High-resolution microarray analysis unravels complex Xq28 aberrations in patients and carriers affected by X-linked blue cone monochromacy.

The human X chromosome contains ∼ 1600 genes, about 15% of which have been associated with a specific genetic condition, mainly affecting males. Blue cone monochromacy (BCM) is an X-linked condition caused by a loss-of-function of both the OPN1LW and OPN1MW opsin genes. The cone opsin gene cluster is composed of 2-9 paralogs with 99.8% sequence homology and is susceptible to deletions, duplications, and mutations. Current diagnostic tests employ polymerase chain reaction (PCR)-based technologies; however, alterations remain undetermined in 10% of patients. Furthermore, carrier testing in females is limited or unavailable. High-resolution X chromosome-targeted CGH microarray was applied to test for rearrangements in males with BCM and female carriers from three unrelated families. Pathogenic alterations were revealed in all probands, characterized by sequencing of the breakpoint junctions and quantitative real-time PCR. In two families, we identified a novel founder mutation that consisted of a complex 3-kb deletion that embraced the cis-regulatory locus control region and insertion of an additional aberrant OPN1MW gene. The application of high-resolution X-chromosome microarray in clinical diagnosis brings significant advantages in detection of small aberrations that are beyond the resolution of clinically available aCGH analysis and which can improve molecular diagnosis of the known conditions and unravel previously unrecognized X-linked diseases.

Genetic and morphological features of human iPSC-derived neurons with chromosome 15q11.2 (BP1-BP2) deletions.

BACKGROUND: Copy number variation on chromosome 15q11.2 (BP1-BP2) causes deletion of CYFIP1, NIPA1, NIPA2 and TUBGCP5; it also affects brain structure and elevates risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered cyfip1 expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from iPSCs (iPSC-neurons). METHODS: iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. RESULTS: CYFIP1, NIPA1, NIPA2 and TUBGCP5 gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD95 protein levels were lower in iPSC-neurons derived from the CNV bearing individuals using Western blot analysis. At 10 weeks post-differentiation, iPSC-neurons appeared to show dendritic spines and qualitative analysis suggested that dendritic morphology was altered in 15q11.2 deletion subjects compared with control cells. CONCLUSIONS: The 15q11.2 (BP1-BP2) deletion is associated with reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated altered iPSC-neuron dendritic morphology.

Application of chromosomal microarray in the evaluation of abnormal prenatal findings.

We performed karyotype and array comparative genomic hybridization (aCGH) analyses on 177 prenatal samples, including 162 (92%) samples from fetuses with sonographic anomalies. Overall 12 fetuses (6.8%) had abnormal karyotype and 42 (23.7%) fetuses had abnormal microarray results: 20 (11.3%) with pathogenic copy number variations (CNVs), 16 with CNVs of uncertain clinical significance, 4 with CNVs establishing carrier status for recessive, X-linked, or susceptibility to late onset dominant disease, and two CNVs with pseudomosaicism due to in vitro cultural artifacts. For 23 pregnancies (13%), aCGH contributed important new information. Our results highlight the interpretation challenges associated with CNVs of unclear significance, incidental findings, as well as technical aspects. Array CGH analysis significantly improved the detection of genomic imbalances in prenatal diagnosis of pregnancies with structural birth defects.

Recurrent triploid and dispermic conceptions in patients with NLRP7 mutations.

To understand the mechanisms leading to hydatidiform mole formation in patients with NLRP7 mutations, we used a combination of various approaches to characterize five products of conception, from two patients, shown by flow cytometry to contain non-diploid cells. We demonstrate that four of these conceptions are triploid and two of them originated from fertilization with more than one sperm. We show that three of these triploid conceptions fulfill the histopathological criteria of partial hydatidiform mole and one fulfills the histopathological criteria of spontaneous abortion. Our data demonstrate that some oocytes from one patient with NLRP7 mutations are not able to prevent polyspermic fertilization and highlight the importance of using several approaches to characterize the genetic complexity of molar tissues and reproductive wastage. Altogether, our previous and current data show the association of NLRP7 mutations with several types of hydatidiform moles and with triploid spontaneous abortions.

Cryptic duplication of 12q24.33 --> qter in a child with Angelman syndrome-simultaneous occurrence of two unrelated cytogenetic events.

Simultaneous occurrence of two unrelated cytogenetic events is rare. We present a case of Angelman Syndrome (AS) deletion and 12q duplication in a child with a history of developmental delay, microcephaly, cerebral palsy, and seizures. Traditional cytogenetic studies showed a normal 46,XY karyotype. Fluorescence in situ hybridization (FISH) using probe D15S10 (AS region/15q11.2) revealed a deletion. In addition, we serendipitously detected 12q24.3 duplication by FISH with 12q subtelomere probe. He inherited this duplication from the mother who presented with a balanced translocation karyotype 46,XX,add(12)(q24.3).ish t(12;13)(q24.3;p11.2)(12qtel-;12qtel+,D13Z1/D21Z1+,RB1+). Array comparative genomic hybridization (array-CGH) revealed a duplication of three bacterial artificial chromosome (BAC) clones (RP11-46H11, RP11-386I8, and RP11-309H3) covering about 423 Kb of DNA sequence. The published 12q terminal duplication cases had a detectable segment by classical banded cytogenetics techniques. To our knowledge, this is the smallest 12q cryptic rearrangement characterized by array-CGH and confirmed by BAC-clone FISH analysis. Based on these findings, we attempted to separate the clinical features associated with AS deletion and those features that are probably due to partial 12q duplication. We then reviewed the genes mapped in the duplicated region using the human genome database to understand the clinical significance. A subsequent pregnancy in the mother revealed an apparently balanced t(12;13) karyotype. We compare our case with the published cases, and discuss the implications of our findings and its relevance in addressing genetic counseling issues.

Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study.

BACKGROUND: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. METHODS: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. RESULTS: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. CONCLUSIONS: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Comparative study of primary and recurrent ovarian serous carcinomas: comparative genomic hybridization analysis with a potential application for prognosis.

OBJECTIVES: The purpose of this study is to comparatively characterize genomic imbalances in primary and recurrent ovarian serous carcinomas and to identify genomic alterations that may be used as a marker for prognosis. METHODS: Twenty ovarian serous carcinomas were studied by comparative genomic hybridization (CGH). RESULTS: Genomic alterations were found in all of the tumors. The most common regions involving gain of DNA copy numbers are 1q41q44, 8q22q24, 19p12q13.1, 20q12q13, 3q26q29, 12p12p13, 2p22p25, 7p14p21, 5p15.2p15.3, and 17q22q25. The most common regions with loss of DNA copy numbers are Xp11.2q13, 4q31q35, Xp21p22.3, 18q22q23, 13q22q31, 9p22p24, and 16q22q24. High-level gains were detected at chromosomal regions of 1q41q44, 2p22p25, 3q26q29, and 19p12q13.1. Comparative analysis of primary and recurrent tumors showed that gains of 2p22p25, 19p12q13.1, and 20q12q13 and loss of 5q14q22 were more common in the recurrent high-grade tumors. About 85% of the tumors showed increases in DNA copy numbers in the regions (2p and 8q) harboring the myc family gene. Patients with tumor containing fewer than seven chromosomal aberrations showed longer survival time. CONCLUSION: The myc oncogene family may play a role in the pathogenesis of ovarian serous carcinomas. Our study suggests that tumors with gains of 2p22p25, 19p12q13.1, and 20q12q13 and loss of 5q14q22 may be at high risk for recurrence. Furthermore, the patients' survival time inversely correlates with the numbers of chromosomal alterations found in their tumors. CGH analysis may have a clinical application in predicting prognosis and risk of recurrence in patients with ovarian serous carcinomas.

Three cases of tetrasomy 9p.

We report three cases of tetrasomy 9p, two of which were confirmed prenatally. All three had characteristic findings on ultrasound and at birth. We also present a review of the literature, which suggests that a recognizable phenotype for this condition is emerging. Common findings on prenatal ultrasound include intrauterine growth restriction, ventriculomegaly, cleft lip or palate, and renal anomalies. These findings can provide a clue toward the prenatal diagnosis of this condition. There is also a clearly recognizable phenotype at birth. Facial characteristics include hypertelorism, broad nasal bridge/bulbous or beaked nose, cleft lip/palate, ear anomalies, and micrognathia. The exact extent of the isochromosome does not seem to predict severity, but mosaic cases are less severe, or at least have a greater probability of survival.

Tetrasomy 15q25.3 --> qter resulting from an analphoid supernumerary marker chromosome in a patient with multiple anomalies and bilateral Wilms tumors.

We describe a girl who had been followed since birth for apparent Shprintzen-Goldberg syndrome (SGS), with macrosomia, long fingers and toes, and craniosynostosis, and presented at 4 years of age with bilateral Wilms tumors (also called nephroblastoma). Cytogenetic analysis of her peripheral blood revealed a de novo supernumerary marker chromosome. This stable marker chromosome is present in 19 of 20 lymphocytes analyzed, as well as in all 40 tumor cells (20 from each tumor) studied. Classical and molecular cytogenetic studies indicate that the marker is derived from an inverted duplication of chromosome 15q25.3 --> qter and contains a neocentromere. The presence of this marker chromosome in our patient results in tetrasomy 15q25.3 --> qter. The relationship between her genotype and phenotype are discussed in light of genes, including IGF1R and FES, mapped to the aneusomic segment.

Genomic alterations in uterine leiomyosarcomas: potential markers for clinical diagnosis and prognosis.

Genomic alterations were analyzed in 21 uterine leiomyosarcomas (ULMSs) by comparative genomic hybridization. DNA copy number changes were detected in all 21 tumors. The most frequent losses were 13q (16/21 = 76%), 10q (13/21 = 62%), 16q (8/21 = 38%), 12p (7/21 = 33%), and 2p (9/21 = 43%). The most common gains were 17p (8/21 = 38%), Xp (7/21 = 33%), and 1q (7/21 = 33%). High-copy-number gains (ratio > 1.5) were identified in Xp, 1q, and 17p. Loss of 13q was identified in both low-grade and high-grade tumors. Inactivation of a tumor suppressor gene in 13q may be an early event in the development of leiomyosarcomas. Loss of 10q, 2p, and 12p and gains of 1q as well as 17p were frequently found in high-grade tumors and recurrent tumors. Inactivation of tumor suppressor genes and activation of oncogenes in these regions may be associated with a more aggressive behavior of ULMS. Patients with only loss of 13q and without the other alterations listed above had longer survival times. Gains of Xp, 17p, and 1q and losses of 13q, 10q, 16q, 12p, and 2p have been reported in extra-uterine leiomyosarcomas. Our findings indicate that the pathogenesis of uterine leiomyosarcomas and extra-uterine leiomyosarcomas follows the same genetic pathways.

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies.

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.

Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole.

The origin of human triploidy is controversial. Early cytogenetic studies found the majority of cases to be paternal in origin; however, recent molecular analyses have challenged these findings, suggesting that digynic triploidy is the most common source of triploidy. To resolve this dispute, we examined 91 cases of human triploid spontaneous abortions to (1) determine the mechanism of origin of the additional haploid set, and (2) assess the effect of origin on the phenotype of the conceptus. Our results indicate that the majority of cases were diandric in origin because of dispermy, whereas the maternally-derived cases mainly originated through errors in meiosis II. Furthermore, our results indicate a complex relationship between phenotype and parental origin: paternally-derived cases predominate among "typical" spontaneous abortions, whereas maternally-derived cases are associated with either early embryonic demise or with relatively late demise involving a well-formed fetus. As the cytogenetic studies relied on analyses of the former type of material and the molecular studies on the latter sources, the discrepancies between the data sets are explained by differences in ascertainment. In studies correlating the origin of the extra haploid set with histological phenotype, we observed an association between paternal-but not maternal-triploidy and the development of partial hydatidiform moles. However, only a proportion of paternally derived cases developed a partial molar phenotype, indicating that the mere presence of two paternal genomes is not sufficient for molar development.

Genomic and functional map of the chromosome 14 t(12;14) breakpoint cluster region in uterine leiomyoma.

A translocation involving chromosomes 12 and 14 [t(12;14)(q15;24.1)] is commonly seen in benign smooth muscle tumor as uterine leiomyoma (UL). A contig of P1-derived artificial chromosome and bacterial artificial chromosome clones on chromosome 14, encompassing a t(12;14) breakpoint cluster region (BCR) in UL, was generated principally using the recently developed HAPPY map of chromosome 14 as a framework (P. H. Dear et al., 1998, Genomics 48: 232-241). Three UL t(12;14) breakpoints have been localized within this contig, showing that a BCR of at least 400 kb exists on chromosome 14. Other studies of tumors with t(12;14) rearrangements similarly show breakpoints within a 475-kb multiple aberration region on chromosome 12. Thus t(12;14) is an example of a translocation in which the breakpoints are located within a BCR on both chromosome 12 and chromosome 14, justifying the identification of expressed sequences that are altered in these BCR regions. A total of four expressed sequences were identified in the BCR on chromosome 14. Two of these were novel cDNAs (D14S1460E and D14S1461E). The chromosome 14 cDNAs were expressed in multiple adult tissues. The identification of a large breakpoint cluster region on chromosome 14 suggests that translocations in this region mediate their effects at a distance and also that elements that predispose this region to recurrent chromosomal translocation may be widely distributed.

Nongestational choriocarcinoma in the postpartum period: a case report.

PURPOSE: To determine the tissue of origin (gestational versus nongestational) of an extensive metastatic choriocarcinoma in an 18-year-old woman to determine prognosis and treatment. METHODS: DNA microsatellite polymorphisms after polymerase chain reaction (PCR) amplification of the tumor tissue and blood from the patient, husband, and daughter were used to determine the tissue of origin. RESULTS: Molecular analyses revealed that the tumor shared the genetic features of only the patient. She responded well to multiagent chemotherapy. CONCLUSIONS: Molecular analysis is a useful tool to determine whether a choriocarcinoma occurring in a female patient of child-bearing age is gestational or nongestational when clinical findings are not clearly indicative of the primary.

Molecular analysis of chromosome 7q21.3 in uterine leiomyoma: analysis using markers with linkage to insulin resistance.

Recent sibling-pair linkage analyses have indicated possible linkage of noninsulin dependent diabetes mellitus (NIDDM) with a number of markers on the long arm of chromosome 7. A coincidental and recent discovery is that specific genetic anomalies identified on chromosome 7 in uterine leiomyoma tumor cells in many cases correspond, cytogenetically, to the same region where genetic linkage to insulin resistance has been identified. In the present study, 15 closely spaced microsatellite markers were used to finely map deletion breakpoints and to test for allelic loss of 7q markers in 12 uterine leiomyoma tumor samples with cytogenetically defined deletions. Of the 9 informative tumor samples, three exhibited breakpoints in the same region where genetic linkage to insulin resistance has been identified (between PON and UT901). Because breakpoints in neoplasias often occur within or adjacent to expressed sequences, these breakpoints may provide a molecular tool to aid in the identification of candidate genes for insulin resistance.

Apparently balanced t(1;7)(q21.3;q34) in an infant with Coffin-Siris syndrome.

Coffin-Siris syndrome is a multiple anomaly/mental retardation syndrome characterized by "coarse" facial appearance, hypoplastic or absent nails on the fifth digits, generalized hirsutism with sparse scalp hair, hypotonia, and developmental delay. Due to several reports of affected sibs with or without a mildly affected parent, both autosomal recessive and autosomal dominant inheritance have been suggested. All previous patients with well-documented Coffin-Siris syndrome are chromosomally normal, and the gene has not been mapped. We report on an infant with typical findings of Coffin-Siris syndrome who also has a de novo apparently balanced translocation of chromosomes 1 and 7, karyotype 46,XY,t(1;7)(q21.3;q34). The parental chromosomes are normal and none of the relatives have signs of Coffin-Siris syndrome. The breakpoints 1q21.3 and 7q34 are suggested as possible locations for a Coffin-Siris gene.

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed.