Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome In

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Article Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function

Tatyana V. Karamysheva 1,* , Tatyana A. Gayner 2,3, Vladimir V. Muzyka 1,4 , Konstantin E. Orishchenko 1,4 and Nikolay B. Rubtsov 1,4

1 Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; [email protected] (V.V.M.); [email protected] (K.E.O.); [email protected] (N.B.R.) 2 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia; [email protected] 3 Center of New Medical Technologies, 630090 Novosibirsk, Russia 4 Department of Genetic Technologies, Novosibirsk State University, 630090 Novosibirsk, Russia * Correspondence: [email protected]; Tel.: +7-(383)-363-49-63 (ext. 1332)

Received: 23 November 2020; Accepted: 15 December 2020; Published: 17 December 2020

Abstract: For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of ﬂuorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.

Keywords: in vitro fertilization (IVF); microdissection; reciprocal translocation; small supernumerary marker chromosomes (sSMC) in humans

1. Introduction Medical genetic counseling’s key goal is to determine the probability of a child’s birth with an unbalanced karyotype in families carrying reciprocal translocations or small supernumerary marker chromosomes (sSMCs). Autosomal reciprocal translocations (ARTs) are complex chromosomal rearrangements involving two or more chromosomes. ARTs are rather frequent and lead to a chromosomal imbalance in the next generation [1]. The de novo formation or inheritance of sSMCs could also lead to genetic stability impairment [2]. sSMCs are relatively rare in the general population, found in 0.043% of newborn infants [3]. It is critical to detect impaired genetic stability and the risk of

Genes 2020, 11, 1511; doi:10.3390/genes11121511 www.mdpi.com/journal/genes Genes 2020, 11, 1511 2 of 19 its manifestation in children carrying ARTs and sSMCs, due to the high pathogenic capacity of these mutations. Timely ART and sSMC detection might prevent a birth with an imbalance of crucial genetic regions. Parent ART or sSMC diagnosis allows them to prepare for a specific molecular cytogenetic karyotyping at the preimplantation stage and during prenatal diagnostic testing. Currently, deletions and translocations of certain chromosomal regions are detected using commercially available DNA probes and Fluorescent In Situ Hybridization (FISH). Comparative genome hybridization on microarrays and mass parallel patient genome sequencing identifies imbalanced genomic regions with high precision. In some cases, karyomapping helps to distinctly analyze the parents’ chromosomes and define which of the parents’ chromosomes the certain chromosomal region comes from [4]. Despite significant molecular cytogenetics progress, differential staining of human metaphase chromosomes remains the most widespread and critical method of primary cytogenetic analysis. In general, any cytogenetic testing begins from the analysis of differentially stained chromosomes because the technique is simple, reliable, flexible, and cost-effective, not requiring the usage of expensive equipment and reagents. At the same time, it enables us to identify balanced chromosomal translocations and inversions, the detection of which is problematic or sometimes impossible (in cases of a preserved balance in euchromatin chromosomal regions) by many cytogenomic diagnostic methods. A special case of chromosomal pathology is complex chromosomal rearrangement (CCR) resulting from breaks and reassociations of more than two chromosomal regions, when the possibility of the formation of gametes, with balanced euchromatin chromosome regions, is dramatically diminished; however, several studies have shown that the birth of healthy progeny is still possible [5]. At the same time, such extreme scenarios are most likely associated with abnormal meiosis [6]. Unfortunately, detailed cytogenetic diagnostics in the case of a CCR could be very problematic. Balanced CCRs could be formed either via a three-step reciprocal exchange between three chromosomes or as a result of complex translocations involving four or five chromosomes, inversions, and insertions. A vast majority of CCRs are combinations of translocations [7]. In some cases, they could lead to the formation of complex variants of rearranged chromosomes. In the current work, we provide an example of such a situation and discuss the cytogenetic testing strategy in the case of a CCR. Another complicated case for diagnostics is sSMC [8]. In the de novo acquiring of sSMC, investigators must answer two goals: determining the origin of sSMCs and an enclosed euchromatin region’s borders. If sSMC possesses a euchromatin region that is detectable by comparative genomic hybridization, it could be simple. In case of its absence, the detection of de novo emerged sSMC requires using other techniques, such as FISH with DNA probes specific for pericentromeric heterochromatin chromosomal regions or the design of an sSMC-bearing microdissectional DNA probe with the following reverse painting on metaphase chromosomes of a patient and a healthy donor [9,10]. Notably, the elucidation of the origin of a de novo sSMC is necessary because, in 10% of cases, it is associated with uni-parental disomy (UPD) for chromosomes bearing the sSMC [11]. Notably, the prenatal cytogenetic diagnostics in identifying an sSMC in a fetus should be performed in a very narrow time window. In the current study, we describe examples of complicated CCR and sSMC. We also discuss its diagnostics strategy and demonstrate the importance of molecular cytogenetic testing in couples with reproductive issues.

2. Materials and Methods Case #1. Peripheral blood of a woman, 26 years of age. Descriptions of clinical findings: after a medical genetic diagnostic testing, no cognitive or physical abnormalities were found. She was phenotypically normal besides three underdeveloped pregnancies in anamnesis. Diagnosis—secondary infertility. Case #2. A couple with reproductive issues: a woman, 30 years of age and a man, 35 years of age, diagnosed with primary infertility. Descriptions of clinical ﬁndings: both the male and female were otherwise phenotypically normal and there was no family history of infertility, congenital abnormalities, or mental retardation. Genes 2020, 11, 1511 3 of 19

For both cases, sample collection was performed at the Institute of Chemical Biology and Fundamental Medicine SB RAS/the Center of new medical technologies, Novosibirsk, Russia.

2.1. Ethics The studies were supervised by the Ethics Committee on Animal and Human Research of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Russia (protocol #03 of 21 September 2019). Patients were enrolled in the study in strict accordance with international standards, including informed consent, acceptance of participation in the study, and conﬁdentiality. All studies follow the ethical standards based on the Helsinki declaration of the International medical association with the year 2000 amendments. Patients involved in the study signed informed consent forms for using their respective biospecimens for scientiﬁc purposes.

2.2. Experimental Design The analysis of chromosomal abnormalities consisted of several steps: (1) GTG-banding (G-bands after trypsin and Giemsa); (2) production of DNA probes via microdissection and consequent ampliﬁcation of dissected material by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR); (3) FISH of microdissection DNA probes on patients’ metaphase chromosomes. If a DNA probe is derived from an abnormal chromosome, the FISH is performed on metaphase chromosomes of both the patient and a healthy volunteer. FISH with a loci-speciﬁc DNA probe was used to identify regions in rearranged chromosomes (Figure S1).

2.3. Metaphase Chromosome Sample and Slide Preparation Lymphocytes from peripheral blood were cultivated according to the standard techniques [12]. Slides with metaphase and prometaphase chromosomes for FISH were prepared as previously described [13]. GTG diﬀerential staining of metaphase chromosomes was performed according to the standard protocol. Diﬀerentially stained chromosomes were analyzed using light microscope OLYMPUS CX41 (Tokyo, Japan). A CCD camera and VideoTesT-Karyo 3.1 software (Ista-Video Test, Saint-Petersburg, Russia) were used for the image acquisition.

2.4. FISH DNA Probes Preparation Microdissectional whole chromosome painting DNA probes for chromosomes 3, 5, 14, 15, and 16; partial chromosome painting DNA probes for the short and the long arms of chromosome 16 and certain segments of chromosomes 3 and 14 were amplified from 5–10 copies of respective chromosomes or chromosomal regions, as described previously [14,15]. DNA probes derived from rearranged chromosomes or sSMC were amplified from one copy using the same protocol. The labeling of DNA derived from microdissectional DNA libraries was performed with 17 additional cycles of degenerate-oligonucleotide-primed polymerase chain reaction (DOP-PCR) with AlexaFluor 488-5-dUTP (MolecularProbes, Eugene, OH, USA) or TAMRA-5-dUTP (5-Tetramethylrhodamine-dUTP, MolecularProbes, Eugene, OH, USA) [16]. Three-color FISH microdissectional DNA libraries were labeled with Alexa-488 dUTP, 5-TAMRA-dUTP, and Cy5-dUTP in 17 additional DOP-PCR cycles. The quality of all DNA probes prepared with chromosome microdissection was tested by Chromosomal in Situ Suppression (CISS) hybridization on metaphase chromosomes of a donor with normal karyotype. FISH of microdissectional DNA probes on metaphase chromosomes of a healthy donor or patient was performed according to a standard protocol of CISS hybridization with suppression of hybridization of repetitive sequences [17]. The general staining of chromosomes was performed using 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, Darmstadt, Germany). Chromosomes and chromosomal regions were identified by analyzing inverted DAPI banding. Karyotypes are reflected according to the international cytogenetic nomenclature [18]. Genes 2020, 11, x FOR PEER REVIEW 4 of 19

Chromosomes and chromosomal regions were identified by analyzing inverted DAPI banding. Genes 2020, 11, 1511 4 of 19 Karyotypes are reflected according to the international cytogenetic nomenclature [18]. Alu-DNA probe was produced as described [19]. FISH of the Alu-DNA probe was performed withoutAlu-DNA suppression probe of was repetitive produced sequences as described hybridization. [19]. FISH ofThe the technique Alu-DNA allowed probe was us performedto stain euchromatinwithout suppression chromosomal of repetitiveregions, but sequences no heterochromatin hybridization. regions. The technique FISH with allowed this DNA us toprobe stain detectedeuchromatin a small chromosomal region with Alu regions, repeats but in nothe heterochromatin satellites of human regions. acrocentric FISH chromosomes with this DNA [19]. probe detectedThe DNA a small probe region used with for Alu visualization repeats in the of satellites18S ribosomal of human DNA acrocentric (rDNA probe) chromosomes was a 3.2 [19 ].kb fragmentThe of DNA human probe 18S used rDNA for visualization in pHr13 [20,21]. of 18S ribosomal It was labeled DNA (rDNA with probe)TAMRA was 5- adUTP 3.2 kb by fragment nick translation.of human To 18S detect rDNA telomeric in pHr13 clusters, [20,21]. the It (TTAGGG)n was labeled probe with TAMRA labeled with 5-dUTP TAMRA by nick 5-dUTP translation. was usedTo detect [22]. telomeric clusters, the (TTAGGG)n probe labeled with TAMRA 5-dUTP was used [22]. AfterAfter performing performing FISH, FISH, chromosomes chromosomes were were sta stainedined with with DAPI. DAPI. Microscopic Microscopic analysis analysis after after FISH FISH waswas performed performed on on AxioPlan AxioPlan 2 2Imaging Imaging microscope microscope (Zeiss, (Zeiss, Jena, Jena, Germany) Germany) with with the the filters ﬁlters kit kit #49 #49 (Zeiss,(Zeiss, Germany), Germany), SP101 SP101 FITC FITC (CHROMA, (CHROMA, Wixom, Wixom, MI, MI, USA), USA), and and SP103v1 SP103v1 Cy3tmv1 Cy3tmv1 (CHROMA, (CHROMA, Wixom,Wixom, MI, MI, USA), USA), CCD CCD camera camera (CV (CV M300, M300, JAI JAI Corporation, Corporation, Miyazaki, Miyazaki, Japan). Japan). For For signal signal registration, registration, ISIS5ISIS5 software software (METASystems(METASystems GmbH, GmbH Altlussheim,, Altlussheim, Germany) Germany was used.) was All used. microscopy All microscopy experiments experimentswere performed were performed at the Core at Facility the Core for Facility microscopic for microscopic analysis of analysis biological of biological objects at theobjects Institute at the of InstCytologyitute of andCytology Genetics and SB Genetics RAS, Russia. SB RAS, Russia.

3.3. Results Results AA primary primary cytogenetic cytogenetic analysis analysis was was performed performed on the on 72 the-h 72-h lymphocyte lymphocyte culture culture derived derived from fromthe patient’sthe patient’s peripheral peripheral blood. blood.GTG differential GTG diﬀ stainingerential of staining metaphase of metaphase chromosomes chromosomes revealed a revealedbalanced a translbalancedocation translocation involving involvingchromosomes chromosomes 3, 5, and 3, 5,16. and Patient’s 16. Patient’s karyotype karyotype was was described described as as 46,46,XX,t(3;16;5)(q25;q21;p15.1)XX,t(3;16;5)(q25;q21;p15.1) (Figure (Figure 1).1).

Figure 1. GTG (G-bands after trypsin and Giemsa) diﬀerentially stained chromosomes of a patient with a complex chromosomal rearrangement (CCR). der(3)t(3;16;5), der(5)t(3;16;5), der(16)t(3;16;5) reﬂect derivatives of chromosomes 3, 5, and 16 involved in the CCR.

Genes 2020, 11, x FOR PEER REVIEW 5 of 19

Figure 1. GTG (G-bands after trypsin and Giemsa) differentially stained chromosomes of a patient with a complex chromosomal rearrangement (CCR). der(3)t(3;16;5), der(5)t(3;16;5), der(16)t(3;16;5) reflect derivatives of chromosomes 3, 5, and 16 involved in the CCR. Genes 2020, 11, 1511 5 of 19 For reassuring the correctness of conclusions made during the analysis of GTG differentially stainedFor chromosomes, reassuring the we correctness performed of additional conclusions cytogenetic made during testing the with analysis WCP3, of WCP5, GTG diandﬀerentially WCP16, whichstained stain chromosomes, the respective we chromosomes. performed additional Whole chromosome cytogenetic testing painting with prob WCP3,es (WCPs) WCP5, were and obtained WCP16, bywhich microdissection stain the respective of metaphase chromosomes. chromosomes Whole chromosome with the painting following probes DOP (WCPs)-PCR wereof the obtained isolated by chromosomalmicrodissection material. of metaphase The quality chromosomes of produced with DNA the following probes was DOP-PCR checked of by the CISS isolated hybridization chromosomal on metaphasematerial. The chromosomes quality of producedof a healthy DNA donor probes (Figure was 2) checked. Obtained by CISSmicrodissected hybridization WCPs on intensively metaphase paintedchromosomes original of chromosomes a healthy donor showing (Figure their2). suitability Obtained for microdissected the analysis of WCPs rearranged intensively chromosomes. painted Aoriginal specific chromosomes signal was showingdetected their on the suitability chromosomes for the analysis homologous of rearranged to the chromosomes.prepared DNA A probes speciﬁc; tsignalherefore, was microdissectio detected on thenal chromosomesDNA probes homologouscould be used to the for prepared the analysis DNA probes;of chromosomal therefore, abnormalities.microdissectional DNA probes could be used for the analysis of chromosomal abnormalities.

(a) (b)

FigureFigure 2. 2. TwoTwo-color-color chromosomal chromosomal in in situ situ suppression suppression (CISS) (CISS) hybridization hybridization with with WCP3 WCP3 (green) (green)//WCP5WCP5 (red)(red) ( (a)) and WCP16 (red) ( b) on metaphase chromosomes of a healthy donor. Whole chromosome paintingpainting probesprobes (WCPs) (WCPs) intensely intensely painted painted original original chromosomes. chromosomes. The general The staining general of chromosomesstaining of chromosomeswith 4’,6-Diamidino-2-phenylindole with 4’,6-Diamidino-2- dihydrochloridephenylindole dihydrochloride (DAPI) in blue. (DAPI) Chromosomes in blue. Chromosomes are marked as arehuman marked chromosome as human 3 (HSA3), chromosome human chromosome3 (HSA3), human 5 (HSA5), chromosome and human chromosome5 (HSA5), and 16 (HSA16). human chromosome 16 (HSA16). Two-color FISH with WCP3 and WCP5 on the patient’s metaphase chromosomes specifically labeledTwo chromosomes-color FISH with 3 and WCP3 5, as well and asWCP5 regions on of the these patient’s chromosomes metaphase into chromosomes rearranged chromosomes. specifically labeledAccording chromosomes to the patient’s 3 and karyotype 5, as descriptionwell as regions based onof thethese analysis chromosomes of GTG di ffintoerential rearrange staining,d chromosomes.WCP3 stained aAccording portion of der(3) to the chromosome patient’s karyotype and a distal description region of the based long armon ofthe der(16) analysis chromosome of GTG in addition to an intact chromosome 3 (Figure3a). WCP5 stained an intact chromosome 5, 5p15.1 qter differential staining, WCP3 stained a portion of der(3) chromosome and a distal region of the→ long armportion of der(16) of der(5) chromosome chromosome in and addition a distal to regionan intact of thechromosome long arm of 3 der(3)(Figure chromosome 3a). WCP5 (Figurestained3 ana). intactWCP16 chromosome labeled an intact 5, 5p15.1 chromosome→qter portion 16, the of short der(5) arm chromosome and a small and proximal a distal region region of of the the long long arm arm of ofder(16) der(3) chromosome, chromosome as(Figure well as3a). a distalWCP16 region labeled of thean intact short chromosome arm of the der(5) 16, the chromosome short arm and (Figure a small3b). proximalBesides the region staining of the pattern, long arm which of der(16) is expected chromosome, after an as analysis well as ofa distal GTG region differential of the chromosomal short arm of thestaining, der(5) WCP3chromosome labeled (Figure a small 3b). region Besides of chromosome the staining 3 pattern, at the short which arm is expected of the der(5) after chromosome an analysis of(Figure GTG 3differentiala). A portion chromosomal of the HSA3 staining, chromosome WCP3 on labeled the der(5) a small chromosome region of does chromosome not match 3 theat the patients’ short armkaryotype of the description der(5) chromosome based on the (Figure analysis 3a). of GTGA portion differentially of the stainedHSA3 chromosome chromosomes. on The the results der(5) of chromosomethe WCP3 painting does suggestnot match that, the during patients the reorganization’ karyotype description of the HSA3 based chromosome, on the analysis it was cleaved of GTG at differentiallyleast in two distinct stained regions, chromosomes. breaking itThe into results three parts. of the The WCP3 identification painting of suggest the chromosome that, during 3 region, the reorganizationre-located to chromosome of the HSA3 der(5), chromosome, and the confirmation it was cleaved of the at contentsleast in two of the distinct translocated regions chromosomal, breaking it regions of chromosomes, required additional experiments.

Genes 2020, 11, x FOR PEER REVIEW 6 of 19

Genes 2020, 11, x FOR PEER REVIEW 6 of 19 into three parts. The identification of the chromosome 3 region, re-located to chromosome der(5), and into threethe parts. confirmation The identification of the contentsof the chromosome of the translocat 3 regioned, re chromosomal-located to chromosome regions of der(5),chromosomes, and required the confirmatioadditionaln of the experiments. contents of the translocated chromosomal regions of chromosomes, required Genes 2020, 11, 1511 6 of 19 additional experiments.

(a) (a) (b) (b)

FigureFigure 3. 3.Chromosome ChromosomeFigure 3. Chromosome paintingpainting of painting patient’s ofchromosomes. chromosomes. patient’s chromoso Two Two-color-colormes. CISS CISSTwo-color hybridization hybridization CISS onhybridization onthe the on the patient’spatient’s metaphase metaphasepatient’s metaphasechromosomes chromosomes with with DNA DNA probes with probes DNWCP3A WCP3 probes (green)/WCP5 (green) WCP3/WCP5 (green)/WCP5 (red) ( (red)a) and ( aWCP16 )(red) and ( WCP16(red,a) and WCP16 (red, (red,(b)). ( bDAPI)). DAPI( bstaining)). DAPI staining is stainingin isblue. in blue.Intactis in Intactblue.complete Intact complete chromosomes complete chromosomes chromosomes are indicated are indicated areas HSA3,indicated as HSA5 HSA3, as, HSA3,and HSA5, HSA5, and andHSA16. HSA16. (a)HSA16. → (a—) arrow¨—arrow (a) shows→—arrow shows the absence shows the absence ofthe a signalabsence of on a signalof the a signalshort on arm theon theof short the short chromosome arm arm of of the the chromosome5 chromosomederivative. 5 5 derivative. derivative.—arrowhead▶—arrowhead indicates the indicates WCP3 signal the WCP3 (green) signal on the (green) short arm onon thethe of the shortshort chromosome armarm ofof thethe 5 chromosomechromosome derivative. 55 derivative. —arrow ➔indicates WCP5 (red) signal at the distal region of the long arm of chromosome 3. (a) derivative.▶➔ —arrow—arrow indicates indicates WCP5 WCP5 (red) (red) signal signal at the at distalthe distal region region of the of long the arm long of arm chromosome of chromosome 3. 3. (a) (a)Chromosome ChromosomeChromosome 3 3material material 3was wasmaterial detected detected was on on detectedchromosomes chromosomes on chromosomes der(5) der(5) and and der(16). der(5) der(16). (andb) ( bChromos )der(16). Chromosomeome (b) 16Chromosome 16 16 materialmaterial was wasmaterial detected detected was on on detected thethe shortshort on armarm the of short chromosome arm of chromosome der(5) der(5).. der(5).

We amplified microdissectional DNA probes from the short arm and C-negative part of the long WeWe amplified amplifiedWe microdissectionalamplifiedmicrodissectional microdissectional DNA DNA probes probes DNA from from probes the theshort from short arm th armande short C and-negative arm C-negative and part C-negative of partthe long of part the of the long arm of chromosome 16 to confirm the translocation of 16q21→qter region to chromosome 5. The long arm ofarm chromosome of chromosome 16 to 16 confirm to confirm the translocation the translocation of 16q21 of 16q21qter→ regionqter region to chromosome to chromosome 5. 5. The specificity of DNA probes was tested on metaphase chromosomes→ of a healthy donor. Partial The specificityspecificity of DNA of probesDNA probes was tested was on tested metaphase on meta chromosomesphase chromosomes of a healthy of a donor. healthy Partial donor. Partial chromosome paints: PCP16p and PCP16q probes labeled C-negative regions of the respective arms chromosomechromosome paints: PCP16p paints: and PCP16p PCP16q and probes PCP16q labeled probes C-negative labeled regionsC-negative of the regions respective of the arms respective of arms of chromosome 16 (Figure 4a,b). chromosomeof chromosome 16 (Figure4a,b). 16 (Figure 4a,b).

(a) (b) Figure 4. Quality control for microdissection(a) DNA probes homologous for short and(b long) arms of chromosome 16. CISS-hybridization of DNA probes PCP16p (a) and PCP16q (b) on metaphase

chromosomes of a healthy donor. DAPI staining is in blue. Both 16p and 16q indicate the short and the long arm of chromosome 16, respectively. Genes 2020, 11, x FOR PEER REVIEW 7 of 19

Figure 4. Quality control for microdissection DNA probes homologous for short and long arms of chromosome 16. CISS-hybridization of DNA probes PCP16p (a) and PCP16q (b) on metaphase chromosomes of a healthy donor. DAPI staining is in blue. Both 16p and 16q indicate the short and the long arm of chromosome 16, respectively. Genes 2020, 11, 1511 7 of 19 The intensity at the proximal portions of stained regions was slightly diminished. Perhaps the number of assembled copies of proximal edge regions was decreased due to the ambiguity of The intensity at the proximal portions of stained regions was slightly diminished. Perhaps identifying C-negative and C-positive region borders and excluding constitutive heterochromatin the number of assembled copies of proximal edge regions was decreased due to the ambiguity of away from the dissection sample. identifying C-negative and C-positive region borders and excluding constitutive heterochromatin PCP16p and PCP16q stained the patient’s chromosomes according to GTG differential staining away from the dissection sample. results and the respective suggested patient’s karyotype. PCP16p labeled the short arms of PCP16p and PCP16q stained the patient’s chromosomes according to GTG diﬀerential staining chromosomes 16 and der(16) (Figure 5a). PCP16q stained the long arm of chromosome 16, a small results and the respective suggested patient’s karyotype. PCP16p labeled the short arms of chromosomes proximal region of the long arm of chromosome der(16) and a distal portion of the short arm of 16 and der(16) (Figure5a). PCP16q stained the long arm of chromosome 16, a small proximal region of the chromosome der(5) (Figure 5b). This staining pattern resembles the initial karyotype description long arm of chromosome der(16) and a distal portion of the short arm of chromosome der(5) (Figure5b). (Figure 1). The low intensity of the 16q12.1→q21 region staining in chromosome der(16) could be This staining pattern resembles the initial karyotype description (Figure1). The low intensity of the explained by both decreased intensity of a proximal C-negative part of 16q and spreading of 16q12.1 q21 region staining in chromosome der(16) could be explained by both decreased intensity preserved→ DNA of 16q12.1→q21 region in chromosome der(16) into neighboring chromosomal of a proximal C-negative part of 16q and spreading of preserved DNA of 16q12.1 q21 region in bands. → chromosome der(16) into neighboring chromosomal bands.

(a) (b)

FigureFigure 5. 5.CISSCISS hybridization hybridization with with PCP16p PCP16p (a) ( aand) and WCP5 WCP5 (green)/PCP16q(red) (green)/PCP16q(red) (b ()b on) on the the patient’s patient’s metaphasemetaphase chromosomes. chromosomes. (a ()a PCP16p) PCP16p DNA DNA probe probe that that is ishomologous homologous to to the the short short arm arm of ofchromosome chromosome 1616 (red). (red). Chromosomal Chromosomal regions regions where where the the specific speciﬁc signal signal was detected are indicated:indicated: 16p—short16p—short armsarmsof ofchromosomes chromosomes 16 16 and and chromosome chromosome 16 derivative. 16 derivati (bve.) Chromosomal (b) Chromosomal regions regions where thewhere speciﬁc the signalspecific was signaldetected was aredetected indicated: are indicated: HSA5, HSA16—intact HSA5, HSA16 chromosomes—intact chromosome 5, 16. der(3),s 5, der(5), 16. der(3), der(16)—chromosomes der(5), der(16)— chromosome3, 5, and 16s respective 3, 5, and 16 derivatives. respective Chromatinderivatives. DAPI Chromatin staining DAPI is in staining blue. is in blue.

ThreeThree-color-color FISH FISH with with WCP3, WCP3, WCP5 WCP5,, and and WCP16 WCP16 probes probes demonstrated demonstrated that that the thechromosome chromosome 3 3 segment, supposedly 3q?25→ 26, is located between regions derived from chromosomes 5 and 16 segment, supposedly 3q?25 26,→ is located between regions derived from chromosomes 5 and 16 (Figure(Figure 66b).b). CISSCISS hybridization hybridization results results with WCP3with /WCP3/WCP5/WCP16WCP5/WCP16 probes onprobes the patient’s on the chromosomes patient’s chromosomesresemble GTG resemble chromosomal GTG chromosomal labeling results, labeling excluding results the, excluding insertion ofthe chromosome insertion of HSA3chromosome material HSA3into chromosomematerial into der(5)chromosome to be localized der(5) to between be localized a sub-band between 5p15.1 a sub and-band a band 5p15.1 16q21. and To a determineband 16q21. the Tolocalization determine ofthe this localization insertion, weof this ampliﬁed insertion, a microdissectional we amplified a DNAmicrodissectional probe from chromosome DNA probe der(5).from chromosome der(5).

GenesGenes 20202020, ,1111, ,x 1511 FOR PEER REVIEW 88 of of 19 19

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FigureFigure 6. Two-color6. Two WCP3(green)-color /WCP3(green)/PCP16q(red)PCP16q(red) (a) and three-color (a WCP3(yellow)) and /WCP5(green)three-color / WCP3(yellow)/WCP5(green)/WCP16(red)WCP16(red) (b) CISS-hybridization on the patient’s(b) CISS metaphase-hybridization chromosomes. on the (patient’sa) —arrow metaphase indicates WCP3 (green) signal at the short arm of chromosome 5 derivative. ¨—arrow indicates the chromosomes. (a) ➔—arrow indicates WCP3 (green) signal at the short arm of chromosome 5 derivative.absence of → a— signalarrow at indicates the long the arm absence of chromosome of a signal 3at derivative. the long arm Three-color of chromosome CISS hybridization3 derivative. ThreeWCP3(green)-color CISS/WCP5(green) hybridization/WCP16(red) WCP3(green)/WCP5(green)/WCP16(red) (b) on metaphase chromosomes of the patient.(b) on Chromosomes metaphase chromand chromosomalosomes of the regions patient. with Chromosomes speciﬁc signals and are indicated.chromosomal (b) ¨regions—arrow with indicates specific breaking signals points are indicated.in 3q25; 16q21; (b) → 5p15.1—arrow loci. indicates Further, breaking 3q?25 points26 is a in chromosome 3q25; 16q21; 3 5p15.1 segment loci. detected Further, by 3q?25 ﬂuorescent→26 is a in → chromosomesitu hybridization 3 segment (FISH) detected in the short by armfluorescent of chromosome in situ hybridization 5 derivative. CISS(FISH) hybridization in the short revealed arm of a chromosomeregion from chromosome5 derivative. 3 CISS in der(5). hybridization Chromatin revealed DAPI staining a region is in from blue. chromosome 3 in der(5). Chromatin DAPI staining is in blue. CISS-hybridization with PCPder(5) labeled 5p15.1 qter, 16q21 qter and 3q25 (Figure7a). Genes 2020, 11, x FOR PEER REVIEW → → 9 of 19 CISS-hybridization PCPder(3) stained 3pter 3q25 and 5p15.1→ 5pter→ (Figure7b). CISS-hybridization with PCPder(5) labeled→ 5p15.1 qter,→ 16q21 qter and 3q25 (Figure 7a). CISS-hybridization PCPder(3) stained 3pter→3q25 and 5p15.1→5pter (Figure 7b). Based on our results, we cannot conclude the presence or the absence of 3q25 region duplication in the patient’s genome. For the completion of the analysis, we made a DNA probe from the der(5) chromosome (PCPder(5)). In the case of a duplication of the material of the 3q25 band, one would expect that CISS hybridization with this DNA probe would label a region in chromosome der(3) besides chromosome der(5), 16q21→qter region in the intact chromosome 16 and 3q25 band in the intact chromosome 3; however, PCPder(5) stained only the regions mentioned above (Figure 7a). The combined in situ hybridization results with two different region-specific microdissectional DNA probes confirmed the presence of 3q25 band in chromosome der(5). Probe 1 was produced from 3q25→q26 ((PCP3q25→q26), and probe 2 from 3q26→qter (PCP3q25→qter). Both probes label the respective chromosome 3 regions (Figure 8).

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FigureFigure 7.7. CISSCISS-hybridization-hybridization of microdissection DNA probesprobes preparedprepared fromfrom singlesingle abnormalabnormal chromosomes:chromosomes: PCPder(5) PCPder(5) (a ()a and) and PCPder(3) PCPder(3) (b) (DNAb) DNA probes probes on metaphase on metaphase chromosomes chromosomes of a healthy of a healthydonor. Chromosomes donor. Chromosomes 3, 5, and 3, 16 5, with and 16a specific with a speciﬁcred signal red are signal indicated. are indicated. Chromatin Chromatin DAPI staining DAPI stainingis in blue. is in blue.

(a) (b)

Figure 8. CISS hybridization with PCP3q25 → q26 (probe1) and PCP3q25 → qter (probe 2) on chromosomes of a healthy individual (a) and the patient (b). Chromosomes with signals are indicated: HSA3—intact chromosome 3, der(3), der(5), and der(16)—derivatives of chromosomes 3, 5, and 16, respectively.

After CISS hybridization on the patient’s chromosomes PCP3q25→q26 and PCP3q25→qter, besides the standard pattern of painting on intact chromosome 3, we identified labeled regions on the rearranged chromosomes der(3), der(5), and der(16) (Figure 8). The painting pattern for der(16) chromosome region was like the pattern of 3q25→qter; however, a region labeled with PCP3q25→ q26 on der(16) chromosome was of a smaller size. PCP3q25→ q26 also stained small portions of der(3) and der(5) chromosomes. Results of FISH with DNA probe EVI1 Breakapart Probe (https://www.cytocell.com/probes/27-evi1-mecom-breakapart) (fig EVI1), which labels 3q26.2 (Figure 9) also match the conclusion that the insertion of chromosome 3 material into der(5)

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Based on our results, we cannot conclude the presence or the absence of 3q25 region duplication in the patient’s genome. For the completion of the analysis, we made a DNA probe from the der(5) chromosome (PCPder(5)). In the case of a duplication of the material of the 3q25 band, one would expect that CISS hybridization with this DNA probe would label a region in chromosome der(3) besides chromosome der(5), 16q21 qter region in the intact chromosome 16 and 3q25 band in the (a) → (b) intact chromosome 3; however, PCPder(5) stained only the regions mentioned above (Figure7a). FigureThe combined 7. CISS-hybridization in situ hybridization of microdissection results with DNA two probes different prepared region-specific from single microdissectional abnormal chromosomes: PCPder(5) (a) and PCPder(3) (b) DNA probes on metaphase chromosomes of a healthy DNA probes confirmed the presence of 3q25 band in chromosome der(5). Probe 1 was produced from donor. Chromosomes 3, 5, and 16 with a specific red signal are indicated. Chromatin DAPI staining 3q25 q26 ((PCP3q25 q26), and probe 2 from 3q26 qter (PCP3q25 qter). Both probes label the is→ in blue. → → → respective chromosome 3 regions (Figure8).

(a) (b)

FigureFigure 8. 8. CISSCISS hybridization hybridization with with PCP3q25 PCP3q25→ q26q26 (probe1) (probe1) and and PCP3q25 PCP3q25→ qterqter (probe (probe 2) 2) on on → → chromosomeschromosomes of of a healthy a healthy individual individual (a) and (a) the and patient the patient (b). Chromosomes (b). Chromosomes with signals with are signalsindicated: are HSA3indicated:—intact HSA3—intact chromosome chromosome 3, der(3), der(5), 3, der(3), and der(5), der(16) and—derivatives der(16)—derivatives of chromosomes of chromosomes 3, 5, and 16, 3, 5, respectively.and 16, respectively.

After CISS hybridization on the patient’s chromosomes PCP3q25→ q26 and PCP3q25→ qter, After CISS hybridization on the patient’s chromosomes PCP3q25 →q26 and PCP3q25 →qter, besidesbesides the the standard standard pattern pattern of ofpainting painting on on intact intact chromosome chromosome 3, we 3, weidentified identified labeled labeled regions regions on theon rearranged the rearranged chromosomes chromosomes der(3), der(3), der(5) der(5),, and der(16) and der(16) (Figure (Figure 8). The8 ).painting The painting pattern patternfor der(16) for der(16) chromosome region was like the pattern→ of 3q25 qter; however, a region labeled with→ chromosome region was like the pattern of 3q25 qter; howe→ver, a region labeled with PCP3q25 PCP3q25 q26 on der(16) chromosome was of a smaller size.→ PCP3q25 q26 also stained small q26 on der(16)→ chromosome was of a smaller size. PCP3q25 q26 also stained→ small portions of der(3)portions and of der(3)der(5) andchromosomes. der(5) chromosomes. Results Resultsof FISH of with FISH withDNA DNA probe probe EVI1 EVI1 Breakapart Breakapart Probe Probe (https://www.cytocell.com/probes/27(https://www.cytocell.com/probes/27-evi1-mecom-breakapart-evi1-mecom-breakapart)) (fig (fig EVI1), EVI1), which which labels 3q26.2labels (Figure3q26.29) (Figurealso match 9) also the match conclusion the conclusion that the insertion that the of insertion chromosome of chromosome 3 material into3 material der(5) chromosomeinto der(5) resembles band 3q25. A specific signal was detected only in 3q26.2 of an intact chromosome 3 and a distal region of chromosome der(16). All this suggests that the patient’s karyotype has undergone two consequent translocations. Genes 2020, 11, x FOR PEER REVIEW 10 of 19 chromosome resembles band 3q25. A specific signal was detected only in 3q26.2 of an intact chromosome 3 and a distal region of chromosome der(16). All this suggests that the patient’s karyotypeGenes 2020, 11 has, 1511 undergone two consequent translocations. 10 of 19

(a) (b)

FigureFigure 9. TheThe analysisanalysis of of 3q26.2 3q26.2 chromosomal chromosomal segment. segment. (a) FISH(a) FISH EVI1 BreakapartEVI1 Breakapart Probe onProbe the patient’son the patient’schromosomes. chromosomes. (b) Scheme (b) of Scheme EVI1 Breakapart of EVI1 Breakapart Probe homology. Probe homology. The EVI1 3q26.2 The EVI1 product 3q26.2 consists product of a consists158 kb probe, of a 158 labeled kb probe, in red, labeled telomeric in red, to thetelomeric D3S4415 to the marker D3S4415 and includingmarker and the including LRRC34 gene,the LRRC34 a green gene,probe a coveringgreen probe a 181 covering kb region, a 181 including kb region, the including entire EVI1 thegene entire and EVI1 ﬂanking gene and regions flanki andng aregions blue probe, and awhich blue probe, covers which the 563 covers kb region the 563 centromeric kb region to centromeric the EVI1 gene, to the including EVI1 gene, the D3S3364including marker. the D3S3364 Signal marker.was detected Signal at was chromosomes detected at 3chromosomes and der(16). Chromatin3 and der(16). DAPI Chromatin staining isDAPI in blue. staining is in blue.

TheThe obtained obtained results results confirm confirm the the notion notion that that the the patient’s patient’s karyotype karyotype forms forms after after two two consequent consequent translocations.translocations. TheThe was,was, probably, probably, the the reciprocal reciprocal translocation translocation t(3;5)(q25;p15.1), t(3;5)(q25;p15.1), and then and the then exchange the exchangebetween 3q25between3qter 3q25 and→3qter 16q21 and16qter 16q21 regions;→16qter however,regions; h aowever, breakpoint a breakpoint in the 3q25 in bandthe 3q25 appeared band → → appearedto be localized to be localized more distally more todistally the breakpoint to the breakpoint for the for first the translocation. first translocation. As a resultAs a result of this of shift,this shift,a small a small region region of chromosome of chromosome 3 was 3 was preserved preserved between between the the material material of of chromosomeschromosomes 5 and and 16 16;; ttherefore,herefore, despite aa complexcomplex chromosomechromosome reorganization, reorganization, a balanceda balanced genotype genotype is preserved.is preserved. Using Using the thecomparative comparative genomic genomic hybridization hybridization (CGH) (CGH microarray) microarray or even or even full genomefull genome sequencing, sequencing, one could one couldmiss themiss abnormalities the abnormalities described described in the above in the section. above Simultaneously,section. Simultaneously the FISH, analysisthe FISH confirmed analysis confirmedthe presence the of pr theesence reciprocal of the translocation reciprocal translocation involving three involving chromosomes three chromosomes (3, 5, and 16) and (3, enabled5, and 16) us andto detect enabled the us insertion to detect of the a small insertion additional of a small portion additional of chromosome portion of 3 atchromosome the short arm 3 at of the chromosome short arm of5 derivative.chromosome 5 derivative. TThehe molecular molecular cytogenetic cytogenetic analysis analysis enabled enabled u uss to to clarify clarify the the organization organization of of der(5) der(5) chromosome chromosome andand to to describe describe the the patient’s patient’s karyotype karyotype 46,XX,der(3)(3pter 46,XX,der(3)(3pter→3q25::5p15.13q25::5p15.1→5pter),der(5)(16qter5pter),der(5)(16qter→ → → → 16q21::3q25::5p15.116q21::3q25::5p15.1→5qter),der(16)(16pter5qter),der(16)(16pter→16q21::3q2516q21::3q25→3qter).3qter). → → → SmallSmall Supernumerary Supernumerary Marker Marker Chromosome Chromosome (sSMC) (sSMC) KaryotypingKaryotyping of of a a couple couple with with reproduction reproduction issues issues by by analyzing analyzing GTG GTG-di-differentiallyfferentially stained stained chromosomeschromosomes can can detect detect an an sSMC sSMC in in all all 15 15 karyotyped karyotyped cells cells of of a a man. man. Any Any other other structural structural or or numeral numeral abnormalitiesabnormalities were were not not detected. detected. The The patient’s patient’s karyotype karyotype (Figure (Figure 10 10) )was was described described as as 47,XY,+mar. 47,XY,+mar. InIn one ofof the the arms arms of theof the sSMC, sSMC a secondary, a secondary construct construct and a satelliteand a couldsatellite be seen.could Ag-NORbe seen. diAgfferential-NOR differentialstaining has staining detected anhas active detected nucleolus an active organizing nucleolus region organizing (NOR) (Figure region 10b), (NOR) suggesting (Figure that sSMC10b), suggestingis derived fromthat sSMC one of is the derived acrocentric from chromosomes.one of the acrocentric chromosomes.

GenesGenes 20202020, ,1111, ,x 1511 FOR PEER REVIEW 1111 of of 19 19

(a) (b)

FigureFigure 10. 10. TheThe karyotype ofof thethe patient. patient. ( a()a GTG-di) GTG-differentiallyﬀerentially stained stained chromosomes chromosomes of the of patientthe patient with withsmall small supernumerary supernumerary marker marker chromosome chromosome (sSMC) (sSMC) in the in karyotype. the karyotype. (b) Metaphase (b) Metaphase plate of plate a patient of a patientwith AgNOR-staining with AgNOR-staining (case 2). (case—arrow 2). → indicates—arrow nucleolusindicates organizernucleolus regionorganizer (NOR) region detected (NOR) on → detectedacrocentric on chromosomesacrocentric chromosomes and sSMC on and the sSMC patient’s on sSMCthe patient’s by AgNOR-staining. sSMC by AgNOR mar—indicates-staining. mar sSMC.— indicates sSMC. Interestingly, most of the sSMCs derived from acrocentric chromosomes are inverted duplications of theInterestingly, short and a partmost of theof initialthe sSMCs chromosome’s derived long from arm. acrocentric The lack of chromosomes nucleolus organizer are regioninverted on duplicationsthe second arm of suggeststhe short that and sSMC a part was of formed the initial after chromosome deleting a big’s part long of arm an acrocentric. The lackchromosome’s of nucleolus organizerlong arm. region Alternatively, on the second it is the arm result suggests of translocation. that sSMC Previously, was formed sSMCs, after whichdeleting include a big apart proximal of an acrocentricregion of the chrom initialosome chromosomes’s long arm. andAlternatively, other regions, it is the have result been of describedtranslocation. [23– Previously,27]. In some sSMCs, cases, whichAg-NOR include staining a proximal detects regionsregion of other the thaninitial the chromosomes nucleolus organizer and other region. regions, FISH have with been labeled described human [23rDNA–27]. (rDNAIn some probe) cases, orAg labeled-NOR staining human detects DNA ofregions a telomere other repeatthan the (TTAGGG)n. nucleolus organizer Tel-DNA region. probe FISH(Figure with 11a,b) labeled was performedhuman rDNA to conﬁrm (rDNA the probe) presence or andlabeled the stabilityhuman ofDNA nucleolus of a telomere organizer repeat region (TTAGGG)nwithinGenes 20 sSMC.20, 11,. xTel FOR-DNA PEER probeREVIEW (Figure 11a,b) was performed to confirm the presence and the stability12 of 19 of nucleolus organizer region within sSMC. Clusters of ribosomal DNA were detected on the short arms of five pairs of acrocentric chromosomes (HSA13, HSA14, HSA15, HSA21 and HSA22), and one of the arms of the sSMC (Figure 11b). FISH with Tel-DNA probe demonstrated the presence of clusters of telomeric repeats at the ends of all chromosomes (Figure 11a).

(a) (b)

FigureFigure 11.11.FISH FISH with with Tel-DNA Tel-DNA probe probe (a) and ( rDNA-probea) and rDNA (b)-probe on the patient’s(b) on the metaphase patient’s chromosomes. metaphase marchromosomes. and arrows mar indicate and arrows sSMC. indicate sSMC.

Because around 80% of the sSMCs derived from acrocentric chromosomes initiate from

chromosome 15 (Liehr, 2013), we tested our hypothesis of sSMC origin from HSA15 by CISS hybridization with WCP15. The suppression of hybridization of repeated sequences was designed in a way that WCP15 completely stains HSA15—its euchromatin and heterochromatin regions (Figure 12). We detected a weak signal at the short arms of other acrocentric chromosomes and a signal of a similar intensity at one of the arms of the sSMC. This signal is due to rDNA clusters and other homologous repetitive sequences at the short arms of acrocentric chromosomes and the sSMC. This result allowed us to conclude that sSMC formation is not associated with chromosome 15.

(a) (b)

Figure 12. CISS hybridization with WCP15 DNA probe on the patient’s metaphase chromosomes. (a) WCP15 DNA probe-stained chromosome 15 (red). A weak signal in nucleolar organizer regions (NORs) of acrocentric chromosomes. (b) Inverted DAPI banding. No. 15—indicates the homologs of chromosome 15. mar—indicates sSMC.

Based on the above information, we produced a DNA probe from one copy of the sSMC to determine its origin. CISS hybridization with this DNA probe labeled sSMC, and the short arms and

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Genes 2020, 11, 1511 12 of 19

(a) (b) Clusters of ribosomal DNA were detected on the short arms of ﬁve pairs of acrocentric Figure 11. FISH with Tel-DNA probe (a) and rDNA-probe (b) on the patient’s metaphase chromosomes (HSA13, HSA14, HSA15, HSA21 and HSA22), and one of the arms of the sSMC chromosomes. mar and arrows indicate sSMC. (Figure 11b). FISH with Tel-DNA probe demonstrated the presence of clusters of telomeric repeats at the ends of all chromosomes (Figure 11a). Because around 80% of the sSMCs derived from acrocentric chromosomes initiate from Because around 80% of the sSMCs derived from acrocentric chromosomes initiate from chromosome 15 (Liehr, 2013), we tested our hypothesis of sSMC origin from HSA15 by CISS chromosome 15 (Liehr, 2013), we tested our hypothesis of sSMC origin from HSA15 by CISS hybridization with WCP15. The suppression of hybridization of repeated sequences was designed in hybridization with WCP15. The suppression of hybridization of repeated sequences was designed in a a way that WCP15 completely stains HSA15—its euchromatin and heterochromatin regions (Figure way that WCP15 completely stains HSA15—its euchromatin and heterochromatin regions (Figure 12). 12). We detected a weak signal at the short arms of other acrocentric chromosomes and a signal of a We detected a weak signal at the short arms of other acrocentric chromosomes and a signal of a similar similar intensity at one of the arms of the sSMC. This signal is due to rDNA clusters and other intensity at one of the arms of the sSMC. This signal is due to rDNA clusters and other homologous homologous repetitive sequences at the short arms of acrocentric chromosomes and the sSMC. This repetitive sequences at the short arms of acrocentric chromosomes and the sSMC. This result allowed result allowed us to conclude that sSMC formation is not associated with chromosome 15. us to conclude that sSMC formation is not associated with chromosome 15.

(a) (b)

FigureFigure 12. 12.CISS CISS hybridization hybridization with with WCP15 WCP15 DNA DNA probe probe on on thethe patient’spatient’s metaphasemetaphase chromosomes. (a) (a) WCP15 DNADNA probe-stainedprobe-stained chromosomechromosome 1515 (red). (red). A A weak weak signal signal in in nucleolar nucleolar organizer organizer regions regions (NORs)(NORs) of acrocentric of acrocentric chromosomes. chromosomes. (b) ( Invertedb) Inverted DAPI DAPI banding. banding. No. No. 15—indicates 15—indicate thes the homologs homologs of of chromosomechromosome 15. 15. mar—indicates mar—indicates sSMC. sSMC.

BasedBased on on the the above above information, information, we we produced produced a DNAa DNA probe probe from from one one copy copy of theof the sSMC sSMC to to determinedetermine its its origin. origin. CISS CISS hybridization hybridization with with thisthis DNADNA probe labeled sSMC, sSMC, and and the the short short arms arms and and pericentromeric regions of chromosomes 14 and 22 (Figure9a). Pericentromeric regions of these chromosomes include a significant number of homologous sequences. Finding conditions suitable for the suppression of hybridization of homologous sequences during CISS hybridization enabled us to decrease unspecific signal intensity at the pericentromeric region of chromosome 22, preserving the specific signal intensity at chromosome 14 (Figure 13a,b). For the confirmation of the sSMC origin from chromosome 14, we produced several region-specific microdissectional DNA probes from chromosome 14 (14pter p11 (PCP 14a), 14p11 q22 (PCP14b), → → 14q22 qter (PCP14c). Such a combination, consisting of three PCP with a respective signal level, → accounts for the multicolor banding (MCB) of chromosome 14 (Figure 14). MCB allows for determining the order and the orientation of chromosomal regions. Notably, the size of regions detected by MCB is always slightly bigger than regions detected via reverse hybridization of chromosome-specific microdissectional DNA probes. To acquire the MCB, we used different classifications of pseudocolors. All of them labeled sSMC and a pericentromeric region of chromosome 14 in a similar fashion, suggesting the originating of the sSMC from chromosome 14 and its pericentromeric region. Genes 2020, 11, x FOR PEER REVIEW 13 of 19

pericentromeric regions of chromosomes 14 and 22 (Figure 9a). Pericentromeric regions of these chromosomes include a significant number of homologous sequences. Finding conditions suitable for the suppression of hybridization of homologous sequences during CISS hybridization enabled us to decrease unspecific signal intensity at the pericentromeric region of chromosome 22, preserving Genesthe specific2020, 11, 1511signal intensity at chromosome 14 (Figure 13a,b). 13 of 19

(a) (b)

FigureFigure 13.13. FISHFISH ofof DNADNA probe, probe, obtained obtained by by microdissection microdissection from from one one copy copy of aof marker a marker chromosome chromosome on Genesmetaphaseon 20 metaphase20, 11, x chromosomesFOR chromosomes PEER REVIEW of the of patient the patient (a) and (a) a and healthy a healthy individual individual (b). mar (b). and mar arrows and arrows indicate indicate sSMC.14 of 19 sSMC.

For the confirmation of the sSMC origin from chromosome 14, we produced several region- specific microdissectional DNA probes from chromosome 14 (14pter→p11 (PCP 14a), 14p11→q22 (PCP14b), 14q22→qter (PCP14c). Such a combination, consisting of three PCP with a respective signal level, accounts for the multicolor banding (MCB) of chromosome 14 (Figure 14). MCB allows for determining the order and the orientation of chromosomal regions. Notably, the size of regions detected by MCB is always slightly bigger than regions detected via reverse hybridization of chromosome-specific microdissectional DNA probes. To acquire the MCB, we used different classifications of pseudocolors. All of them labeled sSMC and a pericentromeric region of chromosome 14 in a similar fashion, suggesting the originating of the sSMC from chromosome 14 and its pericentromeric region.

(a) (b) (c)

FigureFigure 14. 14.Three-color Three-color FISH FISH with with region-speciﬁc region-specific DNA-probes DNA-probes (PCP14a (PCP14a (green), (green), PCP14b PCP14b (red), (red), PCP14c PCP14c (bright(bright blue) blue) on on the the patient’s patient’s metaphase metaphase chromosomes. chromosomes. (a ()a Chromatin) Chromatin DAPI DAPI staining staining in in blue. blue. Inverted Inverted DAPIDAPI banding. banding. (b ()b Patient’s) Patient’s chromosome chromosome 14 14 and and (c ()c MCB) MCB of of sSMCs sSMC ares are marked. marked.

ObtainedObtained results results enable enable to to describe describe sSMC sSMC as as del(14)(p13q11). del(14)(p13q11). A A crucial crucial aspect aspect is is to to identify identify the the localizationlocalization of of a a breakpoint breakpoint in in 14q11.1 14q11.1 and and 14q11.2. 14q11.2. If If the the breakpoint breakpoint is is in in 14q11.1, 14q11.1, then then the the sSMC sSMC doesdoes not not contain contain euchromatin euchromatin material material of of the the long long arm arm of of chromosome chromosome HSA14. HSA14. In In the the case case of of a a breakpointbreakpoint inside inside 14q11.2, 14q11.2, the the genes genes of of a a sub-band sub-band 14q11.2 14q11.2 could could potentially potentially localize localize in in the the sSMC sSMC leadingleading to to the the increase increase in in their their respective respective copy copy numbers. numbers. Sub-band Sub-band 14q11.2 14q11.2 is is G-negative G-negative suggesting suggesting its its enrichment enrichment with with Alu-repeats. Alu-repeats. We We have have performed performed a a hybridization hybridization with with a a labeled labeled Alu-DNA Alu-DNA probe probe onon the the patient’s patient’s metaphase metaphase chromosomes chromosomes to to check check for for the the presence presence of of the the 14q11.2 14q11.2 sub-band sub-band inside inside the the sSMC.sSMC. The The intensity intensity of of the the signal signal from from the the Alu-DNA Alu-DNA probe probe in in the the sSMC sSMC was was considerably considerably lower lower than than the intensity of a signal in G-negative regions of chromosomes and did not exceed the signal intensity in the satellites of acrocentric chromosomes containing a small number of Alu repeats (Figure 15).

(a) (b)

Figure 15. FISH (a) Alu-DNA probe on the patient’s metaphase chromosomes (red). On the right— marker chromosome labeled with a DNA probe detecting euchromatin. Chromosome 14 and the

Genes 2020, 11, x FOR PEER REVIEW 14 of 19

(a) (b) (c)

Figure 14. Three-color FISH with region-specific DNA-probes (PCP14a (green), PCP14b (red), PCP14c (bright blue) on the patient’s metaphase chromosomes. (a) Chromatin DAPI staining in blue. Inverted DAPI banding. (b) Patient’s chromosome 14 and (c) MCB of sSMCs are marked.

Obtained results enable to describe sSMC as del(14)(p13q11). A crucial aspect is to identify the localization of a breakpoint in 14q11.1 and 14q11.2. If the breakpoint is in 14q11.1, then the sSMC does not contain euchromatin material of the long arm of chromosome HSA14. In the case of a breakpoint inside 14q11.2, the genes of a sub-band 14q11.2 could potentially localize in the sSMC leading to the increase in their respective copy numbers. Sub-band 14q11.2 is G-negative suggesting its enrichment with Alu-repeats. We have performed a hybridization with a labeled Alu-DNA probe Geneson the2020 patient’s, 11, 1511 metaphase chromosomes to check for the presence of the 14q11.2 sub-band inside14 of the 19 sSMC. The intensity of the signal from the Alu-DNA probe in the sSMC was considerably lower than the intensity of a signal in G-negative regions of chromosomes and did not exceed the signal intensity the intensity of a signal in G-negative regions of chromosomes and did not exceed the signal intensity in the satellites of acrocentric chromosomes containing a small number of Alu repeats (Figure 15). in the satellites of acrocentric chromosomes containing a small number of Alu repeats (Figure 15).

(a) (b)

Figure 15. FISHFISH (a)( aAlu) Alu-DNA-DNA probe probe on the on patient’s the patient’s metaphase metaphase chromosomes chromosomes (red). On (red). the Onright the— right—markermarker chromosome chromosome labeled labeledwith a DNA with aprobe DNA detecting probe detecting euchromatin. euchromatin. Chromosome Chromosome 14 and the 14 and the sSMC are indicated. (b) Inverted DAPI-banding. Chromatin DAPI staining is in blue. mar(14) and arrows indicate sSMC. Arrowhead indicates chromosome 14.

Our complex molecular cytogenetic analysis of the origin and the contents of the patient’s marker chromosome shows that it is derived from chromosome 14: del(14)(p13q11.1) and does not contain transcriptionally active chromatin.

4. Discussion

4.1. Complex Chromosomal Rearrangements In cytogenetic diagnostics, molecular cytogenomic techniques are becoming increasingly prevalent, and they do not require using a patient’s metaphase chromosomes. One of the most popular strategies to diagnose chromosomal abnormalities includes the analysis of GTG-labeled metaphase chromosomes and the consequent scrutinizing using microarray CGH or mass parallel sequencing. The current study results prove that, sometimes, to identify a chromosomal abnormality, one needs to perform CISS hybridization with whole chromosome DNA probes, which label potentially fragmented chromosomes. Producing microdissectional DNA probes from such chromosomes enables identifying chromosomal abnormalities that are not detected with cytogenomic methods. Moreover, our CCR analysis results suggest that a complex molecular analysis could detect additional chromosomal rearrangements that are critical even in the case of a preserved chromosomal region balance. The alteration of the composition of topology-associated domains (TAD) could potentially affect the regulation of replication and transcription in a chromosomal region with a large number of genes. TAD structural analysis could be performed using Hi-C; however, it all could be much more effective knowing what exact chromosomal regions need to be analyzed thoroughly. The percentage of chromosomal abnormalities detected via molecular cytogenetic or cytogenomic analysis in patients with a normal karyotype according to GTG-differential staining but having phenotypic abnormalities is relatively low, around 5–10% [28,29]. For instance, among patients with idiopathic mental retardation (MR), developmental delay (DD), and/or nonspecific dysmorphic features (NDF) and normal karyotype, described based on GTG-differential analysis, using subtelomeric DNA probes enabled us to detect distinct chromosomal rearrangements. Their complete characterization required performing an Genes 2020, 11, 1511 15 of 19 additional FISH with chromosome-specific and locus-specific DNA probes. In our follow-up study, we detected the insertion of a small region of chromosome 3 into chromosome der(5). This enabled us to determine the sequence of events and to offer a mechanism of formation of insertion 3q25 in chromosome der(5). The formation of a final karyotype potentially proceeded as a two-step process involving multiple cell generations. Unlike GTG banding, FISH diagnostics presented here enabled us to detect the chromosome 3 segment within the der(5) chromosome. Besides describing a translocation, which involves three chromosomes, we could also identify a small region of chromosome 3 on chromosome der(5). That was possible due to the “multiplex” application of WCPs from intact chromosomes, as well as of a WCP from rearranged chromosomes. The detection of a small region of chromosome 3 on chromosome der(5) raised additional questions about the significance of the newly identified chromosomal breakpoints, emphasizing the possible impairment of TAD structures and microdeletion formation in this region. The detection of rearranged chromosomes is of high importance for medical genetic diagnostics and prediction. A probability of gamete formation with balanced chromosomes is extremely low in such a complex chromosomal rearrangement. The family is recommended in vitro fertilization (IVF) using donor oocytes. In the case of spontaneous pregnancy, the patient is recommended to undergo prenatal diagnostic testing. According to the literature, many reciprocal translocation carriers are phenotypically normal, but they have an increased risk of having children with chromosomal imbalance, spontaneous abortion, or infertility [1].

4.2. Small Supernumerary Marker Chromosomes Among chromosomal abnormalities, sSMC represents a special type of chromosomal rearrangement, which has been very problematic in diagnostics until recently. sSMC detection requires an analysis of the patient’s metaphase chromosomes. Cytogenomic techniques (CGH or mass parallel sequencing) can only detect an increase in copy numbers for a specific euchromatin region within sSMC. It is still a question whether it derives from sSMC or one of the chromosomes. If sSMC lacks such a euchromatin region, it escapes detection. Notably, most sSMCs are present only in some of the cells, which could also hamper or even completely block their detection via cytogenomic methods. We suggest that an optimal sSMC identification and characterization strategy is to analyze the patient’s metaphase chromosomes. The determination of its origin and contents should be performed using DNA probes specific for pericentromeric chromosomal regions or making microdissectional DNA probes from sSMC with a consequent reverse painting. These steps help to identify sSMC frequency in a patient’s cells and its origin. In the case of de novo formed sSMCs, researchers can use this information to check for monoparental disomy in the initial chromosome. Amplifying and using microdissectional DNA probe from sSMC also enables us to determine whether it contains any euchromatin. If sSMC is present in all patient’s cells, CGH and mass parallel patients’ genome sequencing allows clarifying what euchromatin region of the initial chromosome is preserved in sSMC. FISH results with a microdissectional DNA probe on metaphase chromosomes could prove complicated to interpret in case of a presence of only a small euchromatin region. At the same time, karyotype mosaicism could question or even exclude CGH or mass parallel sequencing applicability. In such a scenario, FISH with labeled Alu-repeat helps to detect euchromatin material within sSMC more reliably than FISH with microdissectional DNA probe [30]. Another approach to describe sSMC is to sequence the microdissectional DNA library derived from the sSMC. This enables us to detect euchromatin’s presence within sSMC and rather accurately describe its contents [31,32]. However, this approach’s application demands longer periods, which conflicts with the short turnaround time required for prenatal diagnostic testing. Unfortunately, even a detailed description of the sSMC contents does not solve all medical genetic counseling problems. A complete absence of euchromatin within the sSMC suggests that Genes 2020, 11, 1511 16 of 19 the sSMC itself could not lead to the formation of an abnormal phenotype. However, in the case of its de novo formation, one must perform an additional test for UPD in the initial chromosome. It has also proved complicated to assess the pathogenic capabilities of the sSMC, which contains euchromatin, except the sSMCs derived from chromosome 15. Identifying the most frequent locations of chromosomal breakpoints during the formation of such sSMC and many studied cases allows for reliably assessing its clinical implications and consequences. If sSMCs are derived from other chromosomes, such an assessment becomes more problematic. Multiple studies show that sSMCs, in which the size of a euchromatin region does not exceed 3–5 megabase pairs, do not possess any pathogenic potential [33]. Nevertheless, a work by Marle has demonstrated that the risk of an abnormal phenotype could be associated with the size of the euchromatin region (more or less than 1 Mb) or the number of genes (more or less than 10) [34]—these studies need to be continued. The underlined characteristic of sSMCs with small euchromatin regions could potentially be associated with its localization in the interphase nucleus. A small euchromatin material and pericentromeric sSMC heterochromatin could be placed into the peripheric transcriptionally inactive nuclear compartment leading to the suppression of the transcriptional activity. To avoid such suppression, the euchromatin region must potentially exceed a specific size. Indeed, the analysis of B chromosome spatial organization in association with heterochromatin regions of autosomes in Korean field mice Apodemus peninsulae matches well with the above hypothesis [32,35]. On the contrary, in birds, microchromosomes are characterized by high transcriptional activity and are in the inner transcriptionally active nuclear compartment [36]. Concluding, one must mention that besides the high effectiveness and broad capacities of modern cytogenomic methods, performing a well-rounded, complete diagnostic of chromosomal abnormalities requires complex studies utilizing both the cytogenomic techniques as well as traditional cytogenetic methods. Interestingly, copy number variation (CNV) in different sizes is rather frequently detected by CGH, but the interpretation of the results is often problematic. In the current paper, we describe the structural changes in chromosomes, which were detected or expected after GTG banding. The sSMC lacks euchromatin, suggesting that its formation does not lead to the imbalance of the genome’s euchromatin portion. Notably, de novo formed sSMCs are associated with a high risk of uniparental disomy, therefore requiring performing additional tests. Analysis of the complex chromosomal rearrangement also fits well with the notion that it does not cause genetic imbalance. However, identifying the points of breaks and reassociations during the chromosomal rearrangements allows for precisely monitoring these regions, which could be further scrutinized using diverse molecular diagnostic techniques. Significantly, such chromosomal rearrangements cause serious reproductive issues while not leading to genetic imbalance.

5. Conclusions Application of WCPs derived from metaphase chromosomes for the painting of rearranged ones allows the detailed description of their content. In the present study, the approach included applying WCPs derived from abnormal chromosomes; it appeared to be efficient in molecular cytogenetic diagnostics of chromosomes formed with some subsequent translocations and small supernumerary marker chromosomes. CISS hybridization with WCP derived from abnormal chromosomes can precisely define its content, while CISS hybridization with WCPs from intact chromosomes can confirm a conclusion based on a study using WCP generated from the chromosome of interest. Results obtained in the performed analysis of the complex chromosomal abnormality and small supernumerary chromosome showed high efficiency of combining the usage of both types of WCPs and locus-specific probes with studying abnormal karyotypes revealed with the traditional cytogenetic technique of metaphase chromosome GTG-banding in routine karyotype diagnostics.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/12/1511/s1, Figure S1: FISH analysis of a complex chromosomal rearrangement involving three chromosomes and small supernumerary marker chromosome in patients with impaired reproductive function. Genes 2020, 11, 1511 17 of 19

Author Contributions: Conceptualization, T.V.K., T.A.G. and N.B.R.; obtaining of microdissection DNA probes, N.B.R.; data analysis, T.V.K. and K.E.O.; FISH design T.V.K.; writing—manuscript, V.V.M. and N.B.R.; funding acquisition K.E.O. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Ministry of Education and Science of the Russian Federation, grant #2019-0546 (FSUS-2020-0040) and by the Russian Foundation for Basic Research (#19-015-00084a). Acknowledgments: Microscopy was performed in the Core Facility for Microscopy of Biologic Objects, the Institute of Cytology and Genetics SB RAS (reg. No. 3054), Russia. We are grateful to S.A. Romanenko for technical assistance with microdissection with DNA probes derived from abnormal chromosomes. Conﬂicts of Interest: All the authors report no disclosure nor conﬂict of interest.

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