Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

Confined placental mosaicism of Duchenne muscular dystrophy: a case report.

BACKGROUND: Small copy number variations confined to the placenta are extremely rare findings in chorionic villus sampling, nonetheless of great clinical importance. To the best of our knowledge, this is the first reported case of confined placental mosaicism for an intragenic Duchenne muscular dystrophy (DMD) gene deletion. CASE PRESENTATION: We describe a pregnant woman where confined placental mosaicism for an intragenic DMD deletion was detected. She was referred for a chorionic villus sampling due to an increased risk of trisomy 21 derived from combined first trimester screening. Rapid aneuploidy detection showed a male fetus with normal results for chromosomes 13, 18 and 21. A chromosomal microarray demonstrated a deletion of exons 61-62 in the DMD gene in approximately 50% of the cells. A follow-up analysis on amniotic cells showed a normal result for the DMD gene. Hence, confined placental mosaicism was confirmed. CONCLUSIONS: We propose tissue specific fragile sites as a possible theoretical mechanism for the formation of submicroscopic copy number variations and highlight that the finding of DMD deletion mosaicism in a chorionic villus sample might be isolated to the placenta. Therefore, confirmation by amniocentesis is of crucial clinical importance to avoid misdiagnosis of the fetus.

Fetal HLA-G mediated immune tolerance and interferon response in preeclampsia.

BACKGROUND: Fetal immune tolerance is crucial for pregnancy success. We studied the link between preeclampsia, a severe pregnancy disorder with uncertain pathogenesis, and fetal human leukocyte antigen G (HLA-G) and other genes regulating maternal immune responses. METHODS: We assessed sex ratios and regulatory HLA-G haplotypes in population cohorts and series of preeclampsia and stillbirth. We studied placental mRNA expression of 136 genes by sequencing and HLA-G and interferon alpha (IFNα) protein expression by immunohistochemistry. FINDINGS: We found underrepresentation of males in preeclamptic births, especially those delivered preterm or small for gestational age. Balancing selection at HLA-G associated with the sex ratio, stillbirth, and preeclampsia. We observed downregulation of HLA-G, its receptors, and many other tolerogenic genes, and marked upregulation of IFNA1 in preeclamptic placentas. INTERPRETATION: These findings indicate that an evolutionary trade-off between immune tolerance and protection against infections at the maternal-fetal interface promotes genetic diversity in fetal HLA-G, thereby affecting survival, preeclampsia, and sex ratio. We highlight IFNA1 as a potential mediator of preeclampsia and a target for therapeutic trials. FUNDING: Finnish Medical Foundation, Päivikki and Sakari Sohlberg Foundation, Karolinska Institutet Research Foundation, Scandinavia-Japan Sasakawa Foundation, Japan Eye Bank Association, Astellas Foundation for Research on Metabolic Disorders, Japan Society for the Promotion of Science, Knut and Alice Wallenberg Foundation, Swedish Research Council, Medical Society Liv och Hälsa, Sigrid Jusélius Foundation, Helsinki University Hospital and University of Helsinki, Jane and Aatos Erkko Foundation, Academy of Finland, Finska Läkaresällskapet, Novo Nordisk Foundation, Finnish Foundation for Pediatric Research, and Emil Aaltonen Foundation.

Detection rates and residual risk for a postnatal diagnosis of an atypical chromosome aberration following combined first-trimester screening.

OBJECTIVES: To determine the detection rates of all types of chromosome aberrations and the residual risk for postnatal diagnosis of an atypical chromosome aberration depending on the strategy for further investigation with either noninvasive prenatal testing (NIPT) or invasive testing in pregnancies with increased risk following combined first-trimester screening (cFTS). METHODS: A review of all pregnancies examined with cFTS during 2010 to 2017. RESULTS: The cohort consisted of 129 493 pregnancies. There were 852 (0.7%) clinically significant chromosome aberrations, including aberrations detected later on or after birth. A total of 12% were atypical chromosome aberrations. Considering that 40% were detected due to a miscarriage/intrauterine fetal death or a malformation on ultrasound there is a 0.05% (1:2000) background risk of a postnatal diagnosis of a liveborn child with an atypical chromosome aberration if no further invasive test is performed during pregnancy. If all women with an increased risk (≥1:200) had an invasive test and NIPT was performed up to a risk of 1:1000, 95% of common trisomies/sex chromosome aberrations and 55% of atypical aberrations would be detected. CONCLUSIONS: If NIPT was offered to all women with an increased risk following cFTS it would imply that three times as many children would be born with an atypical chromosome aberration.

Preimplantation genetic testing practices in the Nordic countries.

INTRODUCTION: Preimplantation genetic testing (PGT) is growing in importance and volume internationally. International societies such as the European Society for Human Reproduction and Embryology compile international results and these data are published in scientific journals. We present the first compilation of practices, quality measuress and outcome data from Nordic clinics performing PGT. MATERIAL AND METHODS: We conducted a structured online survey of PGT practices in the Nordic countries to compare clinical and laboratory techniques, outcomes and quality measures applied in Nordic clinics. The survey was designed by the authors and answered by the authors and members of the study group. The outcome data represents results from 2018. Results and details were clarified through iteration with responding clinics while maintaining anonymity. Response rate in the study was 80%, with 8 of 10 clinics performing PGT responding. RESULTS: Most of the PGT cycles in the Nordic countries are funded through the public healthcare system with University Hospitals performing the majority of treatments, 716/848, or 84.4%, of oocyte retrievals in this dataset. The genetic analyses are in five cases performed by the affiliated local genetic laboratory, and the remaining three consult with large international private enterprise laboratories. Genetic counseling is widely used. Results in the Nordic clinics compare well with international data. Systematic quality control procedures are in place and the larger clinics and laboratories utilize ISO certification or accreditation in the quality management. Automatic witnessing with detailed electronic documentation of laboratory processes is not utilized in the responding clinics, although a majority uses manual witnessing procedures in the laboratory. The outcome after PGT in terms of clinical pregnancy per transfer is around 40% per embryo transfer and compares well with international data. CONCLUSIONS: Preimplantation genetic testing is organized in rather few clinics in the Nordic countries and most of them use local laboratories for genetic analyses of the biopsies. Laboratory procedures are largely in accordance with international guidelines and the outcome after PGT in terms of clinical pregnancy per transfer is comparable to results in international reports.

Preimplantation genetic testing legislation and accessibility in the Nordic countries.

INTRODUCTION: Assisted reproduction technologies are being rapidly developed and implementation of preimplantation genetic testing (PGT) has allowed patients with genetic disorders to initiate pregnancies while minimizing or eliminating the risk of transmitting these disorders to their offspring. Testing for numeric chromosomal anomalies has been proposed as a way to increase efficacy in assisted reproduction; however, this remains disputed. Legislation is lagging behind the rapid developments in this field. MATERIAL AND METHODS: We conducted a structured online survey of legislation and accessibility to preimplantation genetic testing in the Nordic countries to compare the regulation and uptake of this technique. The survey was designed and answered by the authors. RESULTS: Key elements in the regulation of preimplantation testing for monogenic disorders and structural rearrangements are similar in the Nordic countries, although accessibility varies since only Denmark, Finland, and Sweden have national clinics offering treatment. In addition, Denmark and Finland have private clinics offering PGT. Regulation is the most stringent in Norway where a national board evaluates all couples seeking treatment. Treatment volumes vary between the Nordic countries, with Norway and Finland having lowest treatment numbers. Preimplantation genetic testing for aneuploidy in the embryo varies between the Nordic countries: Finland and Iceland allow this form of treatment, Denmark and Sweden offer it only in the form of a research protocol, and Norway does not allow it at all. Therefore the number of treatment cycles involving testing for embryo aneuploidy are lower in the Nordic countries than in other countries where this treatment option is more common. CONCLUSIONS: Science needs to inform politics regarding the rapidly evolving field of reproductive medicine and we recommend harmonization of legislation and accessibility between the Nordic countries.

From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability.

BACKGROUND: Since different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test. METHODS: We analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure. RESULTS: First, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data. Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively. CONCLUSION: The overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Identification of putative pathogenic single nucleotide variants (SNVs) in genes associated with heart disease in 290 cases of stillbirth.

The incidence of stillbirth in Sweden has essentially remained constant since the 1980's, and despite thorough investigation, many cases remain unexplained. It has been suggested that a proportion of stillbirth cases is caused by heart disease, mainly channelopathies. The aim of this study was to analyze DNA from 290 stillbirth cases without chromosomal abnormalities for pathogenic single nucleotide variants (SNVs) in 70 genes associated with cardiac channelopathies and cardiomyopathies. The HaloPlex Target Enrichment System (Agilent Technologies) was utilized to prepare sequencing libraries which were sequenced on the Illumina NextSeq platform. We found that 12.1% of the 290 investigated stillbirth cases had one (n = 31) or two (n = 4) variants with evidence supporting pathogenicity, i.e. loss-of-function variants (nonsense, frameshift, splice site substitutions), evidence from functional studies, or previous identification of the variants in affected individuals. Regarding identified putative pathogenic variants in genes associated with channelopathies, the prevalence was significantly higher in the stillbirth cohort (n = 23, 7.93%) than the corresponding prevalence of the same variants in the non-Finnish European population of the Exome Aggregation Consortium (2.70%, p<0.001) and SweGen, (2.30%, p<0.001). Our results give further support to the hypothesis that cardiac channelopathies might contribute to stillbirth. Screening for pathogenic SNVs in genes associated with heart disease might be a valuable complement for stillbirth cases where today's conventional investigation does not reveal the underlying cause of fetal demise.

Expanding the Clinical Spectrum of Phenotypes Caused by Pathogenic Variants in PLOD2.

Osteogenesis imperfecta (OI) is a strikingly heterogeneous group of disorders with a broad range of phenotypic variations. It is also one of the differential diagnoses in bent bone dysplasias along with campomelic dysplasia and thanatophoric dysplasia and can usually be distinguished by decreased bone mineralization and bone fractures. Bent bone dysplasias also include syndromes such as kyphomelic dysplasia (MIM:211350) and mesomelic dysplasia Kozlowski-Reardon (MIM249710), both of which have been under debate regarding whether or not they are a real entity or simply a phenotypic manifestation of another dysplasia including OI. Bruck syndrome type 2 (BRKS2; MIM:609220) is a rare form of autosomal recessive OI caused by biallelic PLOD2 variants and is associated with congenital joint contractures with pterygia. In this report, we present six patients from four families with novel PLOD2 variants. All cases had multiple fractures. Other features ranged from prenatal lethal severe angulation of the long bones as in kyphomelic dysplasia and mesomelic dysplasia Kozlowski-Reardon through classical Bruck syndrome to moderate OI with normal joints. Two siblings with a kyphomelic dysplasia-like phenotype who were stillborn had compound heterozygous variants in PLOD2 (p.Asp585Val and p.Ser166*). One infant who succumbed at age 4 months had a bent bone phenotype phenotypically like skeletal dysplasia Kozlowski-Reardon (with mesomelic shortening, camptodactyly, retrognathia, cleft palate, skin dimples, but also with fractures). He was homozygous for the nonsense variant (p.Trp561*). Two siblings had various degrees of Bruck syndrome caused by the homozygous missense variant, p.His687Arg. Furthermore a boy with a clinical presentation of moderate OI had a possibly pathogenic homozygous variant p.Trp588Cys. Our experience of six patients with biallelic pathogenic variants in PLOD2 expands the phenotypic spectrum in the PLOD2-related phenotypes. © 2017 American Society for Bone and Mineral Research.

[NIPT ­ implentation, counselling and ethical issues]. / SFOG ger nationella riktlinjer för fosterdiagnostik med NIPT - Information, genetisk vägledning och informerat val grunden för analys av foster-DNA i blodprov från kvinnan.

NIPT - implentation, counselling and ethical issues It is today possible to analyze cell-free fetal DNA from a blood sample from the pregnant woman, i.e. non-invasive prenatal testing, NIPT. Thus, by a simple blood test from the mother you can detect trisomy 13, 18 and 21 in the fetus with high accuracy. However, NIPT is not a diagnostic test and a positive result should be confirmed by an invasive test, like chorionic villus sampling or amniocentesis. There are national guidelines from the Swedish Society of Obstetrics and Gynaecology (SFOG) on how to implement and use NIPT for trisomies in the Swedish health care. As NIPT is only a blood test there is a risk that it is seen by the expectant parents as a routine test or as a recommendation from the health care. Pre-test counselling is therefore of great importance to enable informed choice.

Knowledge and Attitudes Regarding Non-Invasive Prenatal Testing (NIPT) and Preferences for Risk Information among High School Students in Sweden.

Non-invasive prenatal testing (NIPT) was recently introduced for prenatal testing of genetic disorders. Cell-free fetal DNA is present in maternal blood during pregnancy and enables detection of fetal chromosome aberrations in a maternal blood sample. The public perspective to this new, simple method has not been illuminated. The views of young people (i.e. future parents) are important to develop suitable counseling strategies regarding prenatal testing. The aim was to explore Swedish high school students' attitudes, knowledge and preferences regarding NIPT. A questionnaire was completed by 305 students recruited from one high school in Stockholm, November and December 2014. Most students (80 %) considered prenatal testing as good. The majority (65 %) was positive or very positive towards NIPT and 62 % stated that they potentially would like to undergo the test if they or their partner was pregnant. The vast majority (94 %) requested further information about NIPT. Most students (61 %) preferred verbal information, whereas 20 % preferred information via the Internet. The majority of the high school students was positive towards prenatal testing and most was positive towards NIPT. Further, information was requested by the vast majority before making a decision about NIPT. Most of the students preferred verbal information and to a lesser extent information via the Internet. The attitudes, knowledge and preferences for risk information concerning NIPT in young adults are important, in order to increase knowledge on how to educate and inform future parents.

Analysis of cell-free fetal DNA in maternal blood for detection of trisomy 21, 18 and 13 in a general pregnant population and in a high risk population - a systematic review and meta-analysis.

INTRODUCTION: The aim of this study was to review the performance of non-invasive prenatal testing (NIPT) for detection of trisomy 21, 18 and 13 (T21, T18 and T13) in a general pregnant population as well as to update the data on high-risk pregnancies. MATERIAL AND METHODS: Systematic review and meta-analysis. PubMed, Embase and the Cochrane Library were searched. Methodological quality was rated using QUADAS and scientific evidence using GRADE. Summary measures of diagnostic accuracy were calculated using a bivariate random-effects model. RESULTS: In a general pregnant population, there is moderate evidence that the pooled sensitivity is 0.993 (95% CI 0.955-0.999) and specificity was 0.999 (95% CI 0.998-0.999) for the analysis of T21. Pooled sensitivity and specificity for T13 and T18 was not calculated in this population due to the low number of studies. In a high-risk pregnant population, there is moderate evidence that the pooled sensitivities for T21 and T18 are 0.998 (95% CI 0.981-0.999) and 0.977 (95% CI 0.958-0.987) respectively, and low evidence that the pooled sensitivity for T13 is 0.975 (95% CI 0.819-0.997). The pooled specificity for all three trisomies is 0.999 (95% CI 0.998-0.999). CONCLUSIONS: This is the first meta-analysis using GRADE that shows that NIPT performs well as a screen for trisomy 21 in a general pregnant population. Although the false positive rate is low compared with first trimester combined screening, women should still be advised to confirm a positive result by invasive testing if termination of pregnancy is under consideration.

Positive Attitudes towards Non-Invasive Prenatal Testing (NIPT) in a Swedish Cohort of 1,003 Pregnant Women.

OBJECTIVE: The clinical utilization of non-invasive prenatal testing (NIPT) for identification of fetal aneuploidies is expanding worldwide. The aim of this study was to gain an increased understanding of pregnant women's awareness, attitudes, preferences for risk information and decision-making concerning prenatal examinations with emphasis on NIPT, before its introduction into Swedish healthcare. METHOD: Pregnant women were recruited to fill in a questionnaire, including multiple-choice questions and Likert scales, at nine maternity clinics located in different areas of Stockholm, Sweden. RESULTS: In total, 1,003 women participated in the study (86% consent rate). The vast majority (90.7%) considered examinations aiming to detect fetal abnormalities to be good. Regarding NIPT, 59.8% stated that they had heard about the method previously, yet 74.0% would like to use the test if available. The main factor affecting the women's decision to undergo prenatal chromosomal screening was worry about the baby's health (82.5%), followed by the urge to have as much information as possible about the fetus (54.5%). Most women (79.9%) preferred to receive NIPT information orally. CONCLUSION: The overwhelming majority of a cohort of 1,003 pregnant women considered prenatal examinations good. Moreover, the majority had a positive attitude towards NIPT and would like to use the test if available.

Disomy 21 in spermatozoa and the paternal origin of trisomy 21 Down syndrome.

BACKGROUND: Trisomy 21 Down syndrome is the most common genetic cause for congenital malformations and intellectual disability. It is well known that in the outstanding majority of cases the extra chromosome 21 originates from the mother but only in less than 10 % from the father. The mechanism underlying this striking difference in parental origin of Trisomy 21 Down syndrome is still unknown. However, it seems likely that the main reason is a much higher stringency in the elimination of any trisomy 21 cells during fetal testicular than ovarian development. We have here focussed attention on the paternal gametic output, i.e. the incidence of disomy 21 in spermatozoa. RESULTS: We have used fluorescence in situ hybridisation (FISH) to determine the copy number of chromosome 21 in spermatozoa from 11 men with normal spermiograms. Due to the well-known risk of false positive and false negative signals using a single FISH probe, we have applied two chromosome 21q probes, and we have added a chromosome 18-specific probe to allow differentiation between disomy 21 and diploidy. Analysing a total number of 2000 spermatozoa per case, we documented an average incidence of disomy 21 at 0.13 %, with a range of 0.00-0.25 % and a SD of 0.08. There was no indication of diploidy in this cohort of 22,000 sperm. CONCLUSION: Numerous previous studies on the incidence of disomy 21 in sperm have been published, using FISH. As far as we are aware, none of these have applied more than a single chromosome 21-specific probe. Accepting our mean of 0.13 % of disomy 21, and providing there is no selective fertilisation capability of disomy 21 sperm in relation to the normal, we conclude that around 1 in 800 conceptions is expected to be trisomic for chromosome 21 of paternal origin. Bearing in mind that the maternal origin likely is at least 10 times more common, we tentatively propose that around 1 in 80 oocytes in the maternal ovarian reserve may be disomy 21. One reason for this discrepancy may be a more stringent selection against aberrant chromosome numbers during spermatogenesis than oogenesis. Further work is required to determine the relevant stages of spermatogenesis at which such a selection may take place.

Fetal calcifications are associated with chromosomal abnormalities.

OBJECTIVE: The biological importance of calcifications occasionally noted in fetal tissues (mainly liver) at autopsy or ultrasound is largely unexplored. Previous reports hint at an association to infection, circulatory compromise, malformations or chromosomal abnormalities. To identify factors associated with calcifications, we have performed a case-control study on the largest cohort of fetuses with calcifications described thus far. METHODS: One-hundred and fifty-one fetuses with calcifications and 302 matched controls were selected from the archives of the Department of Pathology, Karolinska University Hospital. Chromosome analysis by karyotyping or quantitative fluorescence-polymerase chain reaction was performed. Autopsy and placenta reports were scrutinized for presence of malformations and signs of infection. RESULTS: Calcifications were mainly located in the liver, but also in heart, bowel, and other tissues. Fetuses with calcifications showed a significantly higher proportion of chromosomal abnormalities than controls; 50% vs. 20% (p<0.001). The most frequent aberrations among cases included trisomy 21 (33%), trisomy 18 (22%), and monosomy X (18%). A similar distribution was seen among controls. When comparing cases and controls with chromosomal abnormalities, the cases had a significantly higher prevalence of malformations (95% vs. 77%, p=0.004). Analyzed the other way around, cases with malformations had a significantly higher proportion of chromosomal abnormalities compared with controls, (66% vs. 31%, p<0.001). CONCLUSION: The presence of fetal calcifications is associated with high risk of chromosomal abnormality in combination with malformations. Identification of a calcification together with a malformation at autopsy more than doubles the probability of detecting a chromosomal abnormality, compared with identification of a malformation only. We propose that identification of a fetal tissue calcification at autopsy, and potentially also at ultrasound examination, should infer special attention towards co-existence of malformations, as this would be a strong indicator for a chromosomal abnormality.

Amplification-free sequencing of cell-free DNA for prenatal non-invasive diagnosis of chromosomal aberrations.

Cell-free DNA has been used for fetal rhesus factor and sex determination, fetal aneuploidy screening, cancer diagnostics and monitoring, and other applications. However current methods of using cell free DNA require amplification, which leads to allelic dropout and bias especially when starting with small amounts of DNA. Here we describe an amplification-free method for sequencing of cell-free DNA, even from low levels of starting material. We evaluated this method in the context of prenatal diagnosis of fetal aneuploidy and compared it with a PCR-based library preparation method as well as a recently described method using unique molecular identifiers (UMI). All methods performed well, however coverage was increased by the amplification-free method and GC-induced bias was reduced by both the amplification-free method and the UMI method. Future diagnostic applications including whole genome sequencing of cell-free DNA will benefit from amplification-free sequencing.

Molecular and cytogenetic analysis in stillbirth: results from 481 consecutive cases.

INTRODUCTION: The underlying causes of stillbirth are heterogeneous and in many cases unexplained. Our aim was to conclude clinical results from karyotype and quantitative fluorescence-polymerase chain reaction (QF-PCR) analysis of all stillbirths occurring in Stockholm County between 2008 and 2012. By screening a subset of cases, we aimed to study the possible benefits of chromosomal microarray (CMA) in the analysis of the etiology of stillbirth. METHODS: During 2008-2012, 481 stillbirths in Stockholm County were investigated according to a clinical protocol including karyotype or QF-PCR analysis. CMA screening was performed on a subset of 90 cases, corresponding to all stillbirths from 2010 without a genetic diagnosis. RESULTS: Chromosomal aberrations were detected by karyotype or QF-PCR analysis in 7.5% of the stillbirths. CMA analysis additionally identified two known syndromes, one aberration disrupting a known disease gene, and 26 variants of unknown significance. Furthermore, CMA had a significantly higher success rate than karyotyping (100 vs. 80%, p < 0.001). DISCUSSION: In the analysis of stillbirth, conventional karyotyping is prone to failure, and QF-PCR is a useful complement. We show that CMA has a higher success rate and aberration detection frequency than these methods, and conclude that CMA is a valuable tool for identification of chromosomal aberrations in stillbirth.

Mutation screening and array comparative genomic hybridization using a 180K oligonucleotide array in VACTERL association.

In order to identify genetic causes of VACTERL association (V vertebral defects, A anorectal malformations, C cardiac defects, T tracheoesofageal fistula, E esophageal atresia, R renal anomalies, L limb deformities), we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a (9;18)(p24;q12) unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases.