Lessons learned from expanded reproductive carrier screening in self-reported Ashkenazi, Sephardi, and Mizrahi Jewish patients.

BACKGROUND: Next-generation sequencing (NGS)-based panels have gained traction as a strategy for reproductive carrier screening. Their value for screening Ashkenazi Jewish (AJ) individuals, who have benefited greatly from population-wide targeted testing, as well as Sephardi/Mizrahi Jewish (SMJ) individuals (an underserved population), has not been fully explored. METHODS: The clinical utilization by 6,805 self-reported Jewish individuals of an expanded NGS panel, along with several ancillary assays, was assessed retrospectively. Data were extracted for a subset of 96 diseases that, during the panel design phase, were classified as being AJ-, SMJ-, or pan-Jewish/pan-ethnic-relevant. RESULTS: 64.6% of individuals were identified as carriers of one or more of these 96 diseases. Over 80% of the reported variants would have been missed by following recommended AJ screening guidelines. 10.7% of variants reported for AJs were in "SMJ-relevant genes," and 31.2% reported for SMJs were in "AJ-relevant genes." Roughly 2.5% of individuals carried a novel, likely pathogenic variant. One in 16 linked cohort couples was identified as a carrier couple for at least one of these 96 diseases. CONCLUSION: For maximal carrier identification, this study supports using expanded NGS panels for individuals of all Jewish backgrounds. This approach can better empower at-risk couples for reproductive decision making.

Experiences from a pilot program bringing BRCA1/2 genetic screening to theUS Ashkenazi Jewish population.

PURPOSE: The notion of offering population-based screening to the Ashkenazi Jewish (AJ) population for the BRCA1/2 founder mutations continues to gain support. A program called the BRCAcommunity initiative was designed to identify the benefits and barriers associated with implementing this screening in a clinical setting. METHODS: Interested AJ individuals were stratified into high-risk (HR) and low-risk (LR) groups based on self-reported cancer histories. Those at HR were offered traditional genetic counseling/testing; those at LR were offered group genetic counseling and subsidized AJ BRCA founder mutation testing. RESULTS: During the pilot year, 62% of initial registrants and 53% of ultimate study participants were classified into the HR group. Among the 101 HR and 88 LR study participants, 8 and 2 BRCA carriers were identified, respectively. The LR carriers would have been missed by current mechanisms. Survey responses provided insight into the motivations and fears associated with pursuing testing, the efficacy of the initiative design, and challenges that exist on multiple levels, including the community, health-care providers, and insurance coverage. CONCLUSION: Although the medical value of identifying presymptomatic BRCA carriers in Ashkenazi Jews is evident, further measures need to be taken before this effort can be accomplished on a large scale.Genet Med advance online publication 13 October 2016.

From Campers to Counselors: a Resource for Prospective Genetic Counseling Students.

When thinking about the future of the genetic counseling field, one place to start is with prospective genetic counseling graduate school applicants. Although resources and mentorship opportunities exist for genetic counselors entering the field, the process of deciding on a career, applying to graduate programs, and being admitted can be daunting. As members of the profession, we should take responsibility for ensuring that individuals have the information and resources necessary to make an educated decision about whether genetic counseling is the correct path for them and to take the initial steps along this path. In this article, we present our Genetic Counseling Boot Camp as a model for other genetic counselors to use in developing their own local programs. This type of program can benefit prospective genetic counselors as they begin their professional journeys and can also provide value for the organizers and presenters who are already seasoned in the field.

The Ashkenazi Jewish carrier screening panel: evolution, status quo, and disparities.

OBJECTIVE: Although prenatal/preconception carrier screening recommendations for individuals of Ashkenazi Jewish descent (AJ) were published by American College of Medical Genetics and Genomics (2008) and American College of Obstetrics and Gynecology (2009), scientific advances have led to widely varied screening panels. Mutation carrier frequencies are sometimes based on small, homogeneous AJ populations. This study sought to update the state of AJ screening for the obstetrician by assessing laboratory screening panel compositions as well as assessing literature and laboratory carrier frequencies for common AJ mutations. METHODS: A literature review (1991-2013) was performed for AJ disease carrier frequencies. AJ screening data from six screening laboratories were collected. AJ panel composition was compared across 16 commercial and academic laboratories. RESULTS: Overall literature and laboratory carrier frequencies of AJ mutations were similar, although the Walker-Warburg syndrome laboratory carrier frequency was almost twice that in the literature. Laboratory AJ disease panel composition varied widely, from 8 to 25 diseases. CONCLUSIONS: Current AJ panels vary widely by laboratory, resulting in disparate levels of screening. Consideration of an updated professional standard for prenatal/preconception AJ screening based on carrier frequency rates, level of disease burden, availability of screening, and cost of technology may be useful in providing equitable and appropriate care for those planning a pregnancy.

Carrier testing for Ashkenazi Jewish disorders in the prenatal setting: navigating the genetic maze.

Exciting developments in the fields of genetics and genomics have facilitated the identification of the etiological basis of many Mendelian disorders. Several of the methods used in gene discovery have focused initially on homogeneous populations, including the Ashkenazi Jewish population. The founder effect is well recognized in this community, in which historical events and cultural behaviors have resulted in a limited number of mutations underlying genetic disorders with substantial health impact. New technologies have made it possible to rapidly expand the test panels, changing testing paradigms, and thereby creating challenges for the physician in deciphering the appropriate approach to genetic screening in this population. The goal of this review is to help primary obstetric health care providers navigate through this quickly moving field so as to better counsel and support their patients of Ashkenazi Jewish heritage.

Detection of carriers in the Ashkenazi Jewish population: an objective comparison of high-throughput genotyping versus gene-by-gene testing.

BACKGROUND: High-throughput genotyping allows rapid identification of targeted mutations at a fraction of the cost of current gene-by-gene testing methodologies. An objective comparison of the two methodologies allows providers to assess the clinical validity/utility of high-throughput carrier screening and establish a comfort level with new genomic technologies. AIM: To verify that high-throughput genotyping accurately determines patient carrier status, DNA samples from previously identified carriers (n=31) of Ashkenazi Jewish genetic diseases were anonymized and submitted for retesting by high-throughput genotyping. RESULTS: The results were 100% concordant (95% CI: 0.998-1), demonstrating that high-throughput genotyping assays accurately identify carriers of targeted mutations in the Ashkenazi Jewish population. In addition, carrier status for diseases and mutations not previously tested was uncovered using the high-throughput assay. CONCLUSIONS: High-throughput genotyping is a cost-effective and clinically valid approach to carrier screening. The use of a broader screen for Ashkenazi Jewish individuals increases the detection of carriers in this population.

The p.L302P mutation in the lysosomal enzyme gene SMPD1 is a risk factor for Parkinson disease.

OBJECTIVE: To study the possible association of founder mutations in the lysosomal storage disorder genes HEXA, SMPD1, and MCOLN1 (causing Tay-Sachs, Niemann-Pick A, and mucolipidosis type IV diseases, respectively) with Parkinson disease (PD). METHODS: Two PD patient cohorts of Ashkenazi Jewish (AJ) ancestry, that included a total of 938 patients, were studied: a cohort of 654 patients from Tel Aviv, and a replication cohort of 284 patients from New York. Eight AJ founder mutations in the HEXA, SMPD1, and MCOLN1 genes were analyzed. The frequencies of these mutations were compared to AJ control groups that included large published groups undergoing prenatal screening and 282 individuals matched for age and sex. RESULTS: Mutation frequencies were similar in the 2 groups of patients with PD. The SMPD1 p.L302P was strongly associated with a highly increased risk for PD (odds ratio 9.4, 95% confidence interval 3.9-22.8, p < 0.0001), as 9/938 patients with PD were carriers of this mutation compared to only 11/10,709 controls. CONCLUSIONS: The SMPD1 p.L302P mutation is a novel risk factor for PD. Although it is rare on a population level, the identification of this mutation as a strong risk factor for PD may further elucidate PD pathogenesis and the role of lysosomal pathways in disease development.

Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry.

Rapid and novel prenatal molecular assay for detecting aneuploidies and microdeletion syndromes.

OBJECTIVES: To develop a targeted aneuploidy and microdeletion detection platform for use in the prenatal setting, to assess the integrity of the platform with a robust validation system, and to prospectively determine the performance of the platform under routine clinical conditions. METHODS: To generate proxies for the various disorders assessed by the assay for analytical validation purposes, cells from ten microdeletion syndromes as well as from common aneuploidies were spiked into cleared amniotic fluid. Genomic DNA was isolated, labeled, and hybridized to microbeads that have been coupled to DNA derived from Bacterial Artificial Chromosome (BAC) from the relevant regions targeted by the array. Beads were read using a flow cytometric multiplex bead array detection system. In the prospective part of the study, 104 amniotic fluid samples were collected and analyzed. RESULTS: All microdeletion syndromes and aneuploidies were validated in a blinded fashion. In the prospective study, the total number of readable samples was 101 of 104 (97%). All sample results were confirmed independently. CONCLUSION: The bead array approach is a rapid and reliable test for detecting aneuploidies and microdeletions. This assay has the potential to provide the benefit of expanded molecular cytogenetic testing to pregnant women undergoing invasive prenatal diagnosis. This approach may be especially useful in parts of the world where cytogenetic personnel and facilities may be limited.

Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice.

Kaiso contributes to DNA methylation-dependent silencing of tumor suppressor genes in colon cancer cell lines.

Aberrant CpG methylation of tumor suppressor gene regulatory elements is associated with transcriptional silencing and contributes to malignant transformation of different tissues. It is presumed that methylated DNA sequences recruit repressor machinery to actively shutdown gene expression. The Kaiso protein is a transcriptional repressor expressed in human and murine colorectal tumors that can bind to methylated clusters of CpG dinucleotides. We show here that Kaiso represses methylated tumor suppressor genes and can bind in a methylation-dependent manner to the CDKN2A in human colon cancer cell lines. The contribution of Kaiso to epigenetic silencing was underlined by the fact that Kaiso depletion induced tumor suppressor gene expression without affecting DNA methylation levels. As a consequence, colon cancer cells became susceptible to cell cycle arrest and cell death mediated by chemotherapy. The data suggest that Kaiso is a methylation-dependent "opportunistic" oncogene that silences tumor suppressor genes when they become hypermethylated. Because Kaiso inactivation sensitized colon cancer cell lines to chemotherapy, it is possible that therapeutic targeting of Kaiso could improve the efficacy of current treatment regimens.

Rybp interacts with Hippi and enhances Hippi-mediated apoptosis.

Rybp (DEDAF) has been shown to interact with DED-containing proteins and to encode pro-apoptotic functions. Here we characterize a novel interaction between Rybp and Hippi, a protein implicated in neuronal apoptosis as well as in the pathogenesis of Huntington's disease. Rybp can synergize with Hippi to enhance Caspase 8-mediated apoptosis and also appears to be essential for Hippi-mediated apoptosis. Moreover, Rybp may mediate or regulate the interaction between Hippi and Caspase 8. Finally, Rybp and Hippi co-localize in a subset of neurons in the developing mouse brain. Together, these findings suggest that Rybp and Hippi may functionally interact in the apoptotic processes that accompany normal murine neural development.

Differential effects of Mxi1-SRalpha and Mxi1-SRbeta in Myc antagonism.

Mxi1 belongs to the Myc-Max-Mad transcription factor network. Two Mxi1 protein isoforms, Mxi1-SRalpha and Mxi1-SRbeta, have been described as sharing many biological properties. Here, we assign differential functions to these isoforms with respect to two distinct levels of Myc antagonism. Unlike Mxi1-SRbeta, Mxi1-SRalpha is not a potent suppressor of the cellular transformation activity of Myc. Furthermore, although Mxi1-SRbeta exhibits a repressive effect on the MYC promoter in transient expression assays, Mxi1-SRalpha activates this promoter. A specific domain of Mxi1-SRalpha contributes to these differences. Moreover, glyceraldehyde-3-phosphate dehydrogenase interacts with Mxi1-SRalpha and enhances its ability to activate the Myc promoter. Our findings suggest that Mxi1 gains functional complexity by encoding isoforms with shared and distinct activities.

Rybp, a polycomb complex-associated protein, is required for mouse eye development.

BACKGROUND: Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. RESULTS: Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- <-> Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of AP-2alpha and Sox2, and reduced levels of betaA4-crystallin gene expression. Ubiquitous transgenic overexpression of Rybp in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development. CONCLUSION: These studies establish Rybp as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as BCOR, CBP, Chx10, Pax2, Pax6, Six3, Ski, Vax1 and Vax2. We propose that the multiple functions for Rybp in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.

The Polycomb-associated protein Rybp is a ubiquitin binding protein.

The Rybp protein has been promoted as a Polycomb group (PcG)-associated protein, but its molecular function has remained elusive. Here we show that Rybp is a novel ubiquitin binding protein and is itself ubiquitinated. The Rybp interacting PcG protein Ring1B, a known ubiquitin E3 ligase, promotes Rybp ubiquitination. Moreover, one target of Rybp's ubiquitin binding domain appears to be ubiquitinated histone H2A; this histone is a substrate for Ring1B's E3 ligase activity in association with gene silencing processes. These findings on Rybp provide a further link between the ubiquitination system and PcG transcriptional repressors.

Yaf2 inhibits caspase 8-mediated apoptosis and regulates cell survival during zebrafish embryogenesis.

Rybp (DEDAF) is a member of the Rybp/Yaf2 protein family and has been shown to encode pro-apoptotic functions and to be essential for mouse embryogenesis. The related Yaf2 protein has not been studied extensively at the cellular or organismal levels. Here we describe zebrafish yaf2 (zyaf2) and show that it is widely expressed during early embryogenesis, with subsequent enrichment of transcripts in the anterior head region. Depletion of zYaf2 during embryogenesis using specific morpholinos activates a wide-spread program of apoptosis and causes developmental arrest before the one somite stage. Partial depletion of Yaf2, achieved by injecting lower dosages of morpholino, circumvents the early arrest but leads to CNS degeneration associated with excessive apoptosis. These phenotypes can be rescued by co-injection of human YAF2 mRNA with the morpholinos or by treatment with a pan-caspase inhibitor or a caspase 8-specific inhibitor. Finally, the observed activation of caspase 8 in the morphants is in accord with the ability of Yaf2 to inhibit caspase 8-mediated apoptosis in cultured cells. Our findings implicate Yaf2 as a survival factor during early zebrafish development and organogenesis. This may suggest that Yaf2 and Rybp can encode opposing functions in the regulation of apoptosis.

Rybp/DEDAF is required for early postimplantation and for central nervous system development.

The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells. Consistent with this result, rybp homozygous null embryos exhibited lethality at the early postimplantation stage. At this time, Rybp was essential for survival of the embryo, for the establishment of functional extraembryonic structures, and for the execution of full decidualization. Through the use of a chimeric approach, the embryonic lethal phenotype was circumvented and a role for Rybp in central nervous system development was uncovered. Specifically, the presence of Rybp-deficient cells resulted in marked forebrain overgrowth and in localized regions of disrupted neural tube closure. Functions for Rybp in the brain also were supported by the finding of exencephaly in about 15% of rybp heterozygous mutant embryos, and by Rybp's distinct neural expression pattern. Together, these findings support critical roles for Rybp at multiple stages of mouse embryogenesis.

Mxi1-SRalpha: a novel Mxi1 isoform with enhanced transcriptional repression potential.

Mxi1 belongs to the Myc/Max/Mad network of proteins that have been implicated in the control of multiple aspects of cellular behavior. Previously, we had reported that the mouse mxi1 gene gives rise to two distinct transcript forms that can encode proteins with dramatically different functional abilities. The Mxi1-SR protein (here termed Mxi1-SRbeta) can interact with Sin3/histone deacetylase and function as a potent transcriptional repressor and growth suppressor, while the Mxi1-WR protein lacks these activities. Here, we describe a new mxi1-derived transcript form (termed mxi1-SRalpha) whose expression is governed by its own promoter, resulting in a spatiotemporally distinct expression profile from that of the highly related mxi1-SRbeta form. Moreover, the Mxi1-SRalpha protein product, with its unique Sin3 interacting domain, has a greater affinity than its Mxi1-SRbeta counterpart for the Sin3 adapter proteins as well as an enhanced potential for transcriptional repression in transient reporter assays. Our identification of this novel Mxi1 isoform that results from alternative 5' exon usage adds an additional layer of complexity to the Mad/Mxi1 family. In addition, our findings warrant re-evaluation of mxi1 expression patterns on the cellular level and its status in human cancer samples, with a renewed focus on the distinct isoforms.

Prostatic intraepithelial neoplasia and intestinal metaplasia in prostates of probasin-RAS transgenic mice.

BACKGROUND: Activation of the RAS pathway has been implicated in the pathogenesis of many types of human cancers, including prostate cancer. Here we employed a transgenic approach to assess the potential contribution of RAS to prostate carcinogenesis. METHODS: Probasin-RAS (Pb-RAS) transgenic mice were generated and shown to express high levels of activated RAS in the prostate lobes. Transgenic prostates were compared to normal controls by histology and immunohistochemistry with relevant markers. RESULTS: Pb-RAS transgenic prostates exhibit neoplastic changes including low-grade prostatic intraepithelial neoplasia, and metaplastic changes towards an intestinal goblet cell phenotype. The finding of high levels of the goblet cell-specific peptide Itf/Tff3 in these transgenic prostates is in accordance with recent microarray studies showing that ITF/TFF3 is upregulated in human prostate cancer samples. CONCLUSIONS: The Pb-RAS mouse model could be useful for elucidating the early events in prostate carcinogenesis, as well as for studying the mechanisms and potential prostate cancer relevance of intestinal metaplasia.