Case report: Whole exome sequencing identified a novel mutation (p.Y301H) of MAF in a Chinese family with congenital cataracts

Background Congenital cataracts stand as the primary cause of childhood blindness globally, characterized by clouding of the eye’s lens at birth or shortly thereafter. Previous investigations have unveiled that a variant in the V-MAF avian musculoaponeurotic-fibrosarcoma oncogene homolog (MAF) gene can result in Ayme-Gripp syndrome and solitary cataract. Notably, MAF mutations have been infrequently reported in recent years. Methods In this investigation, we recruited a Chinese family with non-syndromic cataracts. Whole exome sequencing and Sanger sequencing were applied to scrutinize the genetic anomaly within the family. Results Through whole exome sequencing and subsequent data filtration, a new mutation (NM_005360, c.901T>C/p.Y301H) in the MAF gene was detected. Sanger sequencing validated the presence of this mutation in another affected individual. The p.Y301H mutation, situated in an evolutionarily preserved locus, was not detected in our 200 local control cohorts and various public databases. Additionally, multiple bioinformatic programs predicted that the mutation was deleterious and disrupted the bindings between MAF and its targets. Conclusion Hence, we have documented a new MAF mutation within a Chinese family exhibiting isolated congenital cataracts. Our study has the potential to broaden the spectrum of MAF mutations, offering insights into the mechanisms underlying cataract formation and facilitating genetic counseling and early diagnosis for congenital cataract patients.


Introduction
Congenital cataracts, an ophthalmic disease presented at birth or developing in infancy, can lead to a clouding in the lens of the eye that can cause blurry vision or blindness (1)(2)(3).As the predominant cause of visual impairment and blindness among children, congenital cataracts affect approximately 200,000 children worldwide, with an estimated prevalence  ranging from three to six per 10,000 live births (4,5).The current studies believe that multiple factors including genetics, infections, radiation exposure, toxic agents, and metabolic disturbance underlie the occurrence of congenital cataracts (3).To date, over 40 genes responsible for the molecular etiology of isolated or syndromic congenital cataracts, featuring autosomal dominant or autosomal recessive inheritance patterns, have been identified (1,6).The V-MAF avian musculoaponeurotic-fibrosarcoma oncogene homolog (MAF) gene, also known as MAF bZIP transcription factor (OMIM#177075), is situated on chromosome 16q23.2,spanning approximately 6.9 kilobases (7).As a DNA-binding transcription factor with a leucine zipper motif that can control the activity of target genes, MAF can serve as a transcriptional activator or repressor, regulating diverse cellular processes, including embryonic lens fiber cell development, heightened T-cell susceptibility to apoptosis, and chondrocyte terminal differentiation (8)(9)(10).In 2002, Jamieson et al. (11) initially reported the association of MAF with cataracts, ocular anterior segment dysgenesis, and coloboma.Subsequently, approximately 30 MAF mutations have been detected in patients with Ayme-Gripp syndrome and isolated cataracts (7).
Here, we recruited a Chinese family affected by isolated congenital cataracts.Utilizing whole exome sequencing and Sanger sequencing, we sought to uncover the genetic abnormalities in the affected individuals.

Case presentation
Here, we enrolled the family from Hebei province, China (Figure 1A).The affected proband, a four-year-old boy was admitted to Hebei General Hospital and diagnosed as a total congenital cataract of the right eye (Figure 1B).A medical history survey suggested that the patient suffered from poor vision in dim light at approximately 3 years old but did not arouse parents' attention, and the symptoms of the child developed to lens complete opacification from pupil gray appearance quickly during this year.Finally, the patient agreed to undergo surgery for cataract extraction and implantation for the posterior chamber intraocular lens, and the eye vision is gradually recovering.A study of the family history indicated that his father also suffered from cataracts and underwent cataract extraction in infancy.The proband's grandmother was blind and died several years ago.
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Laboratory investigations
Whole peripheral blood samples of the patient and his family were obtained and stored in EDTA/citrate tubes.Genomic DNA was extracted by GenElute blood genomic DNA extraction kit (Sigma-Aldrich, NA 2010).Whole Exome sequencing (WES) was mainly conducted by Berry Genomics (Beijing, China) and Agilent SureSelect Human All Exon V6 kits and Illumina HiSeq2500 (Illumina Inc., San Diego, United States) and matched to Human Reference Genome (hg19).GATK online software 1 was used to detect SNP, Indel, and variants, and the Annovar (Annotate Variation) tool was used to functionally annotate genetic variant results in detail.
The filtered mutations validation and co-segregation analysis were performed by Sanger sequencing.The primer pairs (the sequence of primers will be provided upon request) were designed by Primer 5.The sequences of the primers were: 5′-3′ TCAGCAAGGAGGAGGTGAT and 3′-5′ CTGCTCACCAACTTC TCGTATT.The sequence of the polymerase chain reaction products was determined using the ABI 3100 Genetic Analyzer (ABI, United States).
After the abovementioned data filtering and Sanger sequencing validation (Figure 1C), a new mutation (NM_005360, c.901T>C/p.Y301H) of MAF was discerned in the proband (Table 1).No other meaningful mutations related to cataracts were detected.Co-segregation analysis indicated that the new mutation was present in the affected family members and was not detected in unaffected family members and healthy controls as well as 200 local control cohorts (Figure 1D).Three programs for analyzing protein functions, polyphen2, SIFT, and MutationTaster, predicted that the p.Y301H variants are probably damaging (1.0), deleterious (0.00), and diseasecausing (0.99), respectively.Cross-species alignment analysis of MAF  amino acid sequences revealed that this mutated site was highly evolutionarily conserved (Figure 2A).Swiss-Model and alphafold2 online software (14) found that the p.Y301H mutation changed the surface hydrophobic area and surface charge of MAF (Figure 2B).

Discussion
Congenital cataracts represent a highly diverse ocular disorder both clinically and genetically (3).Mutations in MAF have been linked to various types and severities of human congenital cataracts (Table 2) (32).MAF proteins play a pivotal role in eyes and lens development, regulating the expression of crystallin genes, MIP (major intrinsic protein of the ocular lens fiber membrane), and other genes expressed in lens fiber cells from the formation of the lens pre-placode to the development of lens fiber cells and lens epithelium (8)(9)(10).In C57BL/6J mice, homozygous deletion of MAF results in embryonic lethality, but the lens fiber cell MAF condition knockout mice (MAF ΔTAM ) develops abnormal lens structure, and the expression of crystallin genes in MAF ΔTAM mice eyes are reduced compared to WT mice (33).The mice data were consistent with human genetic studies, highlighting the essential role of MAF in the differentiation and cell cycle arrest of lens fiber cells.Although the first mutation of MAF was reported in 2002 (11) The MAF protein consists of an N-terminal transactivation domain with a regulatory function and a C-terminal DNA binding domain (18).The C-terminal domain is further divided into three conserved regions (Figure 2A): extended homology region, basic region motif, and leucine zipper motif (15,34).Earlier studies have indicated that the N-terminal variants may result in Ayme-Gripp syndrome which presents with cataracts, hearing loss, epilepsy, intellectual disability, etc., while most of the C-terminal mutation carriers only showed ocular diseases such as cataracts (Table 2) (7).Here, the mutation (p.Y301H) is also situated in the C-terminal of MAF, which provides more evidence that C-terminal mutations are responsible for isolated cataracts.Furthermore, the p.Y301H mutation is in the BR motif of the C-terminal, which is the mutational hot spot region of MAF (7).Previous studies have suggested that the BR motif is responsible for binding the specific target promoters of crystallin genes including CRYGA, CRYAA, CRYBA1, and CRYBA4, which are associated with inherited cataracts (32,35).Additionally, ChIP-seq studies have identified several non-crystallin genes crucial for maintaining lens transparency as direct targets of MAF (36,37).Hence, the p.Y301H mutation may disrupt the bindings between MAF and the target genes such as crystallin genes, and finally disrupt the expression of promoters of crystallin genes, leading to congenital cataracts.Following the ACMG guideline (38), the p.Y301H mutation is likely pathogenic (PM1 + PM2 + PP1 + PP2 + PP3).
Congenital cataracts persist as a leading cause of global blindness, and early surgical intervention, prolonged postoperative amblyopia training, and visual reconstruction constitute the primary therapeutic approaches for the disease (39, 40).Early diagnosis is paramount, particularly in the era of precision medicine, where technologies such as PCR, Sanger sequencing, high-throughput sequencing, and gene editing have facilitated the discovery of numerous pathogenic genes linked to congenital cataract development.This advancement enhances our understanding of the disease's pathogenesis and lays the groundwork for genetics-based treatments (6,28).In the future, we can develop a genetic detection panel that contains all the reported mutations of MAF including the p.Y301H, and the panel may contribute to the genetic counseling and early diagnosis of congenital cataract patients.

Conclusion
Hence, we detected a new mutation (NM_005360, c.901T>C/p.Y301H) of MAF in a Chinese family with congenital cataracts by employing whole exome sequencing and Sanger sequencing.Our study not only explores the genetic lesion of the family and broadens the spectrum of MAF mutations but also confirms that the MAF mutation was linked to non-syndromic total congenital cataracts and facilitates genetic counseling and early diagnosis for congenital cataract patients.Certainly, there were still some limitations in this study, for example, the sample size was small and population specificity was not excluded.

FIGURE 1
FIGURE 1 The clinical and genetic analysis of the family with congenital cataracts.(A) Pedigree of the family.White circles/squares are unaffected family members, and the arrow indicates the proband.(B) The right eye of the proband.(C) Schematic representation of the filter strategies employed in our study.(D) Sanger DNA sequencing chromatogram demonstrates the heterozygosity for an MAF missense mutation (c.901T>C/p.Y301H) in the family.

FIGURE 2
FIGURE 2The bioinformatics analysis the MAF p.Y301H mutation.(A) Alignment of multiple MAF protein sequences across species.The Y301-affected amino acid is located in the highly conserved amino acid region in different mammals (from Ensembl).The red column shows the Y301 site.Blue square: transactivation domain.Red square: extended homology region.Orange square: basic region motif.Green square: leucine zipper motif.(B) The hydrophobic surface area and surface charge of the WT and mutated MAF were predicted by SWISS-MODEL and alphafold2.The red arrow and red square indicate the differences between WT and mutated protein.

TABLE 1
The gene list of Sanger sequencing validation and co-segregation analysis.

TABLE 2
The summary of reported mutations in MAF gene.