Clinical utility of non-invasive prenatal testing in pregnancies with ultrasound anomalies.

OBJECTIVE: To evaluate the application of non-invasive prenatal testing (NIPT) as an alternative to invasive diagnostic prenatal testing in pregnancies with abnormal ultrasound findings. METHODS: This was a retrospective analysis of 251 singleton and multiple pregnancies at high risk for fetal chromosomal abnormality based on findings at sonographic examination, in which NIPT was performed as a first-tier genetic test. NIPT was performed by massively parallel sequencing of cell-free DNA in maternal plasma, allowing genome-wide detection of whole-chromosome, as well as partial, autosomal aneuploidy. Sex chromosomes were not analyzed, according to the current protocol in Dutch laboratories. RESULTS: NIPT was performed at a median gestational age of 20 weeks, indicated by the presence of multiple congenital anomalies (n = 13), isolated structural anomalies (n = 57), increased nuchal translucency ≥ 3.5 mm (n = 58), soft markers (n = 73), growth restriction (n = 40) and other anomalies (n = 10). NIPT results were normal in 224 (89.2%) pregnancies, inconclusive in one (0.4%) and abnormal in 26 (10.4%). Most genetic aberrations detected by NIPT were common whole-chromosome aneuploidies: trisomy 21 (n = 13), trisomy 18 (n = 6) and trisomy 13 (n = 3). Four further NIPT results were abnormal; one was suspected of being confined placental mosaicism and one was of maternal origin. In those with normal NIPT results, sonographic follow-up or examination of the newborn indicated the need for diagnostic genetic testing in 33/224 (14.7%) pregnancies. Clinically relevant genetic aberrations were revealed in 7/224 (3.1%) cases, two of which were whole-chromosome aneuploidies: trisomy 13 and monosomy X. As sex chromosomal aberrations are not included in NIPT analysis, the latter cannot be considered a false-negative result. Other discordant findings were subchromosomal aberrations (< 20 megabases, n = 2) and monogenic aberrations (n = 3). CONCLUSIONS: NIPT should not be recommended for genetic evaluation of the etiology of ultrasound anomalies, as both resolution and sensitivity, or negative predictive value, are inferior to those of conventional karyotyping and microarray analysis. Nonetheless, some pregnant women consider NIPT to be an acceptable alternative to invasive diagnostic testing. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

SNP array analysis in constitutional and cancer genome diagnostics--copy number variants, genotyping and quality control.

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow.

Identification of clinically significant, submicroscopic chromosome alterations and UPD in fetuses with ultrasound anomalies using genome-wide 250k SNP array analysis.

BACKGROUND: The implementation of microarray analysis in prenatal diagnostics is a topic of discussion, as rare copy number variants with unknown/uncertain clinical consequences are likely to be found. The application of targeted microarrays limits such findings, but the potential disadvantage is that relevant, so far unknown, aberrations might be overlooked. Therefore, we explore the possibilities for the prenatal application of the genome-wide 250k single nucleotide polymorphism array platform. METHODS: Affymetrix 250k NspI single nucleotide polymorphism array analysis (Affymetrix, Inc., Santa Clara, California, USA) was performed on DNA from 38 prenatally karyotyped fetuses with ultrasound anomalies. Analyses were performed after termination of pregnancy, intrauterine fetal death or birth on DNA isolated from fetal or neonatal material. RESULTS: Aberrations were detected in 17 of 38 fetuses, 6 of whom with a previously identified chromosomal abnormality and 11 with previously normal or balanced karyotypes. Of the latter, the detected aberration occurred de novo and was considered of clinical relevance in five cases (16%), inherited from a healthy parent in four cases (12%), and de novo yet with unclear clinical relevance in two cases (6%). The clinically relevant abnormalities either were novel copy number variants (n=3) or concerned a uniparental disomy (n=2). CONCLUSION: In at least 16% of fetuses with ultrasound anomalies and a normal or balanced karyotype, causal (submicroscopic) aberrations were detected, illustrating the importance of the (careful) implementation of microarray analysis in prenatal diagnosis. The fact that the identified, clinically relevant, aberrations would have gone undetected with most targeted approaches underscores the added value of a genome-wide approach.

Increased cerebrospinal fluid chitotriosidase index in patients with multiple sclerosis.

OBJECTIVE: To investigate chitotriosidase (CTTS) activity in serum and cerebrospinal fluid (CSF) in multiple sclerosis (MS) patients in relation to disease course and CSF markers for immune activation or inflammation. MATERIALS AND METHODS: We studied 80 patients with relapsing-remitting MS (RRMS), 24 with secondary progressive MS (SPMS), 20 with primary progressive MS (PPMS) and 29 patients with other neurological disorders (OND). We measured CTTS activity and studied the correlation with CSF mononuclear cell count (MNC) and intrathecal IgG production. RESULTS: CTTS activity was significantly higher in CSF, but not in serum, from the total MS group compared with OND and controls. In RRMS and SPMS CTTS, index was increased compared with controls (RRMS, 0.10 +/- 0.21; SPMS, 0.10 +/- 0.15; controls, 0.021 +/- 0.020), but not in PPMS (0.061 +/- 0.052). CTTS index was higher in MS patients with elevated MNC or CSF-restricted oligoclonal IgG bands than in MS patients without these CSF findings. CONCLUSIONS: CTTS index is elevated in RRMS and SPMS. The CTTS index is related to CSF markers of inflammation or immune activation.

CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy.

A homozygous mutation of the CNTNAP2 gene has been associated with a syndrome of focal epilepsy, mental retardation, language regression and other neuropsychiatric problems in children of the Old Order Amish community. Here we report genomic rearrangements resulting in haploinsufficiency of the CNTNAP2 gene in association with epilepsy and schizophrenia. Genomic deletions of varying sizes affecting the CNTNAP2 gene were identified in three non-related Caucasian patients. In contrast, we did not observe any dosage variation for this gene in 512 healthy controls. Moreover, this genomic region has not been identified as showing large-scale copy number variation. Our data thus confirm an association of CNTNAP2 to epilepsy outside the Old Order Amish population and suggest that dosage alteration of this gene may lead to a complex phenotype of schizophrenia, epilepsy and cognitive impairment.

Multiple congenital abnormalities in a newborn with two supernumerary marker chromosomes derived from chromosome 14.

Pure partial duplication or triplication of the proximal part of chromosome 14 has been reported in only 4 patients. Other individuals with a duplication or triplication of this region have additional chromosome imbalances. We present a new case with a supernumerary marker chromosome in all blood cells and in 35% of the cells an additional smaller marker chromosome. Both markers appeared to be derived from chromosome 14 (del(14)(q21.2) in all cells and del(14)(q11.2) in 35% of the cells). This results in a partial duplication of the proximal region of chromosome 14, combined with a mosaic partial triplication of a smaller segment of the same region. In this paper, we compare the clinical features of this case to those of cases from the literature. Although most of the patients from literature were unbalanced translocation carriers, their clinical features were comparable, except from renal abnormalities.

A new case of dup(1)(q21.2q12) in an individual with mild mental retardation.

Proximal duplications of the long arm of chromosome 1 are rare and the few patients that have been described in literature have multiple congenital abnormalities and/or mental retardation. The present paper describes the clinical and cytogenetic findings of an adult patient with only mild mental retardation and some minor malformations. The patient carries an inverted duplication of 1q12q21.2.

A new case of dup(3q) syndrome due to a pure duplication of 3qter.

The characteristic clinical features of the dup(3q) syndrome include typical facial features, mental and growth retardation, and (often) congenital heart anomalies. However, pure duplication of 3qter is rare because most of the reported cases are patients who carry an unbalanced translocation and, in addition to the duplication for 3qter, have a deletion for another chromosomal segment. A new case with a pure duplication of 3q detected in a 2-month-old boy is presented here. Extensive cytogenetic analysis revealed an inverted duplication of the distal part of 3q (chromosomal band 3q26.3 up to the telomere), with no (detectable) loss of the original telomeric sequences. Clinical evaluation revealed several phenotypic hallmarks characteristic for the dup(3q) syndrome. By comparing the duplicated region of this patient with the duplicated regions of the other patients with a pure duplication of 3q, we were able to localize the critical region for the dup(3q) phenotype to band 3q26.3. Alongside this new case with a pure duplication of 3q, an overview of six previous cases is given.

Partial expression of RHc on the RHD polypeptide.

BACKGROUND: In the human Rh blood group system, c is, after D, the most immunogenic antigen. STUDY DESIGN AND METHODS: The background of a new partial c phenotype (D(c)), identified on the RBCs of two unrelated white persons, was studied. This was done by analyzing the reactivity of the RBCs from the donors with anti-c reagents, by performing sequence analysis, and by carrying out transduction studies. RESULTS: Serologic results suggested the existence of a new partial c phenotype. Genomic DNA and cDNA analysis revealed a normal RHCe allele, a normal RHD allele, and an RHD allele that carried two point mutations: 307T>C and 329T>C (the latter known to be associated with the DVII, Tar-positive phenotype). No normal RHc allele was found. Thus, it was most likely that c is encoded by the mutated RHD allele (phenotype DD(c)CCee). Indeed, subsequent transduction of K562 erythroleukemic cells with an RHD cDNA carrying the 307T>C point mutation (leading to S103P) resulted in the expression of c. CONCLUSION: In the human Rh system, P103 is involved in the expression of c. Moreover, c can be expressed in vivo on the D polypeptide.

Short stature as the only presenting feature in a patient with an isodicentric (Y)(q11.23) and gonadoblastoma. A clinical and molecular cytogenetic study.

A 13-year-old phenotypically female patient presented with short stature (height SDS -2.6), but without any Turner stigmata or other dysmorphic features. Chromosome analysis showed mosaicism for an isodicentric (idic) (Y)(q11.23) containing cell line and a 45,X cell line. Subsequent gonadectomy revealed a left streak ovary and a right ovary of abnormal appearance, which on histological examination appeared to contain a gonadoblastoma. DNA analysis showed that the proposed critical region of the gonadoblastoma locus on the Y chromosome was contained within the patient's idic (Y). Conclusion. The case described here shows that patients with 45,X/46,X, isodicentric (Yp) mosaicism and a female phenotype (1) can lack external virilisation but still have a gonadoblastoma and (2) do not necessarily have Turner stigmata but can present with only short stature. This case also underlines the importance of karyotyping patients with unexplained short stature to enable gonadectomy if Y-derived material is detected.

The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study.

The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.

Rhd and C/cE/e genotyping in Slovenian population.

The Rhesus (Rh) blood group system is, after ABO, clinically most important. Alloantibodies directed against Rh antigens are the major cause of a haemolytic disease of newborn (HDN) and of transfusion reactions. In search for novel methods for Rh genotyping we started to compare Rh genotypes identified from different tissues and Rh phenotypes. Genotypes determined from blood samples with PCR based RhD, C/c and E/e genotyping methods were compared with serologically identified phenotypes (N=32). With two exceptions the results of phenotyping and genotyping were in concordance. Two Rh serotypes from a Slovenian family that were unexpected according to the Mendelian laws were characterised genotypically. The two family members were suspected to have a chromosomal deletion on RH gene locus.

[Prenatal typing of Rh- and Kell- blood group system antigens]. / Prenatale typering van de bloedgroepantigenen van het Rh- en het Kell-systeem.

Rhesus (Rh) and Kell blood group immunisations are the most frequent causes of haemolytic disease of the newborn. Recently, the molecular bases of the Rh and Kell antigens have been elucidated. Subsequently, specific polymerase chain reactions (PCRs) could be developed to determine the RhD, RhC/Rhc and RhE/Rhe genotypes as well as the KI genotype (from the Kell blood group) with genomic DNA. The tests were applied to genomically determine the foetal Rh and Kell blood groups with DNA obtained from amniotic fluid cells. The genotypes obtained were compared with the Rh phenotypes established by cord blood red cell serology. The PCRs to determine the RhD, Rhc, RhE and Rhe and KI genotypes were found to be reliable. The test for RhC however, resulted in false-positive C genotypes. Indeed, more than half of the subsequently tested C-negative Negroid donors were false-positive with the DNA test. Thus, except for RhC, it is possible to reliably determine the Rh and KI genotypes of a foetus with DNA isolated from amniotic fluid cells. Amniocentesis, however, carries a risk for the pregnancy and therefore the tests will only be justified in pregnant women in whom an antibody has been detected and the father of the foetus is heterozygous for the specific antigen. Recently foetal RhD genotypes were determined in foetal DNA circulating in the plasma of RhD-negative pregnant women. This could eventually lead to the introduction of assays with which the foetal blood group can be determined without any risk to the foetus.

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons.

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD-specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD-specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D- and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD-specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D- individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD-specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis.

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood. STUDY DESIGN AND METHODS: Red cells from 100 black South Africans and 43 black persons from Amsterdam, the Netherlands, were typed serologically for various Rh system antigens. Allele-specific polymerase chain reaction and sequencing of polymerase chain reaction products were used to analyze C733G (Leu245Val) and G1006T (Gly336Cys) polymorphisms in exons 5 and 7 of RHCE and the presence of a D-CE hybrid exon 3. RESULTS: The respective frequencies of all VS+ and of VS+ V-(r's) phenotypes were 43 percent and 9 percent in the South Africans and 49 percent and 12 percent in the Dutch donors. All VS+ donors had G733 (Val245), but six with G733 were VS- (4 V+w, 2 V-). The four VS- V+w donors with G733 appeared to have a CE-D hybrid exon 5. T1006 (Cys336) was present in 12 percent and 16 percent of donors from the two populations. With only a few exceptions, T1006, a D-CE hybrid exon 3, and a C410T (Ala137Val) substitution were associated with a VS+ V-phenotype ((C)ces or r's haplotype). Two VS+ V-individuals, with the probable genotype, (C)ces/(C)ces), were homozygous for G733 and for T1006. CONCLUSIONS: It is likely that anti-VS and anti-V recognize the conformational changes created by Val245, but that anti-V is sensitive to additional conformational changes created by Cys336.

Lower antigen site density and weak D immunogenicity cannot be explained by structural genomic abnormalities or regulatory defects of the RHD gene.

BACKGROUND: The weak D phenotype is characterized serologically by a weak or negative agglutination reaction with polyclonal anti-D in an immediate-spin test. Agglutination is enhanced in the indirect antiglobulin test. Red cells that are typed weak D have a much lower number of apparently complete D antigens at their cell surface and are associated with considerably weaker immunogenicity than are red cells with normal D. In a previous study, the number of D sites per cell was determined in eight unrelated weak D individuals to range from 490 to 1870 D sites per cell, which corresponded to 4 to 14.2 percent of the number of D sites in CcDee samples. STUDY DESIGN AND METHODS: The RHD gene was investigated for structural abnormalities by Southern blot experiments and polymerase chain reaction-based RHD typing in these individuals. In addition, abnormalities in the transcription process were studied by sequence analysis of RH transcripts and by comparing the relative amounts of RHD mRNA in weak D to those in CcDee, CcDEe, and -D- samples by using a semiquantitative reverse transcriptase-polymerase chain reaction analysis. RESULTS: The RHD gene in weak D phenotypes does not show any abnormalities at either the genomic or the transcriptional level when compared to the RHD gene in normal D phenotypes. CONCLUSION: The weaker immunogenicity of weak D is not explained by structural difference in the RHD gene itself. The weaker expression of D might be caused by factors involved in the Rh-related complex or by an as yet unidentified suppressor gene. This study supports the concept that weak D phenotypes carry complete D polypeptides and reflect a quantitative rather than a qualitative variation of D.

Molecular background of VS and weak C expression in blacks.

BACKGROUND: The Rh system is complex and consists of as many as 45 different antigens. Red cells of about 25 percent of the black population carry VS an Rh-system antigen (Rh20), but this antigen is very rare in whites. VS positivity is always associated with a weak expression of e, and usually also of C. STUDY DESIGN AND METHODS: The RH genes of 11 black VS-positive donors were studied. Transcripts were sequenced for four VS-positive donors, three of whom had red cells with a weak expression of C. In the other donors, only analysis of genomic DNA was carried out. RESULTS: The occurrence of VS was shown to be related to a single-point mutation in exon 5 of the RHCE gene (cytosine 733 guanine, leading to the Leu245Val substitution). The presence of this polymorphism in exon 5 may explain the simultaneously occurring weak e, because the E/e polymorphism is located in the same exon. Study of VS-positive donors with different Rh phenotypes showed that the polymorphism can occur in different alleles of the RHCE gene. In all three donors whose red cells showed a weak expression of C, a hybrid D-CE-D transcript was found, containing exon 4, 5, 6, 7, and (probably) 8 from the RHCE gene. No transcripts were encountered carrying DNA markers normally associated with C expression. CONCLUSION: It is therefore postulated that the hybrid gene is responsible for the weak expression of C in these individuals. The hybrid gene carried a Leu62Phe substitution, as well as the Leu245Val substitution responsible for VS. The gene most probably cosegregates with a C allele encoding Cys 16 (normally encoded only by the C allele) and Val245 (responsible for VS antigenicity when encoded by the RHCE gene). This explains the combination of weak expression of C and VS positivity that is frequently found in blacks.

Involvement of Gly96 in the formation of the Rh26 epitope.

BACKGROUND: The Rh system, a complex blood group system, comprises at least 45 antigens. Red cells expressing c usually express Rh26. Rare cells that are c+ Rh:-26 give variable reactions with anti-c and may have weak expression of f (ce). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed with red cells from persons with the c+ Rh:-26 phenotype occurring in two unrelated Dutch families. Red cells of 11 members of these two families were typed for Rh26, for c (with monoclonal and polyclonal reagents), and for f (ce). The cDNA of three donors was sequenced, while restricted DNA analysis was carried out on material from available members of the two families. RESULTS: Serologic tests showed that the rare c+ Rh:-26 phenotype was associated with a weak expression of c and a normal expression of f. The cDNA analysis of three members of one family revealed a single-point mutation (G286A) in exon 2 of the ce allele. Allele-specific primer amplification, polymerase chain reaction followed by allele-specific restriction analysis, and single-strand conformation polymorphism showed the same polymorphism in all other members of both families, whereas it was absent in 80 control donors. CONCLUSION: The c+ Rh:-26 phenotype, identified in two families, is associated with a single-point mutation at nucleotide 286 (G286A) in the ce allele, which predicts a Gly96Ser amino acid substitution. This substitution also affects c, because all anti-c reagents reacted more weakly. Other polymorphic sites apparently are involved in the formation of the Rh26 epitope as well, because Rh26 is expressed only on the c polypeptide, whereas Gly96 is expressed on all polypeptides.