Two supernumerary marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism

The interpretation of the significance of marker chromosomes, which can be encountered at prenatal diagnosis, is extremely problematic. Various factors contribute to the difficulty of clarifying the phenotypic risks of supernumerary marker chromosomes, including differences in the size, structure, and origin of marker chromosomes, as well as the occurrence of multiple marker chromosomes of different origin in the same proband. Research on marker chromosomes is currently in a data-accumulation phase. We report the presence of two marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism and discuss the related literature.     

Molecular cytogenetic characterization of three familial cases of satellited Y chromosomes

Three families in which a satellited Y chromosome (Yqs) was segregating without apparent phenotypic effect were re-investigated with non-isotopic in situ hybridization methods. Active nucleolus organizer regions were seen in the distal long arm region of all Yqs chromosomes studied and in situ hybridization with the probe D15Z1 showed that, in all three families, the Yqs was the result of a 15p;Yq translocation. In one case, an additional focus of D15Z1 hybridization was seen on 21p.     

Mechanisms and consequences of small supernumerary marker chromosomes: from Barbara McClintock to modern genetic-counseling issues

Supernumerary marker chromosomes (SMCs) are common, but their molecular content and mechanism of origin are often not precisely characterized. We analyzed all centromere regions to identify the junction between the unique chromosome arm and the pericentromeric repeats. A molecular-ruler clone panel for each chromosome arm was developed and used for the design of a custom oligonucleotide array. Of 27 nonsatellited SMCs analyzed by array comparative genomic hybridization (aCGH) and/or fluorescence in situ hybridization (FISH), seven (approximately 26%) were shown to be unique sequence negative. Of the 20 unique-sequence-positive SMCs, the average unique DNA content was approximately 6.5 Mb (range 0.3-22.2 Mb) and 33 known genes (range 0-149). Of the 14 informative nonacrocentric SMCs, five (approximately 36%) contained unique DNA from both the p and q arms, whereas nine (approximately 64%) contained unique DNA from only one arm. The latter cases are consistent with ring-chromosome formation by centromere misdivision, as first described by McClintock in maize. In one case, a r(4) containing approximately 4.4 Mb of unique DNA from 4p was also present in the proband's mother. However, FISH revealed a cryptic deletion in one chromosome 4 and reduced alpha satellite in the del(4) and r(4), indicating that the mother was a balanced ring and deletion carrier. Our data, and recent reports in the literature, suggest that this "McClintock mechanism" of small-ring formation might be the predominant mechanism of origin. Comprehensive analysis of SMCs by aCGH and FISH can distinguish unique-negative from unique-positive cases, determine the precise gene content, and provide information on mechanism of origin, inheritance, and recurrence risk.     

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 22 associated with cat eye syndrome

We present prenatal diagnosis of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 22 associated with cat eye syndrome (CES) using cultured amniocytes in a pregnancy with fetal microcephaly, intrauterine growth restriction, left renal hypoplasia, total anomalous pulmonary venous return with dominant right heart and right ear deformity. The sSMC was bisatellited and dicentric, and was characterized by multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). The SALSA MLPA P250-B1 DiGeorge Probemix showed duplication of gene dosage in the CES region. aCGH showed a 1.26-Mb duplication at 22q11.1–q11.21 encompassing CECR1–CECR7. The sSMC was likely inv dup(22) (q11.21). Prenatal diagnosis of an sSMC(22) at amniocentesis should alert CES. MLPA, aCGH and fetal ultrasound are useful for rapid diagnosis of CES in case of prenatally detected sSMC(22).     

Multicolor FISH used for the characterization of small supernumerary marker chromosomes (sSMC) in commercially available immortalized cell lines

There are only about 30 commercially available cell lines which include small supernumerary marker chromosomes (sSMC). As approximately 2.5 million people worldwide are carriers of an sSMC, this small number of immortalized cell lines is hard to understand. sSMC cell lines provide practically unlimited material for continuing studies e.g. to learn more about marker chromosome formation, or karyotypic evolution. To obtain information about their genetic content, in the present study we analyzed by FISH and multicolor-FISH approaches 19 sSMC cell lines obtained from the European Collection of Cell Cultures (ECACC). Microdissection and reverse painting, (sub-) centromere-specific multicolor-FISH (sub-)cenM-FISH, multicolor banding (MCB) and selected locus-specific FISH probes were applied. Thus, we were able to characterize comprehensively 14 out of 19 sSMC carrying cell lines; in the remaining five cases an sSMC could not be detected. Surprisingly, in six of the nine cell lines with sSMC previously characterized for their chromosomal origin by others, those results had to be revised. This has impact on the conclusions of previous studies, e.g. for uniparental disomy (UPD) in connection with sSMC.     

Incidence and significance of supernumerary marker chromosomes in prenatal diagnosis

The finding of a supernumerary marker chromosome in amniotic fluid cells poses a considerable counseling dilemma. In 6,500 cases referred to our laboratory over a 4 1/2-year period, eight such cases were identified (0.123% of all cases). In five of the eight cases, a diagnosis of true mosaicism between cells with 46 and 47 chromosomes was made. In the remaining three cases, the marker was present in 100% of the cells. In three cases, the marker was determined to be familial in nature with mosaicism present in the parents of two of these cases. Detailed cytogenetic findings for each case are provided. In no cases were abnormalities noted in either abortuses or live borns. The high incidence of mosaicism in these cases seems to indicate a propensity for supernumerary chromosomes to be lost. Familial markers may not be passed on for many generations, and they may arise as new mutations relatively frequently. There is an urgent need for more information on the risks associated with the prenatal detection of supernumerary chromosomes. We recommend that in considering the implications of the prenatal detection of marker chromosomes cases be considered in at least four distinct groups: type 1--familial and nonmosaic; type 2--familial with mosaicism in either the amniotic fluid cells, a parent, or both; type 3--de novo markers and nonmosaic; and type 4--de novo with mosaicism present in the amniotic fluid cells.     

Clinical findings and cytogenetic analysis of small supernumerary ring chromosomes 7: report of two new cases

Two new patients, mosaic for a small supernumerary ring chromosome 7 are described. There are only seven published reported concerning supernumerary ring chromosome 7 and we reviewed the previously reported cases in an attempt to establish genotype-phenotype correlations, which are particularly important for genetic counselling and clinical genetics. Our first case was a 20 months old girl who was referred for a mild motor developmental delay, an asymmetric facial appearance, a plagiocephaly and a short nose with anteverted nostrils. Our second case was a 9 years old boy who was referred for a IQ at the lower end of the normal range (? 80), obesity, hyperactivity and some dysmorphic features including hypertelorism and down slanting palpebral fissures. In both cases, chromosome analysis after G and R banding and FISH showed a small ring chromosome 7 in respectively 76% and 50% of consecutively scored metaphases. Both ring chromosomes were labelled by FISH using the Williams Syndrome locus probe (Elastin Gene D7S486). Comparison between these two cases and previously published cases allowed to delineate frequent clinical findings. A mild mental retardation was found in the majority of patients. which is an important data for genetic counselling.     

Molecular cytogenetic identification and characterization of a de novo supernumerary neocentromeric derivative chromosome 13

We report a young girl with microphthalmia, conductive deafness, aortic isthmus stenosis, laryngomalacia, and laryngeal stenosis carrying a de novo supernumerary neocentromeric derivative chromosome 13. For the precise identification and characterization of the eu- and heterochromatic content of the marker chromosome, straightforward molecular cytogenetic analyses were performed, such as chromosome microdissection, FISH with different probes (e.g. wcp, alphoid centromeric probes, BAC), centromere-specific multicolor FISH (cenM-FISH), and multicolor banding (MCB). The analyses demonstrated that the marker consisted of an inverted duplication (partial tetrasomy) of the distal portion of chromosome 13 that was separated from the endogenous chromosome 13 centromere. Using an all-centromere probe and multicolor cenM-FISH, no alpha-satellite DNA hybridization signal was detectable on any portion of the derivative chromosome. The presence of a functional and active neocentromere on the derivative chromosome 13 was confirmed by positive immunofluorescence signals with CENP-C antibodies. BAC-FISH confirmed the cytogenetic localization of the neocentromere in band 13q31.3. Thus the patient had a mosaic conventional karyotype mos 47,XX,+inv dup(13)(qter-->q21.3::q21.3-->q31.3-->neo-->q31.3-->qter)[6]/46,XX [49].     

Molecular and fluorescence in situ hybridization characterization of the breakpoints in 46 large supernumerary marker 15 chromosomes reveals an unexpected level of complexity

Supernumerary marker chromosomes (SMCs) of chromosome 15, designated "SMC(15)s," are the most common SMC in humans, accounting for as much as 60% of all those observed. We report the characterization of 46 large SMC(15)s, using both fluorescence in situ hybridization and polymerase chain reaction analysis within and distal to the Prader-Willi/Angelman syndrome critical region (PWACR). Our aim was to establish detailed information on origin, content, and breakpoints, to address the formation of SMC(15)s, and to facilitate genotype-phenotype correlations. For all patients in whom we were able to establish the parental origin, the SMC(15)s were maternally derived. Two patients were observed who had familial SMC(15)s, both inherited from the mother; however, in all remaining patients for whom parental samples were available, the SMC(15)s were shown to have arisen de novo. With one exception, all the SMC(15)s were shown to include the entire PWACR. Detailed investigations of the distal breakpoints categorized the SMC(15)s into two groups. Group A, representing approximately two-thirds of the SMC(15)s, had a breakpoint beyond the standard distal PWS/AS deletion breakpoint BP3, at a position close to the microsatellite marker D15S1010 and the bacterial artificial chromosome 10I10. The group B SMC(15)s were shorter, with more variable breakpoints located around BP3. The majority of the SMC(15)s were shown to have asymmetrical breakpoints, with the two inverted arms of the SMC being unequal in length. Our study revealed an unexpected level of complexity and heterogeneity among SMC(15)s that is not seen in other chromosome 15 rearrangements, such as deletions and duplications. This suggests that multiple mechanisms are involved in the formation of large SMC(15)s.     

Neocentric small supernumerary marker chromosomes (sSMC)--three more cases and review of the literature

Here we report on three new patients with neocentric small supernumerary marker chromosomes (sSMC) derived from chromosome 2, 13 and 15, respectively. The sSMC(13) and sSMC(15) had inverted duplicated shapes and the sSMC(2) a ring chromosome shape. All three cases were clinically severely abnormal. A review of the available sSMC literature revealed that up to the present 73 neocentric sSMC cases including these three new cases have been reported. Seven of these cases were not characterized morphologically; in the remainder, 80% had an inverted duplication, 17% a ring and 3% a minute shape. 81% of the reported neocentric sSMC carriers showed severe, 12% moderate and 8% no clinical abnormalities. In summary, we report three more neocentric sSMC cases, provide a review on all up to now published cases, highlight their special characteristics and compare them to centric sSMC.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.     

Characterization of a neocentric supernumerary marker chromosome originating from the Xp distal region by FISH, CENP-C staining, and array CGH

A small supernumerary marker chromosome (SMC) was observed in a girl with severe developmental delay. Her dysmorphism included prominent forehead, hypertelorism, down-slanting palpebral fissures, low-set/large ears, and flat nasal bridge with anteverted nares. This case also presented hypotonia, hypermobility of joints, congenital heart defect, umbilical hernia, failure to thrive, and seizures. The SMC originated from the distal region of Xp as identified by FISH with multiple DNA probes. Staining with antibodies to Centromere Protein C (CENP-C) demonstrated a neocentromere, while FISH with an alpha-satellite DNA probe showed no hybridization to the SMC. A karyotype was described as 47,XX,+neo(X)(pter-->p22.31::p22.31-->pter), indicating a partial tetrasomy of Xp22.31-->pter. This karyotype represents a functional trisomy for Xp22.31-->pter and a functional tetrasomy for the pseudoautosomal region given that there is no X-inactivation center in the marker chromosome. The SMC was further characterized by microarray-based comparative genomic hybridization (array CGH) as a duplicated DNA fragment of approximately 13 megabase pairs containing about 100 genes. We have described here a new neocentromere with discussion of its clinical significance.     

De novo origin of multiple small supernumerary marker chromosomes (sSMCs) in a child with intellectual disability and dysmorphic features

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.     

Mitotic stability of small supernumerary marker chromosomes depends on their shape and telomeres — A long term in vitro study

Mosaicism is present in more than 50% of the cases with small supernumerary marker chromosomes (sSMCs) and karyotype 47,XX,+mar/46,XX or 47,XY,+mar/46,XY. Recently we provided first evidence that the mitotic stability of sSMC is dependent on their structure, i.e. their shape. Thus, here we performed a long term in vitro study on 12 selected cell lines from the Else Kröner–Fresenius-sSMC-cellbank (http://ssmc-tl.com/ekf-cellbank.html) to test mitotic sSMC stability systematically. The obtained results showed that inverted duplicated shaped and also the so-called complex sSMCs (group 1) are by far more stable, than centric-minute- or ring-shaped sSMCs (groups 2). Generally speaking, the percentage of cells with group-1-sSMCs remained stable over 90 days of cell culture, while that of group-2-sSMCs in parts dramatically decreased. In one group-2-cell line the sSMC was even lost completely after 30 days of in vitro culture, in others the sSMC was depleted in up to 40% of the cells. Still the highest rate of sSMC loss was recorded during EBV-transformation. Overall, the major difference between groups 1 and 2 was the number of telomeres per sSMC. In group 1 the sSMCs had “original” telomeres at both of their ends; in group 2 the sSMCs had either no, possibly secondary acquired and/or only one original telomere. This absence of protective telomeric sequences in group 2 seems to make sSMC more susceptible for loss during cell division. Still, also a growth advantage of cells without sSMC cannot be neglected entirely.     

Chromosomal origin of small ring marker chromosomes in man: characterization by molecular genetics.

Ten cases of small ring chromosomes which did not stain with distamycinA/DAPI and did not possess satellite regions associated with nucleolus-organizing regions are described. In situ hybridization with a battery of biotinylated pericentric repeat probes specific either for individual chromosomes or for groups of chromosomes allowed the identification of the chromosomal origin of these marker chromosomes. There was one example of a marker derived from each of chromosomes 1, 3, 6, 14, 16, 18, 20, 13 or 21, and the X, and there were two examples of markers derived from chromosome 12. One case possessed two markers, one derived from chromosome 6, and one derived from the X. The mechanism of generation of ring marker chromosomes is discussed. Five of seven cases who could be phenotypically assessed were abnormal. Three of these--the first with a ring chromosome derived from chromosome 1; the second with two markers, one derived from chromosome 6 and the other from the X chromosome; and the third with a ring chromosome derived from chromosome 20--each possessed distinctive facies. Additional cases with identified rings may allow the delineation of new chromosomal syndromes.     

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and <Emphasis Type="Italic">Apodemus</Emphasis> B chromosomes

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.     

Molecular characterization of pdc2 and mapping of three pdc genes from rice

 The anaerobic fermentation pathway is thought to play an important role under flooding conditions. The pyruvate decarboxylase 2 (pdc2) gene that encodes the first enzyme of this pathway has been cloned and characterized from rice. This gene has an open reading frame that putatively encodes a 603 amino-acid-residue protein with a molecular mass of 64 kDa. pdc2 has five introns dispersed throughout the coding region, which is also true for rice pdc1. Although the length of these introns in rice pdc2 are different from those in rice pdc1, they are located in exactly the same positions based on the deduced amino-acid sequences. The temporal and spatial expression patterns of pdc1 and pdc2 show that pdc2 is induced to a higher level during the early period (1.5–12 h) of anoxia than pdc1, which is induced more after longer time periods (24–72 h) of anoxia in both shoots and roots. The map positions of the three pdc genes have also been determined. Rice pdc1 is located on chromosome 5 between BCD454A and RZ67, pdc2 is located on chromosome 3 between RZ329 and RZ313, and pdc3 is mapped on chromosome 7 distal to RG351. Received: 19 May 1998 / Accepted: 29 September 1998     

Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

Background  

Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications.