Complexity of Phenotype–Genotype Correlations in Spanish Patients with RDH12 Mutations

Abstract

purpose. Several mutations have been described in the RDH12 gene that disturb the activity of the encoded protein, suggesting that RDH12 loss of function disrupts the synthetic pathway of the visual chromophore 11-cis-retinal, therefore resulting in early and progressive retinal degeneration (RD). Mutations in this gene have been related to autosomal recessive Leber congenital amaurosis (LCA) and to a form of autosomal recessive childhood-onset severe retinal dystrophy (CSRD). This study was undertaken to attempt to correlate the genotype and phenotype in Spanish CSRD and LCA patients who harbor RDH12 mutations.

methods. A complete ophthalmic and electrophysiologic examination was performed according to preexisting protocols. A screening for mutations was then performed using denaturing HPLC on a DNA fragment analysis system. Those fragments bearing an abnormal pattern were sequenced.

results. Ten families bearing RDH12 mutations in homozygous or compound heterozygous form were found. All of them corresponded to patients with severe and early-onset retinal dystrophy.

conclusions. The RDH12-associated phenotype is not homogeneous, the position and nature of the mutations clearly influence the pathologic expression of this disease.

Retinol dehydrogenase 12 (RDH12) is a member of the short-chain dehydrogenase/reductase superfamily of proteins that is highly expressed in the retina, specifically in photoreceptors. RDH12 is a component of the visual cycle, where a series of reactions take place, and transport mechanisms are activated between the retinal pigmented epithelium (RPE) and the photoreceptors. This enzyme catalyzes the oxidation and reduction of both all-trans and -cis-retinoids in the presence of NADP⁺ or NADPH as cofactors, ¹ and it has been suggested that RDH12 contributes to the detoxification of medium-chain aldehydes in the cells with active lipid peroxidation. 

The RDH12 gene maps to chromosome 14q23.8 and is composed of seven exons, encoding a protein of 316 amino acids. Several mutations that disturb the activity of this protein have been described, thus pointing out that the loss of function of RDH12 disrupts the cycle of synthesis of the visual chromophore 11-cis-retinal, resulting in early and progressive retinal degeneration. Mutations in this gene have been associated with the autosomal recessive Leber congenital amaurosis (LCA) ² and with a form of juvenile-onset autosomal recessive retinitis pigmentosa (CSRD). ³  

Patients with LCA who have mutations in the RDH12 gene display a specific phenotype defined as a severe congenital progressive cone-rod dystrophy, in contrast with the severe congenital stationary form of LCA. The involvement of RDH12 in the dynamic process of retinal regeneration could explain the progressive nature of the disease. ² This severe macular atrophy causes a serious visual loss before the end of the second decade of life, and patients exhibit mild or no hyperopia and nystagmus. ³  

For individuals with RDH12 mutations and childhood-onset severe retinal dystrophy (CSRD), a rod and cone dystrophy with early onset (2–4 years of age) and progression to legal blindness in early adulthood (18–25 years of age) have been described. These patients display a severe phenotype, with widespread atrophy of the retinal pigment epithelium and with the fundi showing a pronounced attenuation of retinal arterioles and intraretinal bone spicule pigmentation. In several cases, the electroretinogram (ERG) has been described to be extinguished at a very early age. 

Recently, a genotype–phenotype association has been reported in patients with RDH12 mutations. ⁴ The clinical picture described is characterized by poor, although useful, visual function in early life, followed by a progressive decline in visual function, due to both rod and cone degeneration and marked pigmentary retinopathy with bone spicules in the peripheral retina in individuals older than 6 years and pronounced maculopathy in patients older than 7 years old. Likewise, this work shows the genotype–phenotype correlation between Spanish LCA and patients with early-onset RP. 

Methods

A total of 49 Spanish patients with LCA and 311 (210 autosomal recessive and 101 sporadic) unrelated Spanish patients with early-onset RP were studied. The recruitment of patients and relatives was performed through six groups involved in the Retinal Dystrophy Spanish Research Network (EsRetNet). The research adhered to the tenets of the Declaration of Helsinki and it was approved by all the participating institutions. Informed consent was obtained from all the adults and from the children’s parents or tutors after the nature and possible consequences of the study had been explained. 

Ophthalmic and electrophysiological examinations were performed according to preexisting protocols, consisting of the history of the patient and his or her family, funduscopic examination after pupillary dilation, computerized central and peripheral visual field testing, and visual acuity testing with the best correction. The electrophysiological assessment included a full-field ERG, as the protocols by the International Society for Clinical Electrophysiology of Vision and Color on vision testing recommend. ^{5 6} Ophthalmically, the patients were classified into three categories: 150 severe (onset before the age of 10 years), 95 moderate (onset after the age of 10 years and legal blindness before 30), and 66 mild cases (onset before the age of 10 years and legal blindness after 30). 

Blood was collected by venipuncture and genomic DNA was isolated by the salting-out method. The seven exons of the RDH12 gene were amplified by using standard PCR conditions, and oligonucleotide primers were positioned in the intronic regions. Mutation screening was performed by using denaturing HPLC on a DNA fragment analysis system (WAVE; Transgenomic, Omaha, NE). Fragments with an abnormal pattern were sequenced. 

The sequencing reaction was performed with the four-dye terminator cycle sequencing ready reaction kit (dRhodamine DNA Sequencing Kit; Applied Biosystems, Inc. [ABI], Foster City, CA). Sequenced products were purified through thin columns (Sephadex G-501; Princetown Separations, Adelphia, NJ) and resolved in a genetic analyzer (Prism 3100; ABI). Patient haplotypes were determined by analysis of intragenic single nucleotide polymorphisms (SNPs) for chromosomes with the p.L99I mutation. 

Results

The results showed 10 patients presenting RDH12 mutations (Table 1) . All of them corresponded to patients with severe and early-onset retinal degeneration, accounting for 2.78% of the patients analyzed. Table 1summarizes the clinical information about each patient. 

The mutation p.L99I (c.295C>A) appeared most frequently in our series, with an allelic frequency of 40%. We found it in the homozygous state in three patients, two of them consanguineous. SNP analysis confirmed the same haplotype for this mutation: c.187/IVS2+54A, c.187/IVS2+60G, and c.482A/p161Q. In the homozygous state, this mutation is associated with a severe phenotype that includes macular affectation, central scotomata and reduced visual acuity, atrophy of the optic nerve and blood vessels, and hyperpigmentation of the peripheral retina. When this mutation was paired with a frameshift mutation like p.V35fsX62 (c.99_102dupAAAT) or p.A269fsX270 (c.806_810delCCCTG), the phenotype in our patients became more severe with decreased visual acuity of only light perception or counting fingers and a precocious age at onset that varied between birth and 7 years. 

The mutation p.T155I (c. 464C>T) had an allelic frequency of 20% and was the second one to appear with a higher frequency in our series. The two patients with this mutation were homozygous for it, and both reported consanguinity. The phenotype showed age at onset in the first decade of life, with light perception maintained until the fourth decade. It showed a slower progression, with characteristics of a typical retinitis pigmentosa (RP) fundus and extinguished ERG in both eyes. 

For patient S261, who harbored two missense mutations, p.G145E/p.M1? (c.434G>A/c. 2T>C), the associated phenotype showed age at onset in the second decade of life with pigment mobilization, tapetal reflection, and filiform vessels. Visual acuity was poor and its decrease was bilateral (right eye, 0.1; left eye, 0.8). His brother had the same fundus but the reduction in visual acuity was not bilateral; moreover, he had photophobia. 

In the subject homozygous for p.T49M (c. 146C>T), the clinical appearance was that of typical RP, with a preserved central vision and an extinguished ERG at 4 years of age. 

In the compound heterozygous patient M379, p.N125K/p.R234H (c. 375T>A/c.701G>A), a pale papilla with mild vessel constriction and pigmentation in periphery were observed. 

Discussion

The total mutation detection rate described by Thompson et al., ⁷ which includes most of the families in the current study (9/10), was approximately 2.2% of the CSRD families. In our work this rate exhibited an increase to 3.1% of the families studied. We detected the two mutated alleles involved in 2.78% of the families. This major rate could be due to patients receiving more accurate diagnoses or to a major involvement of this gene in the Spanish families studied. 

Most of the mutations reported herein (p.L99I, p.G145E, and p.T155I) show an in vitro reduced ability to convert all-trans-retinal to all-trans retinol in the presence of NADPH, showing just 5% to 18% of the wild-type activity. ⁷  

Homozygous patients for p.L99I mutation presented a phenotype with an early onset (1.5–3 years old), characterized by reduced visual acuity and typical funduscopic characteristics, revealing a hyperpigmentation in middle periphery. However, compound heterozygous individuals for the p.L99I mutation detected in our study and paired with another mutation that produces a stop codon (p.V35fsX62, p.A269fsX270, respectively), manifested a more severe phenotype with a much more reduced visual acuity, counting fingers or light perception. The pigmentation in these cases was dense around the entire retina. The hyperpigmentation of the retina has rarely been seen in young individuals with retinal dystrophy who harbor mutations in genes encoding other visual cycle components. ⁴ Therefore, the localization of the pigmentation is clearly different in patients (e.g., M447) homozygous for p.L99I or p.L99I heterozygous with a stop mutation (B237), as shown in Figure 1 . 

Patients homozygous for the p.L99I mutation presented a phenotype similar to the one described by Janecke et al. ³ But, with regard to compound heterozygous patients, this phenotype appeared to be more severe and with poor visual acuity. 

Patients homozygous for the p.T155I mutation displayed a phenotype with absolute scotomata in both eyes and a fundus typical of that in RP, with bone spicule pigmentation and atrophy. Moreover, one of them had a cataract. 

The compound heterozygous patients S261 and M379 showed a different age at onset, being earlier in M379. The progression differed in both cases. In one patient visual acuity was ∼0.3 in both eyes at the age of 21 years, and in the other patient, at the age of 45, visual acuity was poor and more severe in the right eye. Patient S261 showed a mutation c.2T>C (p.M1?) losing the methionine initiating codon. This change could predict a more severe phenotype in this individual, and so it would be very interesting to check the RNA and protein of this patient, since the loss of the initiation codon may have a dramatic effect. Fundus examination showed a pale optic disc and a strong arteriolar constriction with pigment mobilization at 45 years of age, and the patient reported having photophobia. Patient M379 showed mutation p.N125K, a conserved amino acid residue that it is located in a β-sheet of the Rossman fold of the protein, pairing with the missense mutation p.R234H that retained 44% of the activity level of wild-type protein, as has been described. ⁷ The fundus showed a pale papilla, mild vessel constriction, and periphery pigmentation. The effect resulting from p.N125K loss of function, combined with reduction in p.R234H activity to less than half that of wild-type, results in insufficient RDH12 levels for normal visual cycle function. ⁷  

Although RDH12 is a member of a relatively large protein family, little is known about its tertiary structure. The model proposed by Thompson et al., ⁷ shows an apparently globular form with a core comprising α- and β-sheet secondary structures. All the mutations described are widely distributed across the predicted surface of the protein. RDH12 protein mutational studies suggest a reduced expression and activity and appear to represent disease-associated mutations that generate loss-of function alleles due to disruption of the visual cycle. The sites of missense mutations appear to localize on the protein surface, in areas of unstructured sequence, suggesting that affected residues may disrupt functional domains involved in protein interactions, protein folding and catalytic activity. The clinical phenotype associated with each mutation is quite difficult to establish. The position and nature of the found mutations clearly influence the pathologic expression of this disease, just as has been shown in the p.L99I mutation, where this mutation together with a stop codon is responsible for a more severe phenotype than the p.L99I homozygous mutation. 

In the described model of the retinoid cycle, the all-trans retinal is released from the opsins by hydrolysis of the protonated Schiff base. It is transported out of the rod outer segment by either an ATP-binding cassette transporter or by simple diffusion and finally, is reduced by all-trans retinol dehydrogenase in the reaction that appears the rate-limiting step in pigment regeneration. ⁸ The ABCR protein seems to function as an all-trans-retinal transporter/flippase that delivers all-trans retinal to the RDH12 to convert it into all-trans retinol before to its delivery to the retinal pigment epithelium. ⁹  

As pointed out by Thompson et al., ⁷ in complex alleles that include a pathogenic mutation and a neutral or an apparent polymorphism, the latter could modify the functional consequences of the pathogenic mutation, so it remains unclear how these single nucleotide polymorphisms may have functional consequences for complex alleles. Therefore, every change in every gene should be taken into account to delimitate a perfect correlation genotype–phenotype. 

The complexities of the relations between the distinct proteins involved in the overall process of the visual transduction suggest a real imbricate model of relationship between them. ¹⁰ It is reasonable to suggest that other components of the visual cycle may have an influence on phenotype. The development of high-throughput technologies will allow us to take a more general picture of the mutated genes involved in retinal dystrophies and their role in the phenotype. 

 Supported by Fondo Investigación Sanitaria Grants PI060049 and PI060027; Fundación Mutua Madrileña.

 Submitted for publication March 27, 2008; revised July 4 and October 1, 2008; accepted January 20, 2009.

 Disclosure: D. Valverde, None; I. Pereiro, None; E. Vallespín, None; C. Ayuso, None; S. Borrego, None; M. Baiget, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Phenotype and Genotype Characteristics of RP Patients with RDH12 Gene Mutations

Family	Age at Onset	Age	Genotype			Visual Field	Visual Acuity	Refraction	Fundus	ERG	Other Symptoms
												Alelle 1	Alelle 2
S203	18 mo	20 y	L99I*	L99I*	<10° central	RE 0.08 LE 0.2		Pigment mobilization macular affectation, atrophy of the optic nerve and blood vessels	Abolished ERG (5y)	Nystagmus
M447	3 y	12 y	L99I*	L99I*	<10° central	BE <0.1		Pale discs, arteriolar constriction, RPE abnormalities, alternating areas of atrophy with bone spicule hyperpigmentation in equatorial area; salt and pepper fundus	Extinguished in both eyes	Hypoacusia
B16	3–4 y	30 y	L99I*	L99I*	Absolute scotomas in both eyes	RE <0.1 LE <0.1	RE+2 at 120° LE+2 at 120°	Pale papilla, bone spicule pigmentation in middle periphery, narrow vessels, severe macular atrophy	Extinguished in both eyes	Cataracts
B237 (04–134)	Birth	5 y	L99I*	c.99_102dupAAAT (p. V35fsX62), †	ND	Counting fingers	Not done	See Fig. 1	Extinguished in both eyes	Nystagmus
M131 (1962)	7 y	54 y	L99I*	c.806_810delCCCTG (p. A269fsX270), ‡	<10° central	Light perception BE		Pale discs, arteriolar constriction, dense bone spicule hyperpigmentation in all the retina	Extinguished in both eyes
B19	6 y	39 y	T155I, †	T155I, †	Scotomas absolute both eyes	RE light perception LE hand movements		Pale papilla, bone spicule pigmentation, macular atrophy	Extinguished	Cataracts
S217	2 y	52 y	T155I, †	T155I, †	Scotomas absolute both eyes	Hand movements		Typical RP	Extinguished
S261	11 y	45 y	G145E, †	M1?, †		RE 0.1 LE 0.8		Pigment mobilization, pale optic disc, filiform vessels, tapetal reflection	Extinguished	Photophobia
M184	3 y	28 y	T49M, ‡	T49M, ‡		RE 0.15		Typical RP	Abolished (4 y)
						LE 0.10
M379	3 y	21 y	N125K, †	R234H, †		BE 0.3		Pale papilla, mild vessel constriction, pigmentation in periphery and posterior pole.	NP

*  Perrault et al. ²

†  Thompson et al. ⁷

‡  Janecke et al. ³

Figure 1.

 

Fundus of (A) patient M447 at 13 years of age (p.L991 homozygous) and (B) patient B237 at 5 years of age (heterozygous p.L991/v35fsX62).