Single-cell whole-genome sequencing, haplotype analysis in prenatal diagnosis of monogenic diseases

Abstract

Monogenic inherited diseases are common causes of congenital disabilities, leading to severe economic and mental burdens on affected families. In our previous study, we demonstrated the validity of cell-based noninvasive prenatal testing (cbNIPT) in prenatal diagnosis by single-cell targeted sequencing. The present research further explored the feasibility of single-cell whole-genome sequencing (WGS) and haplotype analysis of various monogenic diseases with cbNIPT. Four families were recruited: one with inherited deafness, one with hemophilia, one with large vestibular aqueduct syndrome (LVAS), and one with no disease. Circulating trophoblast cells (cTBs) were obtained from maternal blood and analyzed by single-cell 15X WGS. Haplotype analysis showed that CFC178 (deafness family), CFC616 (hemophilia family), and CFC111 (LVAS family) inherited haplotypes from paternal and/or maternal pathogenic loci. Amniotic fluid or fetal villi samples from the deafness and hemophilia families confirmed these results. WGS performed better than targeted sequencing in genome coverage, allele dropout (ADO), and false-positive (FP) ratios. Our findings suggest that cbNIPT by WGS and haplotype analysis have great potential for use in prenatally diagnosing various monogenic diseases.

Figure S1.. Pedigrees of the tested hemophilia and LVAS families.

(A) In the hemophilia family, the proband (child) carries a hemizygous mutation of ChrX:F9 gene c.424G>T from the mother. (B) In the LVAS family, the proband (child) carries compound heterozygous mutations of Chr7:SLC26A4 gene c.1975G>C and c.281C>T from the mother and father, respectively.

Figure S2.. Cell staining images and single-cell STR analysis of captured circulating trophoblast cells.

(A) Immunofluorescence images of candidate fetal trophoblast cells. Cells were stained with FITC-anti-CK (green), TRITC-anti-CD45 (yellow), and DAPI (blue). Circles indicate the candidate trophoblast cells selected for downstream analyses. (B) STR analysis of WGA products from single candidate cTBs and the genomic DNA from paired parents. Cells with multiple paternal alleles were scored as cTBs (arrows and Tables S1–S3). Scale bar is 10 μm.

Figure 1.. Sequencing depth test in the healthy family.

(A, B) Genome coverage of different sequencing depths for the father (A) and the mother (B) in the healthy family. (C, D) Genome coverage of different sequencing depths for CFC518 (C) or CFC2282 (D). (E, F) FPR for CFC518 (E) and CFC2282 (F). (G, H) ADO of CFC518 (G) or CFC2282 (H). (I, J) Genome coverage of the regions corresponding to the 67-gene panel from WGS of CFC518 (I) or CFC2282 (J). (K, L) Number of covered 67-gene panel genes in the WGS of CFC518 (K) or CFC2282 (L). (M, N) Genome coverage of the whole-exon region in the WGS of CFC518 (M) or CFC2282 (N). (O, P) Number of covered whole-exon region genes in the WGS of CFC518 (O) or CFC2282 (P). (Q, R) Genome coverage of the OMIM gene panel in the WGS of CFC518 (Q) or CFC2282 (R). (S, T) Number of covered OMIM genes in the WGS of CFC518 (S) or CFC2282 (T).

Figure 2.. Haplotype inheritance surrounding disease-causing genes in the three monogenic disease families.

(A) Deafness family with the haplotype P1/M2 in Chr13:GJB2. (B) Hemophilia family with the haplotype M1 in ChrX:F9. (C) LVAS family with the haplotypes P1/M1 in Chr7:SLC26A4.

Figure S3.. Flow chart of circulating trophoblast cells captured from maternal peripheral blood and characterization.

Peripheral blood was obtained from pregnant women between the 11th and 16th gestational cycles, followed by centrifugation with lymphocyte separation medium to collect the nucleated cells. All nucleated cells were then enriched and stained to acquire candidate cTBs that met the selection criteria. Subsequently, the origin of candidate circulating trophoblast cells was identified by STR analysis after single-cell WGA. Finally, DNA libraries of all confirmed cTBs were sequenced and analyzed.

Figure 3.. Workflow of embryonic haplotype construction using the genotypes of the father, mother, and proband.

The details are described in the Materials and Methods section.