De Novo Noninversion Variants Implicated in Sporadic Hemophilia A: A Variant Origin and Timing Study.

Sporadic hemophilia A (HA) enables the persistence of HA in the population. F8 gene inversion originates mainly in male germ cells during meiosis. To date, no studies have shown the origin and timing of HA sporadic noninversion variants (NIVs); herein, we assume that HA-sporadic NIVs are generated as a de novo variant. Of the 125 registered families with HA, 22 were eligible for inclusion. We conducted a linkage analysis using F8 gene markers and amplification refractory mutation system-quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, which requires further confirmation of the origin in the parent. Nine mothers, four maternal grandmothers, and six maternal grandfathers were confirmed to be the origin of sporadic NIVs, which most likely occurred in the zygote within the first few cell divisions and in single sperm cells, respectively. Three mothers had mosaic variants, which most likely occurred early in postzygotic embryogenesis. All maternal grandparents were free from sporadic NIV. In conclusion, F8 NIVs in sporadic HA were found to be caused primarily by de novo variants. Our studies are essential for understanding the genetic pathogenesis of HA and improving current genetic counseling.

Skewed X-Chromosome Inactivation and Parental Gonadal Mosaicism Are Implicated in X-Linked Recessive Female Hemophilia Patients.

BACKGROUND: Hemophilia A (HA) and B (HB) are X-linked recessive disorders that mainly affect males born from a mother carrier. Females are rarely affected but a number of mechanisms have been suggested in symptomatic females, such as skewed X-chromosome inactivation (XCI), chromosomal rearrangements, and hermaphrodites. Different methodologies are required to elucidate the underlying causes of such diseases in female patients. METHODS: Three families with female hemophilia patients, including two HA and one HB, were enrolled for genetic analyses. Cytogenetics, molecular examinations on F8 and F9 genes, XCI assay, and linkage analysis were performed. RESULTS: All three female patients are demonstrated to be heterozygous for an F8, or F9 mutation: one patient is inherited from her unaffected mother and the other two are sporadic cases. All three patients exhibit skewed XCI. The inherited patient is found to be unmethylated in the maternal X chromosome, which increases the potential for the expression of the mutant allele. The two sporadic cases are hypomethylated or unmethylated in the paternal X chromosome, suggesting that paternal gonadal mosaicism may exist in these families. CONCLUSIONS: In addition to screening for coagulation function, different genetic analyses are mandatory to explore the nature of mechanisms responsible for the X-linked recessive disorders in female patients as shown in this study. Our results confirm that skewed XCI is responsible for hemophilia in heterozygous female patients. Likewise, our results implicate that parental gonadal mosaicism, followed by skewed XCI, contributes to hemophilia in "sporadic" female patients.

Proposal for Practical Approach in Prenatal Diagnosis of Beckwith-Wiedemann Syndrome and Review of the Literature.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a phenotypically and genetically heterogeneous disorder associated with epigenetic/genetic aberrations on chromosome 11p15.4p15.5. There is no consensus criterion for prenatal diagnosis of BWS. METHODS: Three BWS patients with their clinical histories, prenatal ultrasonographic features, and results of molecular diagnosis were presented. Likewise, by incorporating the findings of our cases and literature review, the phenotypic spectrum and genotype-phenotype correlations of fetal BWS were summarized, and a practical approach in prenatal diagnosis of BWS was proposed. RESULTS: A total of 166 BWS cases with prenatal features were included for analysis. Common fetal features include abdominal wall defects (42.8%), polyhydramnios (33.1%), and macrosomia (32.5%). Molecular pathologies include methylation changes in imprinting control region 1 and 2 (ICR1 and ICR2), paternal uniparental disomy of chromosome 11p15.5, copy number change involving 11p15, etc. Some genotype-phenotype correlations were observed. However, the broad phenotypic spectrum but limited features manifested by affected fetuses rendering ultrasonographic diagnosis not easy. CONCLUSIONS: Molecular tests are used for prenatal diagnosis of BWS suspected by ultrasonography. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is recommended as the first-line molecular tool because it simultaneously detects ICR1/ICR2 methylation statuses and copy numbers that solve the majority of clinical cases in the prenatal scenario.

Prenatal Diagnosis of True Fetal Mosaicism with Small Supernumerary Marker Chromosome Derived from Chromosome 16 by Funipuncture and Molecular Cytogenetics Including Chromosome Microarray.

This study examined the molecular characterization of a prenatal case with true fetal mosaicism of small supernumerary marker chromosome 16 (sSMC(16)). A 41-year-old female underwent amniocentesis at 19 weeks of gestation due to advanced maternal age. Chromosomal analysis for cultured amniocytes revealed a karyotype of 47,XY,+mar[4]/46,XY[16]. Spectral karyotyping and metaphase fluorescence in situ hybridization (FISH) demonstrated that the sSMC was derived from chromosome 16 (47,XY,+mar.ish der(16)(D16Z1+)[13/20]). Confined placental mosaicism was initially suspected because the prenatal ultrasound revealed a normal structure and the pregnancy was uneventful. However, interphase FISH of cord blood performed at 28 weeks of gestation showed 20% mosaicism of trisomy chromosome 16 (nuc ish(D16Z2×3)[40/200]). Chromosome microarray analysis further demonstrated 55% mosaicism of an 8.02 Mb segmental duplication at the subcentromeric region of 16p12.1p11.1 (arr[GRCh37] 16p12.1p11.1(27021975_35045499)×3[0.55]). The results demonstrated a true fetal mosaicism of sSMC(16) involving chromosome16p12.1p11.1 that is associated with chromosome 16p11.2 duplication syndrome (OMIM #614671). After non-directive genetic counseling, the couple opted for late termination of pregnancy. This case illustrated the use of multiple molecular cytogenetic tools to elucidate the origin and structure of sSMC, which is crucial for prenatal counseling, decision making, and clinical management.

Genome-Wide Microarray Analysis Suggests Transcriptomic Response May Not Play a Major Role in High- to Low-Altitude Acclimation in Harvest Mouse (Micromys minutus).

The harvest mouse (Micromys minutus) is a small rodent species with a wide range of vertical distribution in Taiwan, extending from the sea level to 3100 m altitude. This species has recently suffered from habitat loss in high-altitude areas due to orchard cultivation, which may have resulted in mouse migration from high to low altitude. To investigate whether there is any physiological mechanism involved in altitude acclimation, rat cDNA microarray was used to compare transcriptomic patterns of the skeletal muscle tissues taken from individuals native to the high-altitude environment and those transferred to the low-altitude captive site. Of the 23,188 genes being analyzed, 47 (33 up-regulated and 14 down-regulated) were found to have differential expression (fold change > 4 or < -4, ANOVA p < 0.05). However, after multiple testing correction with a false discovery rate (FDR), only the result for Tnfrsf12a was found to be statistically significant (fold change = 13, FDR p < 0.05). The result was confirmed by quantitative polymerase chain reaction (q-PCR). The expression of Tnfrsf12a possibly relates to the skeletal muscle biology and thus can be correlated with altitude acclimation. However, finding only one gene transcript with significant alteration suggests that transcriptomic response may not play a major role in high- to low-altitude acclimation in harvest mouse.

Application of molecular cytogenetic techniques to characterize the aberrant Y chromosome arising de novo in a male fetus with mosaic 45,X and solve the discrepancy between karyotyping, chromosome microarray, and multiplex ligation dependent probe amplification.

We present a rare male fetus with karyotype of mosaic 45,X that comprises two types of aberrant Y chromosomes arising de novo (Yq12 deletion and isodicentric Yq11.22). Both types of the aberrant Y chromosomes lack the AZFc region which are expected to result in oligospermia but unaffected male external genitalia. Genetic analyses by karyotyping, chromosome microarray (CMA), and multiplex ligation-dependent probe amplification (MLPA) for the fetus revealed conflicting results. Additional molecular cytogenetics tools including fluorescence in situ hybridization (FISH) and multicolor banding (mBAND) were performed, which help resolving the discrepancy and delineated the composition of the aberrant Y chromosomes. This report highlighted the importance of incorporating multiple genetic technologies for accurate characterization of complex chromosomal rearrangements, which aid in the prenatal diagnosis and genetic counseling.

Noninvasive prenatal diagnosis of fetal aneuploidy by circulating fetal nucleated red blood cells and extravillous trophoblasts using silicon-based nanostructured microfluidics.

BACKGROUND: Noninvasive prenatal testing (NIPT) based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community since 2011 but limitations, such as maternal malignancy and fetoplacental mosaicism, preclude its full replacement of invasive prenatal diagnosis. We present a novel silicon-based nanostructured microfluidics platform named as "Cell Reveal™" to demonstrate the feasibility of capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cell-based noninvasive prenatal diagnosis (cbNIPD). METHODS: The "Cell Reveal™" system is a silicon-based, nanostructured microfluidics using immunoaffinity to capture the trophoblasts and the nucleated RBC (nRBC) with specific antibodies. The automated computer analysis software was used to identify the targeted cells through additional immunostaining of the corresponding antigens. The identified cells were retrieved for whole genome amplification for subsequent investigations by micromanipulation in one microchip, and left in situ for subsequent fluorescence in situ hybridization (FISH) in another microchip. When validation, bloods from pregnant women (n = 24) at gestational age 11-13+6 weeks were enrolled. When verification, bloods from pregnant women (n = 5) receiving chorionic villus sampling or amniocentesis at gestation age 11+4-21 weeks with an aneuploid or euploid fetus were enrolled, followed by genetic analyses using FISH, short tandem repeat (STR) analyses, array comparative genomic hybridization, and next generation sequencing, in which the laboratory is blind to the fetal genetic complement. RESULTS: The numbers of captured targeted cells were 1-44 nRBC/2 ml and 1-32 EVT/2 ml in the validation group. The genetic investigations performed in the verification group confirmed the captured cells to be fetal origin. In every 8 ml of the maternal blood being blindly tested, both fnRBC and EVT were always captured. The numbers of captured fetal cells were 14-22 fnRBC/4 ml and 1-44 EVT/4 ml of maternal blood. CONCLUSIONS: This report is one of the first few to verify the capture of fnRBC in addition to EVT. The scalability of our automated system made us one step closer toward the goal of in vitro diagnostics.

Genomic analyses of the Formosan harvest mouse (Micromys minutus) and comparisons to the brown Norway rat (Rattus norvegicus) and the house mouse (Mus musculus).

The harvest mouse, Micromys minutus (MMIN), has a very wide range of distribution (from the British Isles across the Euroasian continent to Japan and Taiwan). We studied an isolated population of MMIN in Taiwan, which is at the southeastern margin of the species' geographic distribution, and compared its genetic complement with those of the same species previously reported from other geographic locations and with two model rodent species, the house mouse (Mus musculus) and the brown Norway rat (Rattus norvegicus). The diploid number (2N) of MMIN was 68, consistent with that reported for other populations. However, variations were noted in the fundamental number (FN) and the shape and banding patterns of the individual chromosomes among populations. The FN of MMIN was estimated to be 72, including 2 bi-armed autosomes, 31 one-armed autosomes, and one pair of one-armed sex chromosomes. Here, we propose the first ideogram for MMIN. C-banding, Ag-NOR, and the locations of 18S rRNA gene sequences (MMIN chromosomes no. 10, 14, 19, 29, 31, 33, and X) mapped by fluorescence in situ hybridization (FISH) are also reported. Additionally, we compared the 18S rDNA sequences and performed cross-species X chromosome painting (FISH) for M. minutus, M. musculus, and R. norvegicus. The results indicate that both genetic elements are rather conserved across species. Thus, implications for the phylogenetic position of Micromys were limited.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from ring chromosome 2.

OBJECTIVE: To present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from ring chromosome 2 [r(2)]. METHODS AND RESULTS: A 35-year-old woman underwent amniocentesis at 17 weeks of gestation, because of advanced maternal age. Amniocentesis revealed a de novo ring-shaped sSMC in 11 of 23 colonies of cultured amniocytes. Repeated amniocenteses were made. The sSMC was characterized by array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) on uncultured amniocytes. In uncultured amniocytes, aCGH showed a 39.49-Mb genomic gain in chromosome 2 encompassing 2q11.2→q21.2, interphase FISH revealed a mosaic level of 52% (52/100 cells), and QF-PCR manifested a diallelic pattern for chromosome 2, with gene dosage increase in the paternal allele of proximal 2q-specific DNA markers. In cultured amniocytes, the sSMC was characterized by metaphase FISH, spectral karyotyping (SKY) and multicolor banding (MCB) to contain the centromere and proximal 2q, and the karyotype was 47,XX,+r(2)(p11.1q21.2)[14]/46,XX[11]. The pregnancy was terminated. The fetus postnatally manifested facial dysmorphisms. Postnatal cytogenetic analyses revealed the karyotypes of 47,XX,+r(2)[12]/46,XX[28] in cord blood, 47,XX,+r(2)[7]/46,XX[33] in umbilical cord, 47,XX,+r(2)[13]/47,XX,+idic r(2)[3]/46,XX[24] in placenta and 47,XX,+r(2)[8]/47,XX,+idic r(2)[1]/46,XX[31] in amnion. CONCLUSION: Molecular cytogenetic techniques such as aCGH, interphase FISH and QF-PCR on uncultured amniocytes, and SKY, MCB and metaphase FISH on cultured amniocytes are useful for characterization of the nature of a prenatally detected sSMC.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals.

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.

Prenatal diagnosis and molecular cytogenetic characterization of de novo partial trisomy 7p (7p15.3→pter) and partial monosomy 13q (13q33.3→qter) associated with Dandy-Walker malformation, abnormal skull development and microcephaly.

OBJECTIVE: To present the prenatal diagnosis and molecular cytogenetic characterization of de novo partial trisomy 7p (7p15.3→pter) and partial monosomy 13q (13q33.3→qter) associated with Dandy-Walker malformation (DWM), abnormal skull development, microcephaly and multiple congenital anomalies. MATERIALS, METHODS AND RESULTS: A 42-year-old woman, gravida 6, para 1, was referred for amniocentesis at 18 weeks of gestation because of her advanced maternal age. Amniocentesis revealed an aberrant derivative chromosome 13, or der(13). The parental karyotypes were normal. Spectral karyotyping showed that the der(13) was derived from a translocation of chromosomes 7 and 13. Fluorescence in situ hybridization using subtelomeric probes revealed three signals of 7pTEL and only one signal of 13qTEL, indicating a translocation between 7p and 13q in the der(13). Array-based comparative genomic hybridization demonstrated partial trisomy 7p (7p15.3-p22.3) and partial monosomy 13q (13q33.3-q34). The karyotype was 46,XY,der(13)t(7;13)(p15.3;q33.3). Polymorphic DNA marker analysis revealed the paternal origin of the aberrant chromosome. Level II ultrasound at 24 weeks of gestation revealed microcephaly, an irregular-shaped skull, DWM, nuchal edema and transposition of the great arteries. CONCLUSION: Spectral karyotyping, fluorescence in situ hybridization and array-based comparative genomic hybridization are useful for prenatal investigation of the nature of a de novo aberrant derivative chromosome. Partial trisomy 7p (7p15.3→pter) and partial monosomy 13q (13q33.3→qter) can be associated with DWM, microcephaly, abnormal skull development, nuchal edema and cardiovascular defects on prenatal ultrasound.

Complex rearrangements between chromosomes 6, 10, and 11 with multiple deletions at breakpoints.

Here we report on a girl with minor facial anomalies, cleft palate, seizures, microcephaly, psychomotor retardation, and a congenital heart defect. Complex of cytogenetic methods [GTG-banding, spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), multicolor banding (mBAND), and comparative genomic hybridization (array CGH)] showed complex chromosomal rearrangements (CCRs) involving chromosomes 6, 10, and 11 and 4 deletions at the breakpoints. Her father had an unrelated translocation between chromosomes 3 and 16, suggesting the possibility of an autosomal dominant trait that predisposes to complex synapses and recombination between multiple chromosomes during meiosis. This study demonstrates the power of combining available chromosome analysis technologies in resolving CCR.

Interstitial deletion 13q31 associated with normal phenotype: cytogenetic study of a family with concomitant segregation of reciprocal translocation and interstitial deletion.

Gain or loss of a fragment in human chromosomes has been associated with abnormal phenotypes in numerous genetic disorders. However, it is also possible that lack or excess of a particular chromosomal segment is a neutral polymorphism among populations and thus does not cause obvious abnormal phenotype. In this study, conventional GTG-banded karyotyping and molecular cytogenetic analyses (including fluorescence in situ hybridization, spectral karyotyping and comparative genomic hybridization) were applied to study the genotype-phenotype correlation in a Taiwanese family, in which a concomitant segregation of del(13)(q31q31) interstitial deletion and t(13;18)(q32;p11.2) reciprocal translocation in a 2-year-old girl (the proband) was noticed. Two family members (the father and grandmother of the proband) who carried the del(13)(q31q31) but not the translocation t(13;18) both revealed a normal phenotype at adulthood. The finding, which appears novel, that interstitial deletion 13q31 could be associated with a normal phenotype, is therefore valuable in genetic counseling.

Comparison of immunohistochemical and fluorescence in situ hybridization assessment for HER-2/neu status in Taiwanese breast cancer patients.

OBJECTIVE: Accurate diagnostic assessment of human epidermal growth factor receptor-2 (HER-2) is essential and a prerequisite for appropriate application of the humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) to the treatment of patients with breast cancer. Immunohistochemistry (IHC) is the most widely applicable diagnostic modality in studying HER-2 status. Fluorescence in situ hybridization (FISH) is also recognized as a modality in cases with an equivocal IHC status (score, 2+). Some authors claimed that FISH alone is sufficient. The aim of this study was to correlate the test results of IHC and FISH for HER-2 gene amplification in breast cancer patients. FISH for topoisomerase IIalpha (TOP2A) was also studied to see if deletion or amplification of TOP2A has any supplementary role to HER-2, FISH and IHC. MATERIALS AND METHODS: Assessment of HER-2 gene amplification and TOP2A gene amplification/deletion was made by FISH analysis using the LSI TOP2A/HER-2/CEP 17 multicolor probe or the LSI HER-2/CEP dual color probe (Vysis, Downers Grove, IL, USA) in formalin-fixed and paraffin-embedded tissue sections of 54 breast cancer patients who were grouped into stages 1+, 2+ or 3+ based on IHC (HercepTest; DakoCytomation, Carpinteria, CA, USA) observations. RESULTS: None of IHC 1+ breast tumors was HER-2 FISH positive, but three of 18 (17%) IHC 3+ tumors were HER-2 FISH negative. Overall, 53% of the IHC 2+ and 83% of the IHC 3+ cases were HER-2 FISH positive. Only one case with IHC 3+ tumor that was HER-2 FISH positive was found to have TOP2A amplification (>2.0) and no IHC 2+ cases were found to have TOP2A amplification. There were no cases with TOP2A deletion (<0.8) in our whole series. There were also no cases of HER-2 FISH negative tumors, but IHC scored as 2+ or 3+ (0 of 10), to be found with TOP2A amplification. The discordance rates by IHC were high (46.7% in IHC 2+, 16.7% in IHC 3+, 30.3% overall in IHC 2+ or 3+). On the contrary, the discordance rates were zero if by FISH. CONCLUSION: The current algorithm to use HER-2 FISH as a supplementary role to IHC HercepTest 2+ may need some modifications according to the local setting. TOP2A FISH adds little value to HER-2 FISH and IHC staining in our study.