Carsten A. Brandt

Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS).

PRimed IN Situ labeling (PRINS) has become an alternative to traditional fluorescence in situ hybridization (FISH) methods for detection of nucleic acids in situ. PRINS is based on sequence-specific annealing in situ of an unlabeled DNA probe. The probe serves as a primer for chain elongation in situ, catalyzed by a suitable DNA polymerase that uses labeled nucleotides as substrate. The fact that the probe is unlabeled means that high probe concentrations can be utilized, making the hybridization very fast. We describe here a fast method for detection of three different target sequences visualized in different colors with PRINS. An advantage, relative to FISH, is that even probes with different melting temperatures can be detected in the same metaphase with optimal stringency for each probe.

Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS

Brandt CA, Kierkegaard O, Hindkjær J, Jensen PKA, Pedersen S. Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS.

Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primedin situ labelling and chromosome painting (PRINS-painting)

In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primedin situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting)

Sex dependent transmission of Beckwith-Wiedemann syndrome associated with a reciprocal translocation t(9;11)(p11.2;p15.5).

Beckwith-Wiedemann syndrome (BWS), a disorder associated with neonatal hypoglycaemia, increased growth potential, and predisposition to Wilmss tumour (WT) and other malignancies, has been mapped to 11p15. The association with 11p15 duplications of paternal origin, of balanced translocations and inversions with breakpoints within 11p15.4-p15.5 of maternal origin, and the demonstration of uniparental paternal 11p15 isodisomy in some sporadic cases point towards the involvement of genomic imprinting. In agreement with this, we show the paternal origin of a de novo 9;11 translocation in a phenotypically normal mother, whose carrier daughter developed BWS. This supports the fact that BWS associated with balanced chromosome mutations is transmitted in the same sex dependent pattern as non-cytogenetic forms of familial BWS.

Ring chromosome 13: lack of distinct syndromes based on different breakpoints on 13q.

A stillborn male child with anencephaly and multiple malformations was found to have the karyotype 46,XY,r(13) (p11q21.1). The breakpoint at 13q21.1, determined by high resolution banding, is the most proximal breakpoint ever reported in patients with ring chromosome 13. In situ hybridisation with the probe L1.26 confirmed the derivation from chromosome 13 and DNA polymorphism analysis showed maternal origin of the ring chromosome. Our results, together with a review of previous reports of cases with ring chromosome 13 with identified breakpoints, could neither support the theory of distinct clinical syndromes based on different breakpoints on 13q nor correlate the severity of symptoms with instability of the ring.

Application of molecular and cytogenetic techniques to the detection of a de novo unbalanced t(11q;21q) in a patient previously diagnosed as having monosomy 21

Hertz B, Brandt CA, Petersen MB, Pedersen S, König U, Strømkjær H, Jensen PKA. Application of molecular and cytogenetic techniques to the detection of a de novo unbalanced t(11q;21q) in a patient previously diagnosed as having monosomy 21.

A patient with Edwards syndrome caused by a rare pseudodicentric chromosome 18 of paternal origin

We present an unusual case of trisomy 18 due to a pseudodicentric chromosome 18 of paternal origin. The karyotype was: 46,XY,—18, + psu dic(18)(qter→cen→p11.31::p11.31→psucen→qter). The origin of the abnormal chromosome was verified by FISH with a painting probe from chromosome 18. Absence of short‐arm telomeres was shown by multicolor FISH, and the results of DNA analysis showed monosomy for loci D18S59 and D18S170 as well as paternal inheritance of the aberrant chromosome. The childs phenotype was characteristic of trisomy 18.

Charcot-Marie-Tooth disease type 1A: the parental origin of a de novo 17p11.2-p12 duplication

Charcot‐Marie‐Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a DNA duplication on chromosome 17p11.2‐p12 in the majority of cases. Most of the sporadic cases are due to a de novo duplication. We have screened for this duplication in 11 Danish patients with CMT type 1, using four different techniques, and identified a de novo duplication in a sporadic case. Analysis of the fully informative pVAW409R3a alleles in this family showed the duplication to be of paternal origin.

Pseudodicentric chromosome 18 diagnosed by chromosome painting and primed in situ labelling (PRINS).

We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.

A de novo complex t(7;13;8) translocation with a deletion in the TRPS gene region

Abstract Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21). R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality. Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8). Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.