Universal chromosomal microarray analysis reveals high proportion of copy-number variants in low-risk pregnancies.

OBJECTIVES: To evaluate the yield and utility of the routine use of chromosomal microarray analysis (CMA) for prenatal genetic diagnosis in a large cohort of pregnancies with normal ultrasound (US) at the time of genetic testing, compared with pregnancies with abnormal US findings. METHODS: We reviewed all prenatal CMA results in our center between November 2013 and December 2018. The prevalence of different CMA results in pregnancies with normal US at the time of genetic testing ('low-risk pregnancies'), was compared with that in pregnancies with abnormal US findings ('high-risk pregnancies'). Medical records were searched in order to evaluate subsequent US follow-up and the outcome of pregnancies with a clinically relevant copy-number variant (CNV), i.e. a pathogenic or likely pathogenic CNV or a susceptibility locus for disease with > 10% penetrance, related to early-onset disease in the low-risk group. RESULTS: In a cohort of 6431 low-risk pregnancies that underwent CMA, the prevalence of a clinically significant CNV related to early-onset disease was 1.1% (72/6431), which was significantly lower than the prevalence in high-risk pregnancies (4.9% (65/1326)). Of the low-risk pregnancies, 0.4% (27/6431) had a pathogenic or likely pathogenic CNV, and another 0.7% (45/6431) had a susceptibility locus with more than 10% penetrance. Follow-up of the low-risk pregnancies with a clinically significant early-onset CNV revealed that 31.9% (23/72) were terminated, while outcome data were missing in 26.4% (19/72). In 16.7% (12/72) of low-risk pregnancies, an US abnormality was discovered later on in gestation, after genetic testing had been performed. CONCLUSION: Although the background risk of identifying a clinically significant early-onset abnormal CMA result in pregnancies with a low a-priori risk is lower than that observed in high-risk pregnancies, the risk is substantial and should be conveyed to all pregnant women. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Single-nucleotide polymorphism-based chromosomal microarray analysis provides clues and insights into disease mechanisms.

OBJECTIVE: Chromosomal microarray analysis (CMA) is the modality of choice for prenatal diagnosis in pregnancy with fetal malformation, as it has a high diagnostic yield for microdeletion/duplication syndromes. The aim of this study was to demonstrate the additional utility of single-nucleotide polymorphism (SNP)-based CMA in diagnosing monogenic diseases, imprinting disorders and uniparental disomy (UPD). METHODS: CMA was performed using Affymetrix CytoScan array, for all indications in 6995 pregnancies, at a tertiary referral hospital from November 2013 to June 2018. We describe four cases that had a CMA result that provided a more comprehensive understanding of the complex genetic mechanisms underlying the clinical presentation. RESULTS: In the first fetus, CMA was performed due to intrauterine growth restriction and revealed a 75 kbp maternally inherited microdeletion encompassing the Bloom syndrome gene (BLM). A diagnosis of Bloom syndrome was made upon identifying a paternally inherited common Ashkenazi founder mutation. In the second case, CMA was performed due to severely abnormal maternal serum analytes and revealed a deletion in 14q32.2q32.31 on the maternally inherited copy, leading to a diagnosis of Kagami-Ogata syndrome, which is an imprinting disorder. In the third case, amniocentesis was performed because of late-onset fetal macrosomia and mild polyhydramnios. CMA detected a deletion encompassing the locus of Prader-Willi/Angelman syndrome. In the fourth case, amniocentesis was performed due to maternal cytomegalovirus seroconversion. Maternal UPD of the entire long arm of chromosome 11 was detected. CONCLUSION: Prenatal CMA, based on oligo and SNP platforms, increases the diagnostic yield and enables a wider spectrum of disorders to be detected through the identification of complex genetic etiologies beyond only copy number variants. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Role of late amniocentesis in the era of modern genomic technologies.

OBJECTIVE: Traditionally, amniocentesis is performed between 17 and 23 weeks of gestation. This enables decisions regarding the course of pregnancy to be made before viability. Less frequently, amniocentesis is performed in the third trimester. Advanced genomic technologies such as chromosomal microarray analysis (CMA) provide more detailed information about the fetus compared with traditional G-banded chromosomal analysis. The aim of this study was to assess the indications for and safety of late amniocentesis, genetic-test results (especially in the context of CMA technology) and outcome of pregnancies that underwent the procedure after 24 weeks. METHODS: Medical records were analyzed retrospectively of all women in whom amniocentesis was performed at a gestational age of 24 + 0 to 38 + 6 weeks, at Hadassah Medical Center, between June 2013 and March 2017. Parameters investigated included indications for late amniocentesis, complications, CMA results and pregnancy outcome. RESULTS: During the study period, 291 women (303 fetuses, 277 singleton and 14 twin pregnancies; in two twin pairs, one fetus was terminated before amniocentesis) underwent late amniocentesis. CMA was performed in all instances of amniocentesis. The most frequent indication was abnormal sonographic finding(s) (204/303 fetuses, 67%). Preterm delivery occurred in 1.7% and 5.1% of pregnancies within the first week and within 1 month following the procedure, respectively. Aneuploidy was detected in nine (3%) fetuses and nine (3%) others had a pathogenic/likely pathogenic copy number variant, suggesting that CMA doubled the diagnostic yield of traditional karyotyping. Maximal diagnostic yield (17.5%) was achieved for the subgroup of fetuses referred with abnormal sonographic findings in two or more fetal anatomical systems. Variants of uncertain significance or susceptibility loci were found in another nine (3%) fetuses. CONCLUSIONS: In pregnancies undergoing late amniocentesis, CMA increased detection rates of fetal abnormalities and had a shorter turnaround time compared with traditional chromosomal analysis; therefore, late amniocentesis may serve as a helpful tool for detecting fetal abnormalities or reassuring parents following late-appearing abnormal sonographic findings. However, CMA may expose findings of uncertain significance, about which the couple should be precounseled. The procedure appears to be safe. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Uranium series dates from Qesem Cave, Israel, and the end of the Lower Palaeolithic.

Israel is part of a geographical 'out of Africa' corridor for human dispersals. An important event in these dispersals was the possible arrival of anatomically modern humans in the Levant during the late Middle Pleistocene. In the Levant the Lower Palaeolithic ends with the Acheulo-Yabrudian complex, characterized by technological developments, including the introduction of technological innovations such as the systematic production of blades and the disappearance of hand-axes. These reflect new human perceptions and capabilities in lithic technology and tool function. Qesem Cave, discovered in 2000, has a rich, well-preserved Acheulo-Yabrudian deposit holding great promise for providing new insights into the period. Here we report the dates of this deposit obtained by uranium isotopic series on associated speleothems and their implications. The results shed light on the temporal range of the Acheulo-Yabrudian and the end of the Lower Palaeolithic, suggesting a long cultural phase between the Lower Palaeolithic Acheulian and the Middle Palaeolithic Mousterian phases, starting before 382 kyr ago and ending at about 200 kyr ago.

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

Clinical, cellular, and molecular features of an Israeli xeroderma pigmentosum family with a frameshift mutation in the XPC gene: sun protection prolongs life.

An Ashkenazi Jewish Israeli family with two children affected with severe xeroderma pigmentosum was investigated. A son, XP12TA, developed skin cancer at 2 y and died at 10 y. A daughter, XP25TA, now 24 y old, was sun protected and began developing skin cancers at 10 y. Their cultured skin fibroblasts showed reductions in post-ultraviolet survival (11% of normal), unscheduled DNA synthesis (10% of normal), global genome DNA repair (15% of normal), and plasmid host cell reactivation (5% of normal). Transcription-coupled DNA repair was normal, however. Northern blot analysis revealed greatly reduced xeroderma pigmentosum complementation group C mRNA. A plasmid host cell reactivation assay assigned the cells to xeroderma pigmentosum complementation group C. Cells from both parents and an unaffected child exhibited normal post-ultraviolet-C survival and normal DNA repair. Sequencing the xeroderma pigmentosum complementation group C cDNA of XP12TA and XP25TA revealed a homozygous deletion of two bases (del AT 669-670) in exon 5 with a new termination site 10 codons downstream that is expected to encode a truncated xeroderma pigmentosum complementation group C protein. Sequence analysis of the xeroderma pigmentosum complementation group C cDNA in cells from the parents found identical heterozygous mutations: one allele carries both the exon 5 frameshift and an exon 15 polymorphism and the other allele carries neither alteration. Cells from the unaffected brother had two normal xeroderma pigmentosum complementation group C alleles. This frameshift mutation in the xeroderma pigmentosum complementation group C gene led to reduced DNA repair with multiple skin cancers and early death. Sun protection delayed the onset of skin cancer and prolonged life in a sibling with the same mutation.

Identification of the gene causing mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12?15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.

Reversal of nephrotic syndrome due to AA amyloidosis in psoriatic patients on long-term colchicine treatment. Case report and review of the literature.

A case of nephrotic syndrome due to AA amyloidosis in a young woman suffering from erythrodermic psoriasis and psoriatic arthropathy is reported. The nephrotic syndrome regressed completely during long-term (57 months) colchicine treatment. There are 39 case reports in the literature of psoriasis associated with amyloidosis. More than 85% of these patients had concomitant arthropathy. This suggests that arthritis may be an important factor in the appearance of amyloidosis. 59% of psoriatics with amyloidosis had renal failure and 56% of them died shortly after diagnosis of amyloidosis. These observations support the view that amyloidosis associated with psoriasis is an aggressive disease that may be fatal. However, the clinical course of our patient suggests that renal amyloidosis associated with psoriasis may be successfully treated by colchicine.

F-Spondin is required for accurate pathfinding of commissural axons at the floor plate.

The commissural axons project toward and across the floor plate. They then turn into the longitudinal axis, extending along the contralateral side of the floor plate. F-spondin, a protein produced and secreted by the floor plate, promotes adhesion and neurite extension of commissural neurons in vitro. Injection of purified F-spondin protein into the lumen of the spinal cord of chicken embryos in ovo resulted in longitudinal turning of commissural axons before reaching the floor plate, whereas neutralizing antibody (Ab) injections caused lateral turning at the contralateral floor plate boundary. These combined in vitro and in vivo results suggest that F-spondin is required to prevent the lateral drifting of the commissural axons after having crossed the floor plate.

F-spondin and mindin: two structurally and functionally related genes expressed in the hippocampus that promote outgrowth of embryonic hippocampal neurons.

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.

Synthesis of 1,2,3,4-tetrazino[5,6-f]benzo-1,2,3,4-tetrazine 1,3,7,9-tetra-N-oxides.

[reaction: see text] The novel heterocyclic system 10 with the phenanthrene type skeleton, in which the benzene ring is annulated with two 1,2,3,4-tetrazine 1,3-di-N-oxide rings, is of considerable interest in the context of the high nitrogen system stability and from a heteroaromaticity standpoint. The step-by-step synthetic approach to this system involves treatment of 5 with N2O5, resulting in the first 1,2,3,4-tetrazine 1,3-dioxide ring formation. The following displacement of the chlorine atom at the 6-position with ammonia and subsequent repeated treatment with N2O5 results in the second 1,2,3,4-tetrazine 1,3-dioxide ring formation. The structure of 10 was confirmed by a 13C and 14N NMR study.

Accumulation of F-spondin in injured peripheral nerve promotes the outgrowth of sensory axons.

F-spondin, an extracellular matrix protein, is present in peripheral nerve during embryonic development, but its amount diminishes by birth. Axotomy of adult rat sciatic nerve, however, causes a massive upregulation of both F-spondin mRNA and protein distal to the lesion. F-spondin in the distal stump of axotomized nerve promotes neurite outgrowth of sensory neurons, as revealed by protein neutralization with F-spondin-specific antibodies. Thus, F-spondin is likely to play a role in promoting axonal regeneration after nerve injury.

Identification of Sonic hedgehog as a candidate gene responsible for holoprosencephaly.

Holoprosencephaly (HPE) is a genetically and phenotypically heterogenous disorder involving the development of forebrain and midface, with an incidence of 1:16,000 live born and 1:250 induced abortions. This disorder is associated with several distinct facies and phenotypic variability: in the most extreme cases, anophthalmia or cyclopia is evident along with a congenital absence of the mature nose. The less severe form features facial dysmorphia characterized by ocular hypertelorism, defects of the upper lip and/or nose, and absence of the olfactory nerves or corpus callosum. Several intermediate phenotypes involving both the brain and face have been described. One of the gene loci, HPE3, maps to the terminal band of chromosome 7. We have performed extensive physical mapping studies and established a critical interval for HPE3, and subsequently identified the sonic hedgehog (SHH) gene as the prime candidate for the disorder. SHH lies within 15-250 kilobases (kb) of chromosomal rearrangements associated with HPE, suggesting that a 'position effect' has an important role in the aetiology of HPE. As detailed in the accompanying report, this role for SHH is confirmed by the detection of point mutations in hereditary HPE patients.

A role for CdxA in gut closure and intestinal epithelia differentiation.

CdxA is a homeobox gene of the caudal type that was previously shown to be expressed in the endoderm-derived gut epithelium during early embryogenesis. Expression of the CDXA protein was studied during intestine morphogenesis from stage 11 (13 somites) to adulthood in the chicken. The CDXA protein can be detected during all stages of gut closure, from stage 11 to 5 days of incubation, and is mainly localized to the intestinal portals, the region where the splanchnopleure is undergoing closure. In this region, which represents the transition between the open and closed gut, the CDXA protein is restricted to the endoderm-derived epithelium. At about day 5 of incubation, the process of formation of the previllous ridges begins, which marks the beginning of the morphogenesis of the villi. From this stage to day 11 expression of CDXA is localized to the epithelial lining of the intestine. In parallel, a gradual increase in CDXA protein expression begins in the mesenchyme that is close in proximity to the CDXA-positive endoderm. Maximal CDXA levels in the mesenchyme are observed at day 9 of incubation. During days 10 and 11 CDXA levels in the mesenchyme remain constant, and by day 12 CDXA becomes undetectable in these cells and the epithelium again becomes the main site of expression. From day 12 of incubation until adulthood the CDXA protein is present in the intestinal epithelium. Until day 18 of incubation expression can be detected along the whole length of the villus with a stronger signal at the tip. With hatching the distribution along the villi changes so that the main site of CDXA protein expression is at the base of the villi and in the crypts. The transient expression of CDXA in the mesenchyme between days 5 and 11 may be related to the interactions taking place between the mesenchyme and the epithelium that ultimately result in the axial specification of the alimentary canal and the differentiation of its various epithelia. The main CDXA spatial distribution during morphogenesis suggests a tight linkage to the formation and differentiation of the intestinal epithelium itself. CDXA appears to play a role in the morphogenetic events leading to closure of the alimentary canal. During previllous ridge formation the CDXA protein is transiently expressed in the mesenchymal cells thought to provide instructive interactions for the regionalization and differentiation of the gut epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

The chicken CdxA homeobox gene and axial positioning during gastrulation.

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

Unsuspected involvement of the female genitalia in pemphigus vulgaris.

A 56-year-old woman had erosions due to pemphigus vulgaris in the inner thighs and perineum. The cutaneous lesions cleared following intramuscular gold therapy. However, because of complaints of dyspareunia, a colposcopic examination was performed and involvement of the cervix was demonstrated. The need for a vaginal examination in the monitoring of pemphigus vulgaris is emphasized.

Delayed reaction to suture material simulating melanoma.

A twenty-one-year-old woman who had been involved in an accident sustained severe trauma to the left side of her face and scalp. Because of deterioration in her neurologic condition, a number of exploratory burr holes were drilled in her skull. Fifteen years later, a 6 mm black tender nodule was noted on the left temple, close to one of the burr holes. Nodular melanoma was suspected, but results of microscopic examination proved the lesion to be a granuloma with transepidermal elimination of exogenous filamentous material. The relationship between foreign bodies and cutaneous neoplasia, genuine or simulated, is noted briefly.