Severe psychomotor retardation in a boy with a small supernumerary marker chromosome 19p

We describe the clinical case of a nine-year-old boy with psychomotor retardation and a small supernumerary marker chromosome (sSMC) present in mosaic form. Fluorescence in situ hybridization (FISH) using centromere cross-hybridizing probes D1/5/19Z (pZ5.1), the whole chromosome paint probe 19, pool YACs19p (839B1, 872G3, 728C8), and pool YACs19q (767C4, 761C1, 786G6) demonstrated that the sSMC was derived from chromosome 19p. Based on GTG-banding and FISH analyses, the patient's karyotype was interpreted as: 47,XY,+mar.ish der(19) (:p13.3-->p11:)(839B1+, 872G3+,728C8+, D1/5/19Z+) de novo[52]/46,XY[48]. To our knowledge, only two other similar cases have been reported. This case helps to better delineate karyotype-phenotype correlations between sSMC 19p and associated clinical phenomena.     

Characterization of a familial small supernumerary marker chromosome in a patient with adult-onset tongue cancer

Cytogenetic analysis in peripheral blood lymphocytes of a 50-year-old female with tongue cancer showed the presence of one to three copies of a small supernumerary marker chromosome (sSMC) in a mosaic state. Family studies also revealed the marker in mosaic form in four (age <29 years) of eleven clinically normal individuals studied from her family of 16 individuals spanning three generations. Due to the extremely small size of the marker chromosome, identification by classical cytogenetics was not informative. Multicolor FISH followed by whole chromosome painting identified the marker as a derivative of chromosome 21. This is the first report of sSMC21 in an adult-onset tongue cancer patient and some of her family members with no clinical symptoms.     

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).     

Supernumerary marker chromosome 5 diagnosed by M-FISH in a child with congenital heart defect and unusual face

We describe a female patient with a small supernumerary marker chromosome (sSMC) present in mosaic and characterized in detail by fluorescence in situ hybridization (FISH) using all 24 human whole chromosome painting probes, multicolor banding (MCB) and subcentromere specific multicolor FISH (subcenM-FISH). The sSMC was demonstrated to be derived from chromosome 5 and the karyotype of our patient was as follows: 47,XX,+mar.ish r(5)(::p13.2 approximately p13.3-->q11.2::) [60%]/46,XX [40%]. Partial trisomy for the proximal 5p and q chromosomal regions is a rare event. A critical region exists at 5p13 for the phenotype associated with duplication 5p. As far as we know, eight similar cases have been published up to now. We describe a new case which, to our knowledge, is the first characterized in such detail. The role of uniparental disomy (UPD) in cases of SMC is also discussed.     

Unusual small supernumerary marker chromosome (sSMC) 9 in a Klinefelter patient

Small supernumerary marker chromosomes (sSMC) are small additional chromosomes characterizable for their origin only by molecular cytogenetic approaches. sSMC have been reported previously in four types of syndromes associated with chromosomal imbalances: in approximately 150 cases with Turner syndrome, 26 cases with Down syndrome and only one case each with Klinefelter syndrome and "Triple-X"-syndrome. Here we report the second case with an sSMC detected in addition to a Klinefelter karyotype. Molecular cytogenetics applying centromere-specific multicolor FISH (cenM-FISH) and a specific subcentromere-specific (subcenM-FISH) probe set characterized the sSMC as a dic(9)(:p12-->q11.1::q11.1--> p11.1:). The reported patient was described with hypogonadism, gynaecomastia plus a bronchial carcinoma. The patient's clinical features are discussed in connection with other Klinefelter cases and possible consequences of presence of the sSMC(9). Furthermore, a suggestion is made for the mode of sSMC-formation in this case.     

New immortalized cell lines of patients with small supernumerary marker chromosome: towards the establishment of a cell bank.

Sixteen newly established cell lines with small supernumerary marker chromosomes (sSMC) derived from chromosomes 1, 2, 4, 6, 7, 8, 14, 15, 16, 18, 19, 21, and 22 are reported. Two sSMC are neocentric and derived from 15q24.1-qter and 2q35-q36, respectively. Two further cases each present with two sSMC of different chromosomal origin. sSMC were characterized by multicolor fluorescence in situ hybridization for their chromosomal origin and genetic content. Moreover, uniparental disomy of the sister chromosomes of the sSMC was excluded in all nine cases studied for that reason. The 16 cases provide information to establish a refined genotype-phenotype correlation of sSMC and are available for future studies.     

Chromosome 5 derived small supernumerary marker: towards a genotype/phenotype correlation of proximal chromosome 5 imbalances

Small supernumerary marker chromosomes (sSMC) are a morphological heterogeneous group of additional abnormal chromosomes that cannot be characterized alone by conventional banding cytogenetics. Molecular cytogenetic techniques are valuable tools for the accurate identification of sSMC and a prerequisite for sound genetic counseling based on refined genotype/phenotype correlation. We describe a new case of a retarded patient with an sSMC derived from chromosome 5. The characterization of the sSMC was done by subcentromere-specific multicolor (subcenM) fluorescence in-situ hybridization (FISH) and by full tilling resolution array analysis, after microdissection and amplification of the marker DNA. Uniparental disomy for normal sister chromosomes of the sSMC(5) was excluded. The karyotype was mos47,XX,+r(5)(::p11.1 → q12.1::)[70%]/46,XX[30%], being the trisomic region between 46.15 ∼ 49.56 Mb and 61.25 ∼ 61.335 Mb, a region known to harbor ∼45 annotated genes. Together with a review of the previously described cases of sSMC(5) and duplications involving the 5q proximal region, we can conclude that trisomy of the 5q11 region is associated with learning difficulties and speech delay.     

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.     

Mosaic small supernumerary marker chromosome 1 at amniocentesis: Prenatal diagnosis,molecular genetic analysis and literature review

We present prenatal diagnosis and molecular cytogenetic analysis of mosaic small supernumerary marker chromosome 1 [sSMC(1)]. We review the literature of sSMC(1) at amniocentesis and chromosome 1p21.1-p12 duplication syndrome. We discuss the genotype–phenotype correlation of the involved genes of ALX3, RBM15, NTNG1, SLC25A24, GPSM2, TBX15 and NOTCH2 in this case.     

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.     

Five novel locations of Neocentromeres in human: 18q22.1, Xq27.1∼27.2, Acro p13, Acro p12, and heterochromatin of unknown origin

Since the first report in 1993, an ectopic centromere, i.e. neocentromere formation, has been reported in more than 100 small supernumerary marker chromosomes (sSMC), in 7 instances of centromere repositioning, and in about a dozen cases with more complex chromosomal rearrangements. Here we report 2 new cases with centromere repositioning and 3 neocentric sSMC consisting exclusively of heterochromatic material. Yet, no centromere formation was reported for the regions 18q22.1 and Xq27.1～27.2 as it was observed in the 2 cases with centromere repositioning here; in both cases, cytogenetically an inversion was suggested. Two of the 3 neocentric sSMC were derived from a short arm of an acrocentric chromosome. The remainder neocentric sSMC case was previously reported and was stainable only by material derived from itself.     

A new small supernumerary marker chromosome involving 14pter → q12 in a child with severe neurodevelopmental retardation: Case report and literature review

Unstable, gene-rich pericentric regions have been associated with various structural aberrations including small supernumerary marker chromosomes (sSMCs). We hereby report on a new sSMC derived from chromosome 14, generating trisomy 14pter → q12 in a child with severe neurodevelopmental delay. The patient featured facial dysmorphism, generalized hypotonia, transverse palmar creases, structural brain abnormality, and severe cognitive and motor impairment. Literature review indicated this to be a unique case of sSMC 14 which was only composed of pter → q12, and the phenotype secondary to duplications of the similar region partially overlaps with the phenotype reported in this study. The genetic analysis on our case helps to better delineate karyotype–phenotype correlations between proximal trisomy 14 and associated clinical phenomena, and we also propose that the involved chromosomal regions may contain dosage-sensitive genes which are important for the development.     

Possible origin of a small bisatellited additional chromosome

Summary The possible derivation of a small supernumerary marker chromosome was investigated by means of different staining techniques and the frequency of satellite associations. It could be demonstrated that the marker chromosome participates at satellite association more than randomly. The marker chromosome is supposed to derive from 2 of chromosomes 13, 14, 21, or 22.Who died accidentally in 1978     

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.     

Small supernumerary marker chromosomes--progress towards a genotype-phenotype correlation

Small supernumerary marker chromosomes (sSMC) are still a major problem in clinical cytogenetics as they are too small to be characterized for their chromosomal origin by traditional banding techniques, but require molecular cytogenetic techniques for their identification. Apart from the correlation of about one third of the sSMC cases with a specific clinical picture, i.e. the i(18p), der(22), i(12p) (Pallister Killian syndrome) and inv dup(22) (cat-eye) syndromes, most of the remaining sSMC have not yet been correlated with clinical syndromes. Recently, we reviewed the available >1600 sSMC cases (Liehr T, sSMC homepage: http://mti-n.mti.uni-jena.de/~huwww/MOL_ZYTO/sSMC.htm). A total of 387 cases (including the 45 new cases reported here) have been molecularly cytogenetically characterized with regard to their chromosomal origin, the presence of euchromatin, heterochromatin and satellite material. Based on analysis of these cases we present the first draft of a basic genotype-phenotype correlation for sSMC for all human chromosomes apart from the chromosomes Y, 10, 11 and 13.     

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.     

Characterization of an analphoid, neocentromere-positive inv dup 8p marker chromosome using multiplex whole chromosome and sub-telomere FISH analyses

A 30-year-old male patient with mild mental retardation was found to have a small supernumerary marker chromosome (SMC) in 90% of his peripheral blood cells and in 100% of his fibroblast cells. Multiplex whole chromosome and sub-telomere FISH analyses were used to determine that this SMC is an inverted duplicated distal chromosome 8p fragment. Although it was negative for alpha-DNA sequences, this marker had a functional kinetochore (neocentromere) demonstrated by a positive signal with a CENP-C antibody. Apparently intact 8p telomeres at the marker's ends were demonstrated by using a telomere repeat FISH probe. The patient's phenotypically normal mother on G-banding analysis had a small marker chromosome in 8% of her peripheral blood cells in two cultures of the first specimen studied. The marker was not seen in any subsequent maternal peripheral blood or fibroblast specimens. Although it was impossible to further characterize the maternal SMC, it was suggested that the mother had the same marker as the one seen in the proband. Inverted duplicated chromosomal fragments are the most frequent type of analphoid markers. Stable inverted duplicated 8p marker chromosomes were previously reported in three other patients. They all apparently occurred de novo and were found to be positive for kinetochore-associated proteins. Evidence for the possible inheritance of an inverted-duplicated, analphoid SMC was not shown to-date. This study also demonstrates a practical, straightforward approach for analphoid marker characterization in clinical laboratory settings, using whole chromosome multiplex and subtelomere-specific FISH analyses. FISH probes for all sub-telomere chromosomal regions are commercially available and the large majority of analphoid marker chromosomes involve telomere regions.     

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.     

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.