Non-invasive prenatal diagnosis of spinal muscular atrophy by relative haplotype dosage

Abstract

Although technically possible, few clinical laboratories across the world have implemented non-invasive prenatal diagnosis (NIPD) for selected single-gene disorders, mostly owing to the elevated costs incurred. Having previously proven that NIPD for X-linked disorders can be feasibly implemented in clinical practice, we have now developed a test for the NIPD of an autosomal-recessive disorder, spinal muscular atrophy (SMA). Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single-nucleotide polymorphisms across a 6 Mb genomic window on chromosome 5 containing the SMN1 gene. Maternal, paternal and proband DNA samples were also tested for haplotyping purposes. Sequencing data was analysed by relative haplotype dosage (RHDO). Six pregnant SMA carriers and 10 healthy pregnant donors were recruited through the NIPSIGEN study. Inheritance of the maternally and paternally derived alleles of the affected SMN1 gene was determined in the foetus by RHDO analysis for autosomal-recessive disorders. DNA from the proband (for SMA carriers) or an invasively obtained foetal sample (for healthy pregnant donors) was used to identify the maternal and paternal reference haplotypes associated with the affected SMN1 gene. Results for all patients correlated with known outcomes and showed a testing specificity and sensitivity of 100%. On top of showing high accuracy and reliability throughout the stages of validation, our novel test for NIPD of SMA is also affordable and viable for implementation into clinical service.

Non‐invasive prenatal testing for fetal inheritance of maternal β‐thalassaemia mutations using targeted sequencing and relative mutation dosage: a feasibility study

“…Our diagnostic accuracy of 92.6% is comparable to NIPT for monogenic diseases reported in other studies; however, RHDO shows higher levels of concordance, so our test still needs improvement. We obtained three FN results and further work will be required to reduce these.…”

“…However, there have been few studies concerning detection of the maternal mutation. Recent work has demonstrated successful use of RHDO for detection of inheritance of maternal mutations . RHDO has been implemented successfully into some clinical laboratories, but generally remains prohibitively expensive .…”

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis

“…However, this test is less informative than the SNP-haplotype-based approach by next-generation sequencing [25][26][27][28][29]. These previously described methods entail high-cost materials and sophisticated machinery.…”

Development and validation of a haplotype‐free technique for non‐invasive prenatal diagnosis of spinal muscular atrophy

“…Although NIPT for fetal aneuploidies has already been clinically applied, non‐invasive prenatal diagnosis for many single‐gene disorders remains on the developing stage. For NIPD of SMA, a technique by targeted sequencing of cfDNA in maternal plasma and relative haplotype dosage (RHDO) analysis has been previously published . However, this haplotype‐based strategy has several limitations.…”