The development of cytogenetically normal, abnormal and mosaic embryos: a theoretical model

Assisted reproduction and preimplantation genetic diagnosis (PGD) involve various complicated techniques, each of them with its own problems. However, the greatest problem with PGD for chromosome abnormalities is not of a technical nature but is a biological phenomenon: chromosomal mosaicism in the cleavage stage embryo. Here, we present a hypothetical, quantitative model for the development of chromosomally normal, abnormal and mosaic embryos. The arising of mosaicism in 2-8-cell embryos was described by a binomial probability model on the occurrence of mitotic events inducing chromosomal changes in the blastomeres. This model converted the 'mean' rate of mosaicism found in cross-sectional studies (60%) into an equal rate of mosaic embryos at arrival at the 8-cell stage (59.8%). The disappearance of > 90% of the mosaic embryos or the mosaicism itself from surviving embryos during the morula stage was explained by mitotic arrest of most of the mitotically changed cells under increasing cell cycle control. In our model, 25.9 and 14.3% of the embryos at the 8-cell stage are normal and abnormal respectively. The remaining 59.8% of the embryo shows mosaicism: 34.6% of abnormal/normal cells and 25.2% of abnormal/abnormal cells. The high proportion of abnormal and mosaic embryos together explains the high rate of abnormal laboratory findings in PGD for chromosomal abnormalities and aneuploidy screening. The poor representation of a 1- or 2-cell biopsy for the 7- or 6-cell post-biopsy embryo in the case of mosaicism explains the high rate of false-negative and false-positive results.     

Comparison of chromosome analysis and chromosomal microarray analysis: what is the value of chromosome analysis in today’s genomic array era?

PurposeChromosomal microarray analysis enables the detection of microdeletions/duplications and has become the standard in clinical diagnostic testing for individuals with congenital anomalies and developmental disabilities. In the era of genomic arrays, the value of traditional chromosome analysis needs to be reassessed.MethodsWe studied 3,710 unrelated patients by chromosomal microarray analysis and chromosome analysis simultaneously and compared the results.ResultsWe found that chromosomal microarray analysis detected the chromosomal imbalances that were identified by chromosome analysis with the exception of six cases (0.16%) that had mosaic abnormalities. Of note, one case showed mosaicism for two abnormal cell lines, resulting in a balanced net effect and a normal chromosomal microarray analysis. Further structural abnormalities such as unbalanced translocations, rings, and complex rearrangements were subsequently clarified by chromosome analysis in 18% of the cases with abnormal chromosomal microarray analysis results. Apparently balanced rearrangements were detected by chromosome analysis in 30 cases (0.8%).ConclusionOur data demonstrate that although chromosomal microarray analysis should be the first-tier test for clinical diagnosis of chromosome abnormalities, chromosome analysis remains valuable in the detection of mosaicism and delineation of chromosomal structural rearrangements.ConclusionGenet Med 2013:15(6):450–457     

Separation and maintenance of normal cells from human embryonic stem cells with trisomy 12 mosaicism

Human embryonic stem cells (hESCs) are pluripotent and hold great promise as useful tools in basic scientific research and in the field of regenerative medicine. However, several studies have recently reported chromosomal abnormalities such as gains of chromosomes 12, 17 and X in hESCs. This genetic instability presents an obstacle in the application of hESCs as sources of cell therapies. We found that trisomy 12 was correlated with changes in hESC colony morphology during hESC maintenance. In this study, we investigated whether normal and trisomy 12 cells could be separated in hESC cultures displaying trisomy 12 mosaicism with two types of colony morphology using a mechanical transfer technique. Eight sublines were cultured from eight hESC colonies displaying normal or abnormal morphology. Four sublines with normal morphology had normal chromosome 12 numbers, whereas the four sublines with abnormal morphology displayed trisomy 12. These results indicate that a hESC colony with a minor degree of chromosomal mosaicism and normal morphology could proceed to a colony with normal chromosomes after prolonged cultures with mechanical transfer. Therefore, analysis of cultures for chromosomal abnormalities when changes in colony morphology are observed during culture is essential for maintaining normal hESC lines.     

Mosaic paternal uniparental (iso)disomy for chromosome 20 associated with multiple anomalies

Uniparental disomy for a number of human chromosomes is associated with clinical abnormalities. We report a child with a complex chromosomal rearrangement involving chromosome 20 (45,XY,psu dic (20;20)(p13;p13)) and paternal uniparental isodisomy for chromosome 20 in peripheral blood and bone marrow. This patient had multiple congenital abnormalities including microtia/anotia, micrencephaly, congenital heart disease, neuronal subependymal heterotopias, and colonic agangliosis. Molecular studies on DNA from peripheral blood demonstrated paternal uniparental inheritance of chromosome 20. However, fibroblasts demonstrated a mosaic karyotype, with one cell line having 45 chromosomes, including the pseudodicentric chromosome 20 (75% of cells), and a second cell line having 46 chromosomes, including the pseudodicentric chromosome 20, and a normal chromosome 20 (trisomy 20) (25% of cells). FISH experiments using a sub-telomeric probe that maps approximately 120 kb from the 20p telomere, showed that both copies of these sequences were present on the rearranged chromosome, consistent with deletion of a very small interval. This leads us to suggest that in addition to trisomy 20 mosaicism, paternal uniparental disomy for chromosome 20 could contribute to his clinical phenotype.     

Unusual cytogenetic mosaicism involving chromosome 14 abnormalities in a child with an MR/MCA syndrome and abnormal pigmentation

An 8-year-old female child with mental retardation (MR), multiple congenital anomalies (MCA) and irregular pigmentation was shown to have karyotypic mosaicism involving chromosome 14 abnormalities. Four cell lines were found in both peripheral blood lymphocytes and skin fibroblasts and were represented by: a normal karyotype, an isopseudodicentric 14q [iso psu dic(14)], a ring 14 [r(14)], and a monosomy 14 [mono(14)]. Our results are compared with reported cases involving multiple abnormalities of specific chromosomes. Karyotypic mosaicism of comparable chromosome 14 abnormalities is rare, with only one known previous case. Detailed analysis of karyotypic mosaicism of rare chromosomal abnormalities is essential to determine meaningful correlations with specific patterns of malformation.     

Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men

To assess the frequency of chromosomal aberrations in French candidates for intracytoplasmic sperm injection (ICSI), and to explore the existence of a female chromosomal factor in some cases of couple infertility, a collaborative retrospective clinical and cytogenetic study was performed, launched by the Association des Cytogénéticiens de Langue Franciaise (ACLF). The karyotypes of 3208 patients [2196 men (68.4%), 1012 (31.6%) women] included in ICSI programmes over a 3-year period in France were collected. A total of 183 aberrant karyotypes was diagnosed, corresponding to an abnormality frequency of 6.1% (134/2196) for men and 4.84% (49/1012) for women. The following frequencies of abnormalities were observed respectively for men and women: 1.23% (n = 27) and 0.69% (n = 7) for reciprocal translocations, 0.82% (n = 18) and 0.69% (n = 7) for Robertsonian translocations, 0.13% (n = 3) and 0.69% (n = 7) for inversions, 3.32% (n = 73) and 2.77% (n = 28) for numerical sex chromosome aberrations, and 0.59% (n = 13) and 0% for other structural aberrations. Among the male patients of this latter group, 0.40% (n = 9) had a Y chromosome abnormality. Among the male patients with numerical sex chromosome abnormalities, 2.23% (n = 49) were 47,XXY, 0.32% (n = 7) were 47,XYY, and 0.77% (n = 17) had a mosaicism for numerical sex chromosome anomalies. All the female patients with sex chromosome abnormalities (2.77%, n = 28) had mosaicism for numerical sex chromosome anomalies. Even if these cases-the significance of which was sometimes questioned-were disregarded in the analysis, 2.08% (21/1012) of abnormal karyotypes remained in women. An overall increased frequency of chromosomal aberrations was found, and this confirmed that in some cases of poor reproductive outcome there may be a contribution of maternal chromosome aberrations. Indeed, the existence of a chromosome abnormality in the female partner was associated with the group of infertile men in which there was no apparent cause of infertility.     

Distal 8p deletion (8)(p23.1): An easily missed chromosomal abnormality that may be associated with congenital heart defect and mental retardation

We describe the clinical manifestations and molecular cytogenetic analyses of three patients with a similar distal deletion of chromosome 8. Each child had mild developmental delay and subtle minor anomalies. Two had cardiac anomalies but no other major congenital anomalies were present. High resolution G and R banding showed in all three patients del(8)(p23.1), but the breakpoint in case 1 was distal to 8p23.1, in case 2 was in the middle of 8p23.1, and in case 3 proximal to 8p23.1. Fluorescence in situ hybridization (FISH) studies with a chromosome 8 paint probe confirmed that no other rearrangement had occurred. FISH with a chromosome 8-specific telomere probe indicated that two patients had terminal deletions. Chromosome analysis of the parents of case 1 and mother of case 2 were normal; the remaining parents were not available for study. Thirteen individual patients including the three in this study, and three relatives in one family with del(8)(p23.1), have been reported in the past 5 years. Major congenital anomalies, especially congenital heart defects, are most often associated with a breakpoint proximal to 8p23.1. Three patients were found within a 3-year period in this study and five cases were found within 4 years by another group, indicating that distal 8p deletion might be a relatively common chromosomal abnormality. This small deletion is easily overlooked (i.e., cases 1 and 2 were reported as normal at amniocentesis) and can be associated with few or no major congenital anomalies. © 1996 Wiley-Liss, Inc.     

Congenital hyperinsulinism and mosaic abnormalities of the ploidy

Background

Congenital hyperinsulinism and Beckwith‐Wiedemann syndrome both lead to β islet hyperplasia and neonatal hypoglycaemia. They may be related to complex genetic/epigenetic abnormalities of the imprinted 11p15 region. The possibility of common pathophysiological determinants has not been thoroughly investigated.

Objective

To report abnormalities of the ploidy in two unrelated patients with congenital hyperinsulinism.

Methods

Two patients with severe congenital hyperinsulinism, one overlapping with Beckwith‐Wiedemann syndrome, had pancreatic histology, ex vivo potassium channel electrophysiological studies, and mutation detection of the encoding genes. The parental genetic contribution was explored using genome‐wide polymorphism, fluorescent in situ hybridisation (FISH), and blood group typing studies.

Results

Histological findings diverged from those described in focal congenital hyperinsulinism or Beckwith‐Wiedemann syndrome. No potassium channel dysfunction and no mutation of its encoding genes (SUR1, KIR6.2) were detected. In patient 1 with congenital hyperinsulinism and Beckwith‐Wiedemann syndrome, paternal isodisomy for the whole haploid set was homogeneous in the pancreatic lesion, and mosaic in the leucocytes and skin fibroblasts (hemihypertrophic segment). Blood group typing confirmed the presence of two erythroid populations (bi‐parental v paternal only contribution). Patient 2 had two pancreatic lesions, both revealing triploidy with paternal heterodisomy. Karyotype and FISH analyses done on the fibroblasts and leucocytes of both patients were unremarkable (diploidy).

Conclusions

Diploid (biparental/paternal‐only) mosaicism and diploid/triploid mosaicism were present in two distinct patients with congenital hyperinsulinism. These chromosomal abnormalities led to paternal disomy for the whole haploid set in pancreatic lesions (with isodisomy or heterodisomy), thereby extending the range and complexity of the mechanisms underlying congenital hyperinsulinism, associated or not with Beckwith‐Wiedemann syndrome.     

193例遗传咨询病例外周血染色体分析

分析遗传咨询病例１９３人的外周血染色体，发现异常核型２２例，占１１．４％，其中常染色体异常１５例，性染色体异常７例。就诊原因以不良孕产史占多数（６０．１％），智能低下及发育异常次之（２４．９％）。在所发现的２２例异常核型中，以智能低下及发育异常所占的比例最高（２２．９％），性发育异常次之（１８．２％）。     

Familial ring (19) chromosome mosaicism: Case report and review

Ring (19) chromosomal mosaicism has been identified in a 14-month-old girl referred for cytogenetic evaluation due to microcephaly and developmental delay with autistic-like mannerisms. An analysis of her peripheral blood lymphocytes showed a 46,XX,r(19) cell line in 119/121 of cells examined. Of the two remaining cells, one had a normal female chromosome complement and the other showed loss of one of the chromosome 19 homologs. Further analysis by fluorescence in situ hybridization using an all human telomere probe showed the presence of a single hybridization signal on the r(19) chromosome. Subsequent cytogenetic characterization of cells derived from the patient's phenotypically normal mother also demonstrated the presence of a ring 19 chromosome in 4/100 cells. The remaining cells had a normal female chromosome complement. These findings represent the first reported case of familial ring 19 mosaicism. The cytogenetic and clinical findings in these two individuals are discussed in relation to six previously reported cases of de novo ring chromosome 19 mosaicism. © 1996 Wiley-Liss, Inc.     

Low-level mosaicism of trisomy 14: phenotypic and molecular characterization

Trisomy 14 mosaicism is a rare cytogenetic abnormality with a defined and recognizable clinical phenotype. We present a detailed clinical history and physical findings of five patients with low-level mosaicism of trisomy 14 detected by array-based comparative genomic hybridization (array-CGH) analysis or by routine chromosome analysis. These patients exhibited growth and developmental delays with variable severity, congenital anomalies, pigmentary skin lesions, and dysmorphic features. The phenotype of our patients was compared with previously described cases. This report suggests that trisomy 14 mosaicism may be more common than has been previously appreciated and also illustrates the important application of array-CGH to detect low-level mosaic chromosome abnormalities. We predict that a wider application of the array-CGH technology will significantly increase the detection rate of low-level mosaicism and will subsequently improve our ability to provide a diagnosis for patients with dysmorphic features, congenital anomalies, and developmental delay.     

Rothmund-Thomson syndrome in siblings: evidence for acquired in vivo mosaicism

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by skin abnormalities that appear in infancy, skeletal abnormalities, juvenile cataracts and other manifestations of premature aging, and a predisposition to malignancy. The diagnosis is made on clinical grounds as no consistent laboratory test has been identified. Chromosome studies have been reported for only three patients with RTS and in two of these three, trisomy 8 mosaicism was found. We performed a variety of cytogenetic and molecular genetic studies on two siblings with RTS and on their phenotypically normal parents. Two chromosomally abnormal clones involving either trisomy 8 or i(8q) were found in both patients with RTS. These clones were present in vivo , as they were seen in interphase buccal smears and lymphocytes from unstimulated preparations using both conventional cytogenetic studies and fluorescence in situ hybridization (FISH) with a centromere probe for chromosome 8. These results suggest that RTS is associated with in vivo clonal chromosomal rearrangements causing an acquired somatic mosaicism.     

FISH analysis of chromosome X,Y and 18 abnormalities in testicular sperm from azoospermic patients

BACKGROUND: Sperm extracted from testicular biopsies of azoospermic men can successfully be used for ICSI. The concern exists that testicular sperm from azoospermic men suffering from severe testicular failure may have a higher frequency of aneuploidy, which may lead to an increased risk for chromosomally abnormal offspring. METHODS: Testicular sperm from patients showing spermatogenic failure (n = 17) and from patients with normal spermatogenesis (n = 26) were analysed by fluorescence in-situ hybridization (FISH). Numerical chromosomal abnormalities for chromosomes X, Y and 18 were evaluated by FISH in a total of 1697 testicular sperm derived from 43 azoospermic patients. RESULTS: No difference was observed between the frequency of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis (5.6%) and from patients with spermatogenic failure (8.2%). However, the frequency of aneuploidy for chromosome 18 was higher in the group of azoospermic patients with spermatogenic failure than in the group with normal spermatogenesis (3.2 versus 1.3%). Within the obstructive group, sex chromosome aneuploidy (4.5%) occurred more frequently than chromosome 18 aneuploidy (1.3%; P < 0.001). Among testicular sperm derived from patients with spermatogenic failure, sex chromosomal aneuploidy (5.8%) was similar to that for chromosome 18 (3.2%). CONCLUSIONS: So far, no difference in the total frequency of chromosomal abnormalities has been observed between patients with normal spermatogenesis and patients with severe testicular failure. However, aneuploidy for chromosome 18 was higher in the group with spermatogenic failure.     

Detection of submicroscopic subtelomeric chromosome translocations: a new case study

Two sisters presented with multiple congenital abnormalities and developmental delay; abnormalities elsewhere in their extended family suggested that their father carried a balanced translocation. G-banded chromosome analysis showed apparently normal karyotypes. Fluorescence in situ hybridisation (FISH) with whole chromosome paints revealed no apparent abnormality in the father. However, further FISH studies, using multiple subtelomeric probes, demonstrated a derivative chromosome 16 in one sister. Subsequent studies showed that her sister also had a derivative 16 which had been inherited in an unbalanced form from their father, who carried a balanced reciprocal translocation between chromosomes 1 and 16. This report describes the detection of this submicroscopic translocation and the clinical findings in the two sisters.     

Syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation: two additional cases

An apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation was described by Gripp et al. 1996. The authors reported on two unrelated patients with congenital cataracts, sensorineural deafness, distinctive facial appearance, mental retardation, postnatal short stature, and skeletal changes. We report on two additional patients with findings most similar to the reported patients by Gripp et al. 1996, including bilateral congenital cataracts, hearing loss, craniofacial abnormalities, short stature, skeletal abnormalities, and developmental delay. Both of the patients reported herein had chromosome microarray analysis, which showed normal results in Patient 2 but abnormal results in Patient 1 and his mother who both had a chromosome 11q25 subtelomere deletion. Patient 1 and his mother's findings are atypical for the common findings reported in Jacobsen syndrome (11q terminal deletion syndrome), and consistent with the patients reported by Gripp et al. 1996. The etiology for these cases has been unknown. The microarray results on Patient 1 suggest that the other patients with findings of developmental delay, short stature, congenital cataracts, sensorineural hearing loss, and similar craniofacial features may have either a microdeletion of chromosome 11q terminal region or haploinsufficiency of a gene localized to this region.     

浏阳地区178例发育异常儿的细胞遗传学分析及临床研究

目的对本地区8年间178例发育异常儿童进行细胞核型分析,了解本地区染色体畸变类型;结合发病原因和母亲孕期声像图上的遗传标记进行临床研究,评估再发风险,预防染色体畸变患儿的出生。方法本文对自2003年9月至2011年10月的178例、年龄在14周岁以下的发育异常患儿,采用外周血淋巴细胞培养,通过胰酶消化G显带进行染色体核型分析,并收集染色体畸变患儿母亲孕期年龄、是否接触辐射;等有关资料及声像检查异常结果,对染色体畸变患儿进行临床研究。结果在因小儿外貌异常、脑瘫、四肢畸形、身材矮小、智力低下、尿道下裂等发育异常的178例患儿中,共检出异常染色体76例,异常率为42.7%（76/178）,染色体多态19例,所占比率为10.7%（19/178）。在76例染色体畸变患儿中,母亲预产年龄大于35岁的18例,有电子辐射等不良接触史4例,声像学异常结果 15例。结论染色体畸变是导致小儿各种发育异常的重要原因,对这些发育异常患儿进行染体核型分析是非常有必要的。同时应加强孕妇产前筛查,确定产前诊断范围,以期达到预防染色体畸变患儿出生,降低出生缺陷发生的目的。     

Recurrence rate for de novo 21q21q translocation Down syndrome: a study of 112 families

Recurrence of de novo GqGq Down syndrome (DS) in nine reported families and the low frequency of this chromosome abnormality in the population prompted this multicenter study to examine recurrence rate. Pedigrees and cytogenetic findings were analyzed from 112 families ascertained by a DS child with de novo GqGq translocation. Four of 112 sets of parents had abnormal cytogenetic findings. One mother and two fathers had a low proportion of mosaicism for trisomy 21 in peripheral lymphocytes. In one of these fathers, fibroblast studies confirmed the mosaicism (others not similarly studied). Two of these three families have one additional child each, both being normal. In the fourth family, the mother is heterozygous for a pericentric inversion of chromosome 21. Ninety of the 112 probands had a total of 130 full sibs and 34 half sibs. None of these 164 sibs was affected; the recurrence rate could still be as high as 2% (with 95% confidence based upon calculation of a one-tailed confidence interval). Because of the latter, the nine reports of recurrence in the literature, and the abnormal cytogenetic findings in peripheral lymphocytes in 4/112 sets of parents (3.6%), we think that caution should be exercised when counseling that the recurrence risk of this event is remote.     

Fluorescence in situ hybridization detectable mosaicism for Angelman syndrome with biparental methylation

We present a child with mild to moderate global developmental delay including severe speech impairment, inappropriate happy demeanor, wide-based gait, frequent ear infections with mild hearing loss, deep-set eyes, a wide mouth, widely-spaced teeth, normal head circumference, and no seizures. Results of peripheral blood lymphocyte chromosomal analysis with GTG banding were normal. However, fluorescence in situ hybridization (FISH) studies showed mosaicism for a deletion of probes (D15S10 and SNRPN) from the Angelman syndrome (AS) critical region with approximately 40% of peripheral lymphocytes having the deletion. The deleted chromosome 15 also showed centromeric duplication, which was detected with a D15Z1 probe [46,XX, dic(15)(pter-->q11.1::p11.2-->q11. 1::q13-->qter)]. The same duplication pattern was observed in 30% of the nuclei obtained from a buccal smear. Methylation studies using polymerase chain reaction with sodium bisulfite-treated DNA demonstrated a normal biparental methylation pattern. To the best of our knowledge, this is the first case with AS and a FISH detectable deletion in a mosaic pattern. We recommend FISH studies for the detection of mosaicism in the patients with AS clinical findings even if results of the methylation studies are normal.     

Microarray comparative genomic hybridization and cytogenetic characterization of tissue-specific mosaicism in three patients

The presence of more than one cell line in an individual may often be missed by classical cytogenetic analysis due to a low percentage of affected cells or analysis of cells from an unaffected or less affected germ layer. Array comparative genomic hybridization (aCGH) from whole blood or tissue is an important adjunct to standard karyotyping due to its ability to detect genomic imbalances that are below the resolution of karyotype analysis. We report results from three unrelated patients in whom aCGH revealed mosaicism not identified by peripheral blood chromosome analysis. This study further illustrates the important application of aCGH in detecting tissue-specific mosaicism, thereby leading to an improvement in the ability to provide a diagnosis for patients with normal chromosome analysis and dysmorphic features, congenital anomalies, and/or developmental delay.     

5828例遗传咨询者的细胞遗传学分析

目的研究临沂地区遗传咨询患者中染色体异常核型的发生率,探讨染色体异常与疾病发生、发展的关系,为优生优育、降低出生缺陷、提高人口素质服务。方法采用外周血淋巴细胞染色体培养技术,行G显带,必要时进行C显带检查,显微镜下进行核型分析。结果检查5828例遗传咨询患者染色体,就诊原因主要为不良孕产史、不孕不育、原发及继发性闭经、智力低下、先天畸形等,共检出异常染色体核型146种类型805例,检出率为13.81%（805/5828）。检出常染色体结构和数目异常616例,占异常核型的76.52%（616/805）,性染色体结构和数目异常189例,占异常核型的23.48%（189/805）。异常的染色体涉及所有24条染色体。结论染色体异常是导致不良孕产史、不孕不育、性发育异常、智力低下等疾病的重要原因之一,对这些病人进行遗传咨询、生育和康复指导非常必要,以达到提高人口素质的目的。