RGS6: A Novel Gene Associated With Congenital Cataract, Mental Retardation, and Microcephaly in a Tunisian Family

Abstract

Purpose.: The object of this study is to identify the underlying genetic defect in a consanguineous Tunisian family affected with autosomal recessive congenital cataract associated with mental retardation and microcephaly.

Methods.: A whole-genome scan was performed with polymorphic microsatellites in the axiom data for the screened members. Homozygous regions were analyzed with integrated Systems Tool for Eye gene Discovery (iSyTE), to identify candidate genes with lens-enriched expression that were potentially associated with cataract. Then we screened for mutations by direct sequencing. Structure and function of the mutant gene were analyzed by bioinformatics analysis.

Results.: Using whole-genome scanning, we identified six runs of homozygosity shared among affected members of the studied family. Analysis of these regions by iSyTE allowed us to select 3 genes (RGS6, PCNX, and P4HA1) according to their expression in 3 critical stages of lens development. Upon screening for mutations by sequencing analysis, we found a novel mutation in RGS6, the splice-acceptor variant c.1369−1G>C that was not previously reported in congenital cataract phenotypes.

Conclusions.: Our study identified a new gene to be included in the large spectrum of cataract-associated genes. Importantly, the study demonstrated that, in addition to lens-enriched genes that exhibit high expression levels, genes identified by iSyTE that are highly lens-enriched but have lower absolute expression may also represent candidates for potential function in the lens.

Introduction

Congenital or infantile cataract causes 10% to 30% of all blindness in children, with one-third of cases estimated to have a genetic cause.¹ Different clinical aspects can be observed, including cataract with ocular and/or systemic anomalies and polymalformative syndrome, skeletal, dermatological, neurological, metabolic, and genetic or chromosomal diseases.² Although congenital cataract is seen frequently in syndromes including mental retardation and microcephaly accompanied by other abnormalities, this is the first report of inherited congenital cataracts associated only with mental retardation and microcephaly. Isolated bilateral congenital cataract tends to be a highly penetrant Mendelian trait, with autosomal dominant cataracts being more common than autosomal recessive cataracts. Currently, there are approximately 39 loci to which isolated or primary cataracts have been mapped, although the number is constantly increasing.^3–5 In fact, the first early events of lens development will be influenced by genes encoding transcription factors like PAX6, PITX3, MAF, or SOX. If the lens is maturing, mutations affecting the lens membranes (aquaporins/MIP, LIM2, or connexins) or the structural proteins of the cytosol of the lens fiber cells (the crystallins) become more important.⁶ However, in some particular familial cases with congenital cataract, no evident mutation could be found in these specific lens genes. 

Recently, the Integrated Systems Tool for Eye gene Discovery (iSyTE; Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA; http://bioinformatics.udel.edu/research/iSyTE) was established in order to facilitate the identification of genes associated with cataract and other ocular defects.⁷ In this study, we used the iSyTE system to prioritize candidate genes within mapped genomic intervals associated with the studied phenotype (autosomal recessive congenital cataract, mental retardation, and microcephaly) in a Tunisian family, followed by further investigations. 

Methods

Subjects and Sample Collection

Five members (3 normal, 2 affected) of a 4-generation consanguineous Tunisian family with autosomal recessive congenital cataract associated with mental retardation and microcephaly were referred to the department of Congenital and Hereditary Disorders at Charles Nicolle Hospital, Tunis, Tunisia, and were enrolled in a genetic research program in the Laboratory of Human Genetics, Faculty of Medicine, in order to identify genetic defects related to the diagnosed phenotype. 

Patients and parents for minors gave informed consent prior to their inclusion in the study. A detailed medical history was obtained by interviewing family members. All had complete ophthalmic examinations including best-corrected visual acuity, slit-lamp biomicroscopy, and dilated fundus examination. Optical coherence tomography (OCT) and electroretinography (ERG) were performed for the two affected siblings. 

In this study, the research carried out in humans were in compliance with the Helsinki Declaration, and the ethics committee of Charles Nicolle Hospital, Tunis, approved the study. 

Genomic DNA Preparation

Genomic DNA of affected and unaffected members was extracted from peripheral blood leukocytes by using the standard proteinase-K extraction consisting of lysis of red blood cells by red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.5 EDTA, pH 7.5) and white blood cells by a white blood cell lysis buffer (1 mM Na-EDTA, 5 mM Tris HCl, pH 7.5); then treatment of the lysate with a mixture of detergent composed of sodium dodecyl sulfate or sacrosyl and proteinase K in order to liberate the DNA and digest the associated proteins; precipitation of the DNA in the form of filaments by absolute ethanol; and finally, dilution of the DNA in T10E1 buffer (Tris 10 mM, EDTA 0.1–1 mM); samples were stored in 10-mL sterile vacuum tubes containing 100 μL of 0.1 M EDTA.K3. 

Homozygosity Mapping and Linkage Analysis

Genomic DNA samples of selected persons were analyzed using whole-genome nucleotide polymorphism arrays in the Developmental Genetics Unit, King Faisal Specialist Hospital and Research Center, Riyad, Saudi Arabia. The two patients were genotyped using Axiom Hu single nucleotide polymorphism (SNP) chips (Axiom GT1; Affymetrix, Santa Clara, CA, USA), which were scanned and analyzed. Array images were acquired using GeneChip scanner 7G (Affymetrix) with an autoloader that scanned each array. Raw DATA image files were generated using GeneChip Operating System software (GCOS; Affymetrix). Each DATA image was processed by GCOS software to generate a feature extraction. The resulting genotypes were scanned for runs of homozygosity (ROH) with the homozygosity mapper (http://homozygositymapper.org/). 

iSyTE Analysis

Mapped genomic regions identified by homozygosity mapping were investigated using the iSyTE computational tool in combination with Genome Browser (UCSC Genome Informatics Group, Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA, USA) in order to prioritize candidate genes associated with human congenital cataract.⁷ This strategy effectively removed highly expressed but nonspecific housekeeping genes from lens tissue expression profiles and, in turn, allowing identification of genes with highly lens-enriched expression that were potentially associated with lens biology and disease. 

Molecular Analysis

All exons and exon-intron junctions of the three selected genes, pecanex homolog (Drosophila) (PCNX; 36 exons), regulator of G-protein signaling 6 (RGS6; 18 exons), and prolyl 4-hydroxylase, alpha polypeptide I (P4HA1; 16 exons) were amplified from genomic DNA by polymerase chain reaction (PCR) using primers chosen by Primer3 version 4.0.0 (http://primer3.ut.ee/) and checked by UCSC Genome Browser BLAST alignment tool (BLAT; http://genome.ucsc.edu/cgi-bin/hgBlat). Genes' sequences were retrieved from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway; PCNX, accession no. NM_014982; RGS6, accession no. NM_001204416; and P4HA1, accession no. NM_ 000917). 

Amplification reactions were performed by using 100 ng of each patient's genomic DNA as a template, 20 pmol of each primer (Biomatik, Cambridge, ON, Canada), a MgC_l2 concentration depending upon the exon amplified, 1.5 units of Taq DNA polymerase recombinant (Invitrogen, Carlsbad, CA, USA), and 1.25 mM of each dNTP (Bioline, Royaume-Uni, Guyancourt, France) in a total volume of 50 μL. PCR consisted of 35 cycles and was carried out in the automated thermal cycler GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 5 minutes, 95°C for 30 seconds, then 43° to 67°C for 30 seconds, and elongation at 72°C for 1 minute, followed by 1 cycle of extension at 72°C for 7 minutes. The amplified products were purified (DNA Clean and Concentrator 5 kit; Zymo Research, Irvine, CA, USA) and sequenced (Big Dye Terminator cycle sequencing ready reaction DNA sequencing kit Prism 3130; Applied Biosystems) in the forward and reverse directions. Sequencing results were visualized, and data were computer analyzed using Sequencing Analysis version 5.2 and SeqScape software (Applied Biosystems). 

Various Databases and Mutated Protein Analysis

Nucleotide database searches were performed using the Web site of the National Center for Biotechnology Information (NCBI: dbSNP), high-throughput genomic sequencing, and searching in specific databases including 1000 Genomes, Leiden Open (source) Variation Database (LOVD), Ensembl Human Genome Browser, and professional Human Gene Mutation Database (HGMD). 

The functional impact of the splice site variant was predicted by using the online prediction tool Human Slicing Finder version 2.4.1 (HSF; http://www.umd.be/HSF/). The degree of evolutionary conservation of amino acid at the position of interest in other gene orthologs was examined using NCBI (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). 

Results

Clinical Findings

A Tunisian family consisting of two affected siblings was referred for genetic screening. The patients were offspring of a consanguineous marriage. The pedigree is shown in Figure 1 and shows autosomal recessive inheritance of congenital cataract, intellectual disability, and microcephaly phenotype. 

Figure 1

 

Pedigree of the studied family showing autosomal recessive inheritance of the congenital cataract.

The two patients had undergone extracapsular extraction and implantation of the posterior chamber for both eyes. Slit-lamp biomicroscopic examination of anterior segment before surgery revealed the presence of bilateral early posterior polar cataracts. Neither sibling had glaucoma before or after the extraction of cataracts. Although his affected sister had preserved visual acuity and showed normal OCT and ERG, the brother, IV₁₂, exhibited decreased visual acuity with an alteration of retinal pigment epithelium, detected by OCT, which was shown markedly by dilated fundus examination (no photos were kept because the patient was uncooperative). In addition, the brother's ERG results showed normal a wave with extremely reduced b wave (negative type) when recorded with a single bright white stimulus in the dark that supported the notion of night blindness for this patient. 

Regarding the patients' development, the two sibs had delay in gross motor, fine motor, and cognition to a various extents. They did not walk or sit until 15 to 18 months of age and started babbling only after 2 years of age. They were developmentally delayed with measured IQ evidence of moderate mental retardation. Indeed, no signs of dysmorphic features or ataxia were observed. Microcephaly was evident shortly after birth (head circumference = 36 cm at birth). Their magnetic resonance image results were normal. Biological investigations (karyotyping with R-banding) revealed normal karyotypes: 46 XX for females, 46 XY for males [600 bands resolution]) and normal metabolic screening including Fehling reaction and thin layer chromatography of reducing sugars, plasmatic amino acid, and urine organic acid chromatography for all patients. Additional features are shown in Table 1. A total of 50 control individuals, 10 to 30 years of age, were selected, including 20 males and 30 females with no history of congenital cataract, mental retardation, or microcephaly. 

Table 1

 

Clinical Features of the Two Affected Patients Belonging to the Tunisian Studied Family

Patient Characteristic	Sibling IV11	Sibling IV12
Age at referral to Genetic Department	12 y	8 y
Weight (kg)	38	30
Height (cm)	149	131
Cataract	Bilateral with posterior polar type	Bilateral with posterior polar type
Age of cataract	3 y	3 y
OCT	N	Alteration of the pigment epithelium
ERG	N	Notion of night-blindness
Other ocular features	—	Decreased visual acuity
		Leucorea
Microcephaly	±3.8 SD (postnatal)	±5.6 SD (postnatal)
MR	Moderate	Moderate
Developmental delay	Walked and spoke at 2 years of age
MRI	N	N

 ERG, electroretinography; MR, mental retardation; MRI, magnetic resonance image; N, normal; OCT, optical coherence tomography; SD, standard deviation.

Homozygosity Mapping and iSyTE Analysis

Using genome-wide scanning, we identified six ROHs shared between the affected siblings of the studied family. Details about these ROHs are presented in Table 2. These regions of homozygosity were analyzed with the iSyTE database in order to identify lens-enriched genes, both with high and low expressions in the lens. Three critical time points of lens development (from embryonic mouse lens) were considered: at embryonic day 10.5 (E10.5), E11.5, and E12.5, as the lens transitions from the stage of lens placode invagination (E10.5) to that of lens vesicle formation (E11.5) and the onset of lens fiber differentiation (E12.5). Using this approach, we selected three novel lens-specific genes, PCNX (14q24.2), RGS6 (14q24.3), and P4HA1 (10q21.3). An example of the iSyTE tracks for the RGS6 gene is shown in Figure 2. 

Figure 2

 

Visualization of a 14-Mb locus on human chromosome 14 with iSyTE tracks on the UCSC Genome Browser, which confirms a nonsyndromic cataract-associated gene, RGS6, as the most promising candidate in this interval (arrow) for the affected siblings; red indicates highly enriched and blue highly depleted.

Table 2

 

Homozygous Regions Detected in the Studied Tunisian Family, Using Whole-Genome Scan

Chromosome	Physical Position (hg 19)	Chromosomal Position	Size (bp)	Selected Gene(s)
2	95,044,977–98,110,944	q11.1-q11.2	3,065,968	—
7	55,589,884–86,536,829	p11.2-q21.12	30,946,946	—
9	80,703,081–86,268,811	q21.2-q21.32	5,565,731	—
10	37,361,976–39,071,347	p11.21-p11.1	1,709,372	—
10	74,479,756–76,921,715	q22.1-q22.2	2,441,960	P4HA1
14	57,371,544–83,275,084	q22.3-q31.1	25,903,541	PCNX, RGS6

 —, no selected gene; bp, base pairs; hg 19, human genome 19.

Mutation Screening

All exons and flanking regions of the three selected genes PCNX, RGS6, and P4HA1 were screened for mutations. Sanger sequencing revealed a single base substitution in the acceptor splice site of intron 16 of RGS6 (c.1369−1G>C), which segregated with the two affected siblings (IV₁₁, IV₁₂). This mutation was heterozygous in the parents (III₉, III₁₀) and not present in the unaffected member IV₁₃ (Fig. 3). The c.1369−1G>C splice site mutation changed the canonical 3′ acceptor splice site of intron 16 from AG to AC. This substitution was absent in 50 unrelated control individuals, suggesting the sequence change as a disease-cosegregating mutation. 

Figure 3

 

Sequence chromatograms showing a splice site mutation in intron 16 of RGS6 c.1369−1G>C in the two affected siblings (i) and their carrier parents (ii) but not present in their healthy sister and 50 control individuals (iii).

We found no further gene mutations in individuals from the studied families except three nonpathogenic nucleotide polymorphisms, 1 reported SNP (rs3814871) p. Arg578 = in PCNX gene, and 2 other intronic substitutions, c.342+86G>C and c.898−33T>G, in the RGS6 gene. 

Bioinformatics Analysis in Mutated Proteins

Database searches for the RGS6 splice site mutation c.1369−1G>C detected in the two siblings revealed that this variation was not reported before. 

HSF analysis for splicing effects predicted a significant effect on the mRNA splicing compared with the wild-type sequence. This change was predicted to be a broken 3′ acceptor splice site (at position +606,963 nucleotides from the genomic sequence accession no. NM_001204416) and the creation of a new potential branch point, with a consensus value for the wild-type and the mutant sequence at 34.28 and 77.12, respectively. In addition, two putative splice enhancer (ESE) and silencer (ESS) motifs (calculated based on the exonic splicing enhancer finder matrices for SRp40, SC35, SF2/ASF, and SRp55 proteins) between the wild-type reference and the mutant sequence were predicted to be broken, with a score of −100 with creation of a new ESE motif (Fig. 4). Furthermore, the alignment of the AG site in intron 16 of the RGS6 gene in different species proved high conservation (Fig. 5). 

Figure 4

 

Various predicted outcomes of the c.1369−1G>C change in the RGS6 gene according to HSF. The mutation c.1369−1G>C occurred in the invariant AG dinucleotide of intron 16 (reference sequence), and using HSF, we predicted the creation of a new potential branch point downstream, available for use as the alternative acceptor splice site (mutant sequence). Using the exonic splicing enhancer finder matrices for the proteins SRp40, SC35, SF2/ASF, and SRp55, HSF predicted that two putative splice enhancer (ESE) and silencer (ESS) motifs were broken, and we generated a new ESE motif.

Figure 5

 

A multiple sequence alignment from different species of the splice acceptor site AG in the 3′ end of intron 16 of RGS6 (arrow) indicates that the guanine at the 606,936th position of RGS6 genomic sequence and its neighboring nucleotides are highly conserved among many species.

Discussion

The distinctive features of the lens are transparency, crafted shape, and deformability, all of which are critical for proper light refraction. Elucidating the molecular mechanisms that maintain or disrupt this lens transparency is fundamental in preventing cataract. Autosomal recessive nonsyndromic congenital cataract has been assigned to a minority of genes, notably alpha-A crystalline (CRYAA), beta-B1 crystallin (CRYBB1), beta-B3 crystallin (CRYBB3), glucosaminyl (N-acetyl) transferase 2 (GCNT2), lens intrinsic membrane protein (LIM2), galactokinase 1 (GALK1), heat-shock transcription factor 4 (HSF4), and Tudor domain-containing 7 (TDRD7).^8–15 

Nonsyndromic and nonmetabolic origins of congenital cataract were found to be associated with other ocular and/or systemic anomalies in addition to mental retardation and/or microcephaly, unlike the diagnosed phenotype in the studied family. The major goal of this study was to determine the genes responsible for the association between autosomal recessive congenital cataract (ARCC), mental retardation, and microcephaly in a consanguineous Tunisian family with no signs of metabolic or muscular or other anomalies. This family was previously screened for mutations in transcription factors (PAX6, PITX3), heat-shock factor (HSF4), and membrane protein (LIM2); all these genes act early at lens development and have crucial roles in normal brain and lens formation, but no pathogenic mutation was detected.^16,17 In further subsequent work, we investigated four genes, EPHA2, GALK1, GCNT2, and CRYBB1, reported to be responsible for ARCC and highly expressed in fetal and adult brains.^9,11,13,18 Linkage analysis performed for the five screened members led us to exclude a possible linkage for the four analyzed genes.¹⁹ Likewise, three other genes, selected from the shared ROH (detected by homozygosity mapping) between the affected members (SIX homeobox 4 [SIX4]; heat shock 27kDa protein 1 [HSPB1]; and prospero homeobox 2 [PROX2]), were analyzed by sequencing of all exons and exon-intron junctions, and no pathogenic mutation was revealed. Furthermore, linkage analysis was consistent with possible association between visual system homeobox 2 (VSX2) and SIX homeobox 6 (SIX6), but no mutations were found in the coding regions of these genes or flanking intron sequences. 

In the current work, using whole-genome scanning, iSyTE analysis, and sequencing analysis, we identified a novel gene, RGS6, whose detected splice site mutation (c.1369−1G>C) was suspected of being the genetic cause of the ARCC phenotype associated with mental retardation and microcephaly in the two siblings belonging to the Tunisian family. 

The unreported splice site mutation (c.1369−1G>C) of RGS6 intron 16 was identified as the homozygous status for the two affected siblings, whereas their parents were heterozygous, and was not revealed in the healthy sister or in 50 control individuals, confirming the sequence change as a disease-causing, cosegregating mutation. Furthermore, this substitution was conserved in eight tested species (Fig. 5), reflecting the fact that the acceptor splice site AG of RGS6s intron 16-exon17 is likely of primary biological importance. In fact, the RGS6 protein is a member of a protein family called RGS proteins (R7 subfamily) that functions as GTPase-activating proteins (GAPs) for Gα subunits. RGS6 is endowed with disheveled, Egl-10, pleckstrin (DEP), and G protein gamma subunit-like (GGL) domains, in addition to the RGS domain present in all RGS proteins. Twenty splice variants of RGS6 were identified with differences in their subcellular localization patterns related to structural differences in the protein; their expression is detected predominantly in brain.²⁰ 

In addition to its functions in preventing parasympathetic override and severe bradycardia²¹ and its hypothetical role in inhibiting growth of cancer cells via activation of the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2,²² RGS6 is suggested to induce microtubules and neuronal differentiation by a novel mechanism involving interaction of SCG10 (a neuronal growth-associated protein) with complete GGL domain-RGS6.²⁰ This is the first report of a splice site variant on RGS6 in a phenotype associating congenital cataract, mental retardation, and microcephaly. In fact, splice site mutations were reported to result in exon skipping, activation of cryptic splice sites, creation of a pseudo-exon within an intron, or intron retention, among which skipping is the most frequent outcomer.²³ In present study, the mutation c.1369−1G>C occurred in the invariant AG dinucleotide of intron 16 and using HSF we predicted the creation of a new potential branch point downstream available to be used as the alternative acceptor splice site (Fig. 4). Further study is required to confirm the above site. Thus, we hypothesized that the generated protein RGS6 in the two patients from F2 is a nonfunctional truncated protein because of an incomplete GGL domain and a missed RGS domain. Consequently, no interaction will be accomplished with SCG10, leading to an aberrant neuronal differentiation in brain and an abnormal microtubule differentiation in lens, according to iSyTE results, we highlighted the fact that RGS6 is a likely lens-specific gene (Fig. 2), which elucidates the described phenotype in family F2. 

In this phenotype, congenital cataracts caused by the c.1369−1G>C mutation in RGS6 identified in the two sibs are associated with extralenticular abnormalities (mental retardation, microcephaly), suggesting that this gene is directly necessary for both lens and brain, representing a true pleiotropic effect of the mutant gene. 

Our report is the first to describe a new phenotype (congenital cataract, mental retardation, and microcephaly) unrelated to a specific syndrome and linked to a mutation in the RGS6 gene that could be crucial for lens and brain development. 

In conclusion, the present results highlight the genetic heterogeneity of congenital cataract and raise intriguing questions concerning the structural and physiological roles of RGS6 in maintaining lens transparency and normal brain organogenesis. 

Acknowledgments

The authors thank all the patients and their family members for participating in the project. We also thank Pr. Fowzan Alkuraya's team in the Developmental Genetics Unit, King Faisal Specialist Hospital and Research Center, Riyad, Saudi Arabia for their collaboration. 

This study was supported by the Tunisian Ministry of Higher Education and Scientific Research (Laboratory of Human Genetics, Faculty of Medicine of Tunis and Congenital and Hereditary Service of Charles Nicolle's Hospital). The authors declare no conflict of interest. 

Disclosure: M. Chograni, None; F. S. Alkuraya, None; F. Maazoul, None; I. Lariani, None; H. Chaabouni-Bouhamed, None