Consanguinity and pregnancy outcomes in a multi-ethnic, metropolitan European population.

OBJECTIVE: The aim of the present study was to assess the risk of major anomalies in the offspring of consanguineous couples, including data on the prenatal situation. METHODS: Over 20 years (1993-2012), 35,391 fetuses were examined by prenatal sonography. In 675 cases (1.9%), parents were consanguineous, with 307 couples (45.5%) related as first cousins, 368 couples (54.5%) beyond first cousins. Detailed information was retrieved on 31,710 (89.6%) fetuses, (consanguineous 568: 1.8%). RESULTS: Overall prevalence of major anomalies among fetuses with non-consanguineous parents was 2.9% (consanguineous, 10.9%; first cousins, 12.4%; beyond first cousins, 6.5%). Adjusting the overall numbers for cases having been referred because of a previous index case, the prevalences were 2.8% (non-consanguineous) and 6.1% (consanguineous) (first cousin, 8.5%; beyond first cousin, 3.9%). Further adjustment for differential rates of trisomic pregnancies indicated 2.0%/5.9% congenital anomalies (non-consanguineous/consanguineous groups), that is, a consanguinity-associated excess of 3.9%, 6.1% in first cousin progeny and 1.9% beyond first cousin. CONCLUSIONS: The prevalence of major fetal anomalies associated with consanguinity is higher than in evaluations based only on postnatal life. It is important that this information is made available in genetic counselling programmes, especially in multi-ethnic and multi-religious communities, to enable couples to make informed decisions.

Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins.

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Heteromorphic variants of chromosome 9.

BACKGROUND: Heterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers. RESULTS: In this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes. CONCLUSIONS: Based on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.

Noninvasive prenatal detection of chromosomal aneuploidies using different next generation sequencing strategies and algorithms.

OBJECTIVE: Here we describe the successful application of massively parallel sequencing for noninvasive prenatal detection of trisomy 21. In addition, for the detection of a broader spectrum of fetal aneuploidies, a target enrichment approach was successfully tested. METHODS: The circulating cell-free DNA was prepared from 53 maternal blood samples and analysed using Illumina's sequencing systems Genome Analyzer(IIx) and HiSeq2000, respectively. In a first experiment the SureSelect Target Enrichment System was tested. RESULTS: In our initial study analysing 42 samples on the Genome Analyzer(IIx) , all eight samples from women carrying a trisomy 21 fetus were correctly identified. On the basis of our HiSeq2000 sequence data, we discussed new algorithms for detection of fetal trisomy 21. In addition, we successfully used the combination of a target enrichment system followed by sequencing and were able to identify fetal trisomy 13 and fetal trisomy 21. CONCLUSIONS: Our results confirm previous reports that massively parallel sequencing of cell-free fetal DNA allows the reliably noninvasive detection of trisomy 21 from maternal blood with the potential to enhance test selectivity and specificity by bioinformatic means. According to our preliminary results, targeted sequencing might be an alternative strategy to detect chromosomal aneuploidies besides trisomy 21.

'Normal' nuchal translucency: a justification to refrain from detailed scan? Analysis of 6858 cases with special reference to ethical aspects.

OBJECTIVE: To assess the prevalence and detection rate of major anomalies (MAs) by applying first trimester anomaly scan (FTAS) including first trimester fetal echocardiography (FTFE) to all fetuses and discuss ethical implications. METHODS: The study group included 6879 consecutive fetuses with known outcome of pregnancy (follow-up: 98%), 6565 with 'normal' nuchal translucency (NT) (≤ P95), 314 with 'increased' NT (> P95). All fetuses received FTAS/FTFE. As MAs with the potential of being detected at FTAS/FTFE, we defined anomalies present at conception or developed during first trimester. RESULTS: Prevalence of MAs in fetuses with 'normal' NT reached 1.7%. Although 29.8% of chromosomal abnormalities were found in the group of 'normal' NT, 77% of MAs accompanied by a normal karyotype were found in this group. In fetuses with 'normal' NT and MA, diagnosis was made prenatally in 87.4% (FTAS/FTFE: 58.6%). CONCLUSION: A relevant number of MA is present in fetuses with 'normal' NT. More than half will be detected by FTAS/FTFE. As consequence, one should discuss a concept in which also in fetuses with 'normal' NT, FTAS/FTFE should be offered. This concept can also be justified from an ethical point of view, which focuses on the principles of nonmaleficence, justice and respect for autonomy of the pregnant woman.

Small supernumerary marker chromosomes 1 with a normal phenotype.

Small supernumerary marker chromosomes (sSMCs) are a major problem in prenatal cytogenetic diagnostics. Over two-thirds of cases carrying an sSMC derived from chromosome 1 are associated with clinical abnormalities. We report 3 further cases of such sSMCs that did not show any clinical abnormalities. All 3 sSMCs studied were detected prenatally and characterized comprehensively for their genetic content by molecular cytogenetics using subcentromere-specific multicolor fluorescence in situ hybridization, and for a possibly associated uniparental disomy. After exclusion of additional euchromatin due to the presence of sSMCs and a uniparental disomy, parents opted for continuation of the pregnancies and healthy children were born in all 3 cases. It is important to quickly and clearly characterize prenatal sSMCs. Also, all available sSMC cases need to be collected on a homepage such as the Jena Institute of Human Genetics and Anthropology sSMC homepage (http://www.med.uni-jena.de/fish/sSMC/00START.htm).

Molecular cytogenetic characterisation of an interstitial deletion 12p detected by prenatal diagnosis.

OBJECTIVES: Deletions in the short arm of chromosome 12 are rare structural aberrations. Till now only ten patients with interstitial deletions are described in the literature. Here, we report on the first case detected by prenatal diagnosis. Chorionic villi sampling was performed in the 14th week of gestation, indicated by fetal abnormalities detected by ultrasound examination. Conventional cytogenetic and molecular cytogenetic techniques were applied to determine the correct karyotype of the affected fetus. RESULTS: Analysing Giemsa- and QFQ-stained chromosomes of the CVS short-term culture, a structural aberrant chromosome 12 was detected. Fluorescence in situ hybridisation with YAC-probes and CGH analysis with DNA extracted from native chorionic villi proved an interstitial deletion in the aberrant chromosome 12. The GTG-banded chromosomes of the CVS long-term culture confirmed these results. Analyses of the parental chromosomal sets yielded normal results, indicating a de novo aberration. Array CGH analysis was performed to determine accurately the deletion breakpoints. Finally, according to ISCN 2005, the karyotype could be determined as 46,XX.arr cgh 12p13.2p11.21(RP11-77I22-->RP11-144O23)x 1. CONCLUSION: The presented case shows the power of modern cytogenetic methods, allowing a more detailed diagnosis in affected individuals, and therefore, facilitating a more reliable prenatal diagnosis.

Prenatal diagnosis and molecular cytogenetic characterisation of a small de novo interstitial duplication 16q11.2-q13.

We describe the first prenatally detected case of a small de novo interstitial duplication of chromosome 16q. This chromosomal aberration is extremely rare. Amniocentesis was indicated by advanced maternal age only. Ultrasound examinations of the foetus showed no abnormalities. Conventional and molecular cytogenetic analyses on cultured amniocytes by comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) using partial chromosome paints and a locus-specific YAC clone revealed a de novo direct duplication of the chromosomal region 16q11.2-q13 leading to a partial trisomy 16q (46,XX,dup(16)(q11.2q13)). There are only five postnatal reports of comparable duplications involving this chromosomal region. These patients presented with little or no associated dysmorphic features but with significant neurodevelopmental delay and severe behavioural problems. After genetic counselling, the parents opted for termination of pregnancy. Post-mortem examination showed slight facial dysmorphic signs, minor dysgenesis of the ovaries and an atypical outflow of the arteria thyroidea ima.

Slow progression of ataxia-telangiectasia with double missense and in frame splice mutations.

Ataxia-telangiectasia (A-T) is caused by mutations of the ATM gene, the product of which is involved in the regulation of cellular responses to radiation damage. Ataxia usually starts in early childhood but a delayed age at onset and slower rate of neurological deterioration has been found for some patients with variant A-T. Only few patients have been documented to survive into the 4th decade. We report on a patient with an attenuated form of A-T who was diagnosed as having A-T by the age of 52 years and died by the age of 60 years. He was found to be a compound heterozygote for a double missense mutation (D2625E and A2626P) and a novel splicing mutation (496 + 5G --> A) of the ATM gene. Cytogenetic studies of the patient's lymphoblastoid cells revealed modest levels of bleomycin-induced chromosomal instability. Residual ATM protein was found at a level of 10-20% of wildtype. Low residual ATM kinase activity could be demonstrated towards p53, whereas it was poorly detectable towards nibrin. Our results corroborate the view that the clinical variability of A-T is partly determined by the mutation type and indicate that A-T can extend to late adulthood disease.

Prenatal diagnosis of fetal trisomy 6 mosaicism and phenotype of the affected newborn.

The first case of a fetal trisomy 6 mosaicism proven at 25 weeks of gestation by analysis of fetal urine cells is described. Chromosomal analysis was indicated by an ultrasonographically diagnosed heart defect at 21 weeks of gestation. The chromosomal aberration was detected in amniotic fluid cells while fetal blood cells showed a normal chromosome set. At term a boy with normal growth parameter was born. In addition to the expected heart defect, malformations of hands and feet were present.

Small supernumerary marker chromosomes (SMCs): genotype-phenotype correlation and classification.

Small supernumerary marker chromosomes (SMCs) are present in about 0.05% of the human population. In approximately 30% of SMC carriers (excluding the approximately 60% SMC derived from one of the acrocentric chromosomes), an abnormal phenotype is observed. The clinical outcome of an SMC is difficult to predict as they can have different phenotypic consequences because of (1). differences in euchromatic DNA-content, (2). different degrees of mosaicism, and/or (3). uniparental disomy (UPD) of the chromosomes homologous to the SMC. Here, we present 35 SMCs, which are derived from all human chromosomes, apart from chromosome 6, as demonstrated by the appropriate molecular cytogenetic approaches, such as centromere-specific multicolor fluoresence in situ hybridization (cenM-FISH), multicolor banding (MCB), and subcentromere-specific multicolor FISH (subcenM-FISH). In nine cases without an aberrant phenotype, neither partial proximal trisomies nor UPD could be detected. Abnormal clinical findings, such as psychomotoric retardation and/or craniofacial dysmorphisms, were associated with seven of the cases in which subcentromeric single-copy probes were proven to be present in three copies. Conversely, in eight cases with a normal phenotype, proximal euchromatic material was detected as partial trisomy. UPD was studied in 12 cases and subsequently detected in two of the cases with SMC (partial UPD 4p and maternal UPD 22 in a der(22)-syndrome patient), indicating that SMC carriers have an enhanced risk for UPD. At present, small proximal trisomies of 1p, 1q, 2p, 6p, 6q, 7q, 9p, and 12q seem to lead to clinical manifestations, whereas partial proximal trisomies of 2q, 3p, 3q, 5q, 7p, 8p, 17p, and 18p may not be associated with significant clinical symptoms. With respect to clinical outcome, a classification of SMCs is proposed that considers molecular genetic and molecular cytogenetic characteristics as demonstrated by presently available methods.

Ring chromosome 6 in three fetuses: case reports, literature review, and implications for prenatal diagnosis.

Prenatal and postnatal findings in three fetuses with a ring chromosome 6 are presented, and the literature of this rare cytogenetic disorder is reviewed. The described fetuses illustrate the broad spectrum of the clinical manifestation of ring chromosome 6. In one fetus, the disorder was diagnosed incidentally by a routine amniocentesis due to advanced maternal age. The other two fetuses were hydrocephalic and had other congenital anomalies. Remarkably, the ring chromosome 6 tends to disappear in cultured amniotic fluid cells; karyotyping revealed complete or nearly complete monosomy 6. In contrast, the ring was preserved in high proportions of fetal leukocytes. Postnatal growth retardation is the only consistent finding of this chromosomal disorder. Maternal age is not significantly above average. An additional review of 20 literature cases revealed a striking tendency to hydrocephalus, either due to deficient brain growth or secondary to an aqueductal stenosis. Children with hydrocephalus and ring chromosme 6 tend to display facial dysmorphism and may have additional malformations, growth failure, eye anomalies, and seizures. In contrast, there are two reports on children with a ring chromosome 6 who had short stature, normal appearance, and a normal or almost-normal psychomotor development. In such patients at the mild end of the clinical spectrum, the phenotype is basically restricted to what Kosztolányi. [1987: Hum Genet 75:174-179] delineated as "ring syndrome," comprising "severe growth failure without major malformations, without a specific deletion syndrome, with only a few or no minor anomalies, and mild to moderate mental retardation." This "ring syndrome" is considered to occur independently of the autosome involved in the ring formation. The overall impression from our cases and from the literature review of cases with ring chromosome 6 is that the karyotype-genotype correlation is poor. This makes prognostic counseling of parents difficult and unsatisfactory. Serial targeted ultrasound examinations, especially of the brain, are decisive factors in elucidating the prognosis.