Evolution of genome diagnostics in epidermolysis bullosa: Unveiling the power of next-generation sequencing.

BACKGROUND: Genome diagnostics is considered gold standard diagnostics for epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous group of rare disorders characterized by blistering and wounding of mucocutaneous tissues. EB is caused by pathogenic variants in genes encoding proteins of the dermo-epidermal junction. Accurate genetic diagnosis of EB is crucial for prognostication, counselling and precision-medicine. Genome diagnostics for EB started in 1991 with the introduction of Sanger sequencing (SS), analysing one gene at a time. In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel. Several studies have shown a beneficial role for NGS in EB diagnostics, but its true benefit has not been quantified. OBJECTIVES: To determine the benefit of NGS in EB by systematically evaluating the performance of different genome diagnostics used over time based on robust data from the Dutch EB Registry. METHODS: The diagnostic performances of SS and NGS were systematically evaluated in a retrospective observational study including all index cases with a clinical diagnosis of EB in whom genome diagnostics was performed between 01 January 1994 and 01 January 2022 (n = 308), registered at the Dutch EB Expertise Centre. RESULTS: Over time, a genetic diagnosis was made in 289/308 (94%) EB cases. The diagnostic yield increased from 89% (SS) to 95% (NGS). Most importantly, NGS significantly reduced diagnostic turnaround time (39 days vs. 211 days, p < 0.001). The likelihood of detecting variants of uncertain significance and additional findings increased from 5% and 1% (SS) to 22% and 13% (NGS) respectively. CONCLUSIONS: Our study quantifies the benefit of NGS-based methods and demonstrate they have had a major impact on EB diagnostics through an increased diagnostic yield and a dramatically decreased turnaround time (39 days). Although our diagnostic yield is high (95%), further improvement of genome diagnostics is urgently needed to provide a genetic diagnosis in all EB patients.

Estimates of live birth prevalence of children with Down syndrome in the period 1991-2015 in the Netherlands.

BACKGROUND: In Western countries, increasing maternal age has led to more pregnancies with a child with Down syndrome (DS). However, prenatal screening programs, diagnostic testing and termination of pregnancy influence the actual DS live birth (LB) prevalence as well. The aim of this study is to examine these factors in the Netherlands for the period 1991-2015. In our study, we establish a baseline for DS LB prevalence before non-invasive prenatal testing will be made available to all pregnant women in the Netherlands in 2017. METHODS: Full nationwide data from the Dutch cytogenetic laboratories were used to evaluate the actual DS LB prevalence. In addition, nonselective DS prevalence, which is the DS LB prevalence that would be expected in absence of termination of pregnancies, was estimated on the basis of maternal age distribution in the general population. RESULTS: Because of an increase in maternal age, nonselective DS prevalence increased from around 15.6 [95% confidence interval (CI) 13.9-17.4] per 10 000 LBs in 1991 (311 children in total) to around 22.6 (95% CI 20.3-24.9) per 10 000 in 2015 (385), the increase levelling off in recent years. Actual LB prevalence rose from around 11.6 (95% CI 10.9-12.2) per 10 000 in 1991 (230 children) to an estimated peak of 15.9 (95% CI 15.6-16.2) per 10 000 in 2002 (322), gradually decreasing since to 11.1 (95% CI 10.8-11.5) per 10 000 in 2015 (190). Reduction of DS LBs resulting from elective terminations had been fairly constant between 1995 and 2002 at around 28% and rose afterwards from 35% in 2003 to around 50% in 2015. CONCLUSIONS: In spite of expansion of antenatal screening in the Netherlands in the 1990s and early 2000s, actual DS LB prevalence increased during this period. However, after 2002, this trend reversed, probably because of informing all pregnant women about prenatal testing since 2004 and the implementation of a national screening program in 2007.

3p14 deletion is a rare contiguous gene syndrome: report of 2 new patients and an overview of 14 patients.

Interstitial deletions of chromosome 3p14p12 are a rare chromosome rearrangement. Twenty-six patients have been reported in the literature to date, however, a specific clinical phenotype has not yet been delineated. We describe three patients (two new) with overlapping chromosome 3p14p12 deletions and review the clinical and molecular data of 11 well-characterized, published cases. These patients had a number of features in common, such as short stature, failure to thrive, facial dysmorphism, congenital heart defects, urogenital abnormalities, neurological problems, hearing loss, and global developmental delay, suggesting that the interstitial chromosome 3p14p12 deletion gives rise to a multiple congenital anomaly syndrome. Some of the patients show clinical overlap with other complex syndromes such as CHARGE syndrome. Genotype-phenotype analysis revealed candidate genes for parts of the clinical features suggesting that the 3p14 deletion is a contiguous gene syndrome.

Drayer's syndrome of mental retardation, microcephaly, short stature and absent phalanges is caused by a recurrent deletion of chromosome 15(q26.2-->qter).

We reevaluated a unique family with two sibs who had a presumed autosomal recessively inherited syndrome characterized by mental retardation, microcephaly, short stature and absent phalanges. This family was originally described by Drayer et al. in 1977. Using modern molecular techniques, we demonstrated that the syndrome is caused by the recurrence of an apparently de novo 15qter deletion of 5.8 Mb. Analysis of polymorphic markers revealed that the deletion was of maternal origin in both cases, indicating germline mosaicism in the clinically unaffected mother. This study demonstrates the possibility of parental mosaicism and the risk of recurrence in sibs for terminal subtelomeric deletions.

Novel Algorithms for Improved Sensitivity in Non-Invasive Prenatal Testing.

Non-invasive prenatal testing (NIPT) of cell-free DNA in maternal plasma, which is a mixture of maternal DNA and a low percentage of fetal DNA, can detect fetal aneuploidies using massively parallel sequencing. Because of the low percentage of fetal DNA, methods with high sensitivity and precision are required. However, sequencing variation lowers sensitivity and hampers detection of trisomy samples. Therefore, we have developed three algorithms to improve sensitivity and specificity: the chi-squared-based variation reduction (χ2VR), the regression-based Z-score (RBZ) and the Match QC score. The χ2VR reduces variability in sequence read counts per chromosome between samples, the RBZ allows for more precise trisomy prediction, and the Match QC score shows if the control group used is representative for a specific sample. We compared the performance of χ2VR to that of existing variation reduction algorithms (peak and GC correction) and that of RBZ to trisomy prediction algorithms (standard Z-score, normalized chromosome value and median-absolute-deviation-based Z-score). χ2VR and the RBZ both reduce variability more than existing methods, and thereby increase the sensitivity of the NIPT analysis. We found the optimal combination of algorithms was to use both GC correction and χ2VR for pre-processing and to use RBZ as the trisomy prediction method.

Mosaic isodicentric chromosome 18q: sixth report and review.

We describe a girl with a mosaic isodicentric chromosome 18q with discrete features of trisomy 18. She presented with prenatal growth retardation, prominent occiput, small face, high nasal bridge, large nose, thin lips, a perimembranous ventricular septal defect, and subsequent slow psychomotor development and slow growth. Amosaic isopseudodicentric chromosome 18q was detected in cultured lymphocytes: mos 46,XX,psu idic(18)(q23)[74]/ 46,XX[26]. Monosomy of the distal end of 18q23 could not be confirmed by fluorescent in situ hybridization (FISH) with RP 1l-565D23, one of the most telomere located probes of 18q23. Isopseudodicentric chromosome 18q is very rare. Most cases are mosaics. The phenotype varies. More or less distinct features of trisomy 18 and monosomy 18q can be found depending on the degree of mosaicism and the breakpoint in 18q.

Three cases of mosaicism for balanced reciprocal translocations.

Mosaicism for a balanced reciprocal translocation (BRTM) is rare. As far as we know only 26 cases of BRTM, demonstrated in lymphocyte cultures, have been described, five of which had an abnormal phenotype. Prenatally three confirmed cases with a normal phenotypic outcome have been described. Here we present three further cases of BRTM in lymphocyte cultures. The first was detected during a family study, the second after an abnormal karyotype in chorionic villus sampling, and the third because of a history of stillborn children. All three carriers have normal phenotypes. An inventory of the BRTM cases reported so far is made.

[Preimplantation genetic screening for numerical chromosomal abnormalities in embryos from women of 35 years of age and older; first results in The Netherlands]. / Preïmplantatiegenetische screening op numerieke chromosoomafwijkingen bij embryo's van vrouwen van 35 jaar en ouder; de eerste resultaten in Nederland.

OBJECTIVE: To assess the results of preimplantation genetic screening (PGS) for numerical chromosomal abnormalities in embryos from women of 35 years of age and older. DESIGN: Prospective, descriptive. METHOD: Women who were at least 35 years received standard IVF/ICSI treatment including ovarian hyperstimulation, after which matured oocytes were recovered and inseminated. Three days after insemination, one cell was biopsied from each of the available embryos. In these cells, the copy number of 5 (first 21 patients) or 8 chromosomes was determined using fluorescence in situ hybridisation (FISH). Only embryos with a normal or unknown FISH result were implanted in the uterus. Data were collected in an electronic database. RESULTS: PGS was done for 28 IVF- and 22 ICSI-treatments; the average age of the 50 women at the beginning of treatment was 38.5 years. There were 360 embryos generated; of the 288 biopsied embryos 156 (54%) contained an abnormal number of chromosomes. In 45 women, 1 or 2 embryos were transferred. This resulted in 8 ongoing pregnancies (8/50; 16%) and the birth of 9 children, all of whom were found to be healthy on a paediatric examination at 3 to 10 months of age. In 4 cases there was no embryo transfer because all the embryos were chromosomally abnormal. CONCLUSION: In the first 50 patients in The Netherlands, PGS resulted in an ongoing pregnancy rate of 16% per woman. All children showed normal growth and development.

Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded patients without autism and in normal individuals.

Array CGH (comparative genomic hybridization) screening of large patient cohorts with mental retardation and/or multiple congenital anomalies (MR/MCA) has led to the identification of a number of new microdeletion and microduplication syndromes. Recently, a recurrent copy number variant (CNV) at chromosome 16p11.2 was reported to occur in up to 1% of autistic patients in three large autism studies. In the screening of 4284 patients with MR/MCA with various array platforms, we detected 22 individuals (14 index patients and 8 family members) with deletions in 16p11.2, which are genomically identical to those identified in the autism studies. Though some patients shared a facial resemblance and a tendency to overweight, there was no evidence for a recognizable phenotype. Autism was not the presenting feature in our series. The assembled evidence indicates that recurrent 16p11.2 deletions are associated with variable clinical outcome, most likely arising from haploinsufficiency of one or more genes. The phenotypical spectrum ranges from MR and/or MCA, autism, learning and speech problems, to a normal phenotype.

Probability tables for exclusion of mosaicism in prenatal diagnosis.

The decision concerning the number of metaphases that need to be analysed to detect mosaicism of a certain degree depends mainly, for the same confidence levels, on the culture method used (in situ or flask methods). Several probability tables, designed for either the in situ or the flask method, have been reported and can be used to assist laboratories in making the decisions referred to above. However, there are instances where part of the analysis is done using the in situ and flask methods. In such situations, the previously published tables are of limited use. We have generated a new table that can be used in such situations, as well as in cases where only the flask method is used.

Two unbalanced segregation products due to a maternal t(7;16)inv(16).

We report a prenatal case of a maternally inherited abnormal chromosome 16, originally interpreted as a pericentric inversion only, but after family studies re-interpreted as a pericentric inversion (16) accompanied by an unbalanced (7;16) translocation. Because of the inversion 16 and an elder son with developmental delay and craniofacial dysmorphic features, in the past karyotyped as 46,XY, the chromosomes 16 of the mother and son were carefully re-examined. Using a whole chromosome 16 paint and sub-telomere probes of 16p and 16q, the karyotype of the mother was shown to be 46,XX,inv(16)(p11.2q23.2).ish t(7;16)(q36;p13.3)inv(16). Subsequently one chromosome 16 of the elder son appeared to be a der(16)t(7;16)(q36;p13.3). This is probably the result of a meiotic crossover between the chromosomes 16 in the mother. The prenatal karyotype was finally interpreted as 46,XY,inv(16)(p11.2q23.2).ish der(16)t(7;16)(q36;p13.3)inv(16). This is the same cytogenetic imbalance as his elder brother: a partial trisomy of chromosome 7 (q36-->qter) and a partial monosomy of chromosome 16 (p13.3-->pter).

Placental mosaicism is associated with unexplained second-trimester elevation of MShCG levels, but not with elevation of MSAFP levels.

In this patient-control study, we examined the impact of placental mosaicism on the concentrations of maternal serum human chorionic gonadotropin (MShCG) and maternal serum alpha-fetoprotein (MSAFP) in the second trimester of pregnancy. Patient and control groups were selected from 2347 women with a singleton pregnancy, who underwent chorionic villous sampling in the first trimester and from whom second-trimester serum samples had been collected. The concentrations of both serum markers, expressed in multiples of the median (MOM), in 35 women with confined placental mosaicism (CPM) were compared with those in 70 controls with uncomplicated pregnancies. Elevated MSAFP or MShCG was defined as a concentration of > or = 2.0 MOM. Of the 35 pregnancies with CPM, none had an elevated MSAFP level, as opposed to two out of the 70 women (2.9 per cent) in the control group (P = NS). Nine women in the placental mosaicism group (26 per cent) had an MShCG level of > or = 2.0 MOM, compared with five in the control group (7.1 per cent; P = 0.0135). Nineteen women in the placental mosaicism group (54 per cent) were screen-positive for Down's syndrome (cut-off 1:250), compared with 17 women (24 per cent) in the control group (P = 0.0042; relative risk = 2.3). The three highest MShCG levels were found in pregnancies with CPM that involved trisomy 16; all these women delivered a small-for-gestational age (SGA) infant. CPM, especially with trisomy 16, is associated with elevated levels of MShCG, but not with elevated levels of MSAFP. It is an important cause of false-positive results in serum screening programmes for fetal Down's syndrome. It is possible that abnormal MShCG levels in pregnancies with CPM result from dysfunctional placenta, caused by chromosomally abnormal areas. We therefore recommend increased surveillance of pregnancies with unexplained elevated MShCG levels.

46,XY,dup(10q) in direct CVS preparation and mosaic 48,XXXY,dup(10q) in CVS long-term culture and fetal tissue.

Chorionic villus sampling (CVS) was performed on a 40-year-old woman at 9 1/2 menstrual weeks because of advanced maternal age. The direct preparation showed 46,XY,dup(10)(q11.2q23.2). CVS long-term culture and fetal tissue revealed a rare additional abnormality: 48,XXXY,dup(10)(q11.2q23.2). This abnormality represented the major cell line (> 85 per cent in 691 cells) in an (XY)/XXY/XXXY/(XXXXY) mosaic (all cell lines presumably bearing the dup(10q); the presence of XY and XXXXY cell lines is uncertain). To our knowledge, this is the first report of trisomy 10q11-q23 and of prenatally detected 48,XXXY in chorionic villi. The mosaic could have resulted from early post-zygotic non-disjunctions in a 46,XY,dup(10q) or 47,XXY,dup(10q) zygote. The results from DNA studies of four polymorphisms, mapped to Xp and Xq, support this theory. The literature on prenatally detected cases with sex chromosome tetrasomy and pentasomy and those with additional autosomal abnormalities is reviewed. The reported case underlines the problem of false-negative findings when only direct CVS preparations are karyotyped.

Four years' cytogenetic experience with the culture of chorionic villi.

In 1958 chorionic villus samples, investigated by culture method, we found 137 (7%) abnormalities. The abnormal results were classified in certain abnormal (generalised abnormal at high probability) and uncertain abnormal (potentially confined to the placenta) results. Certain abnormal were 73 cases (3.7%). Uncertain abnormal were 64 cases (3.3%), in which confirmation studies were done in 47 cases. In 12 cases of these 47, the abnormality was confirmed and in 35 cases (1.8%) the abnormality was confined to the placenta. Among the latter cases, poor pregnancy outcome [16% intrauterine death (IUD), 6% intrauterine growth retardation (IUGR)] was increased. Total maternal cell contamination was not seen. The positive predictive value of all confirmed abnormal cases was 66%. The positive predictive value was 100% for indications 'ultrasound abnormalities' and 'carrier' and between 50 and 60% for all other indications. Predictive value among uncertain abnormal cases was low (26%). However, the positive predictive value depends of the type of abnormality. Therefore we conclude that the culture method for chorionic villi is a good test for indications 'ultrasound abnormalities' and 'carrier' and reliable for all other indications. Whether or not follow-up investigations should be offered to the parents depends of the type of abnormality. We conclude that the culture method is reliable for prenatal diagnosis and can be used as the sole investigative method.

Prenatal diagnosis in two cases of de novo complex balanced chromosomal rearrangements. Three-year follow-up in one case.

We report two cases of apparently balanced complex de novo chromosomal rearrangements (BCCR) detected prenatally at 17 weeks and 10 weeks of gestation, respectively. Chromosomes were studied using GTG-banding and fluorescent in situ hybridization (FISH). In one case four chromosomes and in the other case three chromosomes were involved in the rearrangements. One of the pregnancies was terminated and no external or internal showed no abnormalities. The child is now 3 years old and has neither congenital anomalies nor evidence of delayed psychomotor development.