Predictors and risk model development for menopausal age in fragile X premutation carriers.

PURPOSE: Women who carry a fragile X mental retardation 1 premutation are at risk for fragile X-associated primary ovarian insufficiency and should be counseled for a potentially reduced fertility. Multiple factors can affect the age of onset and severity of fragile X-associated primary ovarian insufficiency. In this study, we assessed the predictive power of several factors with menopausal age, a surrogate measure of onset of fragile X-associated primary ovarian insufficiency. METHODS: Genetic, environmental, and reproductive factors were analyzed by Cox proportional hazard models in 1068 women, 385 of fragile X families ascertained through the Nijmegen study and 683 of fragile X families or general population families ascertained through the Atlanta study. RESULTS: The highest association with menopausal age among fragile X mental retardation 1 premutation carriers was found for risk index by CGG repeat size (hazard ratio, 1.43) and smoking (hazard ratio, 1.34). Women from the Nijmegen cohort showed an overall lower age at menopause onset, for which a correction was made. A prediction model based on these two parameters, mean menopausal age of first-degree relatives with the same mutation status and the correction for ascertainment site, estimated the probability of becoming postmenopausal for premutation carriers, with a margin of 7-10%. CONCLUSION: We conclude that this model serves as a first step toward clinical application of fragile X-associated primary ovarian insufficiency prediction.

Widespread non-central nervous system organ pathology in fragile X premutation carriers with fragile X-associated tremor/ataxia syndrome and CGG knock-in mice.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder generally presenting with intention tremor and gait ataxia, but with a growing list of co-morbid medical conditions including hypothyroidism, hypertension, peripheral neuropathy, and cognitive decline. The pathological hallmark of FXTAS is the presence of intranuclear inclusions in both neurons and astroglia. However, it is unknown to what extent such inclusions are present outside the central nervous system (CNS). To address this issue, we surveyed non-CNS organs in ten human cases with FXTAS and in a CGG repeat knock-in (CGG KI) mouse model known to possess neuronal and astroglial inclusions. We find inclusions in multiple tissues from FXTAS cases and CGG KI mice, including pancreas, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart, and mitral valve, as well as throughout the associated autonomic ganglia. Inclusions were observed in the testes, epididymis, and kidney of FXTAS cases, but were not observed in mice. These observations demonstrate extensive involvement of the peripheral nervous system and systemic organs. The finding of intranuclear inclusions in non-CNS somatic organ systems, throughout the PNS, and in the enteric nervous system of both FXTAS cases as well as CGG KI mice suggests that these tissues may serve as potential sites to evaluate early intervention strategies or be used as diagnostic factors.

X chromosome inactivation does not define the development of premature ovarian failure in fragile X premutation carriers.

Since only 20% of female fragile X premutation carriers develop premature ovarian failure (POF, i.e., amenorrhea before age of 40 years), and since X chromosome inactivation (XCI) determines the phenotypic severity of full mutation women, we reasoned that the development of POF in fragile X premutation carriers could be due to skewed XCI (XCI ratio >80:20). To determine inactivation ratios and activities of the premutations, inactivation patterns were assessed in peripheral blood samples from 101 fragile X premutation carriers (mean age 47.1 years, range 12-72) through analysis of the AR and FMR1 loci, respectively. In addition, AR inactivation patterns were assessed in peripheral blood samples from 25 women with idiopathic POF (mean age 31.7 years, range 19-48). We addressed the association between age and skewed XCI because older women are prone to XCI skewness. The median XCI ratios were 68% for premutation carriers with POF (N = 37), 67% for premutation carriers without POF (N = 64) and 61% for women with idiopathic POF (N = 25). The incidence of skewing was similar in all groups, that is, 7 of 37 (18.9%) in premutation carriers with POF, 11 of 64 (17.2%) in premutation carriers without POF, and 3 of 25 (12%) in women with idiopathic POF. There was good concordance between inactivation ratios at the two loci tested in 62 premutation carriers (intraclass correlation coefficient = 0.86; P < 0.01). No age-specific skewing was observed. Skewed XCI and activity of the premutation are not associated with POF in fragile X premutation carriers.

Detection of chromosome aneuploidies in chorionic villus samples by multiplex ligation-dependent probe amplification.

The objective of this study was to examine the suitability of multiplex ligation-dependent probe amplification (MLPA) in chorionic villus samples as a replacement for traditional karyotyping for the detection of (an)euploidies of chromosomes 21, 18, 13, X, and Y. Chorionic villus samples were diagnosed by traditional karyotyping using short-term cultures (STC) and long-term cultures (LTC), and by MLPA using kit P095. DNA was extracted after digestion of whole villi with proteinase K and/or trypsin and collagenase. Different cell-dissociation procedures were tested to obtain MLPA results representative of the cytotrophoblast layer and the mesenchymal core. Over 95% of the MLPA results were in concordance with the traditional karyotyping of STC and LTC. Traditional karyotyping revealed seven mosaics. After digestion of whole villi with proteinase K, only abnormal cell lines confined to the STC gave rise to abnormal MLPA results. In one sample, the complete discrepancy between STC and LTC was resolved after enzymatic dissociation of cells from the cytotrophoblast layer and the mesenchymal core. MLPA in chorionic villus samples was found to be a reliable test for the detection of (an)euploidies of chromosomes 21, 18, 13, X, and Y. Whole villi digestion with proteinase K resulted in the over-representation of cytotrophoblasts in the DNA pool. To obtain MLPA results representative for STC and LTC, enzymatic dissociation of cells from the cytotrophoblast layer and mesenchymal core is required.

Lysosomal storage diseases in non-immune hydrops fetalis pregnancies.

BACKGROUND: At least 20 inborn errors of metabolism may cause hydrops fetalis. Most of these are lysosomal storage diseases. The study proposes a diagnostic flowchart for prenatal diagnosis of non-immune hydrops fetalis. METHODS: This study contains a series of 75 non-immune hydrops fetalis pregnancies. Mucopolysaccharides, oligosaccharides, neuraminic acid and 21 lysosomal enzymes were measured in amniotic fluid and cultured amniotic cells. RESULTS: The study gives reference values for mucopolysaccharides and neuraminic acid at various stages of gestation. Four definite and two probable lysosomal diagnoses were found among the 75 investigated cases (=5.3-8%). Fetal death was found to cause false positive values for mucopolysaccharides in amniotic fluid. In the galactosialidosis case, two novel mutations were found in the cathepsin A gene. CONCLUSIONS: Reference values for mucopolysaccharides and neuraminic acid depend on gestational age. In a relatively high percentage of the hydrops foetalis pregnancies, a lysosomal aetiology is found. This study provides a strategy to diagnose lysosomal diseases in hydrops fetalis pregnancies. Awareness of lysosomal storage diseases causing hydrops fetalis is useful as it gives an opportunity for risk evaluation, genetic counseling to parents and targeted prenatal diagnostics for ensuing pregnancies.

A new locus for postaxial polydactyly type A/B on chromosome 7q21-q34.

Postaxial polydactyly (PAP) is the occurrence of one or more extra ulnar or fibular digits or parts of it. In PAP-A, the extra digit is fully developed and articulates with the fifth or an additional metacarpal/metatarsal, while it is rudimentary in PAP-B. Isolated PAP usually segregates as an autosomal dominant trait, with variable expression. Three loci are known for PAP in humans. PAPA1 (including PAP-A/B in one patient) on 7p13 caused by mutations in the GLI3 gene, PAPA2 on 13q21-q32 in a Turkish kindred with PAP-A only, and a third one (PAPA3) in a Chinese family with PAP-A/B on 19p13.1-13.2. We identified a fourth locus in a large Dutch six-generation family with 31 individuals including 11 affecteds. Their phenotype varied from either PAP-A, or PAP-B to PAP-A/B with or without the co-occurence of partial cutaneous syndactyly. We performed a whole-genome search and found linkage between PAP and markers on chromosome 7q. The highest LOD score was 3.34 obtained at D7S1799 and D7S500 with multipoint analysis.

Women's Attitudes towards the Option to Choose between Karyotyping and Rapid Targeted Testing during Pregnancy.

Objectives. Pregnant women, referred because of an increased risk of fetal Down syndrome, who underwent an invasive prenatal procedure were offered a choice between karyotyping and rapid targeted testing. This study aims to assess women's attitudes and experiences towards what option to choose. Methods. A retrospective multicentre survey (2008-2010) was conducted among 1370 women. General questions were asked about decision making issues, followed by personal questions about their experiences in choice making, test preference, influence of others, and possible regrets. Results. In total, 90.1% of the respondents (N = 825) indicated that pregnant women are able to choose, although 33.1% stated that the choice can best be made by a professional. 18.4% indicated that making a choice places a burden on women. In 96.4%, respondents preferred to have the option to choose again in case of a next pregnancy, whereas 2.7% preferred the choice to be made by a professional. Regret was indicated by 1.2%. Decision making was influenced by others in 64.9%. A slightly higher preference for karyotyping was indicated by 52.7% of the respondents. Conclusions. Positive attitudes and experiences were expressed towards the option to choose. Respondents took decisions freely, although sometimes influenced by a partner or a professional, to follow their individual perspectives.

Why do parents prefer to know the fetal sex as part of invasive prenatal testing?

Objectives. The aim of this study was to determine whether prospective parents, primarily referred for prenatal diagnosis to exclude Down syndrome, prefer to know the fetal sex as part of invasive testing. Methods. In this prospective study 400 pregnant women undergoing amniocentesis were invited to answer a questionnaire, including information about demographic factors, current pregnancy, and previous children. In two open-ended questions they were asked why they wanted to know the fetal sex after amniocentesis or ultrasound investigation. Scores were given for reasons that could have played a role in the wish whether or not to know the sex of their unborn child. Results. A total of 210 (52.5%) questionnaires were completed. Overall, 69.0% was interested to know the fetal sex as part of the diagnostic test result. The most important reasons were curiosity (77.8%), "just want to know" (68.0%), and "because it is possible" (66.8%). The overall knowledge of sex chromosomal disorders appeared low and did not seem to affect the parent's wish to know the fetal sex. Almost all women (96.6%) planned to have a 20-week ultrasound scan and 96.2% thought the scan to be reliable in detecting the fetal sex. A minority (28%) was willing to learn the fetal sex by ultrasound examination, whereas 65% preferred to learn the fetal sex only after the amniocentesis. Conclusion. Personal values affect the parental desire to know or not to know the fetal sex. This does not appear to be affected by invasive prenatal testing and/or genetic knowledge of sex chromosomal disorders.

Parent's attitudes towards full-scale prenatal testing for genetic disorders.

OBJECTIVES: Innovations in the field of prenatal diagnostic testing have led to the development of molecular tests that allow the rapid detection of specific genetic defects, such as Down syndrome. In addition, full-scale tests have been developed allowing the detection of many genetic disorders in a single test. Here we examined the attitudes of pregnant women in low risk pregnancies towards full-scale genetic testing and explored relationships between demographic characteristics and the level of interest. METHODS: A prospective study was performed on 115 consecutive pregnant women. They completed the same structured questionnaire at two different time points, before counselling (T1) and after 4 weeks (T2), to assess a possible change of attitude. RESULTS: At T1, 33% of the respondents were in favour of full-scale testing of their unborn child, whereas at T2, this percentage had dropped to 18%. Except for educational level, no significant relationships were noted between the demographic variables and the wish to opt for full-scale testing. A low educational level was significantly related to the interest in full-scale testing. CONCLUSIONS: Low risk pregnant women expressed little interest in full-scale genetic testing. Educational level appeared to affect their views.

Detection of cryptic subtelomeric imbalances in fetuses with ultrasound abnormalities.

It is known from postnatal diagnosis that imbalances of the subtelomeric regions contribute significantly to idiopathic mental retardation. Consequently, subtelomere screening has been incorporated into the recommendations for the evaluation of individuals with unexplained mental retardation and a normal karyotype. Previous studies suggested that for fetuses with ultrasound abnormalities and a normal karyotype, additional screening for submicroscopic imbalances can be relevant for diagnosis and prognosis. In the present paper, we report the detection of such (subtelomeric) imbalances in three fetuses. Prenatally, the three fetuses presented with ultrasound abnormalities highly suspected of a chromosomal aberration. In two of the fetuses, routine karyotyping showed no aberrations but with MLPA or FISH a small subtelomeric imbalance, that could explain the anomalies, was detected. In the third fetus, a chromosomal abnormality was detected with routine cytogenetic analysis (del(X)(p22.1)), but this abnormality could not explain the ultrasound observations and only with subtelomere screening by MLPA a causative chromosomal aberration was detected. As the three fetuses were already prenatally suspected of a chromosomal aberration, this underlines the potential relevance of subtelomere screening in such fetuses, leading to better clinical diagnosis, prognosis and care. Furthermore, when using MLPA, the analysis can be extended to other regions of known clinical importance.

Multiplex ligation-dependent probe amplification (MLPA) as a stand-alone test for rapid aneuploidy detection in amniotic fluid cells.

OBJECTIVE: This study aimed to determine the diagnostic application of multiplex ligation-dependent probe amplification (MLPA) as a stand-alone test for targeted detection of common chromosomal aneuploidies (i.e. 13, 18, 21, X and Y) in amniotic fluid cells in routine prenatal clinical practice. METHODS: In this evaluation study, the MLPA test using kit P095 was performed on 1000 consecutive amniotic fluid samples and the results obtained were compared with traditional karyotyping (TK), the gold standard. RESULTS: The absolute specificity and sensitivity of the MLPA test were 100%. The test yielded a rapid reporting time: 94% within three working days and 5% within seven working days. The test failure rate was 0.8%. The percentage of abnormalities undetectable using this specific test was 2.4%: abnormal foetal ultrasound (N=9), increased risk first trimester screening (N=2), advanced maternal age (N=3) or other reason for referral (N=10). These abnormalities can be categorised in clinically significant (N=8), clinically uncertain (N=4) and clinically nonsignificant (N=12). CONCLUSIONS: MLPA P095 is suitable as a stand-alone test for the rapid and efficient detection of the most common chromosomal aneuploidies in routine prenatal clinical practice. A flow chart for integrating the MLPA test into the cytogenetic laboratory workflow is presented.

Fetal anomaly scan potentially will replace routine AFAFP assays for the detection of neural tube defects.

OBJECTIVES: Introduction of the second-trimester fetal anomaly scan and the decision to offer this scan to every woman in the 18th-22nd week of pregnancy necessitates a re-evaluation of the diagnostic value of the measurement of alpha-fetoprotein (AFP) concentrations in the amniotic fluid (AF) for the detection of neural tube defects (NTDs). METHODS: In this study of 6501 women who underwent amniocentesis, amniotic fluid AFP (AFAFP) concentrations were measured. The women were divided into three categories: group I, without any increased risk of fetal NTD (N = 6188); group II, with an increased risk of fetal NTD (N = 258); and group III, with a clinically diagnosed fetal NTD with known AFAFP concentrations (N = 55). RESULTS: In 27 women of group I (0.4%), the MoM (multiple of the median) level was > 2.5 times the median AFP concentration for the corresponding gestational age, and in two fetuses this was related to NTD. In two pregnancies of group II (0.8%), an increased AFAFP was related to NTD. In group III, 44 of the 55 (80%) samples had an increased AFAFP. CONCLUSION: In the near future, it is likely that imaging will replace AFAFP assays for the detection of fetal NTDs because high quality ultrasound imaging will detect NTDs accurately.