Clinical application of NGS-based SNP haplotyping for PGT-M of methylmalonic acidemia

Figures & data

Figure 1. Molecular diagnosis results of the family. The loci of the mutations c.730insTT and c.1105 C > T are the nucleotides with a black arrow. Compared with the reference sequence of National Center for Biotechnology Information (NCBI), the father had a heterozygous mutation c.1105 C > T in MMUT, the mother had another heterozygous mutation c.730insTT in MMUT, and the proband inherited both c.1105 C > T and c.730insTT.

Figure 2. Quality control of whole genome amplification (WGA). 2, 4, 11, 13, 14, 15, 16, 17, NC and PC represent sample 2, sample 4, sample 11, sample 13, sample 14, sample 15, sample 16, sample 17, negative control and positive control (PC), respectively. All samples were same with PC that means amplified successfully.

Figure 3. Preimplantation genetic testing for aneuploidy (PGT-A) results of all samples (2, 4, 11, 13, 14, 15, 16 and 17). Compared with the hg19 reference genome through PGT-A analysis, the sketch maps shown that sample 4 has a mosaic deletion of the chromosome 6; Sample 13 has a mosaic deletion in the chromosome 2 (q21.2–q32.1) (56.34 Mb); Sample 17 has a deletion in the chromosome 7 (p21.3–q11.23) (67.88 Mb) and a duplication in the chromosome 7 (q22.1–q36.1) (49.99 Mb). The deletions were marked with red arrows and the duplication was marked with blue arrow. Sample 2’s, 11’s, 14’s, 15’s and 16’s chromosomes were balanced.

Table 1. NGS-based SNP haplotyping results of sample 2, 11, 14, 15 and 16

Number of SNP	Father	Mother	Proband		Sample 2		Sample 11		Sample 14		Sample 15		Sample 16
			Paternal haplotype	Maternal haplotype	Paternal haplotype	Maternal haplotype	Paternal haplotype	Maternal haplotype	Paternal haplotype	Maternal haplotype	Paternal haplotype	Maternal haplotype	Paternal haplotype	Maternal haplotype
MUT-14_p	T/T	T/C	T	T	T	C	T	T	T	C	T	T	T	C
MUT −15_p	G/G	G/A	G	G	G	A	G	G	G	A	G	G	G	A
MUT −30_m	A/G	A/A	A	A	A	A	G	A	G	A	A	A	G	A
MUT −33_m	A/G	G/G	A	G	A	G	G	G	G	G	A	G	G	G
MUT −39_m	A/G	G/G	A	G	A	G	G	G	G	G	A	G	G	G
MUT −51_m	A/T	T/T	A	T	A	T	T	T	T	T	A	T	T	T
MUT −52_m	C/T	T/T	C	T	C	T	T	T	T	T	C	T	T	T
MUT −59_p	A/A	T/A	A	T	A	A	A	T	A	A	A	T	A	A
MUT −63_p	T/T	C/T	C	T	T	T	T	C	T	T	T	C	T	T
MUT −66_p	C/C	C/T	A	C	C	T	C	C	C	T	C	C	C	T
MUT −67_m	A/G	A/A	C	A	A	A	G	A	G	A	A	A	G	A
MUT −68_m	A/G	A/A	A	A	A	A	G	A	G	A	A	A	G	A

Figure 4. Sanger sequencing results of preimplantation genetic testing for monogenic disease and prenatal diagnosis. (A) Sanger sequencing results of samples (2, 11, 14, 15 and 16). The loci of the mutation c.730insTT are the nucleotides with a black arrow. Compared with the reference sequence of National Center of Biotechnology Information (NCBI) sample 2 and 15 carried the MMUT mutation c.730insTT, while sample 11, 14 and 16 did not. (B) Prenatal diagnosis results of the fetus (from sample 14) by Sanger sequencing. The loci of the mutations c.730insTT and c.1105 C > T are the nucleotides with a black arrow. Compared with the reference sequences of NCBI the fetus neither carried the MMUT mutation c.730insTT nor c.1105 C > T. These results are same to the PGT-M results of sample 14.

Figure 5. Pedigree of haplotype construction of the MMA family. Circles and square represent females and male, respectively. The half green symbols represent carriers who carried a MMUT mutation and the green symbol represent proband who suffered MMA. The baby blue lines were carrying c.1105 C > T haplotypes, yellow lines were carrying c.730insTT haplotypes, navy blue line and brown line were normal haplotypes. The sites of mutation c.1105 C > T were marked with red points and the sites of mutation c.730insTT were marked with purple points. The carrying c.1105 C > T haplotype of the proband was inherited from her father, and the carrying c.730insTT haplotype was inherited from her mother.