El-Sayed, Y. Y. | Search bioRxiv

bioRxiv2024

In Utero Hematopoietic Stem Cell Transplant for Fanconi Anemia

Fanconi Anemia (FA) is an inherited DNA-repair deficiency caused by mutations in diverse Fanc genes that leads to bone marrow failure and malignancies. FA disease begins at early embryonic stages, and while FA prenatal testing has long been available, no fetal therapies for FA currently exist. Postnatally, FA hematologic disease can be cured through allogeneic hematopoietic stem cell transplantation (HSCT); however, this requires chemotherapy and/or irradiation-based conditioning which amongst various side-effects also increases likelihood of malignancies later in life in these fragile patients. Given fetal immune tolerance and the competitive advantage of healthy hematopoietic stem and progenitor cells (HSPCs) over failing FA HSPCs, in utero HSCT without conditioning may be an alternative approach to stabilization of the hematopoietic system without conventional toxicities. We performed in utero HSCT using HSPCs from wildtype (WT) donors into two FA mouse models (Fancd2-/-, Fanca-/-) and observed robust multi-lineage hematopoietic donor engraftment in homozygous FA mice compared to both heterozygous FA and WT littermates. Upon serial assessments, we also observed increasing donor chimerism up to 94.1%, showcasing the competitive advantage of WT donor HSPCs over FA HSPCs. Given that 1% donor chimerism is predicted to stabilize FA BM, in utero HSCT may be a safe and curative prenatal treatment for all subtypes of FA.

cell biology↗

bioRxiv2017

Nonivasive prenatal diagnosis of single-gene disorders using droplet digital PCR

BackgroundPrenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice.\n\nMethodsWe developed a protocol for noninvasive prenatal diagnosis of inherited single gene disorders using droplet digital PCR (ddPCR) from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction are determined using a panel of Taqman assays targeting high-variability SNPs. Second, the ratio of healthy and diseased alleles in maternal plasma are quantified using Taqman assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented.\n\nResultsWe collected blood samples from 9 pregnancies at risk for different single gene disorders including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, {beta}-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11, and with a fetal fraction as low as 3.7{+/-}0.3%.\n\nConclusionOur method detects single nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders where early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

genetics↗