Diagnosis for choroideremia in a large Chinese pedigree by next‑generation sequencing (NGS) and non‑invasive prenatal testing (NIPT)

Abstract

To develop an effective strategy to isolate and use cell‑free fetal DNA (cffDNA) for the combined use of next‑generation sequencing (NGS) for diagnosing choroideremia and non‑invasive prenatal testing (NIPT) for Y chromosome determination, a large Chinese family with an X‑linked recessive disease, choroideremia, was recruited. Cell‑free DNA was extracted from maternal plasma, and SRY polymerase chain reaction amplification was performed using NIPT. Sanger sequencing was subsequently used for fetal amniotic fluid DNA verification. A nonsense mutation (c.C799T:p.R267X) of the CHM gene on the X chromosome of the proband (IV:7) and another 5 males with choroideremia were detected, while 3 female carriers with no symptoms were also identified. The fetus (VI:7) was identified as female from the cffDNA, and the same heterozygous nonsense mutation present in her mother was also confirmed. At one and a half years of age, the female baby did not present with any associated symptoms of choroideremia. Therefore, cffDNA was successfully used for the combined use of NGS for diagnosing choroideremia in a large Chinese pedigree, and NIPT for Y chromosome determination. This approach should result in a markedly increased use of prenatal diagnosis and improvement, and more sophisticated clinical management of diseases in China and other developing countries. The establishment of a highly accurate method for prenatal gene diagnosis will allow for more reliable gene diagnosis, improved genetic counseling, and personalized clinical management of our patients.

Figure 1.

Large pedigree of choroideremia. The 60-year-old proband (IV:7) is indicated by an arrow. Affected males are denoted by a filled square. Note that the choroideremia carrier (V:11) is a female from the proband's daughter and is pregnant with a fetus (VI:7) that will undergo prenatal diagnosis to demonstrate whether the baby will inherit a normal X chromosome.

Figure 2.

Fundus photograph and OCT images of choroideremia proband. (A) The left eye fundus photograph. (B) The right eye fundus photograph. Fundus photographs revealed that the retinal pigment epithelial layer was patchy with shaded areas of scattered pigmentation; however, the layer appeared transparent in the central area, called the macula (black arrow). The pigment layer was extremely thin, possibly absent, or lacking pigment (blue arrow), leading to its transparent nature, which allowed the sclera and deep choroidal blood vessels (blue arrow) in the areas outside the central macula to be visible (black arrow). The observed blood vessels were markedly smaller in size, and the boundary of the optic-papilla was clear. (C and D) Normal fundus photographs (control) are shown. (E) The OCT images reveal atrophy of the retina at the macular fovea, thinning of the fovea, and loss of choroids. OCT, optical coherence tomography.

Figure 3.

NGS and Sanger sequencing for the family with choroideremia. A nonsense mutation (c.C799T:p.R267X) located in exon 6 of the CHM gene (GenBank no: NM_000,390) on the X chromosome from the proband (IV:7) was detected by NGS. Sanger sequencing confirmed the gene panel result of NGS. (A) The proband (IV:7 in Fig. 1) exhibits a 799 C → T mutation at the first nucleotide position of codon 267 (R267X) on exon 6 of the CHM gene locus (cga-tga). (B) The carrier daughter of the proband, who exhibits heterozygosity. (C and D) The healthy males of the proband's son (V:12 in Fig. 1) and grandson (VI:6 in Fig. 1) harbor normal DNA sequences. Note that the arrows illustrate the position of the point mutation. NGS, next generation sequencing.

Figure 4.

NIPT. (A) NIPT using cffDNA from the mother's blood (V:11 in Fig. 1). Lanes 1–6 are DNA PCR products determined using nested PCR for the mutational diagnosis from the maternal blood plasma (V: 11 in Fig. 1), a known male genomic DNA sample, a known female genomic DNA sample, a blank control without any DNA, a plasma DNA sample from a pregnant woman with a known female fetus, and a plasma DNA sample from a pregnant woman with a known male fetus, respectively. Lanes 2 and 6 show the positive amplification. Lane M indicates the DNA molecular-weight marker DL600, with fragment sizes of 600, 500, 400, 300, 200, and 100 bp. (B) Verification of the SRY gene from the fetal DNA of amniotic fluid in the pregnant woman. Lanes 1–5 represent the diagnosis from fetal amniotic fluid DNA (VI:7 in Fig. 1), a female genomic DNA sample (fetus's mother; V:11 in Fig. 1), a male genomic DNA sample (fetus's father; V:10 in Fig. 1), a male genomic DNA sample (fetus's brother; VI:6 in Fig. 1) and a blank control without any DNA, respectively. Lane M indicates the DNA molecular weight marker DL2000, with fragment sizes of 2000, 1000, 750, 500, 250 and 100 bp. (C and D) The real-time quantitative PCR profiles. (C) The profile for a plasma DNA sample from a pregnant woman and β-ACTIN as control using different dilution of genomic DNA. (D) The profile for a plasma DNA sample from a pregnant woman with the known baby boy and β-ACTIN as control using different dilution of genomic DNA. NIPT, non-invasive prenatal testing; bp, base pairs.

Figure 5.

Sanger DNA sequencing of amniotic fluid DNA from the fetus. The heterozygous nonsense mutation (c.C799T:p.R267X) presenting in the fetus (VI:7) was identified, indicating that the mutation was inherited from her mother since her father possessed a wild type allele.