Clinical and whole exome sequencing findings in children from Yunnan Yi minority ethnic group with retinitis pigmentosa: two case reports

Retinitis pigmentosa (RP, MIM# 268000) is a rare inherited retinal disease, and it causes severe vision impairment due to progressive degeneration of the rod photoreceptor cells in the retina [1]. It might cause photoreceptor cells and RP epithelial cells to be damaged and usually presents a variety of symptoms, including night blindness, decreasing visual fields leading to tunnel vision, and total blindness in some instances [2]. An ophthalmologist in the Netherlands, Franciscus Cornelius, first described RP in 1857 [3]. According to the clinical manifestations, there are two main groups of RP, typical RP or rod-cone dystrophy (RCD), in which the rods are predominantly damaged and which represents 80–90% of total cases, and atypical RP or cone-rod dystrophy (CRD), in which the cones are primarily injured and which represents 10–20% of total cases [4]. According to the various types of pigmentary retinopathies associated with many systemic disorders, RP can be expressed in syndromic and non-syndromic forms [5]. The non-syndromic form occurs alone without any other clinical findings, whereas the syndromic form occurs with other neurosensory disorders, developmental abnormalities, or complex clinical findings [6]. The non-syndromic form accounts for approximately 65% of RP cases [7]. RP prevalence is variable among different ethnic groups, with 1 case per 3000–5000 individuals. The total prevalence of RP is 1:4000 in Europe, 1:3500 in the USA, and 1:3784 in China [6, 8‐10].

Inheritance patterns of RP are autosomal dominant (adRP), autosomal recessive (arRP), X-linked (XLRP), and mitochondrial transmission. These patterns are dependent on the specific RP gene mutations present in the parental generation.

RP1 was the first identified genetic cause of RP in 1999 in Nature Genetics. Over the last 19 years, nearly 100 different genes have been linked to RP, including 27 adRP genes, 58 arRP genes, and 3 XLRP genes [11]. Although the genetic cloning research for RP has achieved remarkable results and nearly 100 genes of RP have been found in patients, 50% of patients with RP still have unexplained genetic causes. [12] Morphological, clinical, and genetic RP assessments indicate complicated ocular impairment, and RP is known as a “fever of unknown origin.”

In the case of the Chinese population, limited studies are available [13], and there is little known about RP in pediatric populations, especially for Yunnan pediatric individuals with RP due to the fact that various populations in Yunnan are relatively “enclosed” in certain areas. These “enclosed” populations have gradually developed into different ethnic groups because Yunnan mountainous areas lead to poor transportation.

Here, we identified and reported the mutations in two Yi minority ethnic group families from Yunnan, which has been inhabited by 26 different ethnicities throughout history.

The first case is of a 6.4-year-old boy who is the only child from non-related parents (Fig. 1). He was born at 39 weeks of gestation without asphyxia by spontaneous vaginal delivery to healthy Yi parents. His birth weight was 2.9 kg. According to his parent’s representation, the proband had a loss of night vision at the age of 5 years old. He was then referred to ophthalmic examination for his poor night vision at 5.8 years old: his cornea was clear, anterior chamber depth was normal, aqueous humor was clean, pupil was round, and reflective light reaction normal. His fundus photography indicated pigmentation around bone cells, narrowing retinal vascular, retinal pigment disorderly distributed, and unclear macular foval reflex (Fig. 2).

The second case is of a 0.5-year-old boy who is the only child from non-related parents (Fig. 1). He was born at 40 weeks of gestation without asphyxia by spontaneous vaginal delivery to healthy Yi parents. His birth weight was 3.1 kg. According to his parent’s representation, the proband had vision problems at the age of 1 month when he had a routine neonatal eye examination. He arrived to our attention after an initial diagnosis was formulated at the age of 4 months. The results of his mobility and blinking activity are presented in Table 1. His fundus photography indicated pigmentation around bone cells and macula foveal reflex was clear (Fig. 2). The detailed clinical data for these two families are presented in Table 1.

WES was conducted on the two probands of these families. The mean read depth of the target regions of each proband sample ranged from 52–66X, and the average throughput depth of the target region in each sample ranged from 90–99X. Raw image files were processed for base calling and raw data generation with Bcl2Fastq software (Bcl2Fastq 2.18.0.12, Illumina, Inc.). In addition, low-quality variations were filtered out to get a quality score ≥ 20.Then, using Short Oligonucleotide Analysis Package (SOAP) aligner software (SOAP2.21, soap.genomics.org.cn/soapsnp.html), the clean reads were aligned to the reference human genome (UCSC hg19, http://​genome.​ucsc.​edu/​). Polymerase chain reaction (PCR) duplicates were removed by the Picard program. The single nucleotide polymorphisms (SNPs) were determined by the SOAPsnp programme. The reads were realigned by Burrows–Wheeler Aligner (BWA) software 0.7.15, and the deletions and insertions (indels) were detected by Genome Analysis Toolkit software 3.7. In addition, the identified indel SNPs were annotated with the Exome-assistant program (http://​122.​228.​158.​106/​exomeassistant). To determine their pathogenicity, non-synonymous variants were evaluated by four algorithms, namely, PolyPhen (http://​genetics.​bwh.​harvard.​edu/​pph2/​), Protein Analysis Through Evolutionary Relationships (PANTHER, www.​pantherdb.​org), Sorting Intolerant from Tolerant [SIFT, (http://​sift.​jcvi.​org/​)] and Pathogenic Mutation Prediction (Pmut; http://​mmb.​pcb.​ub.​es/​PMut/​).

Compared with the variants in the SNP databases, the Consensus Coding Sequence (CCDS), NCBI 36.3, dbSNP (v138), and the 1000 Genomes Project, two mutations were identified in these two families with RP. There is one mutation in gene RDH12 in family 1 and PRPF4 in family 2, respectively (Table 2).

Polymerase chain reaction (PCR) and Sanger sequencing with an ABI3500 sequencer were conducted to validate these mutations in these family members to confirm the accuracy of the mutations identified by whole exome sequencing (WES). The sites of variation were identified to compare the DNA sequences with the corresponding GenBank (www.​ncbi.​nlm.​nih.​gov) reference sequences. The sequences of forward and reverse primers are presented in Additional file 1: Table S1 and were used to confirm potential causative variants in this family. Thermocycling conditions: an initial denaturation of 95 °C for 10 minutes, 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 64 °C for 30 seconds, extension at 72 °C for 45 seconds, and a final extension of 72 °C for 5 minutes. The results of Sanger sequencing showed that parents were carriers of c.524C > T (p.S175L) (Fig. 3) and mother was carrier of c.1273G > A (p.G425S) (Fig. 3) in family 1 and in family 2, respectively. Additionally, these two variants were absent in 40 normal control individuals.

Molecular diagnosis is essential for accurate clinical diagnosis and to provide more precise genetic counseling [14]. To gain insight into the genetic basis of Yunnan pediatric population with RP, which had not been previously studied, two Yi families with RP in Yunnan were recruited and we reported that a WES method was used for the molecular diagnosis of Yunnan children with RP.

In this study, we used WES as an effective tool with potential to improve diagnoses. In addition, we found mutations in two rare genes of RP (The causative genes included two categories: most prevalent genes and rare genes [12]), and are described as follows.

We found mutation c.524C > T in RDH12 in family 1. The c.524C > T mutation in exon 7 was identified in our study. RDH12 is a member of the reductase superfamily/short-chain dehydrogenase of proteins that are expressed in photoreceptor cells [15]. The RDH12 gene encodes a protein composed of 316 amino acids and has a pivotal role in the formation of 11-cis retinal from 11-cis retinol during the regeneration of visual pigments [16]. The mutations of RDH12 have been identified in patients diagnosed with arRP and adRP [17, 18]. Generally, RDH12 retinopathy starts at 2–4 years with impaired visual function [19], but the mutation was found in this proband when retinopathy started at 5.8 years.

In family 2, we discovered by WES and Sanger sequencing that the genetic defects were located in PRPF4. The mutations c.1273G > A (p.G425S, mother) in PRPF4 are causative mutations for family 2. The mutations of PRPF4 result in RP due to the fact that it is a key component of spliceosome, which plays critical roles in pre-mRNA splicing.[20] Up to now, four human heterozygous PRPF4 variants, p.P187A, p.R192H, c.-114_-97del and c.C944T, have been reported to cause retinitis pigmentosa. [21‐23] Among yeast, Caenorhabditis elegans, zebrafish, and humans, PRPF4 is highly conserved.[24] The overexpression of human PRPF4^C944T variant or knockdown of prpf4 showed a functional conservation between human and zebrafish Prpf4 proteins. Mutations in PRPF4 cause adRP due to highly conserved C-terminal region of PRPF4, which interacts with pre-mRNA processing factor 3 [21, 22].

Retinitis pigmentosa causes irreversible blindness, and there is no universal cure for RP [12]. So, our follow-up visits and prognosis and proper management of the disease are missing.

This study is limited by the small number of probands, and further studies are necessary to ascertain the impact of these two mutations on gene functions. It is important to establish a molecular diagnosis, which is an essential prerequisite to identifying causative pathogenic variations and a better understanding of the disease. [25]