Clinical and molecular characterization of an almost complete ring chromosome 4 in two sisters, with recurrence due to gonadal mosaicism.

Autosomal ring chromosomes are rare cytogenetic findings that arise from breakage and fusion of the chromosome ends. Rings are mitotically unstable, usually sporadic and associated with a 'ring syndrome', characterized by a variable phenotype: growth retardation, no significant dysmorphisms and normal to moderately disabled intelligence. We describe the clinical features and molecular characterization of two sisters with ring chromosome 4. Karyotype analysis was performed on both sisters and parents. Chromosome microarray was performed on both sisters to delineate the breakpoint imbalance. Both sisters had a large ring 4 chromosome in the majority of cells analyzed on karyotype. Microarray results were identical in the sisters, showing a 55.8 kb duplication on the terminal 4p arm and a 1.5 Mb deletion on the terminal 4q arm. No genes of interest were identified in these regions. Parental karyotypes on lymphocytes and fibroblasts were normal, with no finding of mosaicism for the ring 4 chromosome. Polymorphic marker analysis revealed the maternal origin of the ring. To our knowledge, this is the first reported instance of a ring 4 chromosome recurring in siblings after extensive parental testing, which suggests this was due to maternal gonadal mosaicism.

Failure of NIPT to detect constitutional chromoanasynthesis involving chromosome 21 in a case of fetal hydrops-A case report.

We report a case of a de novo ring 21 complex chromosomal rearrangement in a fetus presenting with hydrops. Noninvasive prenatal testing (NIPT) failed to detect the imbalance. This case highlights the need to understand the various limitations and strengths of NIPT technology when counseling patients.

A Challenging Prenatal QF-PCR Rapid Aneuploidy Test Result Caused by a Maternally Inherited Triplication within Chromosome Xq26.2.

The aim of this study was to investigate the origin of the biallelic trisomic amplification pattern of the X chromosome microsatellite marker DXS1187 in an otherwise normal male fetus, identified on routine rapid aneuploidy detection (RAD) testing by quantitative fluorescent-polymerase chain reaction (QF-PCR). Amniocentesis was performed on a 35-year-old female at 15 weeks, 2 days gestation for a positive first trimester screen. QF-PCR, metaphase FISH, and chromosomal microarray were carried out on both maternal and fetal DNA. Fetal QF-PCR showed a biallelic trisomic pattern for the X chromosome microsatellite marker DXS1187, with an otherwise normal male amplification pattern at all other sex chromosome markers. Chromosome analysis performed on cultured amniocytes showed a normal male karyotype. Chromosome microarray analysis identified a maternally inherited 304-kb copy number triplication within chromosome Xq26.2 encompassing the DXS1187 marker. The maternally inherited X chromosome harbors an apparently tandem 304-kb triplication that overlaps the DXS1187 marker. As the triplicated region is devoid of clinically relevant genes, it was considered as likely benign in the fetus. Postnatal follow-up reported a healthy male newborn. To our knowledge, this is a unique case demonstrating a "benign" copy number imbalance involving the DXS1187 marker detected by prenatal QF-PCR RAD.

Mosaic trisomy 1q: a recurring chromosome anomaly that is a diagnostic challenge and is associated with a Fryns-like phenotype.

OBJECTIVE: Trisomy of the long arm of chromosome 1 is a very rare cytogenetic anomaly that is difficult to diagnose because of tissue-limited mosaicism. This study aimed to further characterize the prenatal and post-natal findings associated with this anomaly, including the first reported chromosomal microarray finding. METHOD: This is a retrospective study of six cases of mos 46,X,der(Y)t(Y;1)(q12;q21)/46,XY, diagnosed both prenatally and post-natally. Detailed clinical features and pregnancy outcome were documented. RESULTS: Recurrent prenatal and post-natal features of our case series, as well as the previously reported cases, were described, suggesting a Fryns-like phenotype. A diagnosis of mosaic trisomy 1q is difficult to confirm post-natally in some cases because of the tissue provided for analysis, emphasizing the need to study multiple tissue types in cases of fetal loss with a suspected underlying chromosomal imbalance. CONCLUSION: The overlap of clinical features between mosaic trisomy 1q and Fryns syndrome emphasizes the need to obtain appropriate samples for genetic analysis. The present cases and a review of the literature suggest that partial trisomy of the long arm of chromosome 1 is a distinct de novo clinical entity with low recurrence risk. © 2017 John Wiley & Sons, Ltd.

Prenatal Array Comparative Genomic Hybridization in Fetuses With Structural Cardiac Anomalies.

OBJECTIVES: To examine the diagnostic performance of array comparative genomic hybridization (CGH) for fetal cardiac anomalies in two medium-sized Canadian prenatal genetics clinics. METHODS: We prospectively recruited 22 pregnant women with fetal structural cardiac anomalies, normal rapid aneuploidy detection, and FISH for 22q11.2 testing for array CGH analysis. RESULTS: One case had an 8p deletion that was also visible on karyotype and included the GATA4 gene, which has been associated with congenital heart disease. Two cases had inherited pathogenic copy number variants (CNVs) of variable expressivity and penetrance: one was a duplication of 16p11.2 and the other a deletion of 15q11.2. One case had the incidental finding of being a carrier of a recessive disease unrelated to the cardiac anomaly. CONCLUSIONS: Of these prospectively recruited cases of fetal cardiac anomalies, 14% had a pathogenic result on array CGH. Pathogenic CNVs of variable penetrance and expressivity were a significant proportion of the positive results identified. These CNVs are generally associated with neurodevelopmental issues and may or may not have been associated with the fetus' underlying congenital heart disease. Array CGH increases the diagnostic yield in this group of patients; however, certain CNVs remain a challenge for counselling in the prenatal setting.

Review of the recurrent 8q13.2q13.3 branchio-oto-renal related microdeletion, and report of an additional case with associated distal arthrogryposis.

Recurrent 2.65 Mb deletions of 8q13.2q13.3 encompassing EYA1 have been recently described in the literature as a cause of branchio-oto-renal syndrome (BOR). Other clinical features of this recurrent microdeletion syndrome are still being delineated. We describe an additional patient with BOR due to microdeletion of 8q13.2q13.3. In addition to BOR related features, our patient presented with distal arthrogryposis that was detected prenatally, a phenotype that has not previously been described in patients with this deletion. © 2016 Wiley Periodicals, Inc.

A 2q24.3q31.1 microdeletion found in a patient with Filippi-like syndrome phenotype: a case report.

Filippi syndrome is characterized by developmental delay, growth failure, cryptorchidism, bilateral hand and foot syndactyly, and facial dysmorphism. The 2q24q31 contiguous deletion syndrome has similarly been associated with hand and foot anomalies, growth retardation, microcephaly, characteristic facies with a broad prominent nasal root and thin alae nasi, and intellectual disability. We present a patient with this deletion who has a Filippi-like phenotype, which may be the first causative cytogenetic result in this syndrome. This suggests the importance of array comparative genomic hybridization in evaluation of patients with Filippi syndrome, and suggests that the inheritance may not always be autosomal recessive.

Application of microarray-based comparative genomic hybridization in prenatal and postnatal settings: three case reports.

Microarray-based comparative genomic hybridization (array CGH) is a newly emerged molecular cytogenetic technique for rapid evaluation of the entire genome with sub-megabase resolution. It allows for the comprehensive investigation of thousands and millions of genomic loci at once and therefore enables the efficient detection of DNA copy number variations (a.k.a, cryptic genomic imbalances). The development and the clinical application of array CGH have revolutionized the diagnostic process in patients and has provided a clue to many unidentified or unexplained diseases which are suspected to have a genetic cause. In this paper, we present three clinical cases in both prenatal and postnatal settings. Among all, array CGH played a major discovery role to reveal the cryptic and/or complex nature of chromosome arrangements. By identifying the genetic causes responsible for the clinical observation in patients, array CGH has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner.

Nasal epithelial cells of donor origin after allogeneic hematopoietic cell transplantation are generated at a faster rate in the first 3 months compared with later posttransplantation.

Detection of donor-type epithelial cells (ECs) after allogeneic hematopoietic cell transplantation (allo-HCT) using XY chromosome fluorescein in situ hybridization (FISH) has suggested that hematopoietic stem cells carry a degree of developmental plasticity. This is controversial, given artifacts of XY-based detection and the possibility of hematopoietic-nonhematopoietic cell fusion. Moreover, the kinetics of donor-type ECs (quantity at different time points after transplant) is unknown. Here, we document unequivocally the existence of donor-type ECs using a method obviating the artifacts of XY-FISH and study their kinetics. Nasal scrapings and blood specimens were collected from 60 allo-HCT survivors between 7 days and 22 years posttransplantation. DNA extracted from laser-captured nasal ECs (ie, CK(+)CD45(-) cells) and blood leukocytes was polymerase chain reaction-amplified for a panel of 16 short tandem repeat markers. The median percentage of donor-type ECs (among nasal ECs) was 0% on day 7 posttransplantation, 2.8% at 3 months posttransplantation, and 8.5% at 12-22 years posttransplantation. Cell fusion was ruled out by FISH analysis for two autosomes. We conclude that donor-type nasal ECs exist after HCT, and that their percentage rises rapidly in the first 3 months posttransplantation and more slowly thereafter.

Goldenhar phenotype in a child with distal 22q11.2 deletion and intracranial atypical teratoid rhabdoid tumor.

Chromosome-specific low copy repeats (LCRs) are implicated in several clinically significant microdeletion and microduplication syndromes. The well-recognized phenotype of DiGeorge/velocardiofacial syndrome (DG/VCF) results from deletions of the long arm of chromosome 22 (22q11.2) mediated by the proximal LCRs in this region. More recent evidence suggests that the distal LCRs within 22q11.2 are also implicated in microdeletions and microduplications with less characterized phenotypes. Here we report on an infant diagnosed with Goldenhar syndrome (GS) phenotype who developed an atypical teratoid rhabdoid tumor (AT/RT) of the brain due to a distal deletion of the chromosome 22q11.2 region encompassing the INI1/SMARCB1 tumor suppressor. We also discuss the phenotype of patients with germline deletions of this region and the possible implication of the 22q11.2 region in the GS.

Suitability of rapid aneuploidy detection for prenatal diagnosis.

OBJECTIVE: To determine the suitability of replacing full karyotype analysis with molecular genetic rapid aneuploidy detection (RAD) methods, in particular quantitative fluorescence polymerase chain reaction (QF-PCR), for prenatal diagnosis in amniotic fluid samples obtained by amniocentesis. METHODS: We reviewed all fetal karyotypes done at our centre between August 29, 2000, and February 28, 2006. Outcome measures included (1) the proportion of prenatal samples with abnormal karyotypes that would not have been detected by RAD, as a whole and for each indication, and (2) pregnancy outcome for each chromosome abnormality that was predicted to be clinically significant or of uncertain significance and would not have been detected by RAD. RESULTS: Of the 6411 karyotypes reported in the study period, 70 (1.09%) were abnormal karyotypes which would not have been detected by RAD alone. These included 32 cases (0.50%) predicted to confer no increased risk to the fetus, 17 (0.27%) predicted to have a low risk of fetal abnormality, and 21 (0.33%) with an uncertain or high risk of fetal abnormality. If full karyotype was added for nuchal translucency greater than 3.5 mm, structural fetal abnormality on ultrasound, or parental balanced chromosome rearrangement, only five uncertain or high risk cases (0.08%) would not have been detected by RAD alone. CONCLUSION: These results suggest that if the policy of offering full karyotype analysis to all women were to be changed to a policy of offering RAD alone to women without other risk factors such as fetal abnormalities on ultrasound, increased nuchal translucency, or history of chromosome abnormality, this would mean that a chromosome abnormality with a substantial risk of clinically significant fetal abnormality would be missed in fewer than 1/1000 amniocenteses. Our results are similar to others previously reported.

A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture.

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.

Cytogenetic and molecular characterization of a de-novo cryptic deletion of 7p21 associated with an apparently balanced translocation and complex craniosynostosis.

We describe a female infant with complex craniosynostosis, significant craniofacial dysmorphism and developmental delay in which a de-novo apparently balanced translocation between chromosomes 7 and 18 [46,XX,t(7;18)(p15.3;q11.2)] was identified. Additional cytogenetic and molecular investigations identified a cryptic interstitial 7.6-10.6-Mb deletion of the region between bands 7p21.2 and 7p21.3 on the derivative chromosome 18. The deletion was of paternal origin and contained the TWIST1 gene, although her features were not completely characteristic of Saethre-Chotzen syndrome. The phenotype of this patient is likely further complicated by loss of other genes within the deleted region and/or disruption of a critical gene(s) at the sites of the breakpoints on chromosomes 7 and 18. This case illustrates the need for a systematic molecular study of breakpoints and the surrounding chromosomal regions in patients with apparently balanced rearrangements and phenotypic abnormalities.

Unusual segregation products in sperm from a pericentric inversion 17 heterozygote.

Chromosome segregation and interchromosomal effect were studied in spermatozoa from a carrier of a pericentric chromosome 17 inversion, 46,XY,inv(17)(p13.1q25.3). Sperm chromosome segregation, lymphocytes of the inversion carrier, and cells from his offspring were analysed by multicolour fluorescence in situ hybridization. The frequency of balanced sperm was 73%. An unusual segregation of recombinants was observed, viz. deletion of the p arm (14.6%) or duplication of the p arm with the presence of one q arm (8.4%), instead of the expected recombinants, viz. duplication of one arm with deletion of the other and vice versa. These unusual recombinants were explained by the position of the 17q breakpoint, which was between the q arm telomere-associated repeats and the unique q subtelomere region. The offspring of the donor were found to have a 17p deletion including the Miller-Dieker critical region, similar to the most frequent recombinant sperm class. The disomy frequency was significantly increased for chromosome 17 compared with other autosomes, suggesting that pairing and recombination of the inversion may predispose to non-disjunction. There was no significant difference between the frequencies of aneuploidy for chromosomes 13, 21, X and Y in the chromosome inversion heterozygote compared with controls. Thus, this unique pericentric inversion of chromosome 17 produces unusual recombinant products; no evidence was apparent of an interchromosomal effect in any of the tested chromosomes.

Microduplication and triplication of 22q11.2: a highly variable syndrome.

22q11.2 microduplications of a 3-Mb region surrounded by low-copy repeats should be, theoretically, as frequent as the deletions of this region; however, few microduplications have been reported. We show that the phenotype of these patients with microduplications is extremely diverse, ranging from normal to behavioral abnormalities to multiple defects, only some of which are reminiscent of the 22q11.2 deletion syndrome. This diversity will make ascertainment difficult and will necessitate a rapid-screening method. We demonstrate the utility of four different screening methods. Although all the screening techniques give unique information, the efficiency of real-time polymerase chain reaction allowed the discovery of two 22q11.2 microduplications in a series of 275 females who tested negative for fragile X syndrome, thus widening the phenotypic diversity. Ascertainment of the fragile X-negative cohort was twice that of the cohort screened for the 22q11.2 deletion. We also report the first patient with a 22q11.2 triplication and show that this patient's mother carries a 22q11.2 microduplication. We strongly recommend that other family members of patients with 22q11.2 microduplications also be tested, since we found several phenotypically normal parents who were carriers of the chromosomal abnormality.

Duplication of the 22q11.2 region associated with congenital cardiac disease.

The DiGeorge, or velocardiofacial, syndrome has been aetiologically linked to heterozygous deletion of the q11.2 region of chromosome 22. It is the most common of the microdeletion syndromes, and is associated with malformations involving the ventricular outflow tracts. Duplication of the 22q11.2 region has also been reported, adding to a growing list of syndromes involving genomic deletion or duplication that cause disease by decreasing or increasing the gene dosage. We report two cases of congenital cardiac disease associated with microduplications of 22q11.2, and discuss the evidence to date for the potential clinical significance of this genetic defect.

Analysis of chromosome segregation in sperm from a chromosome 2 inversion heterozygote and assessment of an interchromosomal effect.

Using fluorescence in situ hybridization (FISH) analysis, the chromosome segregation of a pericentric inversion of chromosome 2 was studied in spermatozoa. An interchromosomal effect (ICE) was also determined for chromosomes 13, 21, X, and Y. This chromosome inversion included more than 2/3 of the total length of the chromosome and the breaks points were in G-light bands. The frequency of non-recombinant sperm was 55.9%, and that of recombinant sperm was 34.5% (with a 1:1 ratio of duplication of the p arm and deletion of the q arm and vice versa). There was a significantly increased frequency of disomy for chromosome 2 (0.6%) compared to the other autosomes, suggesting that pairing and recombination of the inversion may predispose to nondisjunction. There was no significant difference between the frequencies of aneuploidy for chromosomes 13, 21, X, and Y for the chromosome inversion heterozygote compared to control donors. Thus we did not find evidence for an ICE.