Comparing methods for fetal fraction determination and quality control of NIPT samples.

OBJECTIVE: To compare available analysis methods for determining fetal fraction on single read next generation sequencing data. This is important as the performance of non-invasive prenatal testing (NIPT) procedures depends on the fraction of fetal DNA. METHODS: We tested six different methods for the detection of fetal fraction in NIPT samples. The same clinically obtained data were used for all methods, allowing us to assess the effect of fetal fraction on the test result, and to investigate the use of fetal fraction for quality control. RESULTS: We show that non-NIPT methods based on body mass index (BMI) and gestational age are unreliable predictors of fetal fraction, male pregnancy specific methods based on read counts on the Y chromosome perform consistently and the fetal sex-independent new methods SeqFF and SANEFALCON are less reliable but can be used to obtain a basic indication of fetal fraction in case of a female fetus. CONCLUSION: We recommend the use of a combination of methods to prevent the issue of reports on samples with insufficient fetal DNA; SANEFALCON to check for presence of fetal DNA, SeqFF for estimating the fetal fraction for a female pregnancy and any Y-based method for estimating the fetal fraction for a male pregnancy. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes.

INTRODUCTION: BRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients. METHODS: For this purpose, genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n = 27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n = 14) in a familial cohort of 120 breast carcinomas. RESULTS: Genome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings. CONCLUSION: This study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1-involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis.

Mutations in RARS cause hypomyelination.

Hypomyelinating disorders of the central nervous system are still a diagnostic challenge, as many patients remain without genetic diagnosis. Using magnetic resonance imaging (MRI) pattern recognition and whole exome sequencing, we could ascertain compound heterozygous mutations in RARS in 4 patients with hypomyelination. Clinical features included severe spasticity and nystagmus. RARS encodes the cytoplasmic arginyl-tRNA synthetase, an enzyme essential for RNA translation. This protein is among the subunits of the multisynthetase complex, which emerges as a key player in myelination.