Validation of two-channel sequencing-by-synthesis for noninvasive prenatal testing of fetal whole and partial chromosome aberrations.

OBJECTIVE: To validate Illumina's two-channel NextSeq 500 sequencing system for noninvasive prenatal testing (NIPT) of fetal whole chromosome and partial aberrations. METHODS: A total of 162 plasma samples, previously sequenced for NIPT on a SOLiD 5500xl platform, were sequenced on the NextSeq 500 using 75-bp single-end sequencing, followed by analysis using the WISECONDOR algorithm. RESULTS: For whole chromosome aneuploidy detection, all samples were classified correctly (in total 3× T13, 3× T18, 8× T21 and 145× euploid). Three partial aberrations (36-Mb terminal loss of 5p, 14-Mb gain on 18p and 33-Mb terminal loss of 13q) were also correctly identified. Fetal fractions in 34 male samples sequenced on both the SOLiD 5500xl and NextSeq 500 platform showed no significant difference. To test robustness, two sample sets, containing both euploid and aneuploid samples, were sequenced on different NextSeq 500 machines, revealing identical results. With unchanged laboratory flow, the NIPT turnaround time could be reduced from 15-16 calendar days to 7-8 calendar days, after switching from the SOLiD 5500xl to the NextSeq 500 platform. CONCLUSIONS: The NextSeq 500 platform can be used for NIPT to detect both whole and partial chromosome aberrations. It has fast turnaround times and is suitable for mid-sized laboratories.

Phenotypic spectrum of 20 novel patients with molecularly defined supernumerary marker chromosomes 15 and a review of the literature.

Supernumerary marker chromosomes (SMC) originating from chromosome 15 are the most common SMCs. They encompass clinically irrelevant SMC(15)s containing only heterochromatin and 15p material, and clinically relevant SMC(15)s that consist of both eu- and heterochromatic 15q material. On the basis of size, the clinically relevant SMC(15)s can be subdivided into type A, "large" asymmetric and type B, "small" symmetric SMC(15)s. Type B SMC(15)s contain the triplicated 15pter to BP3 (located at 26.5 Mb) region, while type A SMC(15)s consist of 15pter --> BP4(28.5 Mb)::BP5(30.5 Mb) --> 15pter. In this study, the clinical and molecular features of 18 patients with A and B SMC(15)s and two patients with a partial trisomy 15q were reviewed. Questionnaires (including Child Behavior Check Lists) were used to assess behavior and developmental features in more detail. The marker size and parental origin were determined by multiplex ligation-dependent probe amplification (MLPA). Based on the MLPA results, the majority of patients (14/18) had type A SMC(15)s. The phenotype observed did not show significant differences between types A and B SMC(15)s. A high prevalence of autistic-like behavior, attention problems, aggressive behavior, anxiety, and sleeping problems was reported. Motor and speech development varied extensively among the patients. An association was found between positive seizure history and degree of intellectual disability.

Buccal cell FISH and blood PCR-Y detect high rates of X chromosomal mosaicism and Y chromosomal derivatives in patients with Turner syndrome.

Turner syndrome (TS) is the result of (partial) X chromosome monosomy. In general, the diagnosis is based on karyotyping of 30 blood lymphocytes. This technique, however, does not rule out tissue mosaicism or low grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome. We investigated different approaches to improve the detection of mosaicisms in 162 adult women with TS (mean age 29.9 ± 10.3). Standard karyotyping identified 75 patients (46.3%) with a non-mosaic monosomy 45,X. Of these 75 patients, 63 underwent additional investigations including FISH on buccal cells with X- and Y-specific probes and PCR-Y on blood. FISH analysis of buccal cells revealed a mosaicism in 19 of the 63 patients (30.2%). In five patients the additional cell lines contained a (derivative) Y chromosome. With sensitive real-time PCR we confirmed the presence of this Y chromosome in blood in three of the five cases. Although Y chromosome material was established in ovarian tissue in two patients, no gonadoblastoma was found. Our results confirm the notion that TS patients with 45,X on conventional karyotyping often have tissue specific mosaicisms, some of which include a Y chromosome. Although further investigations are needed to estimate the risk of gonadoblastoma in patients with Y chromosome material in buccal cells, we conclude that FISH or real-time PCR on buccal cells should be considered in TS patients with 45,X on standard karyotyping.

Parental insertional balanced translocations are an important cause of apparently de novo CNVs in patients with developmental anomalies.

In several laboratories, genome-wide array analysis has been implemented as the first tier diagnostic test for the identification of copy number changes in patients with mental retardation and/or congenital anomalies. The identification of a pathogenic copy number variant (CNV) is not only important to make a proper diagnosis but also to enable the accurate estimation of the recurrence risk to family members. Upon the identification of a de novo interstitial loss or gain, the risk recurrence is considered very low. However, this risk is 50% if one of the parents is carrier of a balanced insertional translocation (IT). The apparently de novo imbalance in a patient is then the consequence of the unbalanced transmission of a derivative chromosome involved in an IT. To determine the frequency with which insertional balanced translocations would be the origin of submicroscopic imbalances, we investigated the potential presence of an IT in a consecutive series of 477 interstitial CNVs, in which the parental origin has been tested by FISH, among 14,293 patients with developmental abnormalities referred for array. We demonstrate that ITs underlie ~2.1% of the apparently de novo, interstitial CNVs, indicating that submicroscopic ITs are at least sixfold more frequent than cytogenetically visible ITs. This risk estimate should be taken into account during counseling, and warrant parental and proband FISH testing wherever possible in patients with an apparently de novo, interstitial aberration.