in the splice-site of the probe. This probe is now recognized to detect a polymorphic target sequence (J. Schouten, MRC-Holland, personal communication), and has been replaced in a new generation of subtelomeric probe sets (www. MRC-Holland.com). Thus, the two 3pter deletions are considered to be false positive results caused by a single nucleotide polymorphism (SNP). Recent studies have shown that there is considerable structural variation in the genomes of normal individuals [Iafrate et al., 2004; Sebat et al., 2004]. In the design of subtelomeric MLPA probe sets, a compromise is made between avoiding polymorphisms and selecting target sequences close to the telomeres, in order tomaximize the sensitivity of the screen. In order to reduce the risk of detecting polymorphisms, imbalances detected by only a single probe set were assessed as clinically insignificant. We made this decision for two reasons. First,with regard to small duplications/deletions there is a risk that the clinical significance remains unresolved or a false diagnosis is made. This is distressing for both the family and the medical professionals and genetic counseling becomes inadequate. Second, when submicroscopic imbalances are detected, follow-up is often labor-intensive and costly. In the present study, 15 duplications were detected by one probe set only. Apart from two duplications, all were detected in two, three, or five patients, which is consistent with the assumption that they represent polymorphisms. Indeed, a duplication of *Correspondence to: Dr. Maria Kirchhoff, Rigshospitalet, Department of Clinical Genetics, 4052, Blegdamsvej 9, Copenhagen, Denmark DK-2100. E-mail: markir@rh.dk

Rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization: a one‐year clinical experience with high‐risk and urgent fetal and postnatal samples

Objectives. To evaluate the clinical utility of rapid prenatal and postnatal detection of common chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) analysis with DNA probes.

Twins with mental retardation and an interstitial deletion 7q34q36.2 leading to the diagnosis of long QT syndrome

Long QT syndrome (LQTS) is a potentially lifethreatening condition in which an abnormally prolonged ventricular repolarization predisposes to ventricular arrhythmias, syncope, and sudden death. Beta-blocking agents diminish these risks. Long QT syndrome can be inherited as an autosomal recessive disease associated with deafness or as a more common dominant form [Priori et al., 1999]. Up to five genes encoding potassium (four genes) or sodium (one gene) ion channel proteins have been associated with the latter form. Mutations in these genes reduce the ion channel currents leading to prolonged repolarization, which is expressed in the QT interval of the electrocardiogram. The length of the QT interval depends on age, gender, and heart rate. A QT interval corrected for heart rate (QTc) is considered as prolonged if it exceeds 460 msec [Kass and Moss, 2003]. Furthermore, the morphology of the T wave is different in the dominant types of LQTS [Kanters et al., 2004]. We report a pair of twins inwhomdetection of a deletion of 7q34! 7q36.2 led to the diagnosis and treatment of LQTS. More than50 caseswithdeletionof thedistal part of the long arm of chromosome 7 have been reported [Verma et al., 1992; Schinzel, 2001]. The majority of these patients had terminal deletions and many had holoprosencephaly as a consequenceof a deletion of SHH (sonic hedgehog gene) that maps to band 7q36.3. However, a specific 7q deletion syndrome has not been recognized, probably because of the many different and not always precisely defined breakpoints. Patients with deletions including bands 7q33-35 frequently have low birth weight, postnatal growth retardation, mental retardation, developmental delay, microcephaly, a bulbous nasal tip, and abnormal ears/hearing [Verma et al., 1992; Schinzel, 2001]. Many of these features are unspecific and often seen in patients with a variety of chromosomal aberrations. The twin girls were delivered at 32-week gestation by cesarean because of growth retardation. Birth weights were 1,135/1,162 g; birth lengths 34/35 cm, and head circumferences were 25.5/26.0 cm, respectively; all of thesemeasurements are below 2 SD for gestational age. Twin A was treated for a persistent ductus arteriosuswith indomethacin infusion; twin B received antibiotics due to neonatal sepsis, confirmed by culture of Staphylococcus aureus from the blood. Both twinswere lethargic anddifficult to feed. They gained weight very slowly, and feeding problems continued after the neonatal period. Twin B had meningococcal meningitis at 6 months of age without apparent sequelae. At age 2, a sensorineural hearing impairment was discovered in both girls. The losses were at the 30–50 dB level and treated with hearing aids. Vision was followed from birth because of an iris-retina-nervus opticus coloboma in twin B. Since the age of 7 months both twins had severe hypermetropia (þ8 to þ11) corrected by glasses. Between ages 2 and 3 myringotomy tubes were inserted to treat chronic otitis, and adenotonsillectomy was performed for chronic tonsillitis and recurrent upper airway infections. Psychomotor development was delayed. The twins sat at 12 months and walked at 20 months (age corrected for prematurity). They developed cheerful and trustful personalities, but had severe attention

Prenatal Diagnosis by Fluorescence in situ Hybridization on Chorionic Villus Cells: Nonsignif icance of Maternal Cell Contamination

We assessed the effect of maternal cell contamination on the sensitivity of prenatal diagnosis by fluorescence in situ hybridization (FISH) on overnight attached mesenchymal chorionic villus cells. Double targets FISHs with X and Y chromosome-specific probes were performed on parallel samples from the same pregnancies: (1) samples of assumed fetal tissue retained during dissection, and (2) samples of suspected maternal tissue discarded during dissection. In the karyotypically male samples the tissue retained during dissection contained 0-3% nuclei with two X-specific signals each. In the tissue discarded from the karyotypically male samples 0-50% of the nuclei had two X-specific signals each. We conclude that given thorough dissection of chorionic villi, the sensitivity of prenatal diagnosis of aneuploidies by FISH on overnight attached samples of this tissue is not critically affected by maternal cell contamination.

New rapid test for prenatal detection of trisomy 21 (Down's syndrome): Preliminary report

OBJECTIVE To devise and evaluate a rapid screening method for detecting trisomy 21 (Downs syndrome) in samples of uncultured amniotic fluid cells. DESIGN Non-radioactive in situ hybridisation with HY128, a 500,000 base pair yeast artificial chromosome probe specific for chromosome 21. Blinded study of 12 karyotypically normal amniotic fluid samples and eight samples trisomic for chromosome 21. SETTING Cytogenetic and obstetric services at a tertiary referral centre, Copenhagen. MAIN OUTCOME MEASURES Time necessary to complete the test. Proportion of cell nuclei containing two and three hybridisation signals in karyotypically normal and abnormal amniotic fluid samples. RESULTS The test could be completed within three to four days after amniocentesis. In the normal samples a mean of 73% (range 61-82%) of the amniotic cell nuclei showed two hybridisation signals and 6% (0-18%) showed three signals. By contrast, among the trisomic samples 29% (19-38%) of the nuclei exhibited two signals and 48% (31-60%) showed three signals. CONCLUSION The technique clearly distinguished between normal and trisomic samples. Prenatal diagnosis with in situ hybridisation with chromosome specific probes was fast and may make it possible to screen for selected, aneuploidies. However, the technique is still at a preliminary stage and needs further evaluation and refinement.