Ophthalmic Manifestations and Long-Term Visual Outcomes in Patients with Cobalamin C Deficiency

Abstract

Ocular manifestations of cobalamin C deficiency include a developmental as well as a degenerative phenotype and lack strict correlation to metabolic status, but may be mitigated by prenatal or early treatment.

Purpose

To explore the ocular manifestations of cobalamin C (cblC) deficiency, an inborn error of intracellular vitamin B12 metabolism caused by mutations in the MMACHC gene.

Design

Retrospective, observational case series.

Participants

Twenty-five cblC patients underwent clinical and ophthalmic examination at the National Institutes of Health Clinical Center between August 2004 and September 2012. Patient ages ranged from 2 to 27 years at last ophthalmic visit, and follow-up ranged from 0 to 83 months (median, 36 months; range, 13–83 months) over a total of 69 visits.

Methods

Best-corrected Snellen visual acuity, slit-lamp biomicroscopy, dilated fundus examination, wide-field photography, fundus autofluorescence imaging, sedated electroretinography, spectral-domain optical coherence tomography, and metabolite assessment.

Main Outcome Measures

Visual acuity and presence and degree of retinal degeneration and optic nerve pallor.

Results

Nystagmus (64%), strabismus (52%), macular degeneration (72%), optic nerve pallor (68%), and vascular changes (64%) were present. c.271dupA (p.R91KfsX14) homozygous patients (n = 14) showed early and extensive macular degeneration. Electroretinography showed that scotopic and photopic responses were reduced and delayed, but were preserved remarkably in some patients despite severe degeneration. Optical coherence tomography images through the central macular lesion of a patient with severe retinal degeneration showed extreme thinning, some preservation of retinal lamination, and nearly complete loss of the outer nuclear layer. Despite hyperhomocysteinemia, no patients exhibited lens dislocation.

Conclusions

This longitudinal study reports ocular outcomes in the largest group of patients with cblC deficiency systematically examined at a single center over an extended period. Differences in progression and severity of macular degeneration, optic nerve pallor, and vascular attenuation between homozygous c.271dupA (p.R91KfsX14) patients and compound heterozygotes were noted. The pace and chronicity of ophthalmic manifestations lacked strict correlation to metabolic status as measured during visits. Prenatal or early treatment, or both, may have mitigated ocular disease, leading to better functional acuity, but patients still progressed to severe macular degeneration. The effects of prenatal or early treatment, or both, in siblings, the manifestation of severe disease in infancy, the presence of comorbid developmental abnormalities (e.g., microcephaly, cardiac noncompaction), and the possible laminar structural defect noted in many patients are findings showing that cblC deficiency displays a developmental as well as a degenerative ocular phenotype.

Cobalamin C (cblC) deficiency (Online Medelian Inheritance in Man no., 277400),^1,2 an inborn error of intracellular vitamin B12 metabolism, is caused by mutations in the MMACHC gene³ (Online Medelian Inheritance in Man no., *609831) that leads to impaired intracellular synthesis of 5′-adenosyl- and methylcobalamin, cofactors for the enzymes methylmalonyl-CoA mutase and methionine synthase, respectively. As a result, patients display combined methylmalonic acidemia and hyperhomocysteinemia. Because the enzymatic defect affects the remethylation of homocysteine, hypomethioninemia and a generalized impairment in methyl transfer reactions also are present.

The prevalence of cblC deficiency, as estimated in 2010 by newborn screening studies, is approximately 1 in 100 000 in a United States population,⁴ but can vary widely by ethnicity. For instance, cblC deficiency is reported in as many as 1 in 37 000 persons in a particular Hispanic population in California.⁵ Historically, cblC patients have been categorized based on age of onset, with the early-onset type manifesting symptoms before 4 years of age and the late-onset type manifesting symptoms after 4 years of age.⁶ However, the disorder can present at any point from the prenatal period to adulthood. Prenatal- and infantile-onset patients show the most severe and progressive metabolic, neurologic, and ophthalmic manifestations. They can demonstrate intrauterine growth restriction; chronic failure to thrive; hemolytic uremic syndrome; developmental delay, regression, or both; microcephaly; nystagmus; pigmentary retinopathy; and neurologic dysfunction.^2,6 Patients with noninfantile onset more typically demonstrate developmental delay or regression, or both, characterized by declines in school or work performance, thromboembolic events, progressive encephalopathy, psychiatric symptoms, leukoencephalopathy, or subacute combined degeneration of the spinal cord.^2,6

The phenotype of cblC deficiency seems to correlate with genotype, particularly with regard to the age of onset and disease severity. There are more than 50 known mutations within MMACHC among patients, but the most common is a frameshift mutation c.271dupA (p.R91KfsX14) that accounts for approximately 40% of alleles³ and is associated with early-onset disease, typically in the first year of life.^3,7,8

Cobalamin C deficiency is one of the few disorders associated with infantile maculopathy, which has been documented as early as 3.5 months of age.⁹ Wandering eye movements, inability to fixate, and nystagmus often are the first clinical signs of eye disease in infants with cblC deficiency.^10,11 The maculopathy and progressive retinal dysfunction^11,12 are seen in most patients with early-onset cblC deficiency and rarely in those with late-onset cblC deficiency.^12,13 The first funduscopic signs can include pigmented macular changes that progress to a bull's-eye maculopathy. This maculopathy is characterized by a hypopigmented perimacular zone surrounded by a hyperpigmented ring, typically progresses to the periphery of the retina,^9,10 and is accompanied by measurable dysfunction on electroretinography (ERG) testing.^11,14 Optic nerve atrophy also is noted frequently.^10,15,16

A recent questionnaire-based survey reported a series of 88 European patients with some ocular findings included.¹⁷ However, as noted in a recent comprehensive review of cblC deficiency,¹⁸ publications that detail ophthalmic parameters are sparse. To date, there are fewer than 100 patients described for whom detailed ocular examination results are included (only 61 of these patients are identified by genotype), with the largest studies reporting results from 12 patients.^14,19 Because most patients were examined at different facilities, ophthalmologic data varied between reports, genotypes often were not recorded, and complete metabolic parameters were not measured. Hence, there is a need for a detailed, single-center study that takes into account genotype, metabolic parameters, and ocular phenotypes to advance the understanding of cblC-related ophthalmologic manifestations.

This longitudinal study examined the ophthalmic phenotype of cblC deficiency as ascertained in a large group of patients examined systematically at a single center in the setting of a dedicated natural history study. The cohort included 25 patients ranging in age from 2 to 27 years who were studied serially over a 10-year period. We characterized the natural history of the progression and severity of macular degeneration, optic nerve pallor, and vascular attenuation in the patients. Autofluorescence photography, ocular coherence tomography, and ERG also were used to further our understanding of the structural and functional origins of the ocular manifestations.

We found that the pace and chronicity of the ophthalmic manifestations lacked a strict correlation to metabolic status as measured during protocol visits. The presence of severe disease in infancy, indications of a developmental abnormality in the retinal structure of patients, and the occurrence of comorbid developmental abnormalities (e.g., microcephaly, cardiac defects, or left ventricular noncompaction) suggest that cblC deficiency can feature developmental as well as degenerative phenotypes. Our data present an important set of ocular outcome parameters that will be useful for the community to help examine new therapies for the loss of vision in these patients.

Methods

The patients were evaluated under protocol 04-HG-0127, “Clinical and Basic Investigations of Methylmalonic Acidemia and Related Disorders” (clinicaltrials.gov identifier, NCT00078078). The research adhered to the tenets of the Declaration of Helsinki and was approved by the National Human Genome Research Institute Institutional Review Board. Informed consent from patients, guardians, or both (including consent for use of patient photographs) was obtained.

All patients with a diagnosis of cblC deficiency, determined by cellular enzymologic results, mutation analysis of the MMACHC gene, or both who underwent an ophthalmic examination between August 2004 and September 2012 at the National Institutes of Health Clinical Center were included in this report after data review and abstraction. All ophthalmic examinations were all carried out by one or more of the authors (B.P.B., W.M.Z.).

The complete ophthalmic examinations included visual acuity (VA) and fixation: Snellen visual VA and best-corrected Snellen VA (BCVA); fix and follow (FF); or central, steady, and maintained. In the youngest patients with quantifiable acuity, those younger than 6 years, Teller acuity cards (n = 3) or HOTV (n = 4) were used in acuity assessments. In those patients 6 years of age and older at the time of examination, either Early Treatment Diabetic Retinopathy Study charts (n = 14), Early Treatment Diabetic Retinopathy Study short charts (n = 10), HOTV charts (n = 7), or picture charts (n = 4) were used, with 1 patient older than 20 years (patient 18) assessed using a Teller acuity chart (because of inability to cooperate with other methods). When possible, the evaluation also included slit-lamp biomicroscopy, dilated fundus examination with an indirect ophthalmoscope, wide-field photography (Optos ultra-widefield retinal imaging device), fundus autofluorescence imaging, and sedated ERG with an Espion consol (Diagnosys LLC, Lowell, MA) conducted according to the International Society for Clinical Electrophysiology of Vision Standards.²⁰ Cirrus HD-OCT (Carl Zeiss Meditec, Inc., Dublin, CA) spectral-domain optical coherence tomography scans were obtained by certified technicians. The scans were assessed for quality and were examined for loss of the inner segment–outer segment photoreceptor junction in the macular area and at the edge of the macular lesion.

Treatment regimens varied widely between the different metabolic centers that referred patients to our study. All patients received intramuscular hydroxocobalamin (1–25 mg/day, 1–7 times/week), and variably, betaine (30–300 mg/kg daily), carnitine (10–70 mg/kg daily), and folic or folinic acid (1 or 5–10 mg daily), respectively. Whole-protein consumption ranged from 0.7 to 2.5 g/kg daily. Aspirin also was administered to some patients.^1,2,21 In utero treatment for the 2 younger affected siblings was as follows. For patient 4, the mother received 1 mg hydroxocobalamin per week intramuscularly starting at 20 weeks' gestation after amniocentesis confirming the diagnosis of cblC deficiency. Treatment was increased to 3 mg thrice weekly until delivery. Patient 4 was treated with hydroxocobalamin injections immediately after birth. For patient 5, the mother received 1 mg oral cyanocobalamin daily throughout the pregnancy. Patient 5 received the diagnosis of cblC at 4 days of age and was treated with hydroxocobalamin injections (1 mg/day intramuscularly) starting at 5 days.

Results

A total of 25 patients with cblC deficiency underwent examination by a National Eye Institute ophthalmologist experienced with this disorder as part of the study (Table 1). The patients ranged in age from 2 to 27 years at the most recent ophthalmic visit. Ten patients were female and 15 were male. Infantile onset was seen in 20 of the patients, and these patients were diagnosed in the first year of life; 4 patients were detected by newborn screening (Table 1). Patient 7 had a delayed diagnosis at 2 years of age, but is still classified as having infantile onset because of genotype and medical records from other institutions. Two more infantile-onset patients were younger siblings of already diagnosed individuals and were monitored during pregnancy. The remaining 4 patients in the study, patients 22 through 25, were classified as having noninfantile onset (patients 22 and 23) and were not diagnosed until 3 and 5 years of age, respectively. The patient genotypes are listed in Table 1. Fourteen patients, all with infantile onset, had developmental abnormalities including left ventricular noncompaction cardiomyopathy,²² fetal hydrops, intrauterine growth restriction or small size for gestational age, Ebstein's anomaly, microcephaly, or prenatal microcephaly (Table 1). The developmental abnormalities in patient 3 have been reported previously.²²

Table 1. Metabolic and ophthalmic characteristics of cbIC patients at most recent ophthalmic visit.

Genotype (exon/type of mutation)		Patient #	Gender (Female/Male)	Congenital defect	Leukoencephalopathy	most Recent Ophth Visit, yrs	Study enrollment, yrs	Dx, months of age2	Plasma Hcy, μM3	Serum MMA, μM4	Plasma Methionine, μM5	Ophthalmic Visits, n	Duration Followup, months	Manifest Nystagmus	Strabismus	Best Corrected Va OD, LogMAR6	↓Best-Corrected Va	Macular atrophy first reported age; years, degree7,8	Macular atrophy, most recent visit7,8	Bulls eye visible	Macular coloboma	Vascular Changes7,9	degree of pallor7,10	other notes
c.271dupA p.R91KfsX14 (ex2/duplication, frameshift)	c.271dupA p.R91KfsX14 (ex2/duplication, frameshift)	1	F	none	nr	2	2	5	48	3.3	55	1	n/a	+	-	NoFF	+	>1	y	-	-	n/r	-
		2	M	SGA	nr	3	3	<1NS	34	3.6	40	1	n/a	-	-	0.9*	+	3	y	-	-	+	+D
		3	F	LVNC, fetal hydrops, coloboma	-	6	1	<1	53	5.7	22	7	37	+	+	2.3*	+	>1	+END	-	+	3	+D	14
		41	M	IUGR	nr	10	4	pre	44	2.2	28	7	39	-	+	1	+	4+M	+P	+	-	2	+T
		52	M	none	nr	10	5	pre	70	25	23	5	63	-	-	0.8*	+	5+M	+P	+	-	1	+T
		62	M	none	+	11	6	1	78	31	31	5	63	+	+	1.3	+	6+P	+END	+	-	1	+D
		7	M	none	nr	12	9	26	65.5	1.4	16	2	28	+	+	NoFF	n/a	11	y	-	-	+	+
		81	M	SGA	nr	13	6	2	65	0.5	19	8	83	-	+	FF	n/a	6+P	+P	-	-	2	+T
		9	M	none	-	15	13	<1	56	3	27	3	13	-	-	0.9	+	13+END	+END	hx13	-	3	+D
		10	F	LVNC*, hydrocephalus	-	18	12	2	86	15	18	4	62	+	-	1.3	+	12+END	+END	-	-	3	+D
		11	M	LVNC	-	18	12	3	65	19	28	3	36	+	+	1.5	+	>1	+END	-	+	3	+D	15
		12	M	IUGR/SGA	-	19	19	4	53	1.3	24	1	n/a	+	-	1.4	+	19	y	-	-	+	+
		13	M	LVNC	nr	26	22	4	64	22	18	5	50	+	+	1.6*	+	>1	+P	-	-	2	+D
		14	F	Ebsteins anomaly, LVNC	+	27	26	3	91.7	19	12	2	27	+	+	1.5	+	>1	+END	-	+	2	+D	16
c.271dupA p.R91KfsX14 (ex2/dup, frameshift)	c.331C>T p.R111X (ex3/nonsense)	15	F	congenital microcephaly	nr	4	4	1NS	24	0.9	8	1	n/a	+	+	0.6*	+		n	-	-	n/r	+
		16	M	prenatal microcephaly	nr	10	10	3	82	20	19.5	1	n/a	+	+	0.4*	+		n	-	-	-	-
c.271dupA p.R91KfsX14 (ex2/dup, frameshift)	c.457C>T p.R153X (ex4/nonsense)	17	F	prenatal microcephaly	+	12	9	5	68	4.3	29	4	31	+	+	CUSM	+	9+M	+M	+	-	1	+T
c.271dupA p.R91KfsX14 (ex2/dup, frameshift)	c.600G>A p.W200X (ex4/nonsense)	18	M	microcephaly	nr	22	17	6	94	5.9	20	1	n/a	+	+	1.2*	+		n	-	-	-	-
C.3G>A p.M1? (ex1/start codon)	c.3G>A p.M1? (ex1/start codon)	19	F	none	+	4	4	2	44	3.7	21	1	n/a	+	+	CUSM	n/a	3	y	-	-	+	+
c.440G>A p.G147D (ex4/missense)	c.619dupG p.D207GfsX38 (ex2/unknown)	20	M	none	nr	2	2	<1NS	28	8.7	37	1	n/a	+	-	CUSM	n/a	>1	y	hx	-	n/r	-
c.471G>A p.W157X (ex4/missense)	c.666C>A p.Y222X (ex4/nonsense)	21	M	LVNC*	nr	4	5	<1NS	45	7.5	53	1	n/a	+	-	CUSM	n/a	>1	y	hx	-	+	+
c.271dupA p.R91KfsX14 (ex2/dup, frameshift)	c.482G>A p.R161Q (ex4/missense)	22	F	none	-	8	8	4011	19	6.8	20	1	n/a	-	-	0.1	-		n	-	-	-	-
c.394C>T p.R132X (ex3/nonsense)	c.394C>T p.R132X (ex3/nonsense)	23	M	none	+	7	7	6411	30	5.5	17	1	n/a	-	-	0	-		n12	-	-	-	-
		24	F	none	-	21	21	91	8	0.5	41	1	n/a	-	-	0	-		n	-	-	-	-	17
c.440G>C p.G147A (ex4/missense)	c.440G>C p.G147A (ex4/missense)	25	F	none	-	9	8	nr	4	0.1	31	2	13	-	-	0.6*	+		n	-	-	-	-	18
Patient totals	number	25	10, 15	14	5							69	545	16	13		17		18	4	3	16	17
	median					10	8	3	53	5.5	23	2	37			0.95	1.319
	avg					12	9	10	52.8	8.64	26.3	2.8	42			0.97	1.319
	% of total													64%	52%		68%		72%	16%	12%	64%	68%

Shading indicates values outside of normal range. n/r = not reported. n/a = not applicable. LVNC = left ventricular non-compaction cardiomyopathy LVNC*=prominent trabeculations of the left ventricle suggestive of left ventricular non-compaction. IUGR/SGA = intrauterine growth restriction/small for gestational age.

^{1 and 2}indicate sibling pairs.

³Plasma Hcy, (normal 0-13)

⁴Serum MMA, (normal 0.4)

⁵Plasma Methionine, (normal 7-47)

⁶OD Va reported, as there were not large differences between OD and OS values.

^*= uncorrected Va

⁷As determined by a masked reading of available patient photos.

⁸Macular atrophy key: N = none, M = macular, P = peripheral, END = End stage

⁹Vascular Changes key: +1 = mild, +2 =moderate, +3 = severe

¹⁰Degree of pallor: T = temporal, D = diffuse

^NSIdentified by new born screening

¹¹Some signs or symptoms in infant period, but dx delyed.

¹²Possible sub-clinical macular degeneration not ruled out.

¹³irregular macula at 18 days old, a “grey” macula at 3 months, and a mild “bulls' eye” at 1 year of age

¹⁴“coloboma” first reported at 3 months of age

¹⁵Abnormal ERG wrod and cone abnormalities at 1 month and 22 months old

¹⁶guttae-like formations noted

¹⁷drusen-like deposits noted.

¹⁸Patchy vision not due to macular atrophy, occult maculopathy or occult optic neuropathy.

Thirteen of the patients were seen at least twice, with a median ophthalmic follow-up time of 36.5 months (range, 13–83 months; Table 1) for a collective total of 69 visits. Among the patients were 2 sets of brothers; in one case, the mother, while pregnant with the younger sibling, was treated with intramuscular hydroxycobalamin. Fourteen patients (56%) were homozygous for the c.271dupA (p.R91KfsX14) mutation, with the remainder of the patients distributed across 9 other genotypes.

The metabolites of the patients at the time of last ophthalmic visit are reported in Table 1. Median serum methylmalonic acid (MMA) was 5.5 μM (range, 0.1–31 μM; normal, 0.4 μM), median plasma total homocysteine (tHcy) was 53 μM (range, 4–91.7 μM; normal, 0–13 μM), and median plasma methionine was 23 μM (range, 9–55 μM; normal, 7–47 μM). Only patients 24 and 25, both with non–infantile-onset cblC deficiency, had normal levels of all 3 metabolites at time of last ophthalmic visit (Table 1).

Brain magnetic resonance imaging scans from 13 of 25 patients were available for review. Only 1 individual had a diagnosis of hydrocephalus and had a VP shunt placed at 1 year of age (patient 10). Five patients had evidence of white matter disease (leukoencephalopathy; Table 1). The detailed neuroradiologic findings in this cohort will be the subject of a future report.

Ocular Characteristics

Twenty-three of the 25 patients (92%) showed a degree of ocular abnormality that exceeded the simple need for corrective lenses (Table 1). Seven patients, despite being of ages normally able to cooperate with VA testing, were neurologically impaired and unable to cooperate with VA testing. One patient was simply able to fix and follow (patient 8), 2 patients were unable to fix and follow (patient 1, 2 years of age, and patient 7, 12 years of age), and 4 patients between 2 and 12 years of age (patients 17 and 19–21) exhibited central, unsteady maintained vision.

Manifest nystagmus was seen in 16 patients (64%) and strabismus was seen in 13 patients (52%; Table 1). Although in general anterior segment examinations were unremarkable, in a 27-year-old patient (patient 14), guttae were present in the corneal endothelium of both eyes. Examinations showed a wide range of VAs. The 18 patients who had quantifiable VA had vision ranging from 0.1 to 2.3 logarithm of the minimum angle of resolution (logMAR), with a median acuity of 0.95 (Snellen equivalent, 20/180; Table 1). Lowered BCVA, when quantifiable, ranged from 0.9 to 1.5 logMAR, with a median of 1.3 logMAR (Snellen equivalent, 20/400; Table 1). Because of limitations in patient cooperation, patients were not always assessed for both VA and BCVA. Reduced BCVA was either quantified or reported in the medical record without quantification in 17 patients (68%). Eighty-four percent of patients required corrective lenses.

The box plot in Figure 1 shows all measurable and nonquantifiable (central, steady, and maintained or fix and follow) VAs in the patient population over a range of ages. Depending on the length of follow-up, an individual patient can appear in more than 1 age category. Patient time of onset and genotype are indicated by shape and shading of the symbols. The group as a whole had substantial visual impairment at ages older than 5 to 10 years, with a median VA of 0.9 logMAR, and a number of patients were at or past the 1.0-logMAR (Snellen equivalent, 20/200) legally blind threshold. The median for the age group older than 10 to 15 years was 1.4 logMAR, and VA continued to worsen in the older age groups, with a median value of 1.6 logMAR in the group older than 20 years of age. All of the infantile-onset patients older than 15 years were over the legally blind threshold. Although c.271dupA (p.R91KfsX14) homozygous patients universally were affected severely, c.271dupA (p.R91KfsX14) heterozygous and the other genotype infantile-onset patients also experienced some reduced VA. A number of patients with both c.271dupA (p.R91KfsX14) and other genotypes were affected severely, both neurologically and ophthalmologically, and were able only to fix and follow, 1 patient intermittently (Table 1; Fig 1).

Figure 1.

Box-and-whisker plot showing all recorded acuities at all visits. Visual acuity (VA) of many cobalamin C patients deteriorates to legal blindness, often in the first decade of life. Because of limitations in patient cooperation, patients were assessed for either VA or best-corrected VA (BCVA), and those values are reported. The right eye VA and BCVA are reported, because left eye values were consistent within patients. *Outlier. CUSM = central, unsteady, and maintained; DupA homozyg = ; DupA heterzyg = ; FF = fix and follow only; logMAR = logarithm of the minimum angle of resolution; Non-DupA =.

Although central nervous system damage resulting from the underlying metabolic disease also can limit vision (Table 1), a prominent ophthalmologic feature in 72% of patients is some form of retinal degeneration. This degeneration, shown in Figure 1, is variable in pacing and degree. The most severe patients demonstrate macular atrophy in infancy, as did 7 patients (Table 1). An example of early severe manifestation of cblC is the macular coloboma in a 12-month-old patient (patient 3; Fig 2A). The macular coloboma was documented as being present at 6 months of age or earlier. More typically, macular atrophy is observed beginning in the first decade of life. This may take on a bulls-eye appearance (n = 4 with hx of bulls-eye in 3 additional patients; Table 1; Fig 2B) or a more generalized maculopathy (Fig 2D). Progression continues to involve the remaining macula and periphery (Fig 2B, C, E, F), resulting in end-stage retinal degeneration (which also can take the form of coloboma-like lesions) and legal blindness. Figure 2D shows end-stage degeneration in a 9-year-old patient. This end stage may occur as early as the first decade (patients 3 and 6), although progression to this point more typically takes place early in the second decade of life or later (Fig 1). The macular status of each patient at the time of study entry (as determined either by masked fundus photographs by an experienced reader or according to their medical records from before study entry) and then also at the most recent ophthalmic visit are included in Table 1.

Figure 2.

Macular changes in cobalamin C are early and progressive and may include a developmental component. A, Fundus photograph showing macular coloboma in 12-month-old (patient [pt] 2) homozygous for c.271DupA (p.R91KfsX14). Coloboma was documented at 6 months of age or earlier, but photographs were not available from that time point. B–G, Fundus photographs of sibling pairs homozygous for c.271DupA (p.R91KfsX14) showing degeneration and the effect of prenatal or early treatment, or both, on the course of macular degeneration. B, C, Fundus photographs from patient 5 obtained at (B) 5 years of age and (C) 10 years of age (VA, 0.8 logarithm of the minimum angle of resolution [logMAR]), who was treated with hydroxocobalamin injections starting at 5 days of age. D, Fundus photograph of patient 6, older sibling of patient 5, obtained at 9 years of age (VA, 1.6 logMAR). E, F, Fundus photographs of patient 4, diagnosed and treated prenatally, obtained at (E) 5 years of age (VA, 0.7 logMAR) and (F) 10 years of age (BCVA, 1.0 logMAR). G, Fundus photograph of patient 8, who was not treated prenatally and is the older sibling of patient 4, at 10 years of age (VA, fix and follow). The table below the figure reports the plasma total plasma homocysteine (tHcy), serum methylmalonic acid (MMA), and plasma methionine (Meth) concentrations (μM) for the patients at each time point. *Past history of higher MMA levels, with a high of 4.4 at 8 years of age.

An important corollary to these observations involves prenatal diagnosis, which facilitated treatment either prenatally or soon after birth. In the cases pictured in Figure 2B–G, mothers with a first child diagnosed with cblC deficiency had affected fetuses. The prenatal diagnosis allowed the affected newborns to be treated with intramuscular hydroxocobalamin immediately after birth (patient 4) or within a few days of birth (patient 5). Additionally, the mother of patient 4 was treated with intramuscular hydroxocobalamin as soon as the prenatal diagnosis was confirmed (see “Methods”). Empiric treatment with contemporary vitamin and dietary therapy resulted in children with less cognitive dysfunction (data not shown), better visual function, and less severe retinal findings (Table 1; Figure 2C vs. 2D and Fig 2F vs. 2G). The progression of retinopathy was delayed compared with that of the older sibling; however, it still progressed (Fig 2B, C vs. 2D and Fig 2E, F vs. 2G) and functional acuity still was affected severely. These data suggest that hydroxocobalamin therapy can mitigate, but does not prevent, symptoms or eventual progression of the retinal disease. A potential confounder to this conclusion is that early treatment also may help to prevent other morbidities such as metabolic decompensation or other organ involvement.

Similar to those identified early because of affected older siblings, several patients in this cohort were diagnosed during newborn screening (Table 1; patients 2, 15, 20, and 21) and received empiric contemporary vitamin and dietary treatment. Our examinations showed that all of the newborn screen-positive patients demonstrated symptoms at the first study ophthalmic examination with either clinically apparent macular degeneration (in 3 patients) or lowered BCVA (1 patient; Table 1). Therefore, the treatment did not seem to prevent the ocular manifestations of cblC.

Although c.271dupA (p.R91KfsX14) homozygous patients such as those pictured in Figure 2 show the most extensive retinal degeneration, some degree of degeneration was exhibited by 4 of the other 9 genotypes of patients in the study. These genotypes were: c.271dupA (p.R91KfsX14), c.457C→T (p.R153X); c.3G→A (p.M1L); c.3G→A (p.M1L), c.440G→A (p.G147D), c.619dupG (p.D207GfsX38); and c.471G→A (p.W157X), c.666C→A (p.Y222X). All were patients with infantile-onset disease (Table 1).

The comparison of patients with similar plasma metabolite levels but differing genotypes showed that the genotype rather than metabolite levels were more predictive. Only 2 patients in the study had higher than the normal upper limit of methionine at the last ophthalmic examination, a 2-year old patient (patient 1) homozygous for c.271dupA (p.R91KfsX14) and a 4-year old (patient 21) with c.471G→A (p.W157X), c.666C→A (p.Y222X) genotypes. Both patients had some degree of macular degeneration (Table 1). Photographs were not available because of limited cooperation by the patients. However, 3 patients with similarly high plasma tHcy and MMA levels are shown in Figure 3A–C. The c.271dupA (p.R91KfsX14) homozygous patients (patient 14 [Fig 3A] and patient 11 [Fig 3B]) showed end-stage macular degeneration; however, the patient with c.271dupA (p.R91KfsX14), c.600G→A (p.W200X) (patient 18; Fig 3C) had relatively normal fundus findings despite comparable metabolite profiles as those severely affected. Similarly, patients 15 and 16, with infantile-onset disease and genotype c.271dupA (p.R91KfsX14), c.331C→(p.R111X), had widely variable metabolite levels and no macular atrophy at last ophthalmic visit (Table 1).

Figure 3.

Retinal degeneration occurs in cobalamin C independent of metabolic status and within a variety of genotypes. Metabolite levels (in μM) at the time of examination are listed to the right or left of each panel. A, Fundus photograph showing a macular coloboma in patient 14 at 30 years of age (visual acuity [VA], 1.51 logarithm of the minimum angle of resolution [logMAR] at 27 years of age; c.271DupA (p.R91KfsX14) homozygous). Despite the severe degeneration, a small portion of the retina is preserved. B, Wide-field photograph of patient 11 obtained at 17 years of age (VA, 1.51 logMAR; c.271DupA (p.R91KfsX14) homozygous). Note bone spicules and significant progression despite youth. C, Fundus photograph from patient 18 obtained at 22 years of age, whose genotype is the relatively unusual c.271dupA (p.R91KfsX14), c.600G→A (p.W200X). Although the retina is relatively preserved, central nervous system involvement severely affects vision (right eye VA, 1.2 logMAR). D, Fundus photograph from patient 15 obtained at 14 years of age, with moderate total plasma homocysteine (tHcy) and methylmalonic acid (MMA) levels, showing some optic nerve pallor, but no clinical macular degeneration (c.271dupA (p.R91KfsX14), c331C→T (p.R111X) genotype). E, Fundus photograph from patient 23 obtained at 7 years of age. This patient with later-onset disease had abnormal tHcy and MMA levels and showed no macular degeneration. F, Fundus photograph from patient 24 obtained at 21 years of age. This patient with later-onset disease had normal vision (0 logMAR) and no macular degeneration, but did show drusen-like deposits. Panel inset, Autofluorescence photograph of the same retinal area as in (F). Note the lack of autofluorescence of the drusen-like deposits.

Similar to other types of retinal degeneration, retinal vascular attenuation and optic nerve pallor—particularly temporal pallor involving the papillomacular bundle—also occur in cblC. In most cases, these findings track with the severity of the retinal degeneration, but there are exceptions. For example, 4-year-old patient 15 with genotype c.271dupA (p.R91KfsX14), c.331C→T (p.R111X) exhibited no apparent macular degeneration, but showed temporal pallor clinically (Table 1; Fig 3D). However, patient 16 (10 years of age), who shares the same genotype, did not exhibit optic pallor (Table 1).

None of the later-onset patients in our study (patients 22–25) experienced clinical macular degeneration. Two of these patients are pictured in the final panels of Figure 2. Figure 3E shows patient 23 (7 years of age), who was formally diagnosed just before 5 years of age because of behavioral and cognitive changes in school. This patient had increased plasma tHcy and MMA levels, but showed no macular degeneration. Patient 24 (21 years of age) with late-onset disease showed no functional defect in vision, nystagmus, or strabismus; however, drusen-like deposits were apparent on the fundus examination (Fig 3F). Note the lack of autofluorescence of the deposits in the photograph to the right. True drusen would autofluoresce and also would be atypical in patients of this age.

Fundus autofluorescence imaging, ERG, and OCT were used to characterize further the degeneration in 2 c.271dupA (p.R91KfsX14) homozygous patients. Figure 4A is a wide-field autofluorescence photograph from patient 14, a 27-year-old with end-stage degeneration who is also pictured in Figure 3A. Note the significant hyperautofluorescence in the periphery and macula and the hyperautofluorescence in the center. These autofluorescence results show that retinal pigment epithelium pathologic features coexist with photoreceptor degeneration. Also, despite widespread macular atrophy, there is a clear zone of peripheral sparing. In addition, there is peripapillary sparing around the optic nerve. In the OCT image in Figure 4B, from near the edge of the macular lesion, atrophy and loss of laminar structure extend well beyond the lesion. Also note the abnormal reflectivity, a pattern consistent with end-stage degeneration, and possible indications of developmental abnormalities. The ERG results obtained from this patient at 27 years of age are displayed in Figure 4D. Both scotopic and photopic responses are reduced and delayed (scotopic>photopic), but are remarkably preserved considering the macular condition. Figure 4C shows OCT images of a cut passing through the central macular lesion of patient 11 (also shown in Fig 2B) and shows extreme thinning, some preservation of retinal lamination, and nearly complete loss of the outer nuclear layer.

Figure 4.

Pathologic and functional analysis of c.271DupA (p.R91KfsX14) homozygotes showing preservation of peripheral retinal structure and function and retinal architecture consistent with a developmental defect. A, Autofluorescence photograph from patient (pt) 14 obtained at 27 years of age showing end-stage degeneration. Note the significant hyperautofluorescence in the periphery and macula and the hyperautofluorescence in the center. Despite widespread macular atrophy, there is a clear zone of peripheral sparing. In addition, there is clear peripapillary sparing around the optic nerve. B, Cirrus high-resolution optical coherence tomography (OCT) raster scan from near the edge of the macular atrophic lesion (inset) showing atrophy and loss of retinal laminar structure extending well beyond the macular atrophic lesion. Note abnormal reflectivity and a pattern consistent with end-stage degeneration and possible indications of developmental abnormalities. C, Cirrus high-resolution OCT raster scan from the center of the macular atrophic lesion (inset) showing loss of the inner segment–outer segment photoreceptor junction within macular lesion. The scan shows relatively normal retinal architecture beyond the macular lesion with an abrupt transition (white arrows are used to depict the boundaries of the lesion on this scan). D, Electroretinography (ERG) results obtained from patient 14 showing both scotopic and photopic responses are reduced and delayed (scotopic>photopic), but remarkably preserved considering the macular condition. E, Electroretinography (ERG) results obtained from patient 11 at 17 years of age showing significant attenuation and delay of both scotopic and photopic responses (wide-field photograph in Fig 3B).

Discussion

Our study details the clinical course of 25 cblC deficiency patients ranging in age from 2 to 27 years, with particular focus on the ophthalmic manifestations of the disease over time. Both patients with infantile-onset and later-onset disease were included. Our study includes the oldest c.271dupA (p.R91KfsX14) homozygous patients in the literature to date, with 5 patients 18 through 27 years of age at the last ophthalmic visit (Table 1). Previously, the oldest 2 patients who are c.271dupA (p.R91KfsX14) homozygous reported in the literature were 17 and 23 years of age when the ophthalmic findings were made,^14,18 although there may be older patients in the literature without reported genotypes. In the infantile-onset group, patients are severely affected compared with late-onset patients, and neonatal and infantile presentation of systemic symptoms (e.g., failure to thrive, encephalopathy, acute lethargy) also is accompanied by overt ophthalmic signs. These signs often occur before 1 year of age and include manifest nystagmus (16/21 [76%]), macular degeneration (7/21 [33%]), and irregular macula and reduced ERG results (patient 9 at 3 months of age and patient 11 at 1 month of age). An additional rare and severe early manifestation, first reported recently in a patient in the first year of life,²³ is macular coloboma as seen in patient 3 (3 months of age; Table 1). Although coloboma also is seen in some, but not all, older cblC patients as they progress to end-stage macular degeneration (Table 1; Fig 3A),¹⁸ coloboma seen in the first few months of life could represent either very severe degenerative processes, abnormal ocular development at the time of birth, or both.

A high prevalence of clinically significant structural heart defects have been found in cblC patients,^22,24 and the patients in this study also showed a number of developmental defects (e.g., microcephaly, non–head-sparing intrauterine growth restriction, and cardiac noncompaction; Table 1), perhaps consistent with the recent observations suggesting Mmachc deficiency is critical for preimplantation during early mouse development.²⁵ A role for this enzyme in human ocular development is suggested by the recent documentation, via spectral-domain OCT, of ocular changes from infancy to 13 years of age in one c.271dupA (p.R91KfsX14) homozygous patient in whom photoreceptor outer segment loss, thinning of the outer nuclear layer, and a thickened, delaminated inner retina resembling an immature retina were noted.²³ Other investigators have observed progressive degeneration via OCT imaging performed at 4 and 7 months of age.¹⁸ The fact that we and others also have observed similar changes in retinal structure (Fig 4B, C),^18,23 that patients can demonstrate severe disease in infancy, and that comorbid developmental abnormalities exist reinforces the concept that cblC deficiency comprises a developmental, as well as a degenerative phenotype.

In our series, we did not observe a neuroanatomic retinopathy correlation beyond noting that some patients who had early encephalopathy also had cortical vision problems (patients 6, 14, and 17). However, patients with unrevealing imaging studies also had retinopathy or optic nerve disease (patients 3, 9, 10, and 11). This agrees with the claims of Ricci et al,²⁶ who noted that brain lesions were not the only cause of visual abnormalities in cblC individuals. Conversely, we find that some patients with severe central nervous system impairments also could have largely unaffected retinal structures (patients 18 and 23). Additionally, the ocular examination results of the 1 patient in our cohort with hydrocephalus (patient 10) were very similar to those of same-age nonhydrocephalus patients. We think it is unlikely she experienced pressure effects, but it cannot be completely ruled out. A detailed description of neuroradiologic imaging studies in this cohort is in progress.

In cblC deficiency, as in many metabolic disorders, improvements in treatments can ameliorate systemic symptoms and can improve patient life spans. Therefore, late ocular manifestations with underlying similarities to other diseases with disrupted bioenergetics now are being noted and afford an opportunity to speculate about the pathophysiology underlying the ocular disease manifestations. Guttae found on the corneal endothelium of both eyes of a 27-year-old patient (patient 14) may be one example. These deposits in the corneal endothelial basement membrane associated with endothelial cell loss^27–29 have not been reported in cblC previously. Guttae (as well as retinal dystrophy and optic neuropathy) do appear in other diseases linked to mitochondrial dysfunction such as Kearns-Sayer disease and Leber hereditary optic neuropathy.^30,31 Whether this is the secondary manifestation of mitochondrial dysfunction, well known to be a pathologic characteristic of methylmalonic acidemia,³² is unknown, but is supported by a case study that described a late-onset cblC patient whose muscle biopsy results showed neurodegeneration and a mitochondrial respiratory chain defect with cytochrome oxidase (complex IV) deficiency.³³ Furthermore, a postmortem pathologic study of a 2-year-old cblC patient with macular degeneration¹⁶ documented swollen mitochondria and large vacuoles in the corneal endothelium. This patient also showed an accumulation of mucopolysaccharides in the sclera, potentially because of lysosomal dysfunction, and degenerated mitochondria in the iris pigment epithelium.¹⁶ Whether such an accumulation is a factor in the unusual finding of drusen-like deposits on the macula of late-onset patient 24 (Fig 3F)—which, like the guttae, has not been seen previously in cblC patients—remains unknown but is worthy of exploration in other older cblC patients. As others have described, we found that most late-onset cblC patients do not manifest visible retinal pathologic features. One caveat is that we have conducted limited longitudinal examinations in this small subgroup of patients. Although we do not have repeat visits within our late-onset patient cohort, we do have 2 late-onset patients of the same genotype (patients 22 and 23) who are separated in age by decades and yet have no detectible eye disease. This is worth noting and perhaps an area for future research. Furthermore, it is worth noting that 1 of the 4 late-onset patients reported by Gerth et al¹² showed normal ERG results despite some clinically apparent retinal pathologic features.

We were limited in this study by not being able to examine patients under anesthesia, and therefore we could not assess electrophysiologic measurements or OCT data in many patients. In addition, 12 patients were seen only once, limiting our ability to provide longitudinal follow-up across the cohort.

In our study, pace and chronicity of the ophthalmic manifestations in early-onset cblC lacked strict correlation to metabolic status as measured at the time of visit, because poorly controlled patients could have relatively intact retinas, and conversely, patients with well-controlled disease experienced widespread atrophy. Conversely, crystalline lens dislocation, as seen resulting from cystathionine β-synthase deficiency homocystinuria,³⁴ was not observed in any of our patients despite high levels of tHcy. This implies that the mechanism of lens dislocation is not related to tHcy per se. However, elevated tHcy may increase the amounts of reactive oxygen species, leading to activation of microglia³⁵ and induction of apoptosis in neurons and the vascular endothelium.^36–38 Data obtained by Richard et al³⁹ from fibroblast studies of isolated homocystinuria patients (cblE, cblG, and MTHFR deficient) indicate that the impaired remethylation capacity in patients may increase reactive oxygen species and apoptosis levels. Similarly, patients with isolated MMA (e.g., mut⁰, cblB) sustain much higher levels of methylmalonic acid (mmol/L range) and yet have no macular or retinal degeneration (unpublished observation).⁴⁰

Of the inherited forms of MMA, the cblC retinal atrophy seems to be unique, with 1 reported exception in a patient with cobalamin D.⁴¹ Low methionine levels have been hypothesized to be a pathophysiologic mechanism for cblC ocular disease, either through causing inadequate protein synthesis¹⁰ or a decrease in cysteine synthesis, and therefore decreased levels of RPE-protecting glutathione.^11,42 Because other cobalamin deficiencies (e.g., cobalamin G) have low methionine without eye disease, this mechanism is more likely to be contributory rather than causative. There have been encouraging reports describing the use of methionine to improve retinal function, including rescued rod function in a 3-year-old cblC patient.⁴³ The fact that other forms of cobalamin-associated hyperhomocysteinemia, such as cobalamin E and cobalamin G deficiencies, do not display a retinopathy suggests that a more complex mechanism underlies the eye disease in cblC deficiency, especially because methylenetetrahydrofolate reductase deficiency patients, who also have homocystinuria, do not experience retinal degeneration. In total, the patient observations suggests that the pathogenesis of the retinal findings in cblC is the result of a unique underlying pathophysiologic mechanism or disrupted bioenergetics,⁴⁴ rather than toxic metabolites⁴⁵ per se. However, more data on the ophthalmologic phenotype of patients with these rarer defects is needed. Analyte levels during study visits may or may not correlate with past history, especially because the patient ages varied widely and the disease progression was continuous. Based on the observations that ocular manifestations lacked strict correlation with metabolite levels, we speculate that although metabolic control is desirable, it is probably not the most deterministic factor in cblC-related ocular disease.

In contrast, genotypes were very predictive for ophthalmic prognosis in cblC. c.271dupA (p.R91KfsX14) homozygous patients were severely affected universally, both in our study and in the literature.^14,46,47 Compared with our c.271dupA (p.R91KfsX14) homozygous patients, the results for c.271dupA (p.R91KfsX14) homozygous patients in the literature are very similar.^{14,23,48–51}

Although c.271dupA (p.R91KfsX14) homozygous patients also were the most severely affected over time, certain mutations in trans—c.271dupA (p.R91KfsX14), c.457C→T p.R153X; c.440G→A (p.G147D), c.619dupG (p.D207GfsX38); and c.471G→A (p.W157X), c.666C→A (p.Y222X)—also resulted in severe eye disease, whereas others manifested a comparatively less severe ocular phenotype. Lerner-Ellis⁸ showed through allelic expression studies on human cblC fibroblasts that in early-onset mutations, c.271dupA (ex2/dup, frameshift) and c.331C→T (ex3/nonsense) MMACHC mRNA transcripts were underexpressed compared with control alleles and the later-onset c.394C→T p.R132X mutation. According to current understanding, nonsense mutations would be expected to be subjected to nonsense-mediated decay and loss of protein. However, there is some evidence that prematurely truncated MMACHC proteins can retain some functionality.⁵² This may be the reason behind the unusual phenotype of the c.271dupA (p.R91KfsX14), c.600G→A (p.W200X) genotype patient 18, whose retina is quite preserved despite severe metabolic disease.

Diagnostic and surveillance guidelines for ocular disease in patients with cblC defect have been proposed recently, and more data on early and late manifestations also could improve our understanding of the disease and processes.¹⁸ One outstanding question in particular is perplexing: because biochemical measures ameliorate systemic symptoms of cblC disease, why is the associated macular degeneration treatment resistant? Prenatal or early treatment, or both, with hydroxocobalamin may ameliorate the developmental and behavioral complications of cblC deficiency somewhat, both in our experience (data not shown) and in the literature.^15,53,54 We also observed that prenatal or early treatment, or both, may mitigate the ocular disease partially. Our younger affected siblings had better functional acuity in the early years and as they approached their second decade (Table 1; Fig 2), but still progressed to severe macular atrophy.

Perhaps the water-soluble nature of vitamin B12 is an obstacle to penetration into the ocular compartment, and the use of repeated subconjunctival injection,⁵⁵ small-volume nebulizer,⁵⁶ and more recently, periocular injection of in situ hydrogels⁵⁷ or subconjunctival biodegradable microfilms⁵⁸ may be a more efficient means to deliver the cofactor in these patients. Clearly, research on potential therapeutics would be enhanced significantly if a viable animal model of cblC deficiency could be generated and studied.

Abbreviations and Acronyms

BCVA

best-corrected visual acuity

cblC

cobalamin C

ERG

electroretinography

logMAR

logarithm of the minimum angle of resolution

MMA

methylmalonic acid

MUT

methylmalonyl-CoA mutase

OCT

optical coherence tomography

tHcy

total plasma homocysteine

VA

visual acuity