Deep Sequencing and Phenotyping in an Australian Tuberous Sclerosis Complex "No Mutations Identified" Cohort.

Tuberous sclerosis complex (TSC) is a variable multisystem disorder. The "no mutations identified" (NMI) group are reportedly phenotypically milder than those with an identified molecular cause, and often have mosaic or intronic variants not detected by standard sequencing methods. METHODS: We describe the phenotypes in an Australian TSC NMI group (n = 18) and a molecular testing strategy implementable in a diagnostic laboratory. Massively parallel sequencing (MPS) of the whole genomic regions of TSC1 and TSC2 was performed using DNA extracted from multiple tissue samples per participant. RESULTS: Our study showed that the phenotype in TSC NMI individuals can be similar to those with heterozygous, particularly TSC1, variants. Although neurodevelopmental outcomes can be less severe, the number of organ systems involved was similar to the non-mosaic groups. A diagnostic yield of 72% (13/18) was achieved, with the majority (10/13) being mosaic variants and the remainder heterozygous variants missed on previous testing. CONCLUSION: Testing DNA from multiple tissue samples allowed for validation of otherwise discarded low-level mosaic variants and detection of mosaic variants by MPS without excessive cost or the need for specialised techniques. Implementing this approach in a diagnostic setting is viable and allows optimal clinical care of patients with NMI TSC.

Case report: Deep sequencing and long-read genome sequencing refine prior genetic analyses in families with apparent gonadal mosaicism in PIK3CD-related activated PI3K delta syndrome.

Gonadal and gonosomal mosaicism describe phenomena in which a seemingly healthy individual carries a genetic variant in a subset of their gonadal tissue or gonadal and somatic tissue(s), respectively, with risk of transmitting the variant to their offspring. In families with one or more affected offspring, occurrence of the same apparently de novo variants can be an indicator of mosaicism in either parent. Panel-based deep sequencing has the capacity to detect low-level mosaic variants with coverage exceeding the typical limit of detection provided by current, readily available sequencing techniques. In this study, we report three families with more than one affected offspring with either confirmed or apparent parental gonosomal or gonadal mosaicism for PIK3CD pathogenic variants. Data from targeted deep sequencing was suggestive of low-level maternal gonosomal mosaicism in Family 1. Through this approach we did not detect pathogenic variants in PIK3CD from parental samples in Family 2 and Family 3. We conclude that mosaicism was likely confined to the maternal gonads in Family 2. Subsequent long-read genome sequencing in Family 3 showed that the paternal chromosome harbored the pathogenic variant in PIK3CD in both affected children, consistent with paternal gonadal mosaicism. Detection of parental mosaic variants enables accurate risk assessment, informs reproductive decision-making, and provides helpful context to inform clinical management in families with PIK3CD pathogenic variants.

Genetic counseling of mosaicism for a duplication due to partial trisomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis.

Genetic counseling of mosaicism for a duplication due to partial trisomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis remains difficult because mosaic duplication due to partial trisomy has been reported to be associated with either normal or abnormal phenotype in prenatal diagnosis. This article makes a comprehensive review of the reported cases of mosaicism for a duplication due to partial trisomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis and various counseling issues such as culture artefact, cytogenetic discrepancy between cultured and uncultured amniocytes and among various tissues, perinatal progressive decrease of the abnormal cell line and a possible favorable fetal outcome. The information provided is useful for obstetricians and genetic counselors during genetic counseling of the parents who wish to keep the babies under such a circumstance.

Genetic counseling of mosaic and non-mosaic tetrasomy 9p at prenatal diagnosis.

Genetic counseling of mosaic and non-mosaic tetrasomy 9p remains difficult because of the possible associated congenital abnormalities, cytogenetic discrepancy in various tissues, true-positive and false-positive diagnosis in non-invasive prenatal testing (NIPT), uniparental disomy (UPD) 9, tissue-limited mosaicism, perinatal progressive decrease of the aneuploid cell line, phenotypic normal carriers and possible favorable fetal outcome in the cases with mosaic tetrasomy 9p at amniocentesis. This article presents a comprehensive review of various counseling issues concerning mosaic and non-mosaic tetrasomy 9p at prenatal diagnosis, and the information provided is very useful for genetic counseling under such circumstances.

Genetic counseling of mosaicism for balanced or unbalanced translocation with a normal cell line at amniocentesis.

Genetic counseling of mosaicism for balanced translocation with a normal cell line at amniocentesis is not difficult because most of the reported cases have normal phenotypes. However, genetic counseling of mosaicism for unbalanced translocation with a normal cell line at amniocentesis remains difficult because cases with mosaic unbalanced translocation with a normal cell line at prenatal diagnosis have been reported to be associated with either normal or abnormal phenotype. This article makes a comprehensive review of the reported cases of de novo or familial mosaic unbalanced translocation with a normal cell line and various counseling issues such as meiotic event, post-zygotic mitotic event, culture artefact, chimerism, uniparental disomy (UPD), jumping translocation, cytogenetic discrepancy between cultured and uncultured amniocytes and among various tissues, perinatal progressive decrease of the unbalanced translocation cell line and a possible favorable fetal outcome. The information provided is useful for obstetricians and genetic counselors during genetic counseling of the parents who wish to keep the babies under such a circumstance.

Genetic counseling of mosaicism for a deletion due to partial monosomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis.

Genetic counseling of mosaicism for a deletion due to partial monosomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis remains difficult because mosaic deletion due to partial monosomy has been reported to be associated with either normal or abnormal phenotype in prenatal diagnosis. This article makes a comprehensive review of the reported cases of mosaicism for a deletion due to partial monosomy in a cell line with 46 chromosomes associated with a normal cell line at amniocentesis and various counseling issues such as culture artefact, cytogenetic discrepancy between cultured and uncultured amniocytes and among various tissues, perinatal progressive decrease of the abnormal cell line and a possible favorable fetal outcome. The information provided is useful for obstetricians and genetic counselors during genetic counseling of the parents who wish to keep the babies under such a circumstance.

Low-level mosaic trisomy 14 at amniocentesis in a pregnancy associated with cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes, positive non-invasive prenatal testing for trisomy 14, perinatal progressive decrease of the trisomy 14 cell line and a favorable fetal outcome.

OBJECTIVE: We present low-level mosaic trisomy 14 at amniocentesis. CASE REPORT: A 37-year-old, gravida 2, para 1, woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. This pregnancy was conceived by in vitro fertilization and embryo transfer (IVF-ET). Amniocentesis revealed a karyotype of 47,XX,+14 [4]/46,XX [27], consistent with 12.9% mosaicism for trisomy 14. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed the result of arr (1-22, X) × 2 with no genomic imbalance. Prenatal ultrasound findings were unremarkable. She was referred for genetic counseling at 21 weeks of gestation and was offered expanded non-invasive prenatal testing (NIPT) which was positive for trisomy 14. At 24 weeks of gestation, she underwent repeat amniocentesis which revealed a karyotype of 47,XX,+14 [2]/46,XX [26], consistent with 7% mosaicism for trisomy 14. The parental karyotypes were normal. Simultaneous aCGH analysis on the DNA extracted from uncultured amniocytes revealed no genomic imbalance. Polymorphic marker analysis excluded uniparental disomy (UPD) 14. Interphase fluorescence in situ hybridization (FISH) analysis on 104 uncultured amniocytes detected no trisomy 14 cell. At 35 weeks of gestation, a 2315-g phenotypically normal baby was delivered. The umbilical cord and placenta had the karyotype of 46, XX (40/40 cells). aCGH analysis on the DNA extracted from peripheral blood and buccal mucosal cells at the age of three months revealed no genomic imbalance. The neonate was normal in phenotype and development during postnatal follow-ups. CONCLUSIONS: Low-level mosaic trisomy 14 at amniocentesis can be associated with cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes, perinatal progressive decrease of the trisomy 14 cell line and a favorable fetal outcome.

Mosaic distal 13q duplication due to mosaic unbalanced translocation of 46,XY,der(14)t(13;14)(q32.2;p13)/46,XY at amniocentesis in a pregnancy associated with a favorable fetal outcome, perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes.

OBJECTIVE: We present mosaic distal 13q duplication due to mosaic unbalanced translocation 46,XY,der(14)t(13;14)(q32.2;p13)/46,XY at amniocentesis in a pregnancy associated with a favorable fetal outcome. CASE REPORT: A 37-year-old, gravida 2, para 0, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XY, add(14) (p13)[17]/46,XY[13] (56.6% mosaicism). Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from cultured amniocytes revealed arr 13q32.2q34 × 2∼3, consistent with 45% mosaicism for distal 13q duplication. Repeat amniocentesis at 24 weeks of gestation revealed a karyotype of 46,XY,der(14)t(13;14)(q32.2;p13)[14]/46,XY[16] (46.6% mosaicism). The parental karyotypes were normal. aCGH analysis on the DNA extracted from uncultured amniocytes revealed arr 13q32.2q34 × 2.38, consistent with 30-40% mosaicism for distal 13q duplication. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes detected 22.8% (23/101 cells) mosaicism for distal 13q duplication. Prenatal ultrasound findings were unremarkable. At 39 weeks of gestation, a 3616-g phenotypically normal baby was delivered. The karyotypes of cord blood, umbilical cord and placenta were 46,XY,der(14)t(13;14)(q32.2;p13)[20]/46,XY[20] (50% mosaicism), 46,XY,der(14)t(13;14)(q32.2;p13)[14]/46,XY[26] (35% mosaicism) and 46,XY (40/40 cells) (0% mosaicism), respectively. When follow-ups at the age of 4½ months and the age of one year, the peripheral blood had the karyotype of 46,XY,der(14)t(13;14)(q32.2;p13)[18]/46,XY[22] (45% mosaicism). Interphase FISH analysis on buccal mucosal cells at the age of 4½ months revealed 2.7% (3/110 cells) mosaicism for distal 13q duplication, compared with 1% (1/100 cells) in the normal control. The neonate was normal in phenotype and development. CONCLUSIONS: Mosaic unbalanced translocation at amniocentesis can be associated with a favorable fetal outcome, perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes.

Association of autosomal mosaic chromosomal alterations with risk of bladder cancer in Chinese adults: a prospective cohort study.

Little is known about the prospective association between autosomal mosaic chromosomal alterations (mCAs), a group of large-scale somatic mutations on autosomes, and bladder cancer. Here we utilized data from 99,877 participants who were free of physician-diagnosed cancer at baseline (2004-2008) of the China Kadoorie Biobank to estimate the associations between autosomal mCAs and bladder cancer (ICD-10: C67). A total of 2874 autosomal mCAs events among 2612 carriers (2.6%) were detected. After a median follow-up of 12.4 years, we discovered that participants with all autosomal mCAs exhibited higher risks of bladder cancer, with a multivariable-adjusted hazard ratio (HR) (95% confidence interval [CI]) of 2.60 (1.44, 4.70). The estimate of such association was even stronger for mosaic loss events (HR [95% CI]: 6.68 [2.92, 15.30]), while it was not significant for CN-LOH events. Both expanded (cell fraction ≥10%) and non-expanded autosomal mCAs, as well as mosaic loss, were associated with increased risks of bladder cancer. Of interest, physical activity (PA) significantly modified the associations of autosomal mCAs and mosaic loss (Pinteraction = 0.038 and 0.012, respectively) with bladder cancer. The increased risks of bladder cancer were only observed with mCAs and mosaic loss among participants with a lower level of PA (HR [95% CI]: 5.11 [2.36, 11.09] and 16.30 [6.06, 43.81]), but not among participants with a higher level of PA. Our findings suggest that peripheral leukocyte autosomal mCAs may represent a novel risk factor for bladder cancer, and PA may serve as a potential intervention target for mCAs carriers.

Prenatal diagnosis and molecular cytogenetic analyses of a homozygous Robertsonian translocation family with novel mosaic Robertsonian fission karyotype.

BACKGROUND: Approximately one person in 1000 is a Robertsonian translocation carrier. Errors in the formation of eggs (or more rarely of sperms) may be the cause of Robertsonian translocation. Most Robertsonian translocation carriers are healthy and have a normal lifespan, but do have an increased risk of offsprings with trisomies and pregnancy loss. The fitness of Robertsonian translocation carriers is reduced, but can provide material for evolution. MATERIALS AND METHODS: We have done prenatal diagnosis and molecular cytogenetic analyses on this homozygous Robertson translocation family. We report a homozygous Robertson translocation family with previously undescribed mosaic Robertsonian fission karyotype. RESULTS: We identified six Robertsonian translocation carriers in this family. Four were heterozygous translocation carriers of 45,XX or XY,der(14;15)(q10;q10), one was a homozygous translocation carrier of a 44,XY,der(14;15)(q10;q10),der(14;15)(q10;q10), and one was a previously undescribed Robertsonian fission carrier of 45,XN,der(14;15)(q10;q10)[42]/46,XN[58] with normal phenotype. CONCLUSION: We reported a previously undescribed mosaic Robertsonian fission karyotype. The homozygosity of Robertsonian translocation for speciation may be a potential mechanism of speciation in humans. In theory, the carriers of homologous Robertsonian translocation cannot produce normal gametes, but Robertson fission made it possible for them to produce normal gametes.

Developmental signals control chromosome segregation fidelity during pluripotency and neurogenesis by modulating replicative stress.

Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals-including WNT, BMP, and FGF-converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development.

Morphological Analysis of Neurons and Glia Using Mosaic Analysis with Double Markers.

Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses-soma volume and dendrite characterization-of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses-volume and Sholl analysis-of astrocyte morphology.

Possible new defining presentation of mosaic tetrasomy 9p: multiple and recurrent pilomatrixoma.

Tetrasomy 9p is a rare genetic syndrome resulting from two additional copies of the short arm of chromosome 9. Symptoms often present in the form of congenital abnormalities including cognitive disabilities, growth retardation, abnormal earlobes, congenital heart disease, and dysmorphia of the skull and face. Current literature suggests patients with tetrasomy 9p may exhibit any combination of these symptoms or, in rare instances, none at all. Although karyotyping, chromosomal microarray, and galactose-1-phosphate uridyltransferase activity analyses are the definitive diagnostic methods used, there remains a need for more robust clinical recognition in cases of mild phenotypic expression. Herein, we present a rare case of mosaic tetrasomy 9p in a long-term survival patient with multiple and recurrent pilomatrixomas, rare benign growths more commonly found in individuals under the age of 20. To our knowledge, only two previous reports have noted concurrent tetrasomy 9p with pilomatrixomas. We are the first to identify this phenotype in an adult tetrasomy 9p patient. Dermatopathology evaluation was conducted to verify our diagnoses. Our aim is to present a unique, additional case suggesting multiple pilomatrixomas as a new defining clinical presentation of mosaic tetrasomy 9p and to review the literature underlying the genetic changes associated with this syndrome.

Huntington's disease cellular phenotypes are rescued non-cell autonomously by healthy cells in mosaic telencephalic organoids.

Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.

BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

[Genetic analysis of a fetus with with 45,X/46,X,idic(Y)(q11.2) mosaicism].

OBJECTIVE: To explore the genetic characteristics of a fetus with sex chromosome abnormality indicated by non-invasive prenatal testing (NIPT) at 25+ gestational weeks. METHODS: A pregnant woman who was admitted to the Taizhou Hospital for abnormal NIPT result on January 6, 2023 was selected as the study subject. Relevant clinical data was collected. The fetus was subjected to chromosomal karyotyping analysis, copy number variation sequencing (CNV-seq), fluorescence in situ hybridization (FISH), and multiplex PCR assays. RESULTS: NIPT had suggested monosomy of X chromosome. The fetus was found to have a chromosomal karyotype of 45,X[59]/46,X,del(Y)(q11.2)[17] at 30+ weeks of gestational age. CNV-seq suggested the presence a 7.98 Mb deletion at Yq11.222q12 and a mosaicism 16.92 Mb deletion. FISH suggested that the fetus harbored two SRY genes and a mosaicism sex chromosomal abnormality, and multiplex PCR revealed that its AZF b+c region was completely deleted. C-banded karyotyping showed darkly stained dense mitotic granules at both ends of the Y chromosome. The fetus was ultimately determined as a 45,X/46,X,idic(Y)(q11.2) mosaicism. Following elected abortion, testing of the fetal tissue confirmed the presence of 45,X/46,XY mosaicism, and CNV-seq result of the placental tissue was compatible with that of NIPT. CNV-seq analysis of the couple revealed no obvious abnormality. CONCLUSION: With combined NIPT, karyotyping, CNV-seq, FISH and multiplex PCR assays, the fetus was diagnosed as a 45,X/46,X,idic(Y)(q11.2) mosaicism with deletion of the AZF b+c region. Above finding has enabled prenatal diagnosis for the fetus.