Novel mutations in HSF4 cause congenital cataracts in Chinese families

As part of a genetic screening program for genetic eye disorders, we collected peripheral blood from 42 families with congenital cataracts and 225 related individuals from the southeast China. All the affected individuals and unaffected relatives in their family were performed ophthalmological examinations by slit lamp photography. The study followed the tenets of the Declaration of Helsinki. Informed consents were obtained from each participant except for the children. For any participants that are under the age of 16, the consent to participate was obtained from their parents or legal guardians. 112 samples from ethnically matched control individuals were obtained prior to the study. The experiments were approved by the Ethics Committee of Fujian Medical University. Total genomic DNA was extracted from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Beijing, China) according to the manufacturer’s instructions.

Before this study, we had compiled hot-spot regions of cataract-causing mutations. Briefly, 72 mutant exons of 31 pathogenic genes associated with 299 congenital cataract families or sporadic cases have been reported in 210 selected articles. The 72 exons, account for 34.62% of all the 208 exons in the 31 genes, were ordered by the summary frequency of disease-causing mutations decreasingly across each gene exons, and the top 26 exons in 18 pathogenic genes were selected as the hot-spot mutation regions. The hot-spot regions covered about 80 percentages of mutations in the compiled mutations with only 36.11 percentages (26/72) of all the mutant exons, and 12.5 percentages (26/208) of all the exons.

All the mutations in the 18 common genes causing congenital cataracts were screened for all the probands of 42 families. These genes including CRYAA, CRYAB, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS, GJA8, GJA3, HSF4, MIP, BFSP2, EPHA2, FYCO1 and PITX3. The selected hot spot exons and splice junctions of these genes were amplified by PCR from genomic DNA. The PCR primers and conditions for HSF4 were listed in Table 1, and that for other genes listed in Additional file 1: Table S1. PCR products were purified and directly sequenced on an ABI 3730XL Automated Sequencer (PE Biosystems, Foster City, CA) using the same PCR primers. Intra-familial segregation analysis was performed after identification of HSF4 mutations in probands. The identified HSF4 mutations were also checked in 112 normal unrelated individuals from the same ethnic background.

Mutations description followed the recommendation of the Human Genomic Variation Society (HGVS). The effects of novel missense mutations on the encoded proteins were further evaluated by Polymorphism Phenotyping [5] v2 (PolyPhen-2) and Sorting Intolerant From Tolerant [6] v5.1.1 (SIFT), and the effects of intronic variants on splicing site changes were predicted by the Human Splicing Finder [7] v2.4.1 (HSF). The pathogenicity of all the mutations was evaluated by the standards and guidelines of American College of Medical Genetics and Genomics [8] (ACMG) using InterVar [9] software.

The 42 probands are composed of 30 probands from autosomal dominant families, 8 probands with no family history, and 4 isolated cases. In total, 379 individuals were recruited in this study. All the probands were diagnosed as bilateral cataracts at early childhood. No other ophthalmic or systemic diseases were found for all the patients. Five unrelated families were identified HSF4 mutations. The inheritance pattern of those families is AD (Fig. 1). Based on clinical descriptions, family CAT-02 and family CAT-37 have been diagnosed as congenital perinuclear and nuclear cataracts, respectively`. Other three families including CAT-12, CAT-50 and CAT-51 with congenital total cataracts. The slit-lamp photographs of the patients in two families also showed the phenotype of cataracts (Fig. 2).

In total, five mutations were identified respectively using directly sequencing of the exons and flanking splicing sites of HSF4 (Table 2 and Fig. 3), in five unrelated families with congenital cataracts. There were no variants on other seventeen causing genes detected in these five families. All of the five mutations have not been previously reported. Some mutations identified in other 17 families with congenital cataracts were not reported here. The clinical significances of the five mutations were generated by InterVar [9] software on the basis of the criteria recommended by ACMG/AMP guidelines. The process is automatically done firstly, and then follows a manual adjustment to reclassify the mutations for the criteria that InterVar recommends. These five mutations match the criterion of pathogenic moderate 1 (PM1) since all of them are in the hot spot regions. The five mutations were absent in the public databases including 1000 Genomes, ExAC and Genome Aggregation Database, which indicates they match the criterion of pathogenic moderate 2 (PM2). These five mutations were cosegregation with congenital cataracts in each affected family members (Figs. 1 and 3), indicating that they match the criterion of pathogenic supporting 1 (PP1).

In Family CAT-02, the mutation was identified as a c.314G > C missense mutation, where a Serine was replaced by a Threonine at codon 105 (p.Ser105Thr). In Family CAT-12, a c.218G > T substitution was identified, which result in the replacement of an Arginine at position 73 by Leucine (p.Arg73Leu). In Family CAT-37, a c.233A > G missense mutation was identified, which led to Tyrosine at position 78 replaced by Cysteine (p.Tyr78Cys). In Family CAT-51, a c.187 T > C substitution led to Phenylalanine at position 63 replaced by Leucine (p.Phe63Leu). PolyPhen-2 predicted “probably damaging” of all the four variants except for c.233A > G (benign); while the SIFT method predicted “deleterious” for all the four variants except for c.314G > C (tolerated). These predictions indicated that the four variants may have effect on protein function. In Family CAT-50, a IVS5 c.233-1G > A mutation at 1 bp splice sites of the codon 78 is predicted to alter the WT acceptor site and most probably affect splicing by Human Splicing Finder software, which meet the criterion of pathogenic very strong (PVS1).

Finally, all five novel mutations were classified as “likely pathogenic” for congenital cataracts except for IVS5 c.233-1G > A mutation “pathogenic” using InterVar [9] software in accordance with ACMG standards. PM1, PM2 and PP3 were automated for the four missense mutations. The details of each mutation can be seen in the Table 2.