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LRBA deficiency was first described in 2012 as an autosomal recessive disorder caused by biallelic mutations in the LRBA gene (OMIM #614700). It was initially characterized as producing early-onset hypogammaglobulinemia, autoimmune manifestations, susceptibility to inflammatory bowel disease, and recurrent infection. However, further reports expanded this phenotype (including patients without hypogammaglobulinemia) and described LRBA deficiency as a clinically variable syndrome with a wide spectrum of clinical manifestations. We present the case of a female patient who presented with type 1 diabetes, psoriasis, oral thrush, and enlarged liver and spleen at the age of 8 months. She later experienced recurrent bacterial and viral infections, including pneumococcal meningitis and Epstein Barr viremia. She underwent two consecutive stem cell transplants at the age of 8 and 9 years, and ultimately died. Samples from the patient and her parents were subjected to whole exome sequencing, which revealed a homozygous 1-bp insertion in exon 23 of the patient's LRBA gene, resulting in frameshift and premature stop codon. The patient's healthy mother was heterozygous for the mutation and her father tested wild-type. This finding suggested that either one copy of the paternal chromosome 4 bore a deletion including the LRBA locus, or the patient inherited two copies of the mutant maternal LRBA allele. The patient's sequencing data showed a 1-Mb loss of heterozygosity region in chromosome 4, including the LRBA gene. Comparative genomic hybridization array of the patient's and father's genomic DNA yielded normal findings, ruling out genomic copy number abnormalities. Here, we present the first case of LRBA deficiency due to a uniparental disomy (UPD). In contrast to classical Mendelian inheritance, UPD involves inheritance of 2 copies of a chromosomal region from only 1 parent. Specifically, our patient carried a small segmental isodisomy of maternal origin affecting 1 Mb of chromosome 4.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

To investigate amniotic fluid (AF) samples retrieved in multiple pregnancies by single insertion of the needle, for rapid assessment of chromosome copy number, zygosity, and cross-contamination between fetuses, using Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) amplification of highly polymorphic microsatellite markers. Fifty-two multiple pregnancies were selected (47 twins, 5 triplets) and 108 samples of amniotic fluid were sampled between 12 to 20 weeks of gestation (mean 15.5) using the single-needle technique. Aneuploidy screening by QF-PCR amplification of short tandem repeats (STRs) on chromosomes X, Y, 21, 13, and 18 was carried out within 24 h of collection. Owing to the sampling procedure, the eventual presence of contamination between fetuses was also evaluated in every case. Normal and aneuploid fetuses were readily identified by QF-PCR. Fetal reduction was made available, for trisomic fetuses, without further waiting for completion of fetal karyotyping. In twin gestations, the ultrasound examination of chorionicity was always in agreement with the molecular assessment of zygosity. Contamination between fetuses due to the sampling procedure with a single puncture was never observed. Rapid prenatal diagnosis of aneuploidies by QF-PCR is a sensitive, efficient, and reliable assay. When applied in multiple pregnancies, it has the added value of allowing the assessment of zygosity in all cases, independently of chorionicity and fetal sex. Copyright © 2003 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]