Deletion of conserved non-coding sequences downstream from NKX2-1: A novel disease-causing mechanism for benign hereditary chorea.

BACKGROUND: Benign hereditary chorea (BHC) is an autosomal dominant disorder characterized by early-onset non-progressive involuntary movements. Although NKX2-1 mutations or deletions are the cause of BHC, some BHC families do not have pathogenic alterations in the NKX2-1 gene, indicating that mutations of non-coding regulatory elements of NKX2-1 may also play a role. METHODS AND RESULTS: By using whole-genome microarray analysis, we identified a 117 Kb founder deletion in three apparently unrelated BHC families that were negative for NKX2-1 sequence variants. Targeted next generation sequencing analysis confirmed the deletion and showed that it was part of a complex local genomic rearrangement. In addition, we also detected a 648 Kb de novo deletion in an isolated BHC case. Both deletions are located downstream from NKX2-1 on chromosome 14q13.2-q13.3 and share a 33 Kb smallest region of overlap with six previously reported cases. This region has no gene but contains multiple evolutionarily highly conserved non-coding sequences. CONCLUSION: We propose that the deletion of potential regulatory elements necessary for NKX2-1 expression in this critical region is responsible for BHC phenotype in these patients, and this is a novel disease-causing mechanism for BHC.

A novel NLRP7 protein-truncating mutation associated with discordant and divergent p57 immunostaining in diploid biparental and triploid digynic moles.

NLRP7 is a maternal-effect gene that has a primary role in the oocyte. Its biallelic mutations are a major cause for recurrent diploid biparental hydatidiform moles (HMs). Here, we describe the full characterization of four HMs from a patient with a novel homozygous protein-truncating mutation in NLRP7. We found that some HMs have features of both complete and partial moles. Two HMs expressed p57 in the cytotrophoblast and stromal cells and exhibited divergent and discordant immunostaining. Microsatellite DNA-genotyping demonstrated that two HMs are diploid biparental and one is triploid digynic due to the failure of meiosis II. FISH analysis demonstrated triploidy in the cytotrophoblast and stromal cells in all villi. Our data highlight the atypical features of HM from patients with recessive NLRP7 mutations and the important relationship between NLRP7 defects in the oocyte and p57 expression that appear to be the main contributor to the molar phenotype regardless of the zygote genotype.

Correction: Comprehensive analysis of 204 sporadic hydatidiform moles: revisiting risk factors and their correlations with the molar genotypes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Four children with postnatally diagnosed mosaic trisomy 12: Clinical features, literature review, and current diagnostic capabilities of genetic testing.

Children or adults with mosaic trisomy 12 diagnosed postnatally are extremely rare. Only a small number of patients with this mosaicism have been reported in the literature. The clinical manifestation of mosaic trisomy 12 is variable, ranging from mild developmental delay to severe congenital anomaly and neonatal death. The trisomy 12 cells are not usually able to be detected by phytohemagglutinin stimulated peripheral blood chromosome analysis. The variability of phenotypes and the limited number of patients with this anomaly pose a challenge to predict the clinical outcomes. In this study, we present the phenotypes and laboratory findings in four patients and review the 11 previously reported patients with mosaic trisomy 12 diagnosed postnatally, as well as 11 patients with mosaic trisomy 12 diagnosed prenatally. The findings of this study provide useful information for laboratory diagnosis and clinical management of these patients.

Comprehensive analysis of 204 sporadic hydatidiform moles: revisiting risk factors and their correlations with the molar genotypes.

Hydatidiform mole (HM) is an aberrant human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. HM has two morphological types, complete (CHM) and partial (PHM), and non-recurrent ones have three genotypic types, androgenetic monospermic, androgenetic dispermic, and triploid dispermic. Most available studies on risk factors predisposing to different types of HM and their malignant transformation mainly suffer from the lack of comprehensive genotypic analysis of large cohorts of molar tissues combined with accurate postmolar hCG follow-up. Moreover, 10-20% of patients with one HM have at least one non-molar miscarriage, which is higher than the frequency of two pregnancy losses in the general population (2-5%), suggesting a common genetic susceptibility to HM and miscarriages. However, the underlying causes of the miscarriages in these patients are unknown. Here, we comprehensively analyzed 204 HM, mostly from patients referred to the Quebec Registry of Trophoblastic Diseases and for which postmolar hCG monitoring is available, and 30 of their non-molar miscarriages. We revisited the risk of maternal age and neoplastic transformation across the different HM genotypic categories and investigated the presence of chromosomal abnormalities in their non-molar miscarriages. We confirm that androgenetic CHM is more prone to gestational trophoblastic neoplasia (GTN) than triploid dispermic PHM, and androgenetic dispermic CHM is more prone to high-risk GTN and choriocarcinoma (CC) than androgenetic monospermic CHM. We also confirm the association between increased maternal age and androgenetic CHM and their malignancies. Most importantly, we demonstrate for the first time that patients with an HM and miscarriages are at higher risk for aneuploid miscarriages [83.3%, 95% confidence interval (CI): 0.653-0.944] than women with sporadic (51.5%, 95% CI: 50.3-52.7%, p value = 0.0003828) or recurrent miscarriages (43.8%, 95% CI: 40.7-47.0%, p value = 0.00002). Our data suggest common genetic female germline defects predisposing to HM and aneuploid non-molar miscarriages in some patients.

Phenotypic association of 15q11.2 CNVs of the region of breakpoints 1-2 (BP1-BP2) in a large cohort of samples referred for genetic diagnosis.

In view of conflicting reports on the pathogenicity of 15q11.2 CNVs of the breakpoints 1-2 (BP1-BP2) region and lack of association with a specific phenotype, we collected phenotypic data on 51,462 patients referred for genetic testing at two centers (Magee-Womens Hospital of UPMC and Baylor Genetics Laboratories, Baylor College of Medicine). Using array CGH, 262 patients with deletions and 215 with duplications were identified and tested for their association with four phenotypes (developmental delay, dysmorphic features, autism group of disorders, and epilepsy/seizures). Only association of deletions with dysmorphic features was observed (P = 0.013) with low penetrance (3.8%). Our results, viewed in the context of other reports suggesting the lack of a clear phenotypic outcome, underscore the need for detailed phenotypic studies to better understand the pathogenicity of 15q11.2 (BP1-BP2) CNVs.

Diploid/triploid mixoploidy: A consequence of asymmetric zygotic segregation of parental genomes.

Triploidy is the presence of an extra haploid set of chromosomes and can exist in complete or mosaic form. The extra haploid set of chromosomes in triploid cells can be of maternal or paternal origin. Diploid/triploid mixoploidy is a unique form of triploid mosaicism that requires the aberrant segregation of entire parental genomes into distinct blastomere lineages (heterogoneic cell division) at the earliest zygotic divisions. Here we report on eight cases of diploid/triploid mixoploidy from our institution and conduct a comprehensive review of the literature. The parental origin of the extra set of chromosomes was determined in two cases; and, based on phenotypic evidence we propose the parental origin in the other cases. One case with complex mixoploidy appears to have a digynic origin in addition to the involvement of two different sperm. Of our eight cases, only one resulted in the birth of a live healthy child. The other pregnancies ended in miscarriage, elective termination of pregnancy, intrauterine fetal demise or neonatal death. A review of the literature and the results of our cases show that a preponderance of recognized cases of diploid/triploid mixoploidy has a digynic origin.

Maternal GRB10 microdeletion is a novel cause of cystic placenta: Spectrum of genomic changes in the etiology of enlarged cystic placenta.

INTRODUCTION: The genetics and pathology of diploid complete and triploid partial hydatidiform moles have been well established. Enlarged cystic placenta often indicates an underlying etiology and is frequently associated with adverse pregnancy outcome. Several imprinted genes are strongly expressed in placental tissues and essential for normal placental growth and development. Disruption of these imprinted genes can lead to abnormal placental pathology and placental stunting or overgrowth. We present the genetic etiologies of five unusual mosaic cases of enlarged cystic placentas and report a novel etiology, mosaicism for deletion of the maternal GRB10 gene. METHODS: Five mosaic placental mesenchymal dysplasia cases with discrete populations of "cystic" and "normal" villi and/or atypical p57KIP2 immunostaining were evaluated by genetic analysis; including G-banded karyotyping, fluorescence in situ hybridization (FISH), whole genome CGH + SNP microarray, conventional Sanger sequencing, and STR microsatellite analysis. RESULTS: Genetic etiologies ranged from genome-wide changes, including mosaic androgenetic isodisomy and mosaic diandric triploidy, to a novel microdeletion of the maternally-expressed GRB10 gene. An abnormal mosaic population of cells was also detected in the fetus in two cases. DISCUSSION: Four cases were mosaic for either diandric triploidy or an androgenetic cell population, and the enlarged cystic placentas were likely due to an excess of paternally-expressed growth promoting genes and also the absence of maternally-expressed growth restricting genes. Also we identified mosaicism for a novel microdeletion of the maternal GRB10 allele, a potent growth inhibitor, which resulted in placental overgrowth in the cystic area of one placenta. We advocate the use of ancillary techniques to investigate complex mosaic cases of enlarged cystic placentas to discover atypical genetic etiologies and to increase our understanding of the placental genome.

Chromosome 12q13.13q13.13 microduplication and microdeletion: a case report and literature review.

BACKGROUND: Duplications or deletions in the 12q13.13 region are rare. Only scattered cases with duplications and/or deletions in this region have been reported in the literature or in online databases. Owing to the limited number of patients with genomic alteration within this region and lack of systematic analysis of these patients, the common clinical manifestation of these patients has remained elusive. CASE PRESENTATION: Here we report an 802 kb duplication in the 12q13.13q13.13 region in a 14 year-old male who presented with dysmorphic features, developmental delay (DD), mild intellectual disability (ID) and mild deformity of digits. Comparing the phenotype of our patient with those of reported patients, we find that patients with the 12q13.13 duplication or the deletion share similar phenotypes, including dysmorphic facies, abnormal nails, intellectual disability, and deformity of digits or limbs. However, patients with the deletion appear to have more severe deformity of digits or limbs. CONCLUSIONS: Deletion and duplication of the 12q13.13 region may represent novel contiguous gene alteration syndromes. All seven reported 12q13.13 deletions and three of four duplications are de novo and vary in size. Therefore, these genomic alterations are not due to non-allelic homologous recombination.

Genomic Characterization of a Metastatic Alveolar Rhabdomyosarcoma Case Using FISH Studies and CGH+SNP Microarray Revealing FOXO1-PAX7 Rearrangement with MYCN and MDM2 Amplification and RB1 Region Loss.

Rhabdomyosarcomas (RMS) are rare, heterogeneous, soft tissue sarcomas and a common type of childhood malignancy with a distinct histomorphology. At the molecular level, alveolar rhabdomyosarcoma (ARMS), a subtype of RMS, harbors a signature genetic makeup characterized by specific translocations. The type of translocation and associated genetic aberrations correlate with disease progression, hence we used multiple molecular modalities including high-resolution array comparative genomic hybridization to explore the oncogenic gene fusion and associated copy number variations in a case of metastatic ARMS. We describe a case where traditional cytogenetic and molecular methods yielded inconclusive results in detecting the FOXO1 gene rearrangement. However, microarray analysis identified the essential FOXO1-PAX7 aberration and additional submicroscopic genomic alterations, including amplification of MYCN and MDM2 and deletion of RB1.

Comprehensive genotype-phenotype correlations between NLRP7 mutations and the balance between embryonic tissue differentiation and trophoblastic proliferation.

BACKGROUND: Hydatidiform mole (HM) is a human pregnancy with excessive trophoblastic proliferation and abnormal embryonic development that may be sporadic or recurrent. In the sporadic form, the HM phenotype is driven by an abnormal ratio of paternal to maternal genomes, whereas in the recurrent form, the HM phenotype is caused by maternal-recessive mutations, mostly in NLRP7, despite the diploid biparental origin of the HM tissues. In this study, we characterised the expression of the imprinted, maternally expressed gene, CDKN1C (p57(KIP2)), the genotype, and the histopathology of 36 products of conception (POC) from patients with two defective alleles in NLRP7 and looked for potential correlations between the nature of the mutations in the patients and the various HM features. METHODS/RESULTS: We found that all the 36 POCs are diploid biparental and have the same parental contribution to their genomes. However, some of them expressed variable levels of p57(KIP2) and this expression was strongly associated with the presence of embryonic tissues of inner cell mass origin and mild trophoblastic proliferation, which are features of triploid partial HMs, and were associated with missense mutations. Negative p57(KIP2) expression was associated with the absence of embryonic tissues and excessive trophoblastic proliferation, which are features of androgenetic complete HMs and were associated with protein-truncating mutations. CONCLUSIONS: Our data suggest that NLRP7, depending on the severity of its mutations, regulates the imprinted expression of p57(KIP2) and consequently the balance between tissue differentiation and proliferation during early human development. This role is novel and could not have been revealed by any other approach on somatic cells.

Prenatal detection of del(10)(q11.2) mosaicism in chorionic villus specimens likely caused by a common chromosomal fragile site FRA10G is associated with a normal phenotype.

OBJECTIVE: To summarize the pregnancy outcomes of cases with mosaicism for chromosome 10q11.2 deletion detected by chorionic villus sampling (CVS) and determine whether extensive cytogenetic work-up and follow-up amniocentesis are necessary in such cases. METHODS: CVS was performed at 10-12 weeks of gestation. Chromosome analysis of chorionic villi was performed by standard G-banding techniques. RESULTS: Mosaicism of chromosome 10q11.2 deletion was observed in 24 out of 6063 CVS cases (0.39%). A common fragile site, FRA10G is located at the breakpoint region. The level of mosaicism ranged from 4% to 25%. No evidence of mosaic 10q11.2 deletion was found in follow-up amniocentesis, maternal peripheral blood cells, or from cytogenetic studies of other pregnancies from the same group of patients. All these cases resulted in the live birth of normal healthy infants. CONCLUSION: The presence of del(10)(q11.2) mosaicism in chorionic villus specimens most likely represents an in vitro culture artifact due to FRA10G fragile site in this region without any clinical consequences. If ultrasound results are normal, it is not necessary to perform follow-up amniocenteses and additional laboratory work-up for such cases.

The genetics of gestational trophoblastic disease: a rare complication of pregnancy.

Gestational choriocarcinoma is usually a rapidly spreading fatal disease, but it is curable if diagnosed early and treated. It is a unique malignancy that is a partial or complete allograft with a genotype that is not the same as the host genotype. It is most often preceded by an abnormal molar pregnancy. The surprising and unique androgenetic origin of complete hydatidiform molar pregnancies was first revealed by Kajii and Ohama in 1977. We describe the current understanding of the morphology, epidemiology and genetics of gestational trophoblastic disease that followed the milestone findings by Kajii and Ohama.

Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED12 mutations in majority of uterine leiomyomas.

Uterine leiomyomas (uterine fibroids) arise from smooth muscle tissue in the majority of women by age 45. It is common for these clonal tumors to develop from multiple locations within the uterus, leading to a variety of symptoms such as pelvic pain, abnormal uterine bleeding, and infertility. We performed whole exome sequencing on genomic DNA from five pairs of leiomyomas and corresponding normal myometrium to determine genetic variations unique to leiomyomas. Whole exome sequencing revealed that the gene encoding transcription factor MED12 (Mediator complex subunit 12) harbored heterozygous missense mutations caused by single nucleotide variants in highly conserved codon 44 of exon 2 in two of five leiomyomas. Sanger re-sequencing of MED12 among these five leiomyomas confirmed the two single nucleotide variants and detected a 42 base-pair deletion within exon 2 of MED12 in a third leiomyoma. MED12 was sequenced in an additional 143 leiomyomas and 73 normal myometrial tissues. Overall, MED12 was mutated in 100/148 (67%) of the genotyped leiomyomas: 79/148 (53%) leiomyomas exhibited heterozygous missense single nucleotide variants, 17/148 (11%) leiomyomas exhibited heterozygous in-frame deletions/insertion-deletions, 2/148 (1%) leiomyomas exhibited intronic heterozygous single nucleotide variants affecting splicing, and 2/148 (1%) leiomyomas exhibited heterozygous deletions/insertion-deletions spanning the intron 1-exon 2 boundary which affected the splice acceptor site. Mutations were not detected in MED12 in normal myometrial tissue. MED12 mutations were equally distributed among karyotypically normal and abnormal uterine leiomyomas and were identified in leiomyomas from both black and white American women. Our studies show an association between MED12 mutations and leiomyomas in ethnically and racially diverse American women.

Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings.

Uniparental disomy (UPD) is defined by the inheritance of both copies of a chromosome pair from one single parent. Although 23 cases of paternal UPD6 have been reported earlier, the occurrence of trisomy 6 rescue with paternal UPD6 has not been previously reported. The phenotype of paternal UPD6 results from biallelic expression of the maternally imprinted, paternally expressed ZAC and HYMAI genes, and includes transient neonatal diabetes mellitus (TNDM), intra-uterine growth restriction (IUGR), macroglossia, and minor anomalies. Trisomy rescue has been proposed as a pathogenic mechanism leading to UPD of other chromosomes. We report on the first case of a prenatally diagnosed infant with UPD6 and describe the clinical, cytogenetic, molecular, and novel placental findings in a female infant with paternal UPD6. Low-level trisomy 6 and paternal UPD6 were prenatally diagnosed through amniocentesis. After birth trisomy 6 was documented in the placenta but was not found in three different cell lines from the infant. The placenta was small with a peculiar pattern of vascular proliferation. Our results of trisomy 6 cells predominantly present in the placenta and only in low levels in the amniotic fluid suggest that the distribution and proportion of trisomic and diploid UPD cells contribute to the variability of fetal and placental phenotypes.

NLRP7 in the spectrum of reproductive wastage: rare non-synonymous variants confer genetic susceptibility to recurrent reproductive wastage.

BACKGROUND: NLRP7 mutations are responsible for recurrent molar pregnancies and associated reproductive wastage. To investigate the role of NLRP7 in sporadic moles and other forms of reproductive wastage, the authors sequenced this gene in a cohort of 135 patients with at least one hydatidiform mole or three spontaneous abortions; 115 of these were new patients. METHODS/RESULTS: All mutations were reviewed and their number, nature and locations correlated with the reproductive outcomes of the patients and histopathology of their products of conception. The presence of NLRP7 mutations was demonstrated in two patients with recurrent spontaneous abortions, and some rare non-synonymous variants (NSVs), present in the general population, were found to be associated with recurrent reproductive wastage. These rare NSVs were shown to be associated with lower secretion of interleukin 1ß and tumour necrosis factor and therefore to have functional consequences similar to those seen in cells from patients with NLRP7 mutations. The authors also attempted to elucidate the cause of stillbirths observed in 13% of the patients with NLRP7 mutations by examining available placentas of the stillborn babies and live births from patients with mutations or rare NSVs. A number of severe to mild placental abnormalities were found, all of which are known risk factors for perinatal morbidity. CONCLUSIONS: The authors recommend close follow-up of patients with NLRP7 mutations and rare NSVs to prevent the death of the rare or reduced number of babies that reach term.

Complex X chromosome rearrangement delineated by array comparative genome hybridization in a woman with premature ovarian insufficiency.

OBJECTIVE: To investigate candidate genes affected by a complex X chromosome rearrangement that may play a role in the diagnosis of spontaneous premature ovarian insufficiency (POI). DESIGN: Prospective cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis and oligonucleotide array comparative genome hybridization (CGH). SETTING: University medical center. PATIENT(S): A 36-year-old woman with POI found to have a highly rearrangement X chromosome. INTERVENTION(S): FISH analysis and oligonucleotide array CGH. MAIN OUTCOME MEASURE(S): Oligonucleotide microarray analysis to detect duplicated, deleted, or translocated regions of the X chromosome. RESULT(S): Complex rearrangement of the X chromosome involving ≥12 breakpoints resulting in two deletions, four duplications, and several intrachromosomal translocations. At least 13 genes with possible relevance to POI may be affected by the rearrangement. CONCLUSION(S): Array CGH can reveal candidate genes that may have essential roles in fertility and POI.

Simultaneous detection of imprinted gene expression (p57(KIP2)) and molecular cytogenetics (FICTION) in the evaluation of molar pregnancies.

OBJECTIVE: To simultaneously evaluate the p57(KIP2) antibody expression and genotype of individual cells from paraffin sections of molar pregnancies. STUDY DESIGN: Paraffin sections from 10 typical and unusual molar pregnancies were evaluated with the FICTION technique (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms), using immunofluorescence staining for the p57(KIP2) antibody and enumeration fluorescence in situ hybridization (FISH) probes. The unusual cases included androgenetic/ biparental chimeric complete hydatidiform moles (CHM) and mosaic partial hydatidiform moles (PHM). The unusual molar conceptions provided insight into interpreting atypical p57(KIP2) staining patterns and identifying androgenetic cells. RESULTS: The androgenetic/biparental chimeric CHMs demonstrated a negative p57(KIP2) Staining pattern for the androgenetic cells and positive staining for the biparental cells. Concordantly, the FISH results showed delineation between the androgenetic cells and the biparental cells, indicating 2 distinct genotypes. Also, in the 2 cases of mosaic PHM, the partial loss of p57(KIP2) antibody staining was due to mosaic loss of chromosome 11, assumed to be the maternal copy. This provides a biological explanation as to how false interpretation could occur when evaluating p57(KIP2) immunostaining results. CONCLUSION: The FICTION technique is a valuable ancillary tool for simultaneously evaluating the genotype and p57(KIP2) expression in unusual molar pregnancies.

P57KIP2 immunostaining and molecular cytogenetics: combined approach aids in diagnosis of morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions.

Although molar pregnancies are typically defined by morphological, histologic, and genetic criteria, most cases are diagnosed solely on histologic findings. Recently, several studies have demonstrated the usefulness of p57KIP2 immunostaining as an ancillary diagnostic tool for molar pregnancies. The p57KIP2 gene is paternally imprinted and maternally expressed; therefore, the positive staining of its protein indicates the presence of a functional maternal allele. Because complete hydatidiform moles (CHMs) lack a maternal genome, p57KIP2 immunostaining is absent. Previous studies have validated this staining technique by demonstrating differential nuclear expression in CHMs versus non-CHMs; however, these studies have not included cytogenetic analysis. We report on 58 cases of hydropic placentas, correlating cytogenetic and p57KIP2 immunostaining results. In addition, cases with unusual p57KIP2 staining patterns are discussed. Also included are 2 mosaic conceptions (1 diploid/triploid and 1 diploid/tetraploid), 6 chimeric/mosaic conceptions with androgenetic/biparental cell lines, and 2 cases of placental mesenchymal dysplasia.

The origin of trisomy 13.

Trisomy 13 is one of the most common trisomies in clinically recognized pregnancies and one of the few trisomies identified in liveborns, yet relatively little is known about the errors that lead to trisomy 13. Accordingly, we initiated studies to investigate the origin of the extra chromosome in 78 cases of trisomy 13. Our results indicate that the majority of cases (>91%) are maternal in origin and, similar to other autosomal trisomies, the extra chromosome is typically due to errors in meiosis I. Surprisingly, however, a large number of errors also occur during maternal meiosis II ( approximately 37%), distinguishing trisomy 13 from other acrocentric and most nonacrocentric chromosomes. As with other trisomies, failure to recombine is an important contributor to nondisjunction of chromosome 13.