Exome sequencing of fetal anomaly syndromes: novel phenotype-genotype discoveries.

The monogenic etiology of most severe fetal anomaly syndromes is poorly understood. Our objective was to use exome sequencing (ES) to increase our knowledge on causal variants and novel candidate genes associated with specific fetal phenotypes. We employed ES in a cohort of 19 families with one or more fetuses presenting with a distinctive anomaly pattern and/or phenotype recurrence at increased risk for lethal outcomes. Candidate variants were identified in 12 families (63%); in 6 of them a definite diagnosis was achieved including known or novel variants in recognized disease genes (MKS1, OTX2, FGFR2, and RYR1) and variants in novel disease genes describing new fetal phenotypes (CENPF, KIF14). We identified variants likely causal after clinical and functional review (SMAD3, KIF4A, and PIGW) and propose novel candidate genes (PTK7, DNHD1, and TTC28) for early human developmental disease supported by functional and cross-species phenotyping evidence. We describe rare and novel fetal anomaly syndromes and highlight the diagnostic utility of ES, but also its contribution to discovery. The diagnostic yield of the future application of prenatal ES will depend on our ability to increase our knowledge on the specific phenotype-genotype correlations during fetal development.

Array comparative genomic hybridization in prenatal diagnosis of first trimester pregnancies at high risk for chromosomal anomalies.

OBJECTIVE: To describe the diagnostic performance of array comparative genomic hybridization (aCGH) as a potential first line diagnostic method in first trimester high risk pregnancies. METHOD: In a retrospective study we performed aCGH using a targeted array BAC platform (Constitutional Chip® 4.0, PerkinElmer, Turku Finland, median resolution 600 kB) and the Affymetrix Cytogenetics® Whole Genome 2.7 M array (at a resolution of 400kB) on 100 anonymized prenatal samples from first trimester high risk pregnancies with normal conventional karyotype. We studied the technical feasibility and turn-around-time as well as the detection rate of pathogenic submicroscopic chromosome anomalies and CNVs of unknown significance. RESULTS: We obtained results in 98 of 100 samples in 3 to a maximum of 5 days after DNA extraction. At the given resolution we did not identify any additional pathogenic CNVs but two CNVs of unknown significance in the chromosomal regions 1q21.1q21.2 (deletion) and 5p15.33 (duplication) (2%). CONCLUSION: In accordance with a growing number of reports this study supports the concept that aCGH at a resolution of 400-600kB may be used as a first line prenatal diagnostic test with high diagnostic safety and rapid turn-around time in high-risk first trimester pregnancies. Detection rate of CNVs of unknown significance, considered as a major hindrance for replacing conventional karyotyping by aCGH, is 2%, but the diagnosis of additional submicroscopic anomalies in this heterogeneous group of patients seems to be rare.

Fetal polydactyly: a study of 24 cases ascertained by prenatal sonography.

Records of 24 pregnancies with fetal polydactyly were reviewed for the type of polydactyly, family history, associated sonographic findings, genetic testing, and postnatal/postmortem examination findings. The importance of fetal polydactyly can be mainly elucidated by the family history and absent or associated anomalies on a specialized malformation scan. Fetal karyotyping diagnoses frequent chromosomal anomalies in about half of cases with additional malformations, and array comparative genomic hybridization may be a future means of detecting cryptic chromosomal aberrations. Syndromic disorders of monogenic origin demand a careful interdisciplinary clinical assessment for establishing a clinical diagnosis and prognosis for the outcome of the child.

aCGH on chorionic villi mirrors the complexity of fetoplacental mosaicism in prenatal diagnosis.

OBJECTIVE: To describe the diagnostic performance of array comparative genomic hybridization (aCGH) in the presence of mosaicism in the fetoplacental unit using direct chorionic villi. METHOD: In an ongoing study on the diagnostic performance of aCGH in 80 high-risk pregnancies, we studied three cases of placental mosaicism by carrying out aCGH on DNA of direct chorionic villi and chorionic villi cultures. RESULTS: Case 1: A three- to fourfold dosage gain of the region 18p in aCGH on direct villi was due to two additional isochromosomes 18p confined to the cytotrophoblast. Case 2: aCGH on direct villi revealed a normal result, whereas trisomy 18 mosaicism was present in cultured cells. Case 3: aCGH identifies monosomy X and mosaic disomy of the region Xp11.21-Xq12, whereas this mosaic cell line is not present in the conventional chromosome preparation on the cytotrophoblast. CONCLUSION: Although interpretation of aCGH results may be straightforward in the majority of cases, placental mosaicism may cause misinterpretations of rapid aCGH results on direct chorionic villi due to discrepant chromosomal constitutions of cytotrophoblast and mesenchymal villus core. Further investigations including cultures, fluorescence in situ hybridization and possible amniocentesis will still be required for interpretation of results.

[Pre-eclampsia screening in first and second trimester]. / Präeklampsie-Screening im 1. und 2. Trimenon.

Pre-eclampsia is a pregnancy-associated disease of the second part of the pregnancy, occurring mainly after 20th weeks gestation. The prevalence of hypertension in pregnancy is between 5 to 11% and affects mainly women under 20 years of age. An inadequate invasion of trophoblasts with consequential placental ischemia as a result of insufficiently dilated uterine spiral arteries is thought to be an initial cause in the pathogenesis of pre-eclampsia. The clinical symptoms of pre-eclampsia, such as loss of intravascular volume and edema, are caused by generalized endothelial dysfunction. These symptoms are potentiated by hypertension and reduced colloid osmotic pressure in the plama. The organs being affected by pre-eclampsia are those of the vascular-, hepatic-, renal-, cerebral- and coagulatory systems. The prognosis is much more severe when pre-eclampsia develops very early in the pregnancy. The symptoms include elevated blood pressure (over 140 mmHg systolic, 90 mmHg diastolic) combined with proteinuria. Frequent symptoms are hyperreflexia and edema. The etiology of pre-eclampsia has not been clearly defined. Risk factors/triggers for the development of pre-eclampsia can include chronic hypertension, advanced maternal age at first pregnancy (over 35 y), nephropathy, thrombophilia (heterozygous factor V Leiden mutation, antiphospholipid syndrome, heterozygous prothrombin mutation and homozygous MTHFR), multiple gestation and prior pregnancy with preeclampsia. The incidence of preeclampsia is higher in nulliparous than multiparous women. In many countries pre-eclampsia is still most frequent cause of maternal perinatal mortality. HELLP-Syndrome (haemolysis-elevated liver enzyme- low platelets) is a severe progressive course of this disease. Eclampsia, characterized by generalized tonic-clonic convulsion, is the most dangerous complication of pre-eclampsia, and may develop before or after delivery. This form of pre-eclampsia is associated with higher maternal and fetal mortality. Constant maternal hypertension potentially alter vascular integrity of the placenta with further consequences in fetal blood supply leading to growth restriction or zero growth and subsequently resulting in low birth weight or fetal death. The sooner the disease is detected and confirmed, the better the maternal and fetal prognoses are. This is the reason why it is major importance, together with the employment of preventive measures, to identify patients with risk factors with pre-eclampsia though an adequate screening method, thereby detecting the disease earlier and ensuring better pregnancy outcomes for both mother and child.

Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis.

BACKGROUND: Increased concentrations of cell-free DNA have been found in several disorders and have been interpreted as evidence of increased rates of cell death or turnover. Evidence from in vitro and animal experiments suggests that DNA may play a role in the pathogenesis of rheumatoid arthritis (RA). METHODS: We measured cell-free DNA in plasma and serum from patients with RA and healthy controls by use of quantitative PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA. We used protein G Sepharosetrade mark bead adsorption of plasma and elution to isolate antibody-bound DNA. RESULTS: In paired plasma and serum samples of 16 healthy controls the median GAPDH copies were 4500 genome equivalents (GE)/mL plasma (range 319-21 000) and in 26 RA patients 17 000 GE/mL plasma (2100-2 375 000, P = 0.0001). In the serum from normal controls the median GAPDH copies were 35 000 GE/mL (1700-239 000) and from RA patients 222 000 GE/mL (21 000-2 375 000, P = 0.004). A median of 81% of the cell-free DNA in RA was associated with antibody compared with 9% in healthy controls (P = 0.001). The concentrations of DNA did not vary with the type of therapy patients received. CONCLUSIONS: These results provide new evidence for a role of cell-free DNA-antibody complexes in the etiology of RA, suggest new avenues for basic research, and may prove to be relevant to diagnosis and assessment of therapy.

Cognitive-behavioural group intervention for climacteric syndrome.

BACKGROUND: During peri- and postmenopause there is a high prevalence of psychological symptoms such as emotional instability, depressive moods, anxiety, sleep disorders, and sexual dysfunction. Aetiologically relevant factors for discomfort are decline of sex hormones and psychosocial factors such as lifestyle, attitude towards menopause, pre-menopausal mental health and sociocultural factors. In contrast to the relevance of psychosocial factors, there are few studies on psychological interventions. The present study evaluates an open trial of cognitive-behavioural group intervention consisting of psychoeducation, group discussion and coping skills training for women suffering from climacteric symptoms. METHODS: Thirty women were enrolled in this first trial. Standardised (MRS, HADS-D, Partnership Questionnaire, McCoy Female Sexuality Questionnaire) and especially developed ('Attitudes Towards the Menopause') instruments were administered 3 times, twice before (T1 and T2) and once after the group intervention (T3). General linear model repeated measures were used to analyse changes in questionnaire measures. RESULTS: Taking the average of the two pre-intervention scores, significant improvements were observed in anxiety (p < 0.01), depression (p < 0.02), partnership relations (p < 0.02), overall score of sexuality (p < 0.02), hot flashes (p < 0.01) and cardiac complaints (p < 0.01) from pre- to post-intervention. No changes were found for sexual satisfaction and stressfulness of menopausal symptoms. CONCLUSIONS: This pilot study points at a possible effectiveness of cognitive-behavioural interventions for the treatment of climacteric syndrome. Further studies will have to use randomised trials, comparing different treatments (HRT, phyto-oestrogens, relaxation training, discussion groups) for their effectiveness.

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

BACKGROUND: Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS: Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS: Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (

Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome.

OBJECTIVE: Recent reports have indicated that cell-free fetal DNA can be detected in the urine of pregnant women. We attempted to reproduce those data. METHODS: Urine samples were collected from 18 normal pregnant women (11 with a male fetus). Urinary DNA was examined by Y-chromosome-specific nested polymerase chain reaction (PCR) or real-time PCR. Samples were also examined from two pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which had very high levels of cell-free fetal DNA in the maternal plasma. To validate our data, a quantitative comparison of different DNA extraction procedures used in the previous reports was performed. RESULTS: In no instance were we able to detect any fetal DNA in maternal urine, although copious quantities of cell-free fetal DNA were present in the maternal plasma of those pregnancies affected by HELLP syndrome. Our quantitative analysis of the various extraction procedures used indicated that the commercial column elution method we used was comparable, if not superior, to the noncommercial methods used in previous reports. CONCLUSION: Our data strongly suggest that cell-free fetal DNA is not readily detectable in maternal urine, even under conditions known to increase kidney permeability.