Elisabeth Klein

Microdeletion and Microduplication Syndromes

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted “genomic disorders” or “contiguous gene syndromes” is given.

Clinical Impact of Somatic Mosaicism in Cases with Small Supernumerary Marker Chromosomes

Somatic mosaicism is present in slightly more than 50% of small supernumerary marker chromosome (sSMC) carriers. Interestingly, non-acrocentric derived sSMC show mosaicism much more frequently than acrocentric ones. sSMC can be present in different mosaic rates, which may go below 5% of the studied cells. Also cryptic mosaicism can be present and mosaics may be differently expressed in different tissues of the body. Even though in the overwhelming majority of the cases somatic sSMC mosaicism has no direct clinical effect, there are also cases with altered clinical outcomes due to mosaicism. Also clinically important is the fact that a de novo sSMC, even present in mosaic, may be a hint of uniparental disomy (UPD). As it is under discussion to possibly replace standard karyotyping by methods like array-CGH, the impracticality of the latter to detect low-level sSMC mosaics and/or UPD has to be considered as well. Overall, sSMC mosaicism has to be studied carefully in each individual case, as it can be extremely informative and of importance, especially for prenatal genetic counseling.

Complex small supernumerary marker chromosomes – an update

BackgroundComplex small supernumerary marker chromosomes (sSMC) constitute one of the smallest subgroups of sSMC in general. Complex sSMC consist of chromosomal material derived from more than one chromosome; the best known representative of this group is the derivative chromosome 22 {der(22)t(11;22)} or Emanuel syndrome. In 2008 we speculated that complex sSMC could be part of an underestimated entity.ResultsHere, the overall yet reported 412 complex sSMC are summarized. They constitute 8.4% of all yet in detail characterized sSMC cases. The majority of the complex sSMC is contributed by patients suffering from Emanuel syndrome (82%). Besides there are a der(22)t(8;22)(q24.1;q11.1) and a der(13)t(13;18)(q11;p11.21) or der(21)t(18;21)(p11.21;q11.1) = der(13 or 21)t(13 or 21;18) syndrome. The latter two represent another 2.6% and 2.2% of the complex sSMC-cases, respectively. The large majority of complex sSMC has a centric minute shape and derives from an acrocentric chromosome. Nonetheless, complex sSMC can involve material from each chromosomal origin. Most complex sSMC are inherited form a balanced translocation in one parent and are non-mosaic. Interestingly, there are hot spots for the chromosomal breakpoints involved.ConclusionsComplex sSMC need to be considered in diagnostics, especially in non-mosaic, centric minute shaped sSMC. As yet three complex-sSMC-associated syndromes are identified. As recurrent breakpoints in the complex sSMC were characterized, it is to be expected that more syndromes are identified in this subgroup of sSMC. Overall, complex sSMC emphasize once more the importance of detailed cytogenetic analyses, especially in patients with idiopathic mental retardation.

Multicolor FISH methods in current clinical diagnostics

Multicolor FISH (mFISH) assays are currently indispensable for a precise description of derivative chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting probes in multiplex-FISH and spectral karyotyping. Since then, multiple approaches for chromosomal differentiation based on multicolor-FISH (MFISH) assays have been developed. Predominantly, they are applied to characterize marker or derivative chromosomes identified in conventional banding analysis. Since the introduction of array-based comparative genomic hybridization (aCGH), mFISH is also applied to verify and further delineate aCGH-detected aberrations. For the latter, it is important to consider the fact that aCGH cannot detect or characterize balanced rearrangements, which are important to be resolved in detail in infertility diagnostics. In addition, mFISH is necessary to distinguish different imbalanced situations detectable in aCGH; small supernumerary marker chromosomes have to be differentiated from insertions or unbalanced translocations. This review presents an overview on the available mFISH methods and their applications in pre- and post-natal clinical genetics.

Human Ring Chromosomes – New Insights for their Clinical Significance

Abstract Twenty-nine as yet unreported ring chromosomes were characterized in detail by cytogenetic and molecular techniques. For FISH (fluorescence in situ hybridization) previously published high resolution approaches such as multicolor banding (MCB), subcentromere-specific multi-color-FISH (cenM-FISH) and two to three-color-FISH applying locus-specific probes were used. Overall, ring chromosome derived from chromosomes 4 (one case), 10 (one case), 13 (five cases), 14, (three cases), 18 (two cases), 21 (eight cases), 22 (three cases), X (five cases) and Y (one case) were studied. Eight cases were detected prenatally, eight due developmental delay and dysmorphic signs, and nine in connection with infertility and/or Turner syndrome. In general, this report together with data from the literature, supports the idea that ring chromosome patients fall into two groups: group one with (severe) clinical signs and symptoms due to the ring chromosome and group two with no obvious clinical problems apart from infertility.

How to narrow down chromosomal breakpoints in small and large derivative chromosomes – a new probe set

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ∼10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.

Tetrasomy 9p Mosaicism Associated with a Normal Phenotype in Two Cases

Tetrasomy 9p is a rare chromosomal syndrome and about 30% of known cases exhibit mosaicism. Approximately 50 of the reported cases with tetrasomy 9p mosaicism show a characteristic facial appearance, growth failure, and developmental delay. However, 3 patients with mosaicism for isochromosome 9p and a normal phenotype have also been reported. We report 2 additional cases of clinically normal young females with tetrasomy 9p mosaicism, one of whom also exhibited X chromosome aneuploidy mosaicism leading to an overall of 6 different cell lines. STR analysis performed on this complex mosaic case indicated that the extra isochromosome was of maternal origin while the X chromosome aneuploidy was of paternal origin, indicating a postzygotic event.

Mitotic Stability of Small Supernumerary Marker Chromosomes: A Study Based on 93 Immortalized Cell Lines

Small supernumerary marker chromosomes (sSMC) are known for being present in mosaic form as 47,+mar/46 in >50% of the cases with this kind of extra chromosomes. However, no detailed studies have been done for the mitotic stability of sSMC so far, mainly due to the lack of a corresponding in vitro model system. Recently, we established an sSMC-cell bank (Else Kröner-Fresenius-sSMC-cellbank) with >150 cell lines. Therefore, 93 selected sSMC cases were studied here for the presence of the corresponding marker chromosomes before and after Epstein-Barr virus-induced immortalization. The obtained results showed that dicentric inverted duplicated-shaped sSMC are by far more stable in vitro than monocentric centric minute- or ring-shaped sSMC. Simultaneously, a review of the literature revealed that a comparable shape-dependent mitotic stability can be found in vivo in sSMC carriers. Additionally, a possible impact of the age of the sSMC carrier on mitotic stability was found: sSMC cell lines established from patients between 10-20 years of age were predominantly mitotically unstable. The latter finding was independent of the sSMC shape. The present study shows that in vitro models can lead to new and exciting insights into the biology of this genetically and clinically heterogeneous patient group.

Multiple copies of BCR-ABL fusion gene on two isodicentric Philadelphia chromosomes in an imatinib mesylate-resistant chronic myeloid leukemia patient

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Amplification or duplication of the BCR-ABL gene has been found to be one of the key factors leading to drug resistance to imatinib mesylate (IM). In the present study, we identified the presence of isodicentric Ph chromosomes [idic(Ph)] in an IM-resistant patient. Fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes confirmed the heterogeneity and amplification of the fused BCR-ABL gene. FISH analysis superimposed on G-banding confirmed the presence of idic(Ph) chromosomes. Reverse transcription-polymerase chain reaction (RT-PCR) products revealed the presence of the BCR-ABL fusion transcript b3a2. The idic(Ph) chromosomes in CML were shown to be fused at the satellite regions of the short arms. The patient did not respond to IM chemotherapy and did not achieve remission. In this study, the impact of the idic(Ph) chromosomes on genomic instability, heterogeneity and amplification of the BCR-ABL gene in IM-resistant patients is discussed.

Detailed analysis of an idic(Y)(q11.21) in a mosaic karyotype

Abnormalities involving sex chromosomes account for approximately 0.5% of live births. The phenotypes of individuals with mosaic cell lines that exhibit structural aberrations of the X and Y chromosomes are variable and difficult to predict. Phenotypes associated with sex chromosome mosaicism vary from females with Turner syndrome to males with infertility, and include individuals with ambiguous genitalia. In this study, we report a 17-year-old male with phenotypic features of Klinefelter syndrome with an isodicentric Y chromosome and a final karyotype of 45,X[4]/46,X,idic(Y)(q11.21)[95]/47,XX,+idic(Y)(q11.21)[1]. Application of high resolution molecular cytogenetic techniques as well as molecular studies revealed two copies of the sex-determining region of Y chromosome (SRY) gene and two centromers. Additionally, the breakpoint in Yq11.21 was narrowed down between positions 13.4 and 14.3 MB (hg18). We present a patient with partial disomy of Ypter to Yq11.21 in the majority of the patient cells, showing phenotypic features of Klinefelter syndrome. The syndrome may have occurred due to a more prominent presence of the cell line 47,XX,+idic(Y)(q11.21) detected only once in 1% of the peripheral blood cells. This finding may prove helpful in similar cases with symptoms of Klinefelter syndrome, but which exhibit an absence of the cell line 47,XXY in peripheral blood.