Sirolimus for management of GI bleeding in blue rubber bleb nevus syndrome: A case series.

Blue rubber bleb nevus syndrome (BRBNS) commonly presents with anemia from bleeding gastrointestinal (GI) vascular malformations. Management is highly variable, as no consensus guidelines for medical treatment currently exist. Sirolimus has been used in BRBNS to decrease GI bleeding and seems well tolerated, though questions remain regarding dosing, duration of therapy, and adverse effects. Here, we report our single-center experience of four pediatric patients with BRBNS who were successfully treated with sirolimus and review the existing literature regarding sirolimus for treatment of GI bleeding in BRBNS. Further prospective studies are needed to establish optimal dosage, drug monitoring, and duration.

Fertility and Pregnancy in Women with Transfusion-Dependent Thalassemia.

Because women with transfusion-dependent thalassemia are seeking pregnancy, ensuring the best outcomes for both mother and baby require concerted and collaborative efforts between the hematologist, obstetrician, cardiologist, hepatologist, and genetic counselor among others. Proactive counseling, early fertility evaluation, optimal management of iron overload and organ function, and application of advances in reproductive technology and prenatal screening are important in ensuring a healthy outcome. Many unanswered questions remain requiring further study, including fertility preservation, non-invasive prenatal diagnosis, chelation therapy during pregnancy, and indications and duration of anticoagulation.

Noninvasive Prenatal Test for ß-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

BACKGROUND: Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the ß-hemoglobinopathies to reduce the risk to the fetus. METHOD: Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with ß-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. RESULTS: Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The ß-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. CONCLUSION: This probe capture NGS assay demonstrates the potential of NIPT for ß-hemoglobinopathies.

Fertility and Pregnancy in Women with Transfusion-Dependent Thalassemia.

As more women with transfusion-dependent thalassemia are seeking pregnancy, ensuring the best outcomes for both the mother and baby requires concerted, collaborative efforts between practitioners and the family. Proactive counseling, early fertility evaluation, recent developments in reproductive technology, and optimal management of iron overload, have resulted in more successful pregnancies and the birth of healthy newborns. With advances in technology for prenatal screening and increased awareness to perform screening for hemoglobinopathies, healthy pregnancy outcomes have become the expectation. Topics that require further study include management that allows fertility preservation, improved non-invasive prenatal diagnosis methods for affected fetuses, the use of chelation therapy during pregnancy, and indications for and duration of anticoagulation.

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples.