Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B.

OBJECTIVE: The involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B 'e' antigen (HBeAg)-negative chronic hepatitis B (CHB). DESIGN: Liver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR. RESULTS: Patients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia. CONCLUSIONS: HBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome.

Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles.

STUDY QUESTION: Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer? SUMMARY ANSWER: Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies. WHAT IS KNOWN ALREADY: The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge. STUDY DESIGN, SIZE, DURATION: The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies. LIMITATION, REASON FOR CAUTION: Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable. WIDER IMPLICATION OF THE FINDING: This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available. STUDY FUNDING/COMPETING INTERESTS: No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1000 consecutive clinical cases.

OBJECTIVE: To assess the feasibility of offering array-based comparative genomic hybridization testing for prenatal diagnosis as a first-line test, a prospective study was performed, comparing the results achieved from array comparative genomic hybridization (aCGH) with those obtained from conventional karyotype. METHOD: Women undergoing amniocentesis or chorionic villus sampling were offered aCGH analysis. A total of 1037 prenatal samples were processed in parallel using both aCGH and G-banding for standard karyotyping. Specimen types included amniotic fluid (89.0%), chorionic villus sampling (9.5%) and cultured amniocytes (1.5%). RESULTS: Chromosomal abnormalities were identified in 34 (3.3%) samples; in 9 out of 34 cases (26.5%) aCGH detected pathogenic copy number variations that would not have been found if only a standard karyotype had been performed. aCGH was also able to detect chromosomal mosaicism at as low as a 10% level. There was complete concordance between the conventional karyotyping and aCGH results, except for 2 cases that were only correctly diagnosed by aCGH. CONCLUSIONS: This study demonstrates that aCGH represents an improved diagnostic tool for prenatal detection of chromosomal abnormalities. Although larger studies are needed, our results provide further evidence on the feasibility of introducing aCGH as a first-line diagnostic test in routine prenatal diagnosis practice.

Short tandem repeats haplotyping of the HLA region in preimplantation HLA matching.

Recently, preimplantation genetic diagnosis (PGD) has been considered for several indications beyond its original purpose, not only to test embryos for genetic disease but also to select embryos for a nondisease trait, such as specific human leukocyte antigen (HLA) genotypes, related to immune compatibility with an existing affected child in need of a haematopoetic stem cell (HSC) transplant. We have optimized an indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex. The assay was clinically applied in 60 cycles from 45 couples. A conclusive HLA-matching diagnosis was achieved in 483/530 (91.1%) of the embryos tested. In total, 74 (15.3%) embryos revealed an HLA match with the affected siblings, 55 (11.4%) of which resulted unaffected and 46 (9.5%) have been transferred to the patients. Nine pregnancies were achieved, five healthy HLA-matched children have already been delivered and cord blood HSCs, were transplanted to three affected siblings, resulting in a successful haematopoietic reconstruction.

Chromosomal microarray analysis as a first-line test in pregnancies with a priori low risk for the detection of submicroscopic chromosomal abnormalities.

In this study, we aimed to explore the utility of chromosomal microarray analysis (CMA) in groups of pregnancies with a priori low risk for detection of submicroscopic chromosome abnormalities, usually not considered an indication for testing, in order to assess whether CMA improves the detection rate of prenatal chromosomal aberrations. A total of 3000 prenatal samples were processed in parallel using both whole-genome CMA and conventional karyotyping. The indications for prenatal testing included: advanced maternal age, maternal serum screening test abnormality, abnormal ultrasound findings, known abnormal fetal karyotype, parental anxiety, family history of a genetic condition and cell culture failure. The use of CMA resulted in an increased detection rate regardless of the indication for analysis. This was evident in high risk groups (abnormal ultrasound findings and abnormal fetal karyotype), in which the percentage of detection was 5.8% (7/120), and also in low risk groups, such as advanced maternal age (6/1118, 0.5%), and parental anxiety (11/1674, 0.7%). A total of 24 (0.8%) fetal conditions would have remained undiagnosed if only a standard karyotype had been performed. Importantly, 17 (0.6%) of such findings would have otherwise been overlooked if CMA was offered only to high risk pregnancies.The results of this study suggest that more widespread CMA testing of fetuses would result in a higher detection of clinically relevant chromosome abnormalities, even in low risk pregnancies. Our findings provide substantial evidence for the introduction of CMA as a first-line diagnostic test for all pregnant women undergoing invasive prenatal testing, regardless of risk factors.

Hereditary spastic paraplegia and axonal motor neuropathy caused by a novel SPG3A de novo mutation.

Mutations in the SPG3A gene (atlastin protein) cause approximately 10% of autosomal-dominant hereditary spastic paraplegia. Most patients with an SPG3A mutation present with a pure phenotype and early-onset disease, although complicated forms with peripheral neuropathy are also reported. We report a new heterozygous S398F mutation in exon 12 of the SPG3A gene causing a very early-onset spastic paraplegia in association with motor axonal neuropathy in a 4-year-old girl resembling diplegic cerebral palsy.