Non-Invasive Prenatal Diagnosis of Single Gene Disorders with enhanced Relative Haplotype Dosage Analysis for diagnostic implementation

Non-invasive prenatal diagnosis for single-gene disorders (SGD-NIPD) has been widely adopted by patients, but is mostly limited to the exclusion of paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of maternal allele from cell free DNA (cfDNA) analysis. Based on the study of maternal haplotypes imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has proven to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of analysis have not been fully considered yet. Here we propose a universal and adaptable enhanced-RHDO procedure (eRHDO) through an automated bioinformatics pipeline with a didactical visualization of results that aims to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carriers for CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimal sequencing depth and type 1 and type 2 statistical errors, as well as the quality assessment for intermediate steps and final result through block score and concordance score. Validation was successfully carried out on 56 pregnancies of the test cohort. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained 94.9% conclusive and correctly inferred fetal genotypes, without any false negative or false positive result. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The author(s) received no specific funding for this work. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Pregnant women who are known carriers of cystic fibrosis (CFTR gene), neurofibromatosis type 1 (NF1 gene), Duchenne/Becker muscular dystrophy (DMD gene) or hemophilia (F8 and F9 genes) were recruited nationwide through the DANNI and NID studies, which were ethically approved by the French ’Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé” (ref. 13.386) and ’Comité de Protection des Personnes” (ref. 2014-janvier-13465 and 29BRC18.0055). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All relevant data are within the manuscript and its Supporting Information files. [https://github.com/rdaveau/nipt\_sprt\_erhdo][1] [1]: https://github.com/rdaveau/nipt_sprt_erhdo