Microphthalmos is a rare, inherited or sporadic congenital eye development defection and is responsible for approximately 3–12% visual impairment in children. It is characterized by a short axial length (AL < 20.0–21.0 mm) with thickened choroid and sclera (Hoffman et al. 2015; Verma and Fitzpatrick 2007), a small anterior chamber depth (ACD < 2.2 mm) and a small cornea (horizontal corneal diameter < 11 mm) (Nihalani et al. 2005). According to the anatomical difference and American Society of Cataract and Refractive Surgery (ASCRS) cataract clinical committee, microphthalmos can be divided into four types: simple microphthalmos (or nanophthalmos), microphthalmos with other congenital ocular coloboma, relative anterior microphthalmos (RAM) and axial high hyperopia (Hoffman et al. 2015; Zheng et al. 2017).

Genetic alterations are now a major cause of microphthalmos. The hereditary mode of nanophthalmos is autosomal dominant in many patients and sporadic in others (Plaisancie et al. 2019; Slavotinek 2018). As a developmental eye disease, most of the related genes involved in microphthalmos are also ocular development-associated genes, including SOX2, OTX2, PAX6, and GJA8. Although over 30 genes have been implicated in eye development, we are currently able to explain the genetic bases of these defects in less than half of patients (Plaisancie et al. 2019). Moreover, recent studies on microphthalmia mainly rely on clinical observation, and novel-related genes and mutations in microphthalmos have rarely been explored.

Hence, in our study, we studied the novel mutation genes of microphthalmos in two Chinese families (Family 1: microphthalmos with congenital ocular coloboma and Family 2: simple microphthalmos) and reported three novel mutations. Hopefully, our study will be helpful for microphthalmos prediction, prevention and molecular treatment.

Materials and Methods

Subjects and Clinical Evaluations

To make a precise diagnosis, we performed targeted next-generation sequencing (NGS) of microphthalmos-related genes. Written informed consent of the family members from both families was obtained prior to the collection of 5 mL of their peripheral blood for the following experiment. The study was approved by the ethics committee of Eye and ENT Hospital of Fudan University and was conducted according to the principles of the Declaration of Helsinki.

Our study involved two family members from three generations who underwent detailed history ophthalmic examination, including best corrected visual acuity (BCVA) testing, slit lamp biomicroscopy, IOL master 500 (Carl Zeiss Meditec, Germany), dilated fundus examination, B scan, and SD-OCT (Spectralis HRA + OCT, Heidelberg, Engineering, Inc., Heidelberg, Germany). Family and medical history, including age of onset and other related clinical manifestations, were obtained. Blood samples were collected from the peripheral blood and stored at 4 °C before further analysis.

Genetic Analyses

Genomic DNA from the family was extracted from peripheral blood on Feb 2018 according to the manufacturer's standard procedure using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) (Ma et al. 2015). A capture panel (NimbleGen, Madison, USA) of microphthalmos genes has been previously designed and assessed by our group. The capture panel comprised all exons together with the flanking exon and intron boundaries (±15 bp) of 425 genes that are most frequently involved in common inherited eye diseases. The capture probes (callinonel, scall-in-one) were designed by BGI and produced by Roche.

The library was enriched by array hybridization according to a previously published procedure, followed by elution and post-capture amplification. Then, qualification and NGS targeted sequences were further analyzed on the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA, United States) in collaboration with BGI-Shenzhen (Shenzhen, Guangdong, China) as previously reported (Chen et al. 2015, 2013; Qi et al. 2017). To detect the potential variants in the family, we performed bioinformatics processing and data analysis after receiving the primary sequencing data. We used previously published filtering criteria to generate “clean reads” for further analysis. The “clean reads” (with a length of 90 bp) derived from targeted sequencing and filtering were then aligned to the human genome reference (hg19) using the Burrows–Wheeler Aligner (BWA) Multi-Vision software package (Li and Durbin 2009). After alignment, the output files were used to perform sequencing coverage and depth analysis of the target region, single-nucleotide variants (SNVs) and INDEL calling.

We used the following four databases to test annotation of all identified variants with minor allele frequency (MAF) > 0.1% to eliminate benign variants: dbSNP137, HapMap Project, 1000 Genomes Project and Exome Variant Server. Finally, the variant prioritizations 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 (HGMD) and Online Mendelian Inheritance in Man (OMIM). All mutations and potential pathogenic variants were validated using the conventional Sanger sequencing or spectrum sequencing method. Segregation analysis was performed if DNA from family members was available.

For Sanger verification sequencing, primers were designed on the upstream and downstream of the fragment for all the potential pathogenic mutations. PCR amplification was carried out and Sanger sequencing was done, then we compared the result with the standard sequence of PXDN and CRYBB2 genes to verify the result of next-generation sequencing-based target capture sequencing.

We used the spectrum sequencing method to validate the variant if the common allele was detected by the Sanger sequencing. Agena MassARRAY platform was used for genotyping. It is a robust tool with high accuracy and is cost effective as it involves multiplex PCR and single-base extension. The high-energy laser could make the extended oligonucleotide product have a single charge. The genotype of the corresponding locus could be judged based on different flight time of different oligonucleotides under the action of an electromagnetic field.


Clinical Examination and Pedigree Analysis

A total of 17 members from two three-generation families (Fig. 1) were included in the study. The clinical information of the four affected members is listed in Table 1. The proband of Family 1 (P5), a 29-year-old woman, has suffered from poor vision in both eyes since birth. Ophthalmic examination revealed a BCVA HM in both eyes. The axial length measurement showed the extreme short axial length (OD: 18.36 mm, OS: 19.83 mm). We also found an extremely small cornea diameter (OD: 6.5 mm, OS: 7.2 mm) after corneal diameter evaluation. Fundus examination and B scan both revealed severe choroid coloboma (Fig. 2a). After careful examination of her son, we found that P5 and her son (P9) suffered from the same eye disease: microphthalmos with congenital ocular coloboma and congenital cataract (Fig. 2b).

Fig. 1
figure 1

Proband of the two families

Table 1 Clinical characteristics of the four affected patients
Fig. 2
figure 2

a Slit lamp photograph (left) and B scan (right) of proband (P5) of Family 1. The yellow arrow shows the severe choroid coloboma. b Slit lamp photograph (left) and B scan (right) of proband (P4) of Family 2

The proband of Family 2 (P4), a 31-year-old woman, has suffered from progressive decreased vision in both eyes for over 10 years. Ophthalmic examination also revealed extremely short eyes (axial length: OD: 16.99 mm, OS: 16.02 mm) and small cornea (OD: 9.7 mm, OS: 9.0 mm) with congenital cataract. Fundus examination and a B scan showed moderate normal fundus. After careful examination of her daughter (P7), P4 and her daughter were diagnosed as the same disease: simple microphthalmos.

Genetic Analyses

We performed a targeted NGS approach on the probands (P5 of Family 1 and P4 of Family 2). The targeted gene length was 1,106,466 bp of 425 genes, and the mean depth of the target region was 170.45 with 99.95% coverage. After the data acquisition and bioinformation analysis, two novel heterozygous mutations, PXDN c.3165C > T (p.Pro1055Pro) and PXDN c.2640C>G (p.Arg880Arg), were found in the proband (P5) of Family 1 (Fig. 3; Table 2), and CRYBB2 c.481G>A (p.Gly161Arg) was found in Family 2 (Fig. 4; Table 2). These mutations, PXDN c.3165C>T (p.Pro1055Pro), PXDN c.2640C>G (p.Arg880Arg) and CRYBB2 c.481G>A (p.Gly161Arg), were extremely rare in the control population, with frequencies of 0.0082, 0.0082 and 0 in the 1000 Genomes Project, respectively. Moreover, the three mutations in the SNV database of over 200 Chinese populations were less than 0.01.

Fig. 3
figure 3

PXDN gene mutations found in Family 1. a PXDN c.3165C>T (p.Pro1055Pro). b PXDN c.2640C>G (p.Arg880Arg)

Table 2 Mutations identified in Family 1 and Family 2
Fig. 4
figure 4

CRYBB2 gene mutation found in Family 2

Sanger sequencing was then performed to validate the variants in Family 1. The proband’s son (P9) of Family 1 carried the same heterozygous mutations (c.3165C>T and c.2640C>G) of the PXDN gene (Fig. 3; Table 2). The two variants located in the same chromosome. But, common alleles and the two mutations were not found in the proband’s parents. Moreover, the two mutations did not lead to amino acid changes.

We used the spectrum sequencing method to validate the variant in Family 2 after common allele was detected by the Sanger sequencing method. The proband’s daughter (P7) in Family 2 carried the same heterozygous mutation (c.481G > A) of the CRYBB2 gene. However, common allele was detected and the mutation was not found in the proband’s parents. Multiple orthologous sequence alignment (MSA) using Polyphen revealed that CRYBB2 and its subsequent sequences were highly conserved amino acids across different species (Fig. 5), suggesting that the mutation may lead to a deleterious effect.

Fig. 5
figure 5

Multiple orthologous sequence alignment (MSA) for CRYBB2 p.Gly161Arg mutation


As a high-throughput, low-cost and high-efficiency sequencing technology, NGS technology has been widely used in clinics to identify many rare diseases (Ma et al. 2015; Patel et al. 2019). The mutations and genes identified by NGS have become a powerful tool to explore potential genetic etiology and guide appropriate treatment for eye diseases (Ma et al. 2015; Plaisancie et al. 2016). In our study, we comprehensively screened 425 genes involved in common, inherited eye diseases and successfully identified 3 potentially causative mutations for microphthalmos, PXDN c.3165C>T (p.Pro1055Pro), PXDN c.2640C>G (p.Arg880Arg) and CRYBB2 c.481G>A (p.Gly161Arg), in two Chinese families. Based on the mutation analysis and the clinical measurement, we concluded that these three novel mutations could provide early pre-symptomatic diagnoses and emphasize the significance of the genetic diagnosis of microphthalmos.

The human CRYBB2 (crystalline beta B2) gene is located on chromosome 22q and is encoded in 7 exons. The CRYBB2 gene is a dimer at low concentrations but can form oligomers under physiological conditions. The CRYBB2 gene has contributed to the identical human lens crystalline protein–protein binding (Liu and Liang 2006; Xu et al. 2018), structural constituent of human lens (Chambers and Russell 1993) and structural molecule activity (Zhao et al. 2017). Multiple studies have revealed that CRYBB2 gene mutations [including p.Q155X (Ching et al. 2019; Li et al. 2008; Messina-Baas et al. 2016), p.E167X (Zhou et al. 2016)] were associated with congenital autosomal dominant cataracts. In our study, we also found a CRYBB2 novel mutation (p.Gly161Arg) in simple microphthalmos eyes in Family 2. The proband and her daughter shared the same mutation of the CRYBB2 gene, which adhered to the dominant heredity as previous papers. Furthermore, MSA using Polyphen revealed that CRYBB2 and its subsequent sequences were highly conserved amino acids across different species, suggesting that the CRYBB2 p.Gly161Arg mutation was a deleterious mutation. To the best of our knowledge, the CRYBB2 p.Gly161Arg mutation was originally reported in this study. However, the proband and her daughter had microphthalmos and congenital cataract, and whether the CRYBB2 p.Gly161Arg mutation is a causative mutation of microphthalmos or the congenital cataract needs further clinical and genetic study.

The human PXDN gene is located on chromosome 2p and is encoded in 24 exons. The gene is expressed in corneal epithelium and is secreted into the extracellular matrix. Mutations in the PXDN gene were associated with congenital recessive corneal opacification and other ocular anomalies as well as microphthalmia and anterior segment dysgenesis. Choi et al. reported PXDN Tyr398Thrfs*40 and PXDN Gln316Pro mutations in three families with congenital cataracts, microcornea, sclerocornea and developmental glaucoma (Choi et al. 2015). The defective PXDN gene has been shown to impair sulfilimine bond formation in collagen IV, a constituent of the basement membrane, implying that eye defects result from the loss of basement membrane integrity in the developing eye. In our study, we also found that PXDN gene mutations c.3165C>T (p.Pro1055Pro) and c.2640C>G (p.Arg880Arg) in microphthalmos with congenital ocular coloboma eyes. The two mutations have not been previously reported. Furthermore, the heredity model was prone to be autosomal dominant in Family 1, which implied that the PXDN gene mutations c.3165C>T (p.Pro1055Pro) and c.2640C>G (p.Arg880Arg) might be different from the other PXDN gene mutations. Although the two mutations did not result in amino acid changes, we also concluded that PXDN sequencing should be considered in microphthalmos with anterior segment dysgenesis.

To the best of our knowledge, this study used the first NGS-based assay specifically designed for the confirmation and early diagnosis of microphthalmos in two Chinese families’ pedigrees reported to date. In addition, we found three potentially causative mutations for microphthalmos, PXDN c.3165C>T (p.Pro1055Pro), PXDN c.2640C>G (p.Arg880Arg) and CRYBB2 c.481G>A (p.Gly161Arg), that are likely responsible for microphthalmos. These genetic mutation patterns are novel, and the functions and interactions of the PXDN and CRYBB2 genes should be further investigated. This study not only provides a guide to the attending clinician on the management and prognosis of the patient but also extends the phenotypic spectrum of PXDN- and CRYBB2-associated microphthalmos and enhances our current understanding of the genetic basis of microphthalmos.