Chromosomal microarray as primary diagnostic genomic tool for pregnancies at increased risk within a population-based combined first-trimester screening program.

OBJECTIVE: To evaluate the performance of high-resolution chromosomal microarray (CMA) as the standard diagnostic approach for genomic imbalances in pregnancies with increased risk based on combined first-trimester screening (cFTS). METHODS: This was a retrospective study of genomic findings in a cohort of 575 consecutive pregnancies undergoing invasive testing because of a cFTS risk ≥ 1:300 on a publicly funded population-based screening program in the Central and Northern Regions of Denmark, between September 2015 and September 2016. Women with fetal nuchal translucency thickness ≥ 3.5 mm or opting for non-invasive prenatal testing (NIPT) were excluded. Comparative genomic hybridization was performed using a 180-K oligonucleotide array on DNA extracted directly from chorionic villus/amniocentesis samples. Genomic outcomes were reported in relation to cFTS findings. RESULTS: Of the 575 pregnancies that underwent invasive testing, CMA detected 22 (3.8% (95% CI, 2.5-5.7%)) cases of trisomies 21, 18 and 13, 14 (2.4% (95% CI, 1.4-4.0%)) cases of other types of aneuploidy and 15 (2.6% (95% CI, 1.5-4.3%)) cases with a pathogenic or probably pathogenic copy number variant (CNV). Of the 15 CNVs, three were > 10 Mb and would probably have been detected by chromosomal analysis, but the other 12 would most probably not have been detected using conventional cytogenetic techniques; therefore, the overall detection rate of CMA (8.9% (95% CI, 6.8-11.5%)) was significantly higher than that estimated for conventional cytogenetic analysis (6.8% (95% CI, 5.0-9.1%)) (P = 0.0049). Reducing the cFTS risk threshold for invasive diagnostic testing to 1 in 100 or 1 in 50 would have led, respectively, to 60% or 100% of the pathogenic CNVs being missed. CONCLUSIONS: CMA is a valuable diagnostic technique that can identify an increased number of genomic aberrations in pregnancies at increased risk on cFTS. Limiting diagnostic testing to pregnancies with a risk above 1 in 100 or 1 in 50, as proposed in contingent NIPT/invasive testing models, would lead to a significant proportion of pathogenic CNVs being missed at first-trimester screening. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Case of successful IVF treatment of an oligospermic male with 46,XX/46,XY chimerism.

INTRODUCTION: We present a case of an infertile male with 46,XX/46,XYchimerism fathering a child after ICSI procedure. METHODS: Conventional cytogenetic analysis on chromosomes, derived from lymphocytes, using standard Q-banding procedures with a 450-550-band resolution and short-tandem-repeat analysis of 14 loci. RESULTS: Analysis of 20 metaphases from lymphocytes indicated that the proband was a karyotypic mosaic with an almost equal distribution between male and female cell lines. In total, 12 of 20 (60%) metaphases exhibited a normal female karyotype 46,XX, while 8 of 20 (40%) metaphases demonstrated a normal male karyotype 46,XY. No structural chromosomal abnormalities were present. Out of 14 STR loci, two loci (D18S51 and D21S11) showed four different alleles in peripheral blood, buccal mucosal cells, conjunctival mucosal cells, and seminal fluid. In three loci (D2S1338, D7S820, and vWA), three alleles were detected with quantitative differences that indicated presence of four alleles. In DNA extracted from washed semen, four alleles were detected in one locus, and three alleles were detected in three loci. This pattern is consistent with tetragametic chimerism. There were no quantitative significant differences in peak heights between maternal and paternal alleles. STR-analysis on DNA from the son confirmed paternity. CONCLUSION: We report a unique case with 46,XX/46,XY chimerism confirmed to be tetragametic, demonstrated in several tissues, with male phenotype and no genital ambiguity with oligospermia fathering a healthy child after IVF with ICSI procedure.

Clinical presentations of 23 half-siblings from a mosaic neurofibromatosis type 1 sperm donor.

The Danish sperm donor number 7042 has fathered several offspring with neurofibromatosis type 1 (NF1) worldwide. NF1 is caused by loss-of-function mutations in the NF1 gene and more than 1000 NF1 mutations are identified. Analysis of the donor sperm demonstrated gonosomal mosaicism with an intragenic deletion involving exons 15-29 in the NF1 gene. At the two Danish reference centres for NF1 patients, we evaluated 23 half-siblings from the donor. Nine were diagnosed with NF1. The severity grade of NF1 progressed from minimal to mild/moderate within 3 years of follow-up. The NF1 phenotype shows great variability in intra- and inter-family expressivity and to date only two NF1 genotype-phenotype correlations have been established. This rare possibility of a long-term follow-up of a cohort of half-siblings with NF1 makes further studies including phenotypic variability and search for modifier genes possible. To achieve this goal, we have initiated The International Donor 7042 NF1 Offspring Registry. Research facilitated via this registry may reveal important new knowledge of clinical characteristics and prognostics for the specific NF1 genotype and thereby contribute to future individualised targeted clinical follow-up and treatment.

Chromosomal microarray in fetuses with increased nuchal translucency.

OBJECTIVE: To assess the clinical value of using high-resolution chromosomal microarray (CMA) for the examination of genomic imbalances in prenatal uncultured chorionic villus samples from fetuses with increased nuchal translucency (NT) and a normal quantitative fluorescent polymerase chain reaction (QF-PCR) result, in a clinical setting in which more than 95% of pregnant women receive first-trimester combined screening. METHODS: From January 2013 to July 2014, we included 132 chorionic villus samples from consecutive ongoing pregnancies, with fetal NT ≥ 3.5 mm at 11-13 weeks' gestation, from obstetric units (publicly funded healthcare) in Central and North Denmark Regions. DNA was extracted directly from the samples and examined with QF-PCR (n = 132) and 180 kb oligonucleotide array-based comparative genomic hybridization (n = 94). RESULTS: In 38 cases, aneuploidies for chromosomes 18, 21 or X, or triploidy, were detected by QF-PCR. Among the 94 cases with a normal QF-PCR result, we detected pathogenic copy number variants (CNVs) by CMA in 12 fetuses (12.8% (95% CI, 7.5-21.0%)). In an additional three (3.2%) cases, CNVs with uncertain clinical significance were detected. CONCLUSION: CMA is a valuable diagnostic technique in pregnancies with isolated fetal NT ≥ 3.5 mm.

[Surgical treatment of goiter at a central hospital. A consecutive study with special emphasis on surgical complications]. / Kirurgisk behandling af struma på et centralsygehus. En konsekutiv opgørelse specielt med henblik på kirurgiske komplikatoiner.

The aim of this study was to analyse the frequency of permanent and transient complications following thyroid gland surgery. During a period of four years, from 1991 to 1995, 129 patients were treated operatively for thyroid disease. Sixteen patients were operated for thyrotoxicosis and 113 for nontoxic goitre. Nine patients underwent reoperation for recurrent goitre. Thyroid malignancy was found in two patients. The frequency of permanent complications was 0.7%. Transient unilateral pareses developed postoperatively in two patients. There were no permanent vocal cord paralyses. One patient developed permanent hypoparathyroidism. Five patients had transient postoperative hypocalcaemia requiring calcium supplements. Other complications were haemorrhage (3.8%), wound infection (0.7%) and other postoperative complications (1.5%). It is shown that uncomplicated thyroid surgery may be performed at a district hospital that has a special interest in patient evaluation, indications for surgery and up to date surgical technique.

Circulating serum trefoil factors increase dramatically during pregnancy.

OBJECTIVE: Trefoil factors (TFF1-3) are 7-12 kDa peptides secreted by mucosal surfaces, with changing levels of expression reflected in serum concentrations. The genes for the peptides are located on chromosome 21, the chromosome duplicated in trisomy 21. We studied the levels of circulating TFFs in pregnant women carrying trisomy 21 foetuses and in women with normal pregnancies, throughout pregnancy and postpartum. MATERIAL AND METHODS: Employing ELISA methods, serum collected at gestational weeks (GW) 18, 32, 39 and 8 weeks postpartum from women carrying normal foetuses (n=141) was analysed for TFFs. In addition, serum collected at GW 6-14 (median = 10) from women carrying trisomy 21 foetuses (n=48) or healthy foetuses (n=46) was analysed. RESULTS: Peaking at 39 GW, concentrations of TFF2 and TFF3 were 3.5 and 47 times higher, respectively, than postpartum. Postpartum levels were comparable to concentrations previously measured in nonpregnant women. TFF1 concentrations rose throughout pregnancy and postpartum, being 1.5 times higher postpartum compared to 18 GW. No differences in the levels of TFFs were observed between women carrying trisomy 21 and those with healthy foetuses. To our knowledge, circulating TFF3 has never been reported to reach the levels observed here. Also, the pattern of increase is unusual, as previous reports have shown parallel increases in TFF1 and TFF3 with no alterations in TFF2. CONCLUSIONS: Our results demonstrate that circulating TFFs are not candidate markers of trisomy 21 in first-trimester pregnancies, but raise intriguing questions concerning the origin of TFFs produced during pregnancy and their physiological function.

Clinical aspects of altered glycosylation of glycoproteins in cancer.

Alteration of the expression of carbohydrate structures is frequently observed in tumor cells. This review summarizes the different changes of O- and N-linked glycoproteins observed in cancer cells, the impact of the tumor-related carbohydrate phenotypes on the clinical outcome of the cancer disease, and the various ways in which carbohydrate structures can interact with different carbohydrate-detecting adhesion molecules, selectins, and sialoadhesins. Various ways of inhibiting the formation of cell adhesion-engaged carbohydrates on the cell surface, or inhibiting the binding are discussed. Carbohydrate structures which are in clinical use as circulating tumor markers and the effect of genotypes on tumor marker concentrations are reviewed.

Increased concentrations of genotype-interpreted Ca 19-9 in urine of bladder cancer patients mark diffuse atypia of the urothelium.

We investigated the use of genotype-interpreted measurements of the tumor marker Ca 19-9 in the urine of bladder cancer patients as a marker of the extent of urothelial disease. Ca 19-9 in urine (sialyl-Le(a)/creatinine ratio) was measured in 81 bladder cancer patients and correlated to T-category, histologic grade, and presence of urothelial dysplasia. As reference group, Ca 19-9 ratio was measured in urine from 21 apparently healthy individuals. The amount of sialyl-Le(a) expressed is influenced by the Lewis genotype and secretor status. Accordingly, secretor status was determined in urine by a novel ELISA method, and the Lewis genotypes of all of the individuals were determined by PCR cleavage methods. Ca 19-9 concentrations in urine were higher (P < 0.01) in bladder cancer patients than in healthy individuals and significantly (P = 0.02) higher in cancer patients with concomitant urothelial dysplasia than in those with normal urothelium. For individuals Lewis-genotyped as homozygous wild-type, Ca 19-9 concentrations in urine were higher, both in cancer patients (P = 0.06) and in healthy individuals (P = 0.004), than in the heterozygous individuals. Furthermore, nonsecretor cancer patients had higher (P < 0.01) Ca 19-9 concentrations in urine. Attention is drawn to the possibility of a general genotype interpretation of a result in clinical chemistry.

Immunoassays of human trefoil factors 1 and 2: measured on serum from patients with inflammatory bowel disease.

BACKGROUND: The trefoil factors (TFF1-3) are cysteine-rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up-regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross-sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD). METHODS: The TFF1-ELISA was based on two polyclonal rabbit antibodies and the TFF2-ELISA on a monoclonal mouse antibody and a polyclonal rabbit antibody. RhTFF1 and 2 were employed to prepare the calibrators. TFF1-3 were assayed in serum from IBD patients (n=41) and controls (n=13). RESULTS: The TFF1- (TFF2-) ELISA had a detection limit of 3 pmol/L (6 pmol/L) and an analytical imprecision (CV(A)) of 7.0-8.8 for mean concentrations of 24-120 pmol/L (6.1-8.0 for mean concentrations of 17-77 pmol/L). The central reference intervals (n=300) were 140-1400 pmol/L (37-190 pmol/L). There was no variation with age and menstrual cycle. Food intake reduced concentrations of TFF1 by approximately 15%, but did not influence concentrations of TFF2. TFF1 and TFF3 were increased in serum from IBD patients. CONCLUSIONS: We have developed assays for measuring TFF1 and TFF2. Finding increased TFF concentrations in serum from IBD patients suggests that measurements of trefoil peptides may be of clinical relevance in IBD.

Reference values and biological variation for tumor marker CA 19-9 in serum for different Lewis and secretor genotypes and evaluation of secretor and Lewis genotyping in a Caucasian population.

The concentration of the tumor marker CA 19-9 is influenced by the patient's secretor status and Lewis genotype. The aim of this study was to establish novel reference intervals for CA 19-9 in serum based on secretor and Lewis genotypes, to investigate the biological variation of CA 19-9, and to evaluate the utility of Lewis and secretor genotyping on a group of individuals with serologically defined Lewis phenotypes. CA 19-9 was measured in serum of 500 healthy individuals. Secretor and Lewis genotypes were determined by sequencing and PCR-cleavage methods. Significant differences were found between subgroups with different Lewis and secretor genotypes. Genotype-based reference intervals for CA 19-9 are presented. The upper reference limit for all individuals was 28.7 kilounits/L; for secretors and nonsecretors, the upper reference limits were 12.4 and 61.2 kilounits/L, respectively. The analytical imprecision (CVA) was 9.8%, the within-subject variability (CVI) was 15.8%, and the between-subject variability (CVG) was 102.2%. Good agreement was found between Lewis and secretor genotyping and conventional blood grouping. Genotype-based reference intervals may be a way to increase the clinical utility of CA 19-9. On the basis of the calculation of a critical difference for sequential values (significant at P

Lewis antigen mediated adhesion of freshly removed human bladder tumors to E-selectin.

PURPOSE: Twenty fresh surgical specimens of human bladder tumors were tested for their ability to adhere to recombinant P and E-selectin. The adhesion was correlated to immunological detection of carbohydrate structures. MATERIALS AND METHODS: A static titertray assay with immobilized selectins and appropriate controls was used for bladder tumor cell adhesion. On the same tumors expression of carbohydrate structures was examined by immunohistochemistry and Western blotting. RESULTS: No tumor bound to P-selectin. Nine tumors showed a high number of cells binding to E-selectin, 5 showed intermediate binding, and 6 showed only rare binding. The specificity of the binding was verified by inhibition with EDTA, by blocking antibodies to E-selectin, and by an acrylamide based sLe(x) (Galbeta1-4 [Fucalpha1-3]GlcNAc-) polymer. The binding was significantly more frequent (p <0.045) in superficial tumors than in invasive tumors. The binding property was correlated to the detection of carbohydrate structures in Western blots and tissue sections of the same tumors, using six different monoclonal antibodies: anti-sLe(a), anti-sLe(x), anti-Le(a), anti-Le(x) (two different clones) and anti-Le(b). Most blot-stainings were smeared indicating a mucin-type carrier molecule, but 115, 55 and 40 kDa bands carrying Le(a) and/or Le(b) epitopes were present in all tumors that bound. The Le(a) structure, as detected by blotting, was the only structure necessary for binding in the center of the wells (p <0.001), and was correlated to number of bound cells (p <0.006). A weaker correlation was found between Le(b) and number of bound cells (p <0.032), whereas it was remarkable that no correlation was found with Le(x) or sLe(x). Immunohistological staining of Le(a) on cell membranes correlated with frequent binding (p <0.003), whereas no correlation was found to secretor and Lewis genotypes. CONCLUSIONS: These data on clinical specimens indicate that Lewis antigen mediated E-selectin adhesion may play a role in the human bladder cancer disease.

Influence of Lewis alpha1-3/4-L-fucosyltransferase (FUT3) gene mutations on enzyme activity, erythrocyte phenotyping, and circulating tumor marker sialyl-Lewis a levels.

Fucosylated glycoproteins carrying alpha1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known alpha1-4-fucosyltransferase (FucT), and individuals who are deficient in this enzyme type as Lewis-negative on erythrocytes. We examined the mutational spectrum of the Lewis gene in Denmark and found 6 different mutations. Five, T59G, T202C, C314T, G508A, and T1067A, were frequent, and one, C445A, was only detected in one out of 40 individuals. Allele-specific polymerase chain reaction as well as cloning of FUT3 alleles showed that the 202 and 314 mutations were co-located on the same allele. COS7 cells transfected with an allele having the 202/314 mutations lacked enzyme activity. Polymerase chain reaction-cleavage assays were established for the genotyping of healthy individuals as well as 20 genuine Lewis-negative cancer patients and 10 non-genuine. The latter have Lewis-negative erythrocytes but saliva alpha1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3 alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower alpha1-4FucT activity in saliva than did homozygous wild-type individuals. The lower enzyme level in heterozygous individuals resulted in a significantly (p < 0.04) lower level of circulating sialyl-Lewis a structure in serum. This has the clinical impact that cut-off levels in tumor marker assays should be defined on the basis of genotyping. In the group of non-genuine Lewis-negative cancer patients, whose erythrocytes convert from Lewis-positive to Lewis-negative during the disease, FUT3 heterozygosity was significantly (p < 0.05) more common.