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Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1F
CLINICAL STUDIES
Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in
CACNAJF
Kym M. Boycott,* PhD; William G. Pearce, t MD; N. Torben Bech-Hansen,* PhD ABSTRACT • RESUME Background: Incomplete X-linked congenital stationary night blindness (CSNB) is a clinically variable condition that has been shown to be caused by mutations in the calcium-channel CACNA IF gene. We assessed the clinical variability in the expres sion of the incomplete CSNB phenotype in a subgroup of patients of Mennonite ancestry with the same founder mutation. Methods: Sixty-six male patients from 15 families were identified with a common mutation in exon 27 of CACNA IF (LI 056insC). Clinical variability in night blindness, reduced visual acuity, myopia, nystagmus and strabismus was examined. Results: At least one of the major features of CSNB (night blindness, myopia and nys tagmus) was absent in 72% of the patients. All the examined features varied wide ly, both between and within families. Interpretation: Although the patients shared a common CACNA IF muta
tion, there was considerable variability in the clinical expression of the incomplete CSNB phenotype. These findings suggest the presence of other genetic factors modifying the phenotype of this disorder.
Contexte: La cecite nocturne stationnaire congenitale partielle, liee au chromo
some X (CSNB), est une condition cliniquement variable et attribuable a des mutations du gene porteur de calcium CACNA IF. Nous avons etudie la vari abilite clinique de !'expression du phenotype de la CSNB partielle chez un sous groupe de patients de souche mennonite ayant la meme mutation originelle. Methodes : Nous avons retenu 66 patients du sexe masculin provenant de 15 families ayant une mutation commune de l'exon 27 du CACNA IF (LI 056insC). Nous avons examine la variabilite clinique de la cecite nocturne, de la baisse d'acuite visuelle, de la myopie, du nystagmus et du strabisme.
From *the Department of Medical Genetics, Faculty of Medicine, University of Calgary, Calgary, Alta., and tthe Department of Oph thalmology, Faculty of Medicine, University of Alberta, Edmonton, Alta.
Reprint requests to: Dr. N. Torben Bech-Hansen, Department of Medical Genetics, Faculty of Medicine, HMRB311, 3330 Hospital Dr. NW, Calgary AB T2N 4Nl; fax (403) 210-8119, [email protected]
Accepted for publication Feb. 12, 2000
Can j Ophthalmol 2000;35:204-13
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Clinical variability-Boycott et al
Clinical variability-Boycott et al Resultats : Au total, 72% des patients ne presentaient pas au mains une des prin cipales caracteristiques de la CSBN (cecite nocturne, myopie ou nystagmus). Les caracteristiques etudiees variaient considerablement tant au sein des families qu'entre elles. Interpretation : Bien que les patients partagent une mutation com mune du CACNA IF, I'expression clinique du phenotype de la CSNB partielle varie considerablement. Ces constatations incitent a penser que d'autres facteurs genetiques modifient le phenotype de cette maladie.
X
-linked congenital stationary night blindness (CSNB) is a heterogeneous nonprogressive reti nal disorder characterized clinically by night blindness, decreased visual acuity, myopia, nystagmus and stra bismus.1-3 Clinical heterogeneity among families has led to the classification of X-linked CSNB into com plete and incomplete forms. 4 This division is based fundamentally on abnormalities in the electroretino gram (ERG) (e.g., the scotopic b-wave, the oscillatory potentials and cone function) and in the psychophysi cal dark-adapted thresholds. In patients with complete CSNB the scotopic b-wave evoked by dim blue flash es and the early oscillatory potential wavelets are unrecordable. The night blindness is profound, as doc umented by the absence of rod dark adaptation. The photopic system in these patients is mildly abnormal. In patients with incomplete CSNB the scotopic b-wave is recordable although subnormal in amplitude, the scotopic oscillatory potentials are often recordable but may show abnormal implicit times, 5 and the psycho physical dark-adapted thresholds are variably eleva ted above normal. Under photopic conditions the b-wave is barely recordable, no oscillatory potentials are elicited, 5 and the 30-Hz flicker is reduced. 4 In addi tion, when the eye is dark adapted and stimulated con tinuously for 12 to 15 minutes with a 30-Hz flicker, there is an exaggerated increase in amplitude and a characteristic change in ERG shape. 3·6 X-linked CSNB is not readily identified clinically owing to the lack of specificity of the main clinical fea tures (night blindness, reduced visual acuity, myopia and nystagmus). Another contributing factor is the absence of a clinically identifiable structural abnormal ity of the retina. 2 Part of the clinical variability seen in X-linked CSNB can now be attributed to genetic het erogeneity, the gene for complete X-linked CSNB (CSNBl) mapping to Xpl 1.4, and the gene for incom plete X-linked CSNB (CSNB2) mapping to Xpl 1.23. 7
Efforts to isolate the gene responsible for incom plete CSNB were based on a positional cloning approach 8 and have culminated in the discovery of mutations in a new retina-specific voltage-gated L type calcium-channel a 1-subunit gene, designated CACNAJF. 9 The CACNAJF gene contains 48 exons 9 and has a predicted protein length of 1977 amino acids in the larger isoform (N.T.B.H., unpublished observa tions, 1999). Analysis of CACNAJF in 20 families with incomplete CSNB showed six different muta tions, all of which were predicted to cause premature protein truncation. 9 Parallel studies by Strom and col leagues10 confirmed these observations. These find ings established that mutations in CACNAJ F cause incomplete CSNB, making this disorder an example of a human calcium-channel disease of the retina. One of the mutations identified in CACNAJF, an insertion of a C nucleotide in exon 27 (Ll056insC), predicted to cause a frameshift and premature trunca tion of the protein product, was present in 15 different families of Mennonite ancestry. To address the ques tion of clinical variability within a set of subjects with the same underlying molecular defect, we examined the clinical findings in a large set of patients from these families with the L1056insC mutation in the CACNAJF gene. METHODS
Fifteen families (families 50, 60, 60B, 80, 130, 150, 160, 170, 180, 190, 200, 240, 250, 330 and 340) shared part of a common Mennonite haplotype. The same mutation in CACNAJF was subsequently shown to segregate in these families. 7•9 The family structure and clinical findings in three of these kindreds have previously been reported (families 50 [B], 2 80 [I] 2 and 60 [C] 2·11 •12). The one base pair insertion in exon 27 of CACNAJF (L1056insC) was initially identified by
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Table I-Normative electroretinography data* Light-adapted Feature Time, ms a-wave b-wave Amplitude, µV a-wave b-wave
Dark-adapted
Bright white
White flicker
Dim blue
Bright white
12-20 35-40
30-35
65-90
15-25 45-55
50-100 120-180
80-130
90-170
100-175 200-400
*Within 2 standard deviations of the mean. Source: Retinal Testing Unit, Department of Ophthalmology, University of Alberta, Edmonton.
direct DNA sequencing of the exon, amplified by polymerase chain reaction (PCR), from a patient with incomplete CSNB from family 60. 9 Detection of the L1056insC mutation in other families sharing the Mennonite haplotype and segregation analysis within families were based on the separation of radiolabelled PCR products in 6% polyacrylamide gels. 9 The L1056insC mutation was originally designated as L991insC, 9 but the designation was revised after splice variants were identified in this gene (N.T.B.H., unpublished observations, 1999). Members of each family underwent an ophthalmo logic examination, and the diagnosis was made as described elsewhere. 2•11 •12 Briefly, we recorded the ERG using the Nicolet CA 2000 electrodiagnostic sys tem with a Nicolet Ganzfeld photostimulator and gold foil electrodes (C.H. Electronics, London). The ear lobe electrodes were Grass Gold Cup with Nicolet electrolyte gel, and the surface electrodes were Nicolet gold surface electrodes with Ten 20 electroencepha lography paste. Following maximum dilation of the pupils with 1% tropicamide drops and topical anesthe sia with 0.5% proparacaine drops, gold foil electrodes were inserted into the lower fomix of each eye. The earlobes served as ground. The recording gold foil electrodes were referenced to the forehead electrode. Cone responses were obtained after 5 minutes of light adaptation with a Ganzfeld background light of 10 foot lambert. Bright white flashes with a relative strobe intensity of 1.50 (10.02 cd · m2/s) were pre sented. This was followed by a white flash of strobe intensity 0.75 (2.11 cd · m2/s) flickered at 31.1 Hz. Low and high filter frequencies of 1 Hz and 100 Hz respectively were used with a sensitivity of 500 µV. Rod responses were obtained after 25 minutes of dark
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adaptation. Dim blue flashes were presented with the use of Wratten filters #47, #47A and #47B in com bination with a relative strobe intensity of 0.25 (0.81 cd · m2/s) at a rate of 0.9/s. A single bright white flash with a relative strobe intensity of 0.75 (2.11 cd · m2/s) was then presented. Low and high filter fre quencies of 1 Hz and 100 Hz respectively were used. Sensitivity was set at 500 µV for the dim blue flash and 1000 µV for the bright white flash. Normative data are shown in Table 1. We performed dark adaptometry using the Goldmann-Weekers adaptometer. After the pupils were maximally dilated with 1% tropicamide drops, the patient underwent preadaptation for 5 minutes with the adaptometer calibrated at a luminosity of 3400 lx. Illumination was then extinguished, and, with the Goldmann-Weekers dim white circular target of 5.5, responses were obtained at a rate of at least two per minute for 30 minutes. Normative data are not shown. RESULTS
Mutation analysis
Mutation screening by direct DNA sequencing in an affected male from family 60 identified the insertion of a C nucleotide in the DNA sequence of exon 27 of the CACNAIF gene. 9 Screening of the remaining members of family 60 and the 14 other families by PCR amplification of exon 27 showed the presence of a one base pair insertion, consistent with segregation of the mutation in the 15 families. Segregation of this L1056insC mutation in a part of family 60 is shown in Fig. 1, top. This portion of the family was also of inter est because of the identification of two female mem
Clinical variability-Boycott et al
C4
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Fig. I-Top: Analysis of the LI056insC mutation in members of family 60 with incomplete X linked congenital stationary night blindness. Exon 27 from CACNA IF was amplified by poly merase chain reaction (PCR), and the products were separated by gel electrophoresis accord ing to size. The LI 056insC mutation results in the insertion of a C nucleotide within exon 27, making the amplified product one base larger in affected members. Therefore, affected mem bers have a 235 base pair (bp) PCR product, whereas unaffected members have a 234 bp prod uct. Carrier females have both a 234 bp and a 235 bp product, representing the alleles on the two different X chromosomes. The clinical findings in patients C4, EI 0 I and EI 07 are described in Table 2. For the remaining family members the phenotype is inferred from the genotype. An asterisk indicates that haplotype construction using polymorphic X chromosome markers (unpublished observations) has shown that three of the males in the third generation inherited the mutant chromosome from their maternal grandfather (also indicated with an asterisk), whereas the two other males inherited the mutant chromosome from their carrier maternal grandmother. Bottom: Direct DNA sequencing of the exon 27 PCR product in one of the females from family 60 homozygous for the LI 056insC mutation. An extra C nucleotide is inserted in exon 27 on both copies of her X chromosome. Homozygosity of the mutation is shown by the absence of bands representing two different X chromosomes, as would be seen in a carrier female. The extra C nucleotide at this position produces a frameshift mutation. At codon I056 the leucine (Leu) seen in the normal protein has been changed to a proline (Pro). The amino acid sequence continues to be altered until codon I066, which is translated into a stop codon, and synthesis of the protein is terminated at this point. Arg =arginine, Asp =as perigine.
bers homozygous for the L1056insC mutation. To explain this finding, it is assumed that the deceased father of these females was also affected with incom
plete CSNB due to the L1056insC mutation. The DNA sequence of this region of exon 27 from one of the homozygous females is shown in Fig. 1, bottom.
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Left eye
Visual acuity
-I 2.S0+4.00 x IOS -2.SO -9.SO+ 1.00 x as -6.7S+2.2S x I00 -2.2S+ 1.00 x I6S -7.7S+ I.7S x ISS --0.2S+ I.7S x I6S -4.7S+ 1.00 x 7S -3.00+0.7S x 180 -6.2S+ I.2S x 90 + I .00+0.7S x 70 + 1.00+ 1.00 x I4S -7.00+ 1.00 x I2S +4.SO+O.SO x 90 +0.2S+0.2S x 90 +O.S0+0.2S x 180 +2.00+0.SO x 90 -12.2S+l.7SX 120 -I.SO+ 1.7S x 120 -3.00+2.SO x I2S -4.2S+ 3.7S x 90 +O.SO+ I.2S x I00 -9.SO+ 3.2S x 90 -2.S0+2.7S x 11 S -8.7S+4.2S x I00 -6.00+ I.7S x 9S -8.2S+3.00X9S -7.S0+2.00 x 9S -8.00+ l.7S x I00 Plano+2.00 X 90 --0.2S+2.00 x 90 -4.7S+3.2S x I I0 -7.2S+2.SO x 90 + 1.00+ I .2S x 9S +I .S0+2.00 x 80
Right eye - I2.S0+400 x 80 -3.SO -9.SO+O.SO x 90 -7.2S+2.SO x 70 -3.7S+3.00X 180 -7.7S+ I.SO x IS -7.00+ I.2S X IS -2.SO -3.2S -6.2S+ I.3S x 90 +2.S0+0.2S x 90 +2.00+0.7S x 3S -S.7S+ 1.00 x so +4.SO+ I.2S x as --0.2S+O.SO x 90 -0.7S+l.2SX IOS +I .SO+ 1.00 X90 - I0.7S+ I.SOX SS -I .SO+ I.7S x 70 -4.S0+2.00 x as -2.7S+2.2S x 90 + 1.00+ I.2S x 70 - I0.00+2.7S x 90 -2.2S+2.SO x 6S -6.00+ 3.00 x 80 -6.2S+2.2S x 90 -8.2S+ 3.00 x 90 -6.2S+ 1.00 x as -S.7S -I .2S+2.00 x 90 --0.7S+2.00 x 90 -4.SO+ 3.SO x 90 -8.00+3.00 x 90 + 1.00+ I.2S x as +2.2S+2.2S x 100
Left eye
Refractive error Present Present Present Absent Present Present Present Present Present Present Absent Present Present Absent Absent Absent NA Present Present Absent Present Absent Present Present Absent Absent Present Absent Present Present Absent Present NA Absent Absent
Night blindness symptoms
1.0 O.S
0.2S 1.0 N 1.0
3.0 2.0 2.0 l.S N
1.0 2.0 O.S 1.0 l.S
2.S 3.0 2.0 2.S
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Dark adaptation (rod), log units elevation Present Present Absent Absent Present Absent Absent Present Present Present Absent Absent Absent Absent Absent Absent Present Absent Absent Present Present Absent Absent Absent Absent Absent Absent Absent Present Absent Absent Absent Present Absent Latent
Congential nystagmus
Table 2-Clinical findings in 66 members of 15 families with incomplete X-linked congenital stationary night blindness*
(Continued)
Esotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Esotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Exotropia Exotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Exotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Esophoria
Strabismus
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240
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Visual acuity
Age, yr
Table 2-Continued
-l.00+0.7S x IOS - I8.7S+ 1.00 x I I S -3.S0+2.SO x IOS -I l.S0+2.7SX 100 -16.00+ l.2S x IOS - I I .S0+2.00 x I2S -6.S0+4.00 x I00 -6.2S+0.7S x IS -7.00+ 1.00 x I00 - IO.S0+0.7S x I3S -3.00+0.SO x 90 - I0.2S+ 2.2S x 8S -3.00 -8.S0+2.00 x 9S -9.00+ I.2S x 80 -4.S0+2.00 x 90 Plano+ I .2S X 140 -2.00+0.SO x I00 -4.00+ 1.00 x 170 - IO.SO+ 3.00 x S -1.00+ 1.00 x 90 -6.2S+ I.7S x 90 -S.00+ 3.00 x 90 -6.00+0.SO x 90 -3.2S+ 3.2S x I IS -S.00+3.00 x 90 -l.2S+ l.7S x 8S -7.7S+l.SOX 110 -2.00+2.00 x 90 -2.SO+ l.7S x IOS +0.2S+0.7S x 80
Right eye -0.SO+ I.2S x 7S - I 8.7S+0.7S x 60 -3.7S+2.2S x 90 - I0.00+ 2.SO x 70 -I S.2S+2.2S x I2S - IO.SO+2.SO x 60 -4.SO+3.2S x 60 -S.SO+O.SO x 6S -S.2S -9.SO+ 1.00 x 30 -4.SO+O.SO x 90 -9.2S+2.2S x 80 -2.SO -8.00+4.00 x 90 -9.00+2.2S x 8S -4.00+2.2S x 90 -0.2S+ I.2S x 6S -2.2S+O.SO x so -3.7S+O.SO x 180 - I I.SO+ 3.SO X 170 +0.2S+ 1.00 x 90 -4.00+ I.SO x 90 -S.00+3.00 x 90 -S.SO+O.SO x 90 -I .7S+2.2S x 90 -6.00+4.SO x 9S -0.2S+O.SO x 180 -6.7S+ l.2S x 7S -2.S0+2.00 x 90 -2.S0+0.7S x 6S -l.7S+l.2SX9S
Left eye
Refractive error Present Absent Present Present Present Present Absent Present Absent Absent Absent Present Present Absent Absent Absent Present Present Present Present Present Absent NA NA Present Present Absent Absent Absent Present Present
Night blindness symptoms
1.0
1.0 1.0
1.0 1.0
2.0 2.0
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1.0 l.S 1.0 1.0 l.S l.S l.S l.S l.S
O.S 2.0
Dark adaptation (rod), log units elevation Absent Absent Present Present Absent Absent Present Absent Absent Absent Present Present Present Present Absent Absent Absent Absent Absent Present Present Present Present Present Absent Present Absent Present Present Present Present
Congential nystagmus
Orthophoria Orthophoria Exotropia Orthophoria Orthophoria Exotropia Exotropia Orthophoria Orthophoria Orthophoria Exotropia Orthophoria Esotropia Exotropia Orthophoria Orthophoria Orthophoria Exophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria NA Orthophoria Orthophoria Orthophoria Orthophoria Exotropia
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Clinical variability-Boycott et al Clinical findings
The clinical findings in the 66 patients are shown in Table 2. Of the 66 patients 32 were from family 60, and 34 were from the 14 other families. Important clinical features were absent in 45 of 62 patients: in 26 patients one major feature was absent (night blindness in 7, and nystagmus in 19), 1 had neither night blindness nor myopia, 13 had neither night blindness nor nystagmus, 1 had neither myopia nor nystagmus, and 4 did not have night blindness, myopia or nystagmus.
ily 190 [Dl5] and one patient from family 340 [A3400]) the refractive error was plano (2 eyes) or hyperopic (18 eyes). In one patient from family 60 [E80] the hyperopia was +4.50 dioptres in both eyes. Fifty-one eyes had low myopia (-0.25 D to -4.75 D), 44 had moderate myopia (-5.00 D to -9.75 D), and 17 had high myopia (-10.00 D or more). Considerable variation was present even within the same family. A difference in refractive error of about 10 D was found between two sets of brothers in family 60 (E37/E77 and E42/E49).
Nystagmus
In 62 cases it was possible to obtain information about visual performance in a dim or dark environ ment. Thirty-seven patients had a history of impaired night vision, and in 25 there was no suggestion of a night vision problem on specific questioning.
Congenital nystagmus was present in 29 patients (including latent nystagmus in 1) and absent in 37 patients. Table 3 shows the relation of nystagmus to visual acuity in the 60 patients in whom visual acuity could be assesed with the Snellen eye chart. The pres ence of nystagmus was associated with a reduction in visual acuity.
Dark adaptometry
Strabismus
Dark adaptometry was performed in 42 patients, of whom 40 showed variable elevation of the rod seg ment. Of the 40, 25 reported difficulty with vision in a dim environment: in 11 the elevation was mild (1.0 log unit or less), in 9 it was moderate (1.5 to 2.0 log units), and in 5 it was marked (more than 2.0 log units). The remaining 15 patients had no histo ry of visual difficulties in a dim environment; 8 had mild elevation of the rod segment, and 7 had moderate elevation. The rod segment was not elevated in two of the patients from family 60 (E4 and E42), although both demonstrated the exon 27 founder mutation and had other features consistent with CSNB, most notably a negative ERG (bright flash elicits a normal a-wave and ab-wave that has less amplitude than the a-wave). Neither patient reported impaired night vision.
Of the 65 patients who underwent testing for stra bismus, 9 had exotropia, 3 had esotropia, 1 had exo phoria, and 1 had esophoria.
Impaired night vision
Visual acuity Visual acuity was reduced in all 60 patients in whom this clinical feature could be determined. The impairment ranged from 20/25 to 20/400.
Myopia In 20 eyes (both eyes of seven patients from family 60 [El4, E16, E42, E80, E84, E130 and E144] and one eye of three patients from family 60 [E73, E81 and E120], one patient from family 180 [G3], one patient from fam
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Funduscopy No specific or pathognomonic fundus abnormalities were seen. In patients with lower degrees of myopia Table 3-Visual acuity of 60 patients (right eye) by presence or absence of congenital ny stagmus No. of eyes
Visual acuity 20/25 20/30 20/40 20/50 20/60 20/70 20/80 20/100 20/200 20/400
Nystagmus present (n = 24)
Nystagmus absent (n = 36)
0 0 2 6 4 2* 4 3 2
I 9 12 10 2 2 0 0 0 0
*Latent in one patient (patient E16 from family 60)
Clinical variability-Boycott et al
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Fig. 2-Full-field electroretinography (ERG) responses in a normal subject (left) and two patients with incomplete congenital sta tionary night blindness and the exon 27 founder mutation in CACNA IF. Case IV-40 is patient E36 from family 60, and case Ill-A is patient D 174 from family 60B. In both patients a bright flash elicits a normal a-wave and a b-wave that has less amplitude than the a-wave (negative ERG). The scotopic dim blue response is reduced but detectable, and both the b-wave of the cone response and the 30-Hz flicker response are also reduced (reproduced from reference 11 with permission from Springer-Verlag, Berlin).
the fundus appeared essentially normal. In some of the patients with moderate or high myopia the fundus showed characteristic myopic changes, including tem poral sector pallor of the optic nerve head and variable attenuation of the retinal pigment epithelium.
Electroretinography Scotopic or photopic ERG, or both, was performed in 56 patients, including at least 1 member from each of the 15 families. The results were consistent with a diag nosis of incomplete CSNB. All patients had a subnor mal scotopic b-wave or a negative ERG or both. The flicker and photopic responses in all 56 patients were significantly abnormal. Representative ERG findings in an unaffected male and two affected males from family 60 (IV-40) and 60B (III-A) are shown in Fig. 2. INTERPRETATION
Sixty-six affected male patients from 15 different
families with incomplete CSNB were shown to have the L1056insC mutation in exon 27 of the CACNAJ F gene. All 15 families are of Mennonite descent and are presumed to share a common ancestor, although we cannot currently demonstrate their relationship on a unifying pedigree.7· 9 Between 1874 and 1880 near ly 7000 Mennonites immigrated to Manitoba from Russia. 13 Our suspicion that the L1056insC mutation originated before this wave of immigration is sup ported by the observation that the same mutation has been shown to be present in a family with incomplete CSNB in Germany. 10 It is likely that other descen dants of the Anabaptist movement and members of the Mennonite community in Canada, the United States and other parts of the world are also affected with incomplete CSNB due to the L1056insC muta tion in CACNAJ F. Although this disease is called CSNB, 40% of the patients in our study did not have a history of diffi culty with vision in conditions of dim illumination. However, almost all of these patients were found to
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Clinical variability-Boycott et al have defective rod function on dark adaptometry, although none showed marked elevation. We found considerable variation in refractive error, even within families (+4.50 D to -18.75 D); in two instances there was a difference of more than 10 D between brothers. Visual acuity ranged from 20/25 to 20/400 and varied widely within families. Nystagmus was present in 44% of the patients, and its presence was associated with a greater decrease in visual acuity. However, when nystagmus was absent, the visual acuity was also reduced. These findings confirm that cone as well as rod function is impaired in patients with incomplete X-linked CSNB. Strabismus was not as common, being present in 22% of the patients. Although our patients share the common CACNAJF mutation, one or more of the three charac teristic features of night blindness, myopia and nys tagmus were absent in over 72% of cases. The most notable clinical feature uniformly present in all 66 patients was impaired visual acuity. All affected patients had at least 1 line of decrease from 20/20 in visual acuity. Numerous patients were reexamined over several years, and no instances of unaccounted reduction in vision were noted. Interestingly, several youngsters showed what we felt was an age-related improvement in visual acuity as they got older. The general decrease in visual acuity seen in our patients, although at times minimal, was found to be a more consistent feature than the history of night vision problems by which this disorder is defined. This unexpected observation became, over the course of the study, the most valuable means of identifying possibly affected family members. The clinical vari ability seen in our patients also suggests the presence of other genetic factors modifying the phenotype of this disorder. Since incomplete CSNB is an X-linked disorder, the clinical findings generally reported have been for male patients. Three female subjects from family 60 with clinical manifestations were described previous ly.12 After identification of the gene, all three were found to be homozygous for the L1056insC muta tion.9 In this report we describe the identification of two additional female subjects homozygous for the L1056insC mutation from family 60. Given the apparently high frequency of this specific mutation in the Mennonite community, manifesting female sub jects homozygous for the L1056insC mutation are not entirely unexpected and would be the product of a union between two people with a common ancestor. The L1056insC mutation in CACNAJF seen in this
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set of patients with incomplete X-linked CSNB disrupts the reading frame of the protein. This is pre dicted to cause the truncation of the protein at amino acid position 1066, resulting in the loss of function of the a 1F-subunit and, consequently, the calcium chan nel. We have suggested that loss of function of the calcium channel will impair the flux of calcium into both rods and cones that is required for neurotrans mitter release from presynaptic terminals in the dark adapted retina. 9 As a result, the ON-bipolar cells will be inadequately stimulated and will remain in a chron ic light-adapted state, rendering the patient unable to respond to low-level light changes (night blindness). In the past, X-linked CSNB has been defined by what was believed to be the characteristic feature, night blindness. It has also been referred to as "X linked night blindness with high myopia." However, in the families that we studied, 40% of 62 subjects had no complaints of night vision difficulties, and 15% of 132 eyes had no myopia. We found that the most valuable clinical finding for identifying family members who were likely to be affected was an impairment in visual acuity. Confirmation of the final diagnosis required observation of the specific ERG changes characteristic of incomplete CSNB. In light of these findings, incomplete CSNB would be more accurately defined as X-linked amblyopia with myopia, night blindness and nystagmus, or, perhaps more appropriately given the variability in these major features, as X-linked visual impairment. We thank the participating families and especially the large Mennonite kindred that prompted our interest in identifying the incomplete congenital stationary night blindness gene. This research was supported in part by the Retinitis Pigmentosa Research Foundation (Canada) and the l.D. Bebensee Foundation. Dr. Bech-Hansen was supported by the Department of Ophthalmology, University of Alberta, and the Alberta Children's Hospital Foundation and is the Roy Allen Investigator in Eyesight Research, Faculty of Medicine, University of Calgary. Dr. Boycott is the recipi ent of an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship. REFERENCES
1. Carr RE. Congenital stationary night blindness. Trans Am
Ophthalmol Soc 1974;72:449-87. 2. Pearce WG, Reedyk M, Coupland SG. Variable expressiv ity in X-linked congenital stationary night blindness. Can J Ophthalmol 1990;25:3-10. 3. Heon E, Musarella MA. Congenital stationary night blind ness: a critical review of molecular approaches. In: Wright
Clinical variability-Boycott et al
4.
5. 6.
7.
8. 9.
10.
11.
12.
13.
AF, Jay B, editors. Molecular genetics of inherited eye dis orders. Chur (Switzerland): Harwood Academic Pub lishers; 1994. p. 277-301. Miyake Y, Yagasaki K, Horiguchi M, Kawase Y, Kanda T. Congenital stationary night blindness with negative elec troretinogram: a new classification. Arch Ophthalmol 1986;104:1013-20. Tremblay F, LaRoche RG, De Becker I. The electroretino graphic diagnosis of the incomplete form of congenital sta tionary night blindness. Vision Res 1995;35:2383-93. Miyake Y, Horiguchi M, Ota I, Shiroyama N. Charac teristic ERG flicker anomaly in incomplete congenital stationary night blindness. Invest Ophthalmol Vis Sci 1987;28:1816-23. Boycott KM, Pearce WG, Musarella MA, Weleber RG, Maybaum TA, Birch DG, et al. Evidence for genetic het erogeneity in X-linked congenital stationary night blind ness. Am J Hum Genet 1998;62:865-75. Collins FS. Positional cloning: let's not call it reverse any more. Nature Genet 1992;1:3-6. Bech-Hansen NT, Naylor MJ, Maybaum TA, Pearce WG, Koop B, Fishman GA, et al. Loss-of-function mutations in a calcium-channel o: 1-subunit gene in Xpl 1.23 cause incomplete X-linked congenital stationary night blindness. Nature Genet 1998;19:264-7. Strom TM, Nyakatura G, Apfelstedt-Sylla E, Hellebrand H, Lorenz B, Weber BHF, et al. An L-type calcium-channel gene mutated in incomplete congenital stationary night blindess. Nature Genet 1998;19:260-3. Bech-Hansen NT, Boycott KM, Gratton K, Ross DA, Pearce WG. Localization of a gene for incomplete X linked congenital stationary night blindness to the interval between DXS6849 and DXS8023 in Xpl 1.23. Hum Genet 1998;103: 124-30. Bech-Hansen NT, Pearce WG. Manifestations of X-linked congenital stationary night blindness in three daughters of an affected male: demonstration of homozygosity. Am J Hum Genet 1993;52:71-7. Epp FH. Mennonites in Canada, 1786-1920. Toronto: Macmillan of Canada; 1974. p. 183-206.
Key words: Mennonite, incomplete congenital stationary night blindness, night blindness, visual acuity, myopia, nystagmus
lesions. It could be the little boy with bilaterally sub normal vision of apparently unknown origin. It could also be the little boy with bilaterally reduced vision and a high refractive error, who does not necessarily have "bilateral refractive amblyopia" but may have incomplete CSNB. Unlike the experience of Boycott and colleagues, we have seen patients with X-linked incomplete CSNB with unusually pale optic discs, who even had delayed implicit times on visual evoked potential testing. Confirming the diagnosis of incomplete CSNB is important because we can tell the patient this is a non progressive disorder and we can explain the X-linked mode of transmission. The ERG remains the ultimate diagnostic tool for confirming incomplete CSNB. The classic criteria for CSNB, described by Miyake and associates, 1 are the negative appearance of the scotopic bright-flash response as well as a severely attenuated photopic re sponse. They base the differential diagnosis between the complete and incomplete forms on the absence or presence respectively of rod-related activity. This cri terion is sometimes difficult to evaluate, particularly in children, in whom contaminated recordings are com mon. We find that it is easier to detect the absence of oscillatory potentials (OPs) in photopic conditions and their large amplitude in scotopic conditions. In con trast, patients with complete CSNB always present with a large OP4 in photopic and scotopic conditions, which corresponds to the exclusive activation of the OFF system. 2 Finally, we agree that the abnormal scotopic retinal function does not translate clearly into a significant complaint of night blindness and that bilaterally sub normal visual acuity remains the one constant feature of incomplete CSNB. Inge De Becker, MD, FRCSC Franfois Tremblay, PhD Department of Ophthalmology Dalhousie University Halifax, NS
Discussion
I
n this paper Kym Boycott and colleagues report clinical information pertinent to the clinician, who likely sees patients with X-linked incomplete CSNB but doesn't know it. Who are these patients? It could be the male with high bilateral myopia and slightly reduced visual acu ity, who is being examined for peripheral retinal
REFERENCES
1. Miyake Y, Yagasaki K, Horiguchi M, Kawase Y, Kanda T.
Congenital stationary night blindness with negative elec troretinogram: .a new classification. Arch Ophthalmol 1986;104:1013-20. 2. Tremblay F, LaRoche RG, De Becker I. The electroretino graphic diagnosis of the incomplete form of congenital sta tionary night blindness. Vision Res 1995;35:2383-93.
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Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in
CACNAJF
Kym M. Boycott,* PhD; William G. Pearce, t MD; N. Torben Bech-Hansen,* PhD ABSTRACT • RESUME Background: Incomplete X-linked congenital stationary night blindness (CSNB) is a clinically variable condition that has been shown to be caused by mutations in the calcium-channel CACNA IF gene. We assessed the clinical variability in the expres sion of the incomplete CSNB phenotype in a subgroup of patients of Mennonite ancestry with the same founder mutation. Methods: Sixty-six male patients from 15 families were identified with a common mutation in exon 27 of CACNA IF (LI 056insC). Clinical variability in night blindness, reduced visual acuity, myopia, nystagmus and strabismus was examined. Results: At least one of the major features of CSNB (night blindness, myopia and nys tagmus) was absent in 72% of the patients. All the examined features varied wide ly, both between and within families. Interpretation: Although the patients shared a common CACNA IF muta
tion, there was considerable variability in the clinical expression of the incomplete CSNB phenotype. These findings suggest the presence of other genetic factors modifying the phenotype of this disorder.
Contexte: La cecite nocturne stationnaire congenitale partielle, liee au chromo
some X (CSNB), est une condition cliniquement variable et attribuable a des mutations du gene porteur de calcium CACNA IF. Nous avons etudie la vari abilite clinique de !'expression du phenotype de la CSNB partielle chez un sous groupe de patients de souche mennonite ayant la meme mutation originelle. Methodes : Nous avons retenu 66 patients du sexe masculin provenant de 15 families ayant une mutation commune de l'exon 27 du CACNA IF (LI 056insC). Nous avons examine la variabilite clinique de la cecite nocturne, de la baisse d'acuite visuelle, de la myopie, du nystagmus et du strabisme.
From *the Department of Medical Genetics, Faculty of Medicine, University of Calgary, Calgary, Alta., and tthe Department of Oph thalmology, Faculty of Medicine, University of Alberta, Edmonton, Alta.
Reprint requests to: Dr. N. Torben Bech-Hansen, Department of Medical Genetics, Faculty of Medicine, HMRB311, 3330 Hospital Dr. NW, Calgary AB T2N 4Nl; fax (403) 210-8119, [email protected]
Accepted for publication Feb. 12, 2000
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Clinical variability-Boycott et al
Clinical variability-Boycott et al Resultats : Au total, 72% des patients ne presentaient pas au mains une des prin cipales caracteristiques de la CSBN (cecite nocturne, myopie ou nystagmus). Les caracteristiques etudiees variaient considerablement tant au sein des families qu'entre elles. Interpretation : Bien que les patients partagent une mutation com mune du CACNA IF, I'expression clinique du phenotype de la CSNB partielle varie considerablement. Ces constatations incitent a penser que d'autres facteurs genetiques modifient le phenotype de cette maladie.
X
-linked congenital stationary night blindness (CSNB) is a heterogeneous nonprogressive reti nal disorder characterized clinically by night blindness, decreased visual acuity, myopia, nystagmus and stra bismus.1-3 Clinical heterogeneity among families has led to the classification of X-linked CSNB into com plete and incomplete forms. 4 This division is based fundamentally on abnormalities in the electroretino gram (ERG) (e.g., the scotopic b-wave, the oscillatory potentials and cone function) and in the psychophysi cal dark-adapted thresholds. In patients with complete CSNB the scotopic b-wave evoked by dim blue flash es and the early oscillatory potential wavelets are unrecordable. The night blindness is profound, as doc umented by the absence of rod dark adaptation. The photopic system in these patients is mildly abnormal. In patients with incomplete CSNB the scotopic b-wave is recordable although subnormal in amplitude, the scotopic oscillatory potentials are often recordable but may show abnormal implicit times, 5 and the psycho physical dark-adapted thresholds are variably eleva ted above normal. Under photopic conditions the b-wave is barely recordable, no oscillatory potentials are elicited, 5 and the 30-Hz flicker is reduced. 4 In addi tion, when the eye is dark adapted and stimulated con tinuously for 12 to 15 minutes with a 30-Hz flicker, there is an exaggerated increase in amplitude and a characteristic change in ERG shape. 3·6 X-linked CSNB is not readily identified clinically owing to the lack of specificity of the main clinical fea tures (night blindness, reduced visual acuity, myopia and nystagmus). Another contributing factor is the absence of a clinically identifiable structural abnormal ity of the retina. 2 Part of the clinical variability seen in X-linked CSNB can now be attributed to genetic het erogeneity, the gene for complete X-linked CSNB (CSNBl) mapping to Xpl 1.4, and the gene for incom plete X-linked CSNB (CSNB2) mapping to Xpl 1.23. 7
Efforts to isolate the gene responsible for incom plete CSNB were based on a positional cloning approach 8 and have culminated in the discovery of mutations in a new retina-specific voltage-gated L type calcium-channel a 1-subunit gene, designated CACNAJF. 9 The CACNAJF gene contains 48 exons 9 and has a predicted protein length of 1977 amino acids in the larger isoform (N.T.B.H., unpublished observa tions, 1999). Analysis of CACNAJF in 20 families with incomplete CSNB showed six different muta tions, all of which were predicted to cause premature protein truncation. 9 Parallel studies by Strom and col leagues10 confirmed these observations. These find ings established that mutations in CACNAJ F cause incomplete CSNB, making this disorder an example of a human calcium-channel disease of the retina. One of the mutations identified in CACNAJF, an insertion of a C nucleotide in exon 27 (Ll056insC), predicted to cause a frameshift and premature trunca tion of the protein product, was present in 15 different families of Mennonite ancestry. To address the ques tion of clinical variability within a set of subjects with the same underlying molecular defect, we examined the clinical findings in a large set of patients from these families with the L1056insC mutation in the CACNAJF gene. METHODS
Fifteen families (families 50, 60, 60B, 80, 130, 150, 160, 170, 180, 190, 200, 240, 250, 330 and 340) shared part of a common Mennonite haplotype. The same mutation in CACNAJF was subsequently shown to segregate in these families. 7•9 The family structure and clinical findings in three of these kindreds have previously been reported (families 50 [B], 2 80 [I] 2 and 60 [C] 2·11 •12). The one base pair insertion in exon 27 of CACNAJF (L1056insC) was initially identified by
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Clinical variability-Boycott et al
Table I-Normative electroretinography data* Light-adapted Feature Time, ms a-wave b-wave Amplitude, µV a-wave b-wave
Dark-adapted
Bright white
White flicker
Dim blue
Bright white
12-20 35-40
30-35
65-90
15-25 45-55
50-100 120-180
80-130
90-170
100-175 200-400
*Within 2 standard deviations of the mean. Source: Retinal Testing Unit, Department of Ophthalmology, University of Alberta, Edmonton.
direct DNA sequencing of the exon, amplified by polymerase chain reaction (PCR), from a patient with incomplete CSNB from family 60. 9 Detection of the L1056insC mutation in other families sharing the Mennonite haplotype and segregation analysis within families were based on the separation of radiolabelled PCR products in 6% polyacrylamide gels. 9 The L1056insC mutation was originally designated as L991insC, 9 but the designation was revised after splice variants were identified in this gene (N.T.B.H., unpublished observations, 1999). Members of each family underwent an ophthalmo logic examination, and the diagnosis was made as described elsewhere. 2•11 •12 Briefly, we recorded the ERG using the Nicolet CA 2000 electrodiagnostic sys tem with a Nicolet Ganzfeld photostimulator and gold foil electrodes (C.H. Electronics, London). The ear lobe electrodes were Grass Gold Cup with Nicolet electrolyte gel, and the surface electrodes were Nicolet gold surface electrodes with Ten 20 electroencepha lography paste. Following maximum dilation of the pupils with 1% tropicamide drops and topical anesthe sia with 0.5% proparacaine drops, gold foil electrodes were inserted into the lower fomix of each eye. The earlobes served as ground. The recording gold foil electrodes were referenced to the forehead electrode. Cone responses were obtained after 5 minutes of light adaptation with a Ganzfeld background light of 10 foot lambert. Bright white flashes with a relative strobe intensity of 1.50 (10.02 cd · m2/s) were pre sented. This was followed by a white flash of strobe intensity 0.75 (2.11 cd · m2/s) flickered at 31.1 Hz. Low and high filter frequencies of 1 Hz and 100 Hz respectively were used with a sensitivity of 500 µV. Rod responses were obtained after 25 minutes of dark
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adaptation. Dim blue flashes were presented with the use of Wratten filters #47, #47A and #47B in com bination with a relative strobe intensity of 0.25 (0.81 cd · m2/s) at a rate of 0.9/s. A single bright white flash with a relative strobe intensity of 0.75 (2.11 cd · m2/s) was then presented. Low and high filter fre quencies of 1 Hz and 100 Hz respectively were used. Sensitivity was set at 500 µV for the dim blue flash and 1000 µV for the bright white flash. Normative data are shown in Table 1. We performed dark adaptometry using the Goldmann-Weekers adaptometer. After the pupils were maximally dilated with 1% tropicamide drops, the patient underwent preadaptation for 5 minutes with the adaptometer calibrated at a luminosity of 3400 lx. Illumination was then extinguished, and, with the Goldmann-Weekers dim white circular target of 5.5, responses were obtained at a rate of at least two per minute for 30 minutes. Normative data are not shown. RESULTS
Mutation analysis
Mutation screening by direct DNA sequencing in an affected male from family 60 identified the insertion of a C nucleotide in the DNA sequence of exon 27 of the CACNAIF gene. 9 Screening of the remaining members of family 60 and the 14 other families by PCR amplification of exon 27 showed the presence of a one base pair insertion, consistent with segregation of the mutation in the 15 families. Segregation of this L1056insC mutation in a part of family 60 is shown in Fig. 1, top. This portion of the family was also of inter est because of the identification of two female mem
Clinical variability-Boycott et al
C4
*
*
*E107 E101
235 bp 234 bp
T
1066 A
G
Normal male G
A
A
G
T
Female
:
/
·.C _ / 1056~G 11T
1055
ft
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Pro
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Fig. I-Top: Analysis of the LI056insC mutation in members of family 60 with incomplete X linked congenital stationary night blindness. Exon 27 from CACNA IF was amplified by poly merase chain reaction (PCR), and the products were separated by gel electrophoresis accord ing to size. The LI 056insC mutation results in the insertion of a C nucleotide within exon 27, making the amplified product one base larger in affected members. Therefore, affected mem bers have a 235 base pair (bp) PCR product, whereas unaffected members have a 234 bp prod uct. Carrier females have both a 234 bp and a 235 bp product, representing the alleles on the two different X chromosomes. The clinical findings in patients C4, EI 0 I and EI 07 are described in Table 2. For the remaining family members the phenotype is inferred from the genotype. An asterisk indicates that haplotype construction using polymorphic X chromosome markers (unpublished observations) has shown that three of the males in the third generation inherited the mutant chromosome from their maternal grandfather (also indicated with an asterisk), whereas the two other males inherited the mutant chromosome from their carrier maternal grandmother. Bottom: Direct DNA sequencing of the exon 27 PCR product in one of the females from family 60 homozygous for the LI 056insC mutation. An extra C nucleotide is inserted in exon 27 on both copies of her X chromosome. Homozygosity of the mutation is shown by the absence of bands representing two different X chromosomes, as would be seen in a carrier female. The extra C nucleotide at this position produces a frameshift mutation. At codon I056 the leucine (Leu) seen in the normal protein has been changed to a proline (Pro). The amino acid sequence continues to be altered until codon I066, which is translated into a stop codon, and synthesis of the protein is terminated at this point. Arg =arginine, Asp =as perigine.
bers homozygous for the L1056insC mutation. To explain this finding, it is assumed that the deceased father of these females was also affected with incom
plete CSNB due to the L1056insC mutation. The DNA sequence of this region of exon 27 from one of the homozygous females is shown in Fig. 1, bottom.
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Left eye
Visual acuity
-I 2.S0+4.00 x IOS -2.SO -9.SO+ 1.00 x as -6.7S+2.2S x I00 -2.2S+ 1.00 x I6S -7.7S+ I.7S x ISS --0.2S+ I.7S x I6S -4.7S+ 1.00 x 7S -3.00+0.7S x 180 -6.2S+ I.2S x 90 + I .00+0.7S x 70 + 1.00+ 1.00 x I4S -7.00+ 1.00 x I2S +4.SO+O.SO x 90 +0.2S+0.2S x 90 +O.S0+0.2S x 180 +2.00+0.SO x 90 -12.2S+l.7SX 120 -I.SO+ 1.7S x 120 -3.00+2.SO x I2S -4.2S+ 3.7S x 90 +O.SO+ I.2S x I00 -9.SO+ 3.2S x 90 -2.S0+2.7S x 11 S -8.7S+4.2S x I00 -6.00+ I.7S x 9S -8.2S+3.00X9S -7.S0+2.00 x 9S -8.00+ l.7S x I00 Plano+2.00 X 90 --0.2S+2.00 x 90 -4.7S+3.2S x I I0 -7.2S+2.SO x 90 + 1.00+ I .2S x 9S +I .S0+2.00 x 80
Right eye - I2.S0+400 x 80 -3.SO -9.SO+O.SO x 90 -7.2S+2.SO x 70 -3.7S+3.00X 180 -7.7S+ I.SO x IS -7.00+ I.2S X IS -2.SO -3.2S -6.2S+ I.3S x 90 +2.S0+0.2S x 90 +2.00+0.7S x 3S -S.7S+ 1.00 x so +4.SO+ I.2S x as --0.2S+O.SO x 90 -0.7S+l.2SX IOS +I .SO+ 1.00 X90 - I0.7S+ I.SOX SS -I .SO+ I.7S x 70 -4.S0+2.00 x as -2.7S+2.2S x 90 + 1.00+ I.2S x 70 - I0.00+2.7S x 90 -2.2S+2.SO x 6S -6.00+ 3.00 x 80 -6.2S+2.2S x 90 -8.2S+ 3.00 x 90 -6.2S+ 1.00 x as -S.7S -I .2S+2.00 x 90 --0.7S+2.00 x 90 -4.SO+ 3.SO x 90 -8.00+3.00 x 90 + 1.00+ I.2S x as +2.2S+2.2S x 100
Left eye
Refractive error Present Present Present Absent Present Present Present Present Present Present Absent Present Present Absent Absent Absent NA Present Present Absent Present Absent Present Present Absent Absent Present Absent Present Present Absent Present NA Absent Absent
Night blindness symptoms
1.0 O.S
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3.0 2.0 2.0 l.S N
1.0 2.0 O.S 1.0 l.S
2.S 3.0 2.0 2.S
2.S
1.0
Dark adaptation (rod), log units elevation Present Present Absent Absent Present Absent Absent Present Present Present Absent Absent Absent Absent Absent Absent Present Absent Absent Present Present Absent Absent Absent Absent Absent Absent Absent Present Absent Absent Absent Present Absent Latent
Congential nystagmus
Table 2-Clinical findings in 66 members of 15 families with incomplete X-linked congenital stationary night blindness*
(Continued)
Esotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Esotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Exotropia Exotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Exotropia Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Esophoria
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2SO 330 340
240
200
190
180
160 170
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80 130
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20/40 20/SO 20/80 20/60 20/40 20/SO 20/SO 20/SO 20/30 20/40 20/60 20/400 20/100 20/60 20/40 20/40 20/30 20/40 20/40 20/60 20/100 20/SO Follows Poor fix. 20/60 20/200 20/40 20/SO I-mm 20/70 20/40
8 9 37 17 27 24 27 12 10 9 9 7 4 IS 12
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Left eye
Visual acuity
Age, yr
Table 2-Continued
-l.00+0.7S x IOS - I8.7S+ 1.00 x I I S -3.S0+2.SO x IOS -I l.S0+2.7SX 100 -16.00+ l.2S x IOS - I I .S0+2.00 x I2S -6.S0+4.00 x I00 -6.2S+0.7S x IS -7.00+ 1.00 x I00 - IO.S0+0.7S x I3S -3.00+0.SO x 90 - I0.2S+ 2.2S x 8S -3.00 -8.S0+2.00 x 9S -9.00+ I.2S x 80 -4.S0+2.00 x 90 Plano+ I .2S X 140 -2.00+0.SO x I00 -4.00+ 1.00 x 170 - IO.SO+ 3.00 x S -1.00+ 1.00 x 90 -6.2S+ I.7S x 90 -S.00+ 3.00 x 90 -6.00+0.SO x 90 -3.2S+ 3.2S x I IS -S.00+3.00 x 90 -l.2S+ l.7S x 8S -7.7S+l.SOX 110 -2.00+2.00 x 90 -2.SO+ l.7S x IOS +0.2S+0.7S x 80
Right eye -0.SO+ I.2S x 7S - I 8.7S+0.7S x 60 -3.7S+2.2S x 90 - I0.00+ 2.SO x 70 -I S.2S+2.2S x I2S - IO.SO+2.SO x 60 -4.SO+3.2S x 60 -S.SO+O.SO x 6S -S.2S -9.SO+ 1.00 x 30 -4.SO+O.SO x 90 -9.2S+2.2S x 80 -2.SO -8.00+4.00 x 90 -9.00+2.2S x 8S -4.00+2.2S x 90 -0.2S+ I.2S x 6S -2.2S+O.SO x so -3.7S+O.SO x 180 - I I.SO+ 3.SO X 170 +0.2S+ 1.00 x 90 -4.00+ I.SO x 90 -S.00+3.00 x 90 -S.SO+O.SO x 90 -I .7S+2.2S x 90 -6.00+4.SO x 9S -0.2S+O.SO x 180 -6.7S+ l.2S x 7S -2.S0+2.00 x 90 -2.S0+0.7S x 6S -l.7S+l.2SX9S
Left eye
Refractive error Present Absent Present Present Present Present Absent Present Absent Absent Absent Present Present Absent Absent Absent Present Present Present Present Present Absent NA NA Present Present Absent Absent Absent Present Present
Night blindness symptoms
1.0
1.0 1.0
1.0 1.0
2.0 2.0
O.S 2.0
1.0 l.S 1.0 1.0 l.S l.S l.S l.S l.S
O.S 2.0
Dark adaptation (rod), log units elevation Absent Absent Present Present Absent Absent Present Absent Absent Absent Present Present Present Present Absent Absent Absent Absent Absent Present Present Present Present Present Absent Present Absent Present Present Present Present
Congential nystagmus
Orthophoria Orthophoria Exotropia Orthophoria Orthophoria Exotropia Exotropia Orthophoria Orthophoria Orthophoria Exotropia Orthophoria Esotropia Exotropia Orthophoria Orthophoria Orthophoria Exophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria Orthophoria NA Orthophoria Orthophoria Orthophoria Orthophoria Exotropia
Strabismus
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Clinical variability-Boycott et al Clinical findings
The clinical findings in the 66 patients are shown in Table 2. Of the 66 patients 32 were from family 60, and 34 were from the 14 other families. Important clinical features were absent in 45 of 62 patients: in 26 patients one major feature was absent (night blindness in 7, and nystagmus in 19), 1 had neither night blindness nor myopia, 13 had neither night blindness nor nystagmus, 1 had neither myopia nor nystagmus, and 4 did not have night blindness, myopia or nystagmus.
ily 190 [Dl5] and one patient from family 340 [A3400]) the refractive error was plano (2 eyes) or hyperopic (18 eyes). In one patient from family 60 [E80] the hyperopia was +4.50 dioptres in both eyes. Fifty-one eyes had low myopia (-0.25 D to -4.75 D), 44 had moderate myopia (-5.00 D to -9.75 D), and 17 had high myopia (-10.00 D or more). Considerable variation was present even within the same family. A difference in refractive error of about 10 D was found between two sets of brothers in family 60 (E37/E77 and E42/E49).
Nystagmus
In 62 cases it was possible to obtain information about visual performance in a dim or dark environ ment. Thirty-seven patients had a history of impaired night vision, and in 25 there was no suggestion of a night vision problem on specific questioning.
Congenital nystagmus was present in 29 patients (including latent nystagmus in 1) and absent in 37 patients. Table 3 shows the relation of nystagmus to visual acuity in the 60 patients in whom visual acuity could be assesed with the Snellen eye chart. The pres ence of nystagmus was associated with a reduction in visual acuity.
Dark adaptometry
Strabismus
Dark adaptometry was performed in 42 patients, of whom 40 showed variable elevation of the rod seg ment. Of the 40, 25 reported difficulty with vision in a dim environment: in 11 the elevation was mild (1.0 log unit or less), in 9 it was moderate (1.5 to 2.0 log units), and in 5 it was marked (more than 2.0 log units). The remaining 15 patients had no histo ry of visual difficulties in a dim environment; 8 had mild elevation of the rod segment, and 7 had moderate elevation. The rod segment was not elevated in two of the patients from family 60 (E4 and E42), although both demonstrated the exon 27 founder mutation and had other features consistent with CSNB, most notably a negative ERG (bright flash elicits a normal a-wave and ab-wave that has less amplitude than the a-wave). Neither patient reported impaired night vision.
Of the 65 patients who underwent testing for stra bismus, 9 had exotropia, 3 had esotropia, 1 had exo phoria, and 1 had esophoria.
Impaired night vision
Visual acuity Visual acuity was reduced in all 60 patients in whom this clinical feature could be determined. The impairment ranged from 20/25 to 20/400.
Myopia In 20 eyes (both eyes of seven patients from family 60 [El4, E16, E42, E80, E84, E130 and E144] and one eye of three patients from family 60 [E73, E81 and E120], one patient from family 180 [G3], one patient from fam
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Funduscopy No specific or pathognomonic fundus abnormalities were seen. In patients with lower degrees of myopia Table 3-Visual acuity of 60 patients (right eye) by presence or absence of congenital ny stagmus No. of eyes
Visual acuity 20/25 20/30 20/40 20/50 20/60 20/70 20/80 20/100 20/200 20/400
Nystagmus present (n = 24)
Nystagmus absent (n = 36)
0 0 2 6 4 2* 4 3 2
I 9 12 10 2 2 0 0 0 0
*Latent in one patient (patient E16 from family 60)
Clinical variability-Boycott et al
Case IV-40
Normal
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4882t_/ 48.82t~
µV/d
µV/d
µV/d
Case Ill-A
97.65t~ µV/d
ERG
48.82t~ µV/d
48.82t~
Scotopic response
48.82t~ µV/d
48.82t~ µV/d
Cone response
µV/d
Bright-flash
µV/d
I
48.82 [ " n. -~ _ µV/d [ \ _ / ' \ _ / \_,,/ '-.../
48.82 [ µVid[
25 ms/d
30-Hz flicker
25 ms/d
25 ms/d
Fig. 2-Full-field electroretinography (ERG) responses in a normal subject (left) and two patients with incomplete congenital sta tionary night blindness and the exon 27 founder mutation in CACNA IF. Case IV-40 is patient E36 from family 60, and case Ill-A is patient D 174 from family 60B. In both patients a bright flash elicits a normal a-wave and a b-wave that has less amplitude than the a-wave (negative ERG). The scotopic dim blue response is reduced but detectable, and both the b-wave of the cone response and the 30-Hz flicker response are also reduced (reproduced from reference 11 with permission from Springer-Verlag, Berlin).
the fundus appeared essentially normal. In some of the patients with moderate or high myopia the fundus showed characteristic myopic changes, including tem poral sector pallor of the optic nerve head and variable attenuation of the retinal pigment epithelium.
Electroretinography Scotopic or photopic ERG, or both, was performed in 56 patients, including at least 1 member from each of the 15 families. The results were consistent with a diag nosis of incomplete CSNB. All patients had a subnor mal scotopic b-wave or a negative ERG or both. The flicker and photopic responses in all 56 patients were significantly abnormal. Representative ERG findings in an unaffected male and two affected males from family 60 (IV-40) and 60B (III-A) are shown in Fig. 2. INTERPRETATION
Sixty-six affected male patients from 15 different
families with incomplete CSNB were shown to have the L1056insC mutation in exon 27 of the CACNAJ F gene. All 15 families are of Mennonite descent and are presumed to share a common ancestor, although we cannot currently demonstrate their relationship on a unifying pedigree.7· 9 Between 1874 and 1880 near ly 7000 Mennonites immigrated to Manitoba from Russia. 13 Our suspicion that the L1056insC mutation originated before this wave of immigration is sup ported by the observation that the same mutation has been shown to be present in a family with incomplete CSNB in Germany. 10 It is likely that other descen dants of the Anabaptist movement and members of the Mennonite community in Canada, the United States and other parts of the world are also affected with incomplete CSNB due to the L1056insC muta tion in CACNAJ F. Although this disease is called CSNB, 40% of the patients in our study did not have a history of diffi culty with vision in conditions of dim illumination. However, almost all of these patients were found to
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Clinical variability-Boycott et al have defective rod function on dark adaptometry, although none showed marked elevation. We found considerable variation in refractive error, even within families (+4.50 D to -18.75 D); in two instances there was a difference of more than 10 D between brothers. Visual acuity ranged from 20/25 to 20/400 and varied widely within families. Nystagmus was present in 44% of the patients, and its presence was associated with a greater decrease in visual acuity. However, when nystagmus was absent, the visual acuity was also reduced. These findings confirm that cone as well as rod function is impaired in patients with incomplete X-linked CSNB. Strabismus was not as common, being present in 22% of the patients. Although our patients share the common CACNAJF mutation, one or more of the three charac teristic features of night blindness, myopia and nys tagmus were absent in over 72% of cases. The most notable clinical feature uniformly present in all 66 patients was impaired visual acuity. All affected patients had at least 1 line of decrease from 20/20 in visual acuity. Numerous patients were reexamined over several years, and no instances of unaccounted reduction in vision were noted. Interestingly, several youngsters showed what we felt was an age-related improvement in visual acuity as they got older. The general decrease in visual acuity seen in our patients, although at times minimal, was found to be a more consistent feature than the history of night vision problems by which this disorder is defined. This unexpected observation became, over the course of the study, the most valuable means of identifying possibly affected family members. The clinical vari ability seen in our patients also suggests the presence of other genetic factors modifying the phenotype of this disorder. Since incomplete CSNB is an X-linked disorder, the clinical findings generally reported have been for male patients. Three female subjects from family 60 with clinical manifestations were described previous ly.12 After identification of the gene, all three were found to be homozygous for the L1056insC muta tion.9 In this report we describe the identification of two additional female subjects homozygous for the L1056insC mutation from family 60. Given the apparently high frequency of this specific mutation in the Mennonite community, manifesting female sub jects homozygous for the L1056insC mutation are not entirely unexpected and would be the product of a union between two people with a common ancestor. The L1056insC mutation in CACNAJF seen in this
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set of patients with incomplete X-linked CSNB disrupts the reading frame of the protein. This is pre dicted to cause the truncation of the protein at amino acid position 1066, resulting in the loss of function of the a 1F-subunit and, consequently, the calcium chan nel. We have suggested that loss of function of the calcium channel will impair the flux of calcium into both rods and cones that is required for neurotrans mitter release from presynaptic terminals in the dark adapted retina. 9 As a result, the ON-bipolar cells will be inadequately stimulated and will remain in a chron ic light-adapted state, rendering the patient unable to respond to low-level light changes (night blindness). In the past, X-linked CSNB has been defined by what was believed to be the characteristic feature, night blindness. It has also been referred to as "X linked night blindness with high myopia." However, in the families that we studied, 40% of 62 subjects had no complaints of night vision difficulties, and 15% of 132 eyes had no myopia. We found that the most valuable clinical finding for identifying family members who were likely to be affected was an impairment in visual acuity. Confirmation of the final diagnosis required observation of the specific ERG changes characteristic of incomplete CSNB. In light of these findings, incomplete CSNB would be more accurately defined as X-linked amblyopia with myopia, night blindness and nystagmus, or, perhaps more appropriately given the variability in these major features, as X-linked visual impairment. We thank the participating families and especially the large Mennonite kindred that prompted our interest in identifying the incomplete congenital stationary night blindness gene. This research was supported in part by the Retinitis Pigmentosa Research Foundation (Canada) and the l.D. Bebensee Foundation. Dr. Bech-Hansen was supported by the Department of Ophthalmology, University of Alberta, and the Alberta Children's Hospital Foundation and is the Roy Allen Investigator in Eyesight Research, Faculty of Medicine, University of Calgary. Dr. Boycott is the recipi ent of an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship. REFERENCES
1. Carr RE. Congenital stationary night blindness. Trans Am
Ophthalmol Soc 1974;72:449-87. 2. Pearce WG, Reedyk M, Coupland SG. Variable expressiv ity in X-linked congenital stationary night blindness. Can J Ophthalmol 1990;25:3-10. 3. Heon E, Musarella MA. Congenital stationary night blind ness: a critical review of molecular approaches. In: Wright
Clinical variability-Boycott et al
4.
5. 6.
7.
8. 9.
10.
11.
12.
13.
AF, Jay B, editors. Molecular genetics of inherited eye dis orders. Chur (Switzerland): Harwood Academic Pub lishers; 1994. p. 277-301. Miyake Y, Yagasaki K, Horiguchi M, Kawase Y, Kanda T. Congenital stationary night blindness with negative elec troretinogram: a new classification. Arch Ophthalmol 1986;104:1013-20. Tremblay F, LaRoche RG, De Becker I. The electroretino graphic diagnosis of the incomplete form of congenital sta tionary night blindness. Vision Res 1995;35:2383-93. Miyake Y, Horiguchi M, Ota I, Shiroyama N. Charac teristic ERG flicker anomaly in incomplete congenital stationary night blindness. Invest Ophthalmol Vis Sci 1987;28:1816-23. Boycott KM, Pearce WG, Musarella MA, Weleber RG, Maybaum TA, Birch DG, et al. Evidence for genetic het erogeneity in X-linked congenital stationary night blind ness. Am J Hum Genet 1998;62:865-75. Collins FS. Positional cloning: let's not call it reverse any more. Nature Genet 1992;1:3-6. Bech-Hansen NT, Naylor MJ, Maybaum TA, Pearce WG, Koop B, Fishman GA, et al. Loss-of-function mutations in a calcium-channel o: 1-subunit gene in Xpl 1.23 cause incomplete X-linked congenital stationary night blindness. Nature Genet 1998;19:264-7. Strom TM, Nyakatura G, Apfelstedt-Sylla E, Hellebrand H, Lorenz B, Weber BHF, et al. An L-type calcium-channel gene mutated in incomplete congenital stationary night blindess. Nature Genet 1998;19:260-3. Bech-Hansen NT, Boycott KM, Gratton K, Ross DA, Pearce WG. Localization of a gene for incomplete X linked congenital stationary night blindness to the interval between DXS6849 and DXS8023 in Xpl 1.23. Hum Genet 1998;103: 124-30. Bech-Hansen NT, Pearce WG. Manifestations of X-linked congenital stationary night blindness in three daughters of an affected male: demonstration of homozygosity. Am J Hum Genet 1993;52:71-7. Epp FH. Mennonites in Canada, 1786-1920. Toronto: Macmillan of Canada; 1974. p. 183-206.
Key words: Mennonite, incomplete congenital stationary night blindness, night blindness, visual acuity, myopia, nystagmus
lesions. It could be the little boy with bilaterally sub normal vision of apparently unknown origin. It could also be the little boy with bilaterally reduced vision and a high refractive error, who does not necessarily have "bilateral refractive amblyopia" but may have incomplete CSNB. Unlike the experience of Boycott and colleagues, we have seen patients with X-linked incomplete CSNB with unusually pale optic discs, who even had delayed implicit times on visual evoked potential testing. Confirming the diagnosis of incomplete CSNB is important because we can tell the patient this is a non progressive disorder and we can explain the X-linked mode of transmission. The ERG remains the ultimate diagnostic tool for confirming incomplete CSNB. The classic criteria for CSNB, described by Miyake and associates, 1 are the negative appearance of the scotopic bright-flash response as well as a severely attenuated photopic re sponse. They base the differential diagnosis between the complete and incomplete forms on the absence or presence respectively of rod-related activity. This cri terion is sometimes difficult to evaluate, particularly in children, in whom contaminated recordings are com mon. We find that it is easier to detect the absence of oscillatory potentials (OPs) in photopic conditions and their large amplitude in scotopic conditions. In con trast, patients with complete CSNB always present with a large OP4 in photopic and scotopic conditions, which corresponds to the exclusive activation of the OFF system. 2 Finally, we agree that the abnormal scotopic retinal function does not translate clearly into a significant complaint of night blindness and that bilaterally sub normal visual acuity remains the one constant feature of incomplete CSNB. Inge De Becker, MD, FRCSC Franfois Tremblay, PhD Department of Ophthalmology Dalhousie University Halifax, NS
Discussion
I
n this paper Kym Boycott and colleagues report clinical information pertinent to the clinician, who likely sees patients with X-linked incomplete CSNB but doesn't know it. Who are these patients? It could be the male with high bilateral myopia and slightly reduced visual acu ity, who is being examined for peripheral retinal
REFERENCES
1. Miyake Y, Yagasaki K, Horiguchi M, Kawase Y, Kanda T.
Congenital stationary night blindness with negative elec troretinogram: .a new classification. Arch Ophthalmol 1986;104:1013-20. 2. Tremblay F, LaRoche RG, De Becker I. The electroretino graphic diagnosis of the incomplete form of congenital sta tionary night blindness. Vision Res 1995;35:2383-93.
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