Next-generation sequencing reveals a novel NDP gene mutation in a Chinese family with Norrie disease.

PURPOSE: Norrie disease (ND) is a rare X-linked genetic disorder, the main symptoms of which are congenital blindness and white pupils. It has been reported that ND is caused by mutations in the NDP gene. Although many mutations in NDP have been reported, the genetic cause for many patients remains unknown. In this study, the aim is to investigate the genetic defect in a five-generation family with typical symptoms of ND.
METHODS: To identify the causative gene, next-generation sequencing based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members using Sanger sequencing.
RESULTS: We identified a novel missense variant (c.314C>A) located within the NDP gene. The mutation cosegregated within all affected individuals in the family and was not found in unaffected members. By happenstance, in this family, we also detected a known pathogenic variant of retinitis pigmentosa in a healthy individual.
CONCLUSION: c.314C>A mutation of NDP gene is a novel mutation and broadens the genetic spectrum of ND.

Norrie disease (ND; OMIM 310600) is a rare X-linked genetic disorder, the main symptoms of which are congenital blindness and white pupils.[12] It can cause abnormal development of the retina, while some infants will have a yellow-white massive microstructure with well vasculature in the posterior vitreous at birth or soon after birth. In addition, as the disorder progresses, shrinking of the eyeballs may occur, then the lens appears cloudy and eventually becomes covered by cataracts. About one-third of individuals with ND may develop progressive hearing loss due to vascular and cochlear abnormalities, and more than half experience developmental delays with cognitive impairment and/or behavioral disorders.[3] The exact, annual incidence and prevalence of ND are unknown, but more than 400 cases have been detailed in the literature. It is not associated with any specific racial or ethnic group, but affected patients are almost always male, while females are typically carriers.

ND is caused by mutations in the NDP (Xp11.3) gene, which encodes a secreted protein with a cystine-knot motif, which activates the Wnt/beta-catenin pathway. A diagnosis of ND is based on a combination of characteristic, clinical ocular findings, and molecular genetic testing of the NDP gene. No biochemical or functional assays are available as disease markers. Thus, identifying new mutations for clinical, genetic diagnosis of ND is crucial.

The purpose of this study was to identify the underlying genetic defect in a Chinese family with ND, and to provide clinical guidance for birth defect prevention. Targeted sequencing enables us to identify a novel NDP gene mutation (p. Ala105Glu) in two affected patients. Surprisingly, we observed a false-positive mutation in SLC7A14 gene related to retinitis pigmentosa (RP) when analyzing data generated from an unaffected family member.

Methods

Patients

A five-generation Chinese family, consisting of five affected males, was recruited. Complete clinical diagnosis was carried out on each of them. Diagnosis of ND, with typical symptoms, was established based on ophthalmic examination. Family pedigrees are shown in Fig. 1, wherein is depicted the two affected and six unaffected family members participating in the study. All the participants had given written and informed consent and were also involved in publication. All procedures were approved by the ethics committee of the hospital and adhered to the tenets of the Declaration of Helsinki.

Figure 1

Pedigree of the Norrie disease family, as reported in this study

Capture panel

A gene capture panel was designed to encompass the exons and UTR region of 366 eye disease-related genes, with a target region size of 1.7 M. The capture probes were custom designed and produced by BGI. Before the study, we tested the sensitivity of our method using four samples, by sequence capture performed on two platforms (Illumina's HiSeq sequencer, and BGI's BGISEQ-500 sequencer, respectively). Briefly, all samples had an average depth of more than ×200, and the coverage of target region was around 96% using BGISEQ-500.

Next-generation sequencing on BGISEQ-500

Genomic DNA of two affected (III-1, IV-13) and one unaffected (II-1) family member was extracted from peripheral blood, using a standard protocol. Paired-end DNA libraries were quantified and sequenced on BGISEQ-500. Using DNA nanoballs and combinational probe-anchor synthesis developed from Complete Genomics sequencing technology, it generates short reads on a large scale which can help fulfill the growing demands for sequencing.[4]

Bioinformatics analysis

WES data were mapped to the human reference genome (UCSC hg19) using Burrows-Wheeler aligner (BWA-MEM, version 0.7.10).[5] Variants calling were performed using Genome Analysis Tool Kit (GATK, version 3.3).[6] All variants were annotated by Annovar[7] and SnpEff.[8] Then, the variants identified through the above pipeline were further filtered to eliminate benign variants with minor allele frequency (MAF) >0.1% in 1000 Genomes,[9] dbSNP,[10] EXAC,[11] ESP6500[12] database, and internal data. Finally, variant prioritization and selection were performed combining total depth, quality score, MAF, potential deleterious effect, and the existence of mutation reports in common databases such as the Human Gene Mutation Database,[13] the Retinal Information Network,[14] ClinVar,[15] or Online Mendelian Inheritance in Man[16] to evaluate variant calling confidence.

Sanger validation

Primer3 was used to design all polymerase chain reaction primers for validation. Sanger sequencing was used to validate the identified mutations. Segregation studies were undertaken using eight family members (affected individuals III-1 and IV-13; unaffected individuals II-1, III-2, III-18, IV-1, IV-2, and IV-14).

Results

Clinical characteristics

Two males from this family–the proband and his uncle–were affected by the disease. Patient III-1 is a 44-year-old man with congenital blindness, who has been taking medication since the age of 16 after presenting with epilepsy. The examination revealed that he suffers from microphthalmia and atrophy. Besides this, previous clinical reports had shown that he had a shallow anterior chamber and iris atrophy.

Patient IV-13 was born with the ability to sense light; however, as he grew older, the binocular pupils of his eyes began to turn white, and yellow-white lump tissues were found in the posterior vitreous tissue. When he was 1-year-old, both of his eyes had become entirely blind.

Mutation identification

To identify the causative variants in the ND family cohort, we performed targeted capture sequencing of 366 retina disease genes, using our capture panel described in the methods. High-quality results were obtained [Table 1], with mean coverage within the target region in excess of 94%.

Table 1

Quality of datasets

Following bioinformatics analysis, a hemizygous missense mutation c.314C>A in Exon3/CDS2 of NDP completely segregated in both of the two affected family members (III-1, IV-13) as well as in one unaffected family member (II-1). This mutation causes an amino acid change from alanine to glutamic acid at position 105 (p. Ala105Glu). Both SIFT and PolyPhen predicted p. Ala105Glu to be damaging, with high hazard scores. Following this result, we applied a further mutation validation strategy, by Sanger sequencing, with additional family members [Fig. 2]. The results confirmed that the disease in this family was due to this mutation (III-1 and IV-13 were hemizygous patients; III-2, IV-1, and IV-2 were unaffected; II-1, III-17, and IV-14 were heterozygous carriers). Multiple orthologous sequence alignment revealed that 105 codon alanine of NDP was highly conserved amino acids across different species [Fig. 3]. It suggests that any mutation at those codons may have a deleterious effect.

Figure 2

Detected mutations in the NDP genes in patients with Norrie disease. Partial sequences of NDP from the Norrie disease patients (III-1, IV-13: Hemizygous for the c.314C>A genotype), controls (IV-1, III-2, IV-2), and carriers (II-1, III-17, IV-14: Heterozygous for the genotype)

Figure 3

Protein sequence alignment of human NDP with its orthologs. Protein alignment showing conservation of residues NDP Ala105 across ten species. The mutation occurred at evolutionarily conserved amino acids (in the red box)

Interestingly, in an unaffected individual (II-1) without any retina disease symptoms present at the clinical examination, we found an SLC7A14 mutation[17] c.1391G>T (p. Cys464Phe) by sequencing, which has been reported to be a RP pathogenic variant in four cases. In addition, Sanger sequencing was used to further identify the variation [Fig. 4].

Figure 4

Detected a mutation in the SLC7A14 genes in II-1

Discussion

BGISEQ-500 achieves the international standard of excellence for sequencing, and in particular, meets the demands of clinical application. It integrates automatic sample preparation, sequencing process, and data analysis, performing several applications with a one-touch operation. It has proven that it can produce data of the highest standard, with performance concordant to other, highest standard platforms, such as Sanger sequencing.

Besides this evaluation of the BGISEQ-500, we have developed a targeted sequencing-based diagnostic gene panel for ophthalmic diseases, which can offer a precise and cost-effective detection of disease-causing gene variants for clinical use, encompassing both known and novel mutations.

ND is a rare X-linked genetic disorder that leads to blindness in males. According to the statistics, of more than 60% of ND patients with point mutations (>60%), about 20% of patients have deletions of the NDP gene.[18] NDP encodes Norrin protein which contains the cystine-knot domain.[19] Most mutations of NDP gene associated with ND are related to this domain, such as P98 L (c.293C>T) and S111X (c.332C>A).[20] In this study, we report upon a family with a history of typical ND. A novel mutation (c.314C>A, p. Ala105Glu) in the NDP was found in two hemizygous, affected male individuals (III-1, IV-13) and in one heterozygous, unaffected individual (II-1). This mutation in exon 3 at codon 105 involves in the cystine knot. Previously, the same missense mutation was reported in one patient with X-linked familial exudative vitreoretinopathy (FEVR).[21] This result suggests that X-linked FEVR and ND may be caused by the same mutation in the NDP gene.

Indeed, FEVR is characterized by abnormal vascularization of the peripheral retina. In contrast, for this family cohort, the affected individuals were congenitally blind, which is the typical symptom of ND. Thus, genetic testing is necessary to ensure correct diagnosis, and our work may provide new data and clues for additional research helping with the diagnosis of this disease.

According to pedigree analysis and determination of the mutation, ND can be localized to be an X-linked, recessive genetic disease. According to the fetal karyotype analysis, the proband's sister (III-2) was pregnant with a boy, who might also suffer from congenital blindness. To determine whether the male fetus (IV-2) inherited the pathogenic mutation, we used amniotic fluid to detect the mutation (c.314C>A) in NDP. The test result showed that IV-2 did not carry this mutation. So far, no symptoms of ND have been detected in this boy after his birth, providing some corroboration with our findings. From this case, it may be inferred that our work should be of great significance in the clinical prevention of birth defects for affected families such as this one.

Notably, when we analyzed the proband mother's data (II-1), we found her not only to be carrying a heterozygous mutation of the NDP gene (c.314C>A), but also to be carrying a known, pathogenic homozygous mutation of the SLC7A14 gene (c.1391G>T), which was previously reported to be present in four patients with RP. However, clinical examination of the individual showed that she did not have any detectable symptoms of RP. Therefore, we suggest that the clinical significance, or at least the penetrance of this genotype in causing the RP phenotype, requires further research.

Conclusion

We identified a novel mutation c.314C>A in NDP gene caused Norrie disease in a Chinese family. Our finding broadens the genetic spectrum of ND and indicates the feasibility of panel-based next generation sequencing for inherited disease.

Financial support and sponsorship

This work is supported by the Shenzhen Municipal Government of China (No. KJYY20151116165726645).

Conflicts of interest

There are no conflicts of interest.