I-16 Zdrav Vestn Supl | junij 2012 | Letnik 81 Children with complete or incomplete congenital stationary night blindness IzvIrnI članek/OrIgInaL artIcLe Children with complete or incomplete congenital stationary night blindness: ophthalmological findings, standard erGs and On-OFF erGs for differentiation between types Otroci s prirojeno stacionarno nočno slepoto: oftalmološke značilnosti, standardni erg ter On-OFF erg razlikovanje med kompletno in nekompletno obliko Maja Šuštar, Branka Stirn-Kranjc, Jelka Brecelj abstract Purpose: Congenital stationary night blindness (CSNB) is a group of retinal disorders with di- verse clinical characteristics. The most common types are complete and incomplete CSNB. Both can have normal fundus appearance and an elec- tronegative waveform of the electroretinogram (ERG). Our aim was to define ERG differences between complete and incomplete CSNB in a pe- diatric population. Subjects and methods: In 12 children (5–18 years old) with clinical signs of stationary night blindness, standard full-field ERGs and ON-OFF ERGs were recorded. These were abnormal if the implicit time was above 95 % of the upper confi- dence limit and the amplitude below 5 % of the lower confidence limit of the normative data. Comparisons between the data were performed with Mann-Whitney U tests, with p < 0.01 con- sidered as significant. Results: According to the ERG characteristics, complete CSNB was diagnosed in 8 of the chil- dren, and incomplete CSNB in the remaining 4 children. Dark-adapted ERGs showed negative waveforms of the combined rod–cone response in all 12 children, with normal a-waves and re- duced b-waves, which indicated post-photore- ceptor dysfunction. No rod response was detect- able in the children with complete CSNB, with reduced rod response in those with incomplete CSNB. Light-adapted ERGs showed normal or subnormal amplitudes of the cone response and the 30-Hz flicker response in complete CSNB, where the cone response a-waves were also distinctly broadened. In the children with in- complete CSNB, the light-adapted ERGs were significantly reduced. In complete CSNB, the ON-OFF ERGs showed alterations of only the ON-response component (ON-bipolar cell dys- function), while in incomplete CSNB, both the ON- and OFF-responses were reduced (ON- and OFF-bipolar cell dysfunction). Comparisons of the ERG amplitudes between the children with complete and incomplete CSNB demonstrated significant differences in rod responses, cone responses, 30-Hz flicker responses, and OFF- responses. Conclusion: Distinct electrophysiological char- acteristics can be used to differentiate between complete and incomplete CSNB. Moreover, ON- OFF ERGs are important for precise localization of the retinal bipolar cell dysfunction, and these can also be reliably recorded in children. Izvleček Namen: Prirojena stacionarna nočna slepota (‘Congenital stationary night blindness’–CSNB) je mrežnična bolezen, za katero je značilen nor- malen izgled očesnega ozadja in elektronegativ- Eye Clinic, University Medical Centre Ljubljana, Ljubljana, Slovenia korespondenca/ Correspondence: Maja Šuštar, e-mail: sustar.majchi@gmail. com ključne besede: kompletna oblika cSnB, elektroretinogram, nekompletna oblika cSnB, nočna slepota, On-OFF erg, otroci key words: complete cSnB, electroretinogram, incomplete cSnB, night blindness, On-OFF erg, children Citirajte kot/Cite as: Zdrav Vestn 2012; 81: I-16–28 Prispelo: 9. jan. 2012, Sprejeto: 2. feb. 2012 Zdrav Vestn Supl | children with complete or incomplete congenital stationary night blindness I-17 IZVIrnI čLaneK/OrIgInaL artIcLe lovanja bipolarnih celic. Odgovor paličnic je bil neizziven pri kompletni obliki CSNB in izziven, a pomembno znižan pri otrocih z nekompletno obliko CSNB. Fotopični ERG je pri kompletni obliki CSNB pokazal normalno ali mejno abnor- mno amplitudo odgovora čepnic in odgovora 30 Hz, odgovor čepnic pa je kazal značilno razširje- no izoblikovanost vala a. Pri otrocih z nekomple- tno obliko CSNB je bil fotopični ERG pomemb- no znižan. ON-OFF ERG je pri kompletni obliki CSNB pokazal znižanje zgolj ON-odgovora (dis- funkcija ON-bipolarnih celic), medtem ko sta bila pri nekompletni obliki CSNB znižana tako ON- kot tudi OFF-odgovor (disfunkcija ON- in OFF-bipolarnih celic). Primerjava med komple- tno in nekompletno obliko CSNB je pokazala signifikantne razlike v amplitudi odgovora palič- nic, odgovora čepnic, 30 Hz odgovora in OFF- -odgovora. Zaključek: Značilne elektrofiziološke spremem- be v delovanju mrežnice omogočajo prepoznavo in razlikovanje kompletne in nekompletne obli- ke prirojene stacionarne nočne slepote. ON-OFF ERG je uporaben za natančno umestitev dis- funkcije mrežničnih bipolarnih celic pri nočni slepoti in ga je mogoče zanesljivo zabeležiti tudi pri otrocih. a nonspecific fundus appearance and are currently classified into complete (cCSNB) or incomplete (icCSNB) types.5 CSNB with autosomal dominant inheritance has a de- fect at the level of rod phototransduction. This type is associated with mutations in the GNAT1, PDE6B or RHO genes, and its clinical presentation can include a normal fundus or bone-spicule pigmentation in the peripheral retina for the specific locus of the RHO mutation (Gly90Asp).1 Another form of night blindness with photoreceptor dys- function is known as Oguchi disease, an au- tosomal recessive night blindness disorder with a diffuse grayish fundus appearance that normalizes after a long period of dark adaptation. Oguchi disease is associated with mutations in the SAG or GRK1 genes, which are involved in the phototransducti- on cascade.6,7 A similar autosomal recessive disorder of night vision that is caused by im- pairment of rodopsin regeneration is known as fundus albipunctatus. This disorder is as- Introduction Congenital stationary night blindness (CSNB) is a clinically and genetically hete- rogeneous retinal disorder that is characte- rized by vision impairment under dim-light conditions. CSNB can be classified accor- ding to ophthalmological findings, mode of inheritance, genetic testing, and specific cha- racteristics of the electroretinogram (ERG).1 A schematic representation of the different forms of night blindness according to mode of inheritance, genetic diversity and level of dysfunction is shown in Figure 1. The most common forms of CSNB are those associated with mutations in genes subsequent to the phototransduction ca- scade, which can be inherited as either X- -linked (NYX or CACNA1F gene mutations) or autosomal recessive (GRM6, TRPM1 or CAPB4 gene mutations). These forms have been previously described as the Schubert– Bornschein type of CSNB,4 and they have na izoblikovanost elektroretinograma (ERG). Glede na elektrofiziološke značilnosti ter genet- sko variabilnost je mogoče prepoznati komple- tno in nekompletno obliko nočne slepote. Cilj raziskave je bil preučiti elektrofiziološke razlike med kompletno in nekompletno obliko nočne slepote v slovenski pediatrični populaciji. Metode in preiskovanci: V študijo je bilo vklju- čenih 12 otrok (5–18 let), katerih klinični znaki so bili v soglasju s stacionarno nočno slepoto. Pri vseh otrocih je bil opravljen skotopični in foto- pični ERG po standardu Mednarodne zveze za klinično elektrofiziologijo vida (ISCEV) ter ON- -OFF ERG. Rezultati so bili prepoznani kot ab- normni, če je čas do vrha posameznega odgovora presegal 95 % zgornje meje zaupanja, amplituda pa je bila nižja od 5 % spodnje meje zaupanja normativnih podatkov. Primerjava med rezul- tati je bila izvedena s testom Mann-Whitney U in prepoznana kot statistično pomembna pri p < 0,01. Rezultati: Glede na vzorec elektrofizioloških abnormnosti je bila kompletna oblika CSNB prepoznana pri 8 otrocih in nekompletna obli- ka pri 4 otrocih. Skotopični ERG je pri vseh 12 otrocih pokazal elektronegativno izoblikovanost maksimalnega odgovora, z normalnim valom a in znižanim valom b, ki kaže na prizadetost de- I-18 Zdrav Vestn Supl | junij 2012 | Letnik 81 IZVIrnI čLaneK/OrIgInaL artIcLe Figure 1: classification of stationary night blindness disorders according to mode of inheritance [2–3]. cSnB, congenital stationary night blindness; ar, autosomal recessive; aD, autosomal dominant; *, night blindness with abnormal fundus appearance. Figure 2: the fundus appearance in the 8 children with ccSnB and the 4 children with iccSnB. the findings were symmetrical across both eyes, with the fundus of the right eye shown here for all of the children. response of dark-adapted ERGs.4,5 These ERG features indicate that photoreceptor function is preserved, and thus that there is a block in the signal transmission between the photoreceptors and the bipolar cells.9,10 ERG classification into cCSNB and icCSNB is based on the complete absence or the residual presence of rod function, respec- tively. Moreover, cCSNB has been shown to be associated with a defect in the post- -photoreceptor retinal ON-pathway, which can be seen in abnormal ON- and normal OFF-components of the ON-OFF ERGs. In contrast, in icCSNB, there is a partial defect of both the ON- and OFF-pathways, which sociated with mutations in the RDH5 gene, and fundoscopy reveals distinct multiple yellow-white dots that are located at the le- vel of the retinal pigment epithelium in the mid-periphery.8 ERGs are relevant for confirmation of these disorders, as they can indicate the le- vel of dysfunction and reveal specific ERG abnormalities in CSNB. The negative wa- veform of ERGs is a highly characteristic sign in both cCSNB and icCSNB, where dysfunction occurs at the post-photorecep- tor level. This can be seen as larger ampli- tudes of the a-wave compared with those of the b-wave in the combined rod–cone Zdrav Vestn Supl | children with complete or incomplete congenital stationary night blindness I-19 IZVIrnI čLaneK/OrIgInaL artIcLe Ta bl e 1: S um m ar y of th e cl in ic al fi nd in gs in th e 8 ch ild re n w ith c cS n B an d th e 4 ch ild re n w ith ic cS n B. Ch ild Se x ag e va re fr . e In it ia l c om pl ai nt n ys . D ar k ad ap t. Fu nd us a pp ea ra nc e In he ri t. Complete CSnB 1 M 8 0. 6 0. 6 -5 .0 -5 .5 Lo w v is ua l a cu ity + M ild o pt ic d is c pa llo r, pe ri pa pi ll ar y at ro ph y ar ,X ? 2 M 9 0. 9 0. 8 -4 .7 5 -6 Fa m ily h is to ry o f c om pr om is ed n ig ht vi si on + M yo pi c X 3 M 10 0. 9 0. 8 -1 .2 5 -3 .2 5 Lo w v is ua l a cu ity , p ro bl em s w ith n ig ht vi si on + M ild o pt ic d is c pa llo r ar ,X ? 4 M 10 0. 3 0. 1 -7 -8 Pr ob le m s w ith n ig ht v is io n + ab no rm al O pt ic d is c pa llo r ar ,X ? 5 F 10 0. 6 0. 6 -0 .2 5 -0 .2 5 Lo w v is ua l a cu ity + ab no rm al M ild o pt ic d is c pa llo r ar ? 6 M 10 0. 7 0. 5 -7 .5 -6 .5 Lo w v is ua l a cu ity , p ro bl em s w ith n ig ht vi si on ab no rm al te m po ra l o pt ic d is c pa llo r, pe ri pa p. at ro ph y ar ,X ? 7 M 17 0. 7 0. 8 -1 1 -1 1 Fa m ily h is to ry o f c om pr om is ed n ig ht vi si on + O pt ic d is c pa llo r, pe ri pa pi ll ar y at ro ph y X 8 M 17 0. 7 0. 8 -1 0 -8 Fa m ily h is to ry o f c om pr om is ed n ig ht vi si on M yo pi c X Incomplete CSnB 9 M 5 0. 4 0. 6 -6 .0 –7 .0 Fa m ily h is to ry o f l ow v is ua l a cu ity n or m al X 10 M 10 0. 4 0. 4 -3 .2 5– 1. 75 Lo w v is ua l a cu ity M ild a tt en ua tio n of th e ve ss el s ar ,X ? 11 M 11 0. 6 0. 6 -2 .0 –2 .0 Fa m ily h is to ry o f l ow v is ua l a cu ity te m po ra l o pt ic d is c pa llo r, til te d op tic d is c X 12 M 18 0. 9 0. 8 -3 .2 5– 6. 0 Lo w v is ua l a cu ity ab no rm al O pt ic d is c pa llo r ar ,X ? VA – v is ua l a cu ity ; R ef r.E – re fr ac tiv e er ro r; N ys . – p re se nc e of n ys ta gm us ; D ar k ad ap t. – da rk a da pt om et ry ; I nh er it. – p at te rn o f i nh er ita nc e; M – m al e; F – fe m al e. I-20 Zdrav Vestn Supl | junij 2012 | Letnik 81 IZVIrnI čLaneK/OrIgInaL artIcLe Figure 3: Standard full-field ergs and On- and OFF-responses, as a comparison of an age-matched control child (left) with a representative child (child 8) with ccSnB (middle) and a representative child (child 12) with iccSnB (right). the same patterns of changes were seen in the other children with ccSnB and iccSnB. to determine whether ON-OFF ERGs are significant for the differentiation of children with CSNB. Subjects and methods Subjects This study was performed according to the tenets of the Declaration of Helsinki, and it was approved by the National Ethics Committee. All of the children and their pa- rents were informed about the investigation protocol. The study included 8 children with a diagnosis of cCSNB (7 ♂, 1 ♀), which were 8–17 years old (mean age: 10.6 years), and 4 children with icCSNB (4 ♂), aged from 5–18 years (mean age: 11.0 years). Three of the patients with CSNB were adolescents at the time of inclusion in this study (17–18 years old), although they were followed from a yo- unger age, and for the sake of convenience they were considered here as children. The main opthalmological findings of these children with CSNB are summarized in Table 1, and these include: variable visu- al loss (cCSNB: 0.1–0.9, mean: 0.6; icCSNB: 0.4–0.9, mean: 0.6; visual acuitiy Snellen equiv.), refractive error ranged from mo- derate to high myopia (spherical equivalent range, cCSNB: -0.25 to -11.00 D; icCSNB: -1.75 to -7.00 D), abnormal dark adaptation, is seen as reductions in both the ON- and OFF-components of the ON-OFF ERGs.5 This classification, which was first defined according to these electrophysiological fin- dings, was subsequently confirmed by ge- netic analysis. In cCSNB with an X-linked mutation of the NYX gene, which encodes a proteoglycan protein known as nyctalopin, promotion of the retinal ON-pathway inter- connections is most probably disturbed.11 In cCSNB with an autosomal-recessive mutati- on of the GRM6 gene12 or the recently disco- vered mutation of the TRPM1 gene,13 there is a defect in glutamate transmission betwe- en the photoreceptors and the ON-bipolar cells. IcCSNB is associated with either an X- -linked mutation of the CACNA1F gene14 or, more rarely, an autosomal-recessive mutati- on of the CAPB42 and CACNA2D415 genes. All three of these mutations lead to disrup- tion of glutamate release from the photore- ceptors to the ON- and OFF-bipolar cells.7 However, impaired night vision is not always the major complaint for those with CSNB, and the fundus appearance can be indistinctive in some forms of CSNB. There- fore, diagnosis of CSNB can be challenging, especially in children. The aim of the present study was to screen the Slovene population of children with CSNB to distinguish bet- ween cCSNB and icCSNB using standard ERGs versus recently developed ON-OFF ERGs. Furthermore, the purpose was also Zdrav Vestn Supl | children with complete or incomplete congenital stationary night blindness I-21 IZVIrnI čLaneK/OrIgInaL artIcLe The pupils were dilated with 1 % tropicami- de (Mydriacyl®). Full-field ERGs were re- corded using a Ganzfeld stimulator of the RETIport unit (Roland Consult, Wiesba- den, Germany). The dark-adapted ERG re- sponses were recorded after 20 min of dark adaptation, and the light-adapted responses after 10 min of light adaptation (backgro- und luminance, 22 cd/m2). The stimulus intensities were consistent with the ISCEV standard values,16 the intensity of the stan- dard flash (as combined rod–cone respon- se, oscillatory potentials, cone response and 30-Hz flicker) was 2.4 cd s/m2, and the in- tensity of the attenuated dark-adapted flash (rod response) was 0.03 cd s/m2. ON-OFF ERGs were recorded from one eye, as descri- bed previously,18 with a full-field hand-held Espion ColorBurst stimulator (Diagnosys LLC, Littleton, MA, USA), which was powe- red by white LEDs. The ON-OFF ERG re- sponses were elicited with 200 ms stimuli of 1.9 log cd s/m2 intensity, which were presen- ted on a photopic background of 50 cd/m2 luminance. The flashes were delivered at 2 Hz and the means were taken over 20 to 30 responses. At least two measurements were nystagmus (six of these 12 children), normal anterior eye segment, clear optic media, nor- mal fundi (Figure 2) or temporal optic disc pallor or attenuated vessels, normal color vi- sion, and variable constriction of the visual field. No clinical progression was identified by follow-up over at least 3 years. According to their family histories, X-linked inheritan- ce was recognized in five of these 12 children, while with the others, none of their family members had any symptoms of night blin- dness. All of the children were included in the molecular genetic procedures, although to date these results have been obtained for only one of these children (Table 1, child 10). electroretinography Full-field ERGs were recorded simulta- neously from both eyes following the stan- dards of the International Society of Clinical Electrophysiology of Vision (ISCEV).16 The recording electrode was a HK-loop, placed in the fornix of the lower eyelid.17 The sil- ver-chloride reference electrode was placed on the ipsilateral temple, and the ground electrode was positioned on the forehead. Table 2: Summary of the erg data and statistical comparisons between ccSnB and iccSnB (Mann-Whitney U tests). Comb. rod-cone resp. – a-wave Comb. rod-cone resp. – b-wave rod resp. – b-wave amplitude [mv] Impl. time [ms] amplitude [mv] Impl. time [ms] amplitude [mv] Impl. time [ms] complete cSnB 133 ±23 18.4 ±1.3 69.7 ±21 31.6 ±1.7 0 - Incomplete cSnB 125 ±17 18.5 ±1.2 92.1 ±24 33.9 ±4.3 51.4 ±20 107 ±7 Difference nS nS nS nS P < 0.001 P < 0.001 Cone resp. – a-wave Cone resp. – b-wave 30 Hz – b-wave complete cSnB 23.8 ±6.8 16.7 ±0.9 98.9 ±34 32.1 ±1.1 87.2 ±22 28.4±1.5 Incomplete cSnB 14.6 ±3.4 16.3 ±1.2 16.9 ±4.3 28.4 ±1.6 17.4 ±4.7 25.8±1.0 Difference P = 0.003 nS P < 0.001 P < 0.001 P < 0.001 P< 0.001 ON resp. – b-wave OFF resp. – d-wave complete cSnB 12.6 ±5.6 37.5 ±2.7 26.7 ±6.9 216 ±1.6 Incomplete cSnB 6.9 ±5.1 34.0 ±6.7 8.7 ±4.2 225 ±1.2 Difference nS nS P < 0.001 P < 0.001 NS: No significant difference between the groups I-22 Zdrav Vestn Supl | junij 2012 | Letnik 81 IZVIrnI čLaneK/OrIgInaL artIcLe repeated and averaged for each of the full-fi- eld and ON-OFF ERG responses. All of the responses were differentially amplified and stored on the hard disc of a computer. The flash intensity and background luminance were calibrated with a photometer/ radi- ometer (IL-1700; International Light INC, Newburyport, USA) using a photopic light detector. analysis The amplitude of the a-wave was measu- red from the stimulus onset to the middle of the first negative trough. The amplitude of the b-wave was measured from the trough of the a-wave to the first positive peak. The amplitude of the d-wave was measured from the stimulus offset to the following positive peak. The results were considered as abnor- mal if the implicit time was above 95 % of the upper confidence limits and the ampli- tude was below 5 % of the lower confidence limits of the normative data.19 Compari- sons between the data were performed with Mann-Whitney U tests, and were conside- red significant for p < 0.01. The data were analyzed using Origin 7.0 (OriginLab Corp., Northampton, USA) and SPSS, version 12.0 (SPSS Inc., Chicago, USA). results Representative ERGs of a healthy control child and children with cCSNB and icCSNB are shown in Figure 3. Both types of CSNB were characterized by an electronegative combined rod–cone response of the dark- -adapted ERG, which can be recognized as the smaller b-wave amplitude compared with the a-wave amplitude. Rod respon- ses to dim stimuli were not detectable with cCSNB, although residual responses remai- ned with icCSNB. There were no peaks in the oscillatory potentials with cCSNB, with some seen with icCSNB. Abnormalities were also present in the light-adapted ERGs, which were more pro- minent with icCSNB, where both the cone response and the 30-Hz flicker response were severely reduced. On the contrary, the- se two responses appeared nearly normal Figure 4: Individual data of dark-adapted ergs (rod–cone responses and rod responses, as indicated) where the amplitudes from both eyes were plotted against the implicit time; normative limits indicate abnormality of the values when located in the area marked with red. ccSnB – black ■ symbols; iccSnB – blue ● symbols. Zdrav Vestn Supl | children with complete or incomplete congenital stationary night blindness I-23 IZVIrnI čLaneK/OrIgInaL artIcLe with cCSNB, except that the a-wave of the cone response was broadened. The ON- and OFF-responses elicited with long duration flashes under light-adapted conditions were also changed in both types of CSNB. The ON-response (b-wave) was severely redu- ced in both types of CSNB, while the OFF- -response (d-wave) was abnormal only in icCSNB. Figures 4, 5 and 6 show the response data of all of these children with CSNB, where the amplitudes measured from both eyes were plotted against the implicit time. The limits were also plotted, which indicate the normal and abnormal ranges of the data. Fi- gure 4 shows the plots of the full-field ERGs recorded under dark-adapted conditions. The a-wave of the combined rod–cone re- sponse had a normal amplitude and implicit time in all of these children with cCSNB and icCSNB. The b-wave of the combined rod– cone response was significantly reduced in both CSNB types, while the time to peak of the b-wave remained within the normal range. The rod response was not detectable with cCSNB (the values are not plotted), while the rod response amplitude was redu- ced in the children with icCSNB; one child also showed a delayed implicit time of the rod response. The plots summarizing the data of the light-adapted ERG responses in children with cCSNB and icCSNB are shown in Fi- gure 5. The amplitude of the cone response a-wave was normal to borderline subnormal in cCSNB, while it was borderline subnor- mal to reduced in the children with icCSNB. The a-wave implicit time was normal in the majority of these children with both CSNB types; only one child with cCSNB showed borderline prolongation of the implicit time. The b-wave of the cone response had nor- mal to borderline subnormal amplitudes with cCSNB, while a reduction in the am- plitude was distinct in the children with icCSNB. The b-wave implicit time was wi- thin normal limits with both CSNB forms. The 30-Hz flicker response had normal to borderline amplitudes and implicit times in the children with cCSNB, while a severe reduction of its amplitude was seen in the children with icCSNB. Figure 5: Individual data of light-adapted ergs (cone responses and 30-Hz flicker responses, as indicated) where the amplitudes from both eyes were plotted against the implicit time; normative limits indicate abnormality of the values when located in the area marked with red. ccSnB – black ■ symbols; iccSnB – blue ● symbols. I-24 Zdrav Vestn Supl | junij 2012 | Letnik 81 IZVIrnI čLaneK/OrIgInaL artIcLe Figure 6: Individual data of the On-OFF ergs (On- and OFF-responses, as indiacted) where the amplitudes from both eyes were plotted against the implicit time; normative limits indicate abnormality of the values when located in the area marked with red. ccSnB – bblack ■ symbols; iccSnB – blue ● symbols. In iccSnB, data from only 6 eyes are plotted, because in one child On-OFF erg was contaminated with blinking artifacts. CSNB were the most significant for the rod response, the b-wave of the cone response, the 30-Hz flicker response, and the d-wave or OFF-response, which was elicited with long-duration stimuli. The combined rod– cone responses were similar with both types of CSNB. Discussion The present study reveals distinct ERG features that can be used to differentiate bet- ween children with cCSNB and icCSNB, in- herited as either X-linked or autosomal re- cessive trait. Standard full-field ERGs show an electronegative waveform of the combi- ned rod–cone responses with both types of CSNB. cCSNB was characterized by comple- te rod dysfunction and borderline cone dys- function, while with icCSNB, there was pre- servation of rod function and severe cone dysfunction seen. ON-OFF ERGs revealed ON-bipolar cell dysfunction with cCSNB, while with icCSNB, there was both ON- and OFF-bipolar cell dysfunction. According to Boycott et al.,20 clinical di- agnosis of CSNB can be made with the fol- lowing findings: a history of night blindness, reduced visual acuity, characteristic findings on ERGs, myopia or hyperopia, nystagmus and strabismus in 50 % to 70 % of cases, and in general, normal color vision and normal fundus appearance. A positive family history is seen in cases of X-linked inheritance. All of the clinical findings of the children in- cluded in the present study were consistent with these criteria. With the exception of the ERGs, all of the other clinical findings were of very similar appearance with both cCSNB and icCSNB. All of these children were myo- pic, and they all manifested a similar loss of visual acuity, which appeared to be the ma- jor complaint in both of these CSNB groups. Both CSNB types also had nonspecific fun- dus changes, which varied from a normal appearance to myopic changes and mild optic disc pallor. Similar descriptions have also been reported in other studies.1,10,20,21 Nystagmus, squint, tilted optic discs and pa- radoxical pupil responses have been repor- ted in association with both conditions.22 In the group of children in the present study, The alterations in the ON-OFF ERG data with cCSNB and icCSNB are summarized in Figure 5. The ON-response (b-wave) was markedly abnormal in both types of CSNB, as it was reduced in all of these children. Prolongation of the implicit time was seen in some of these children with cCSNB and icCSNB. The OFF-response (d-wave) am- plitude and implicit time were normal or borderline subnormal in the children with cCSNB, while they were severely reduced and prolonged with icCSNB. The statistical comparisons between the- se data for cCSNB and icCSNB are summa- rized in Table 2. The differences in the values of the responses between these two types of Zdrav Vestn Supl | children with complete or incomplete congenital stationary night blindness I-25 IZVIrnI čLaneK/OrIgInaL artIcLe Figure 7: ergs according to the gOSH protocol at the age of 6 years and the standard full-field ergs at the age of 7 years in a child (child 6) with ccSnB. although the ISCEV currently aims to in- clude these in the standards, together with some other extended ERG protocols. Cones synapse onto ON-bipolar and OFF-bipolar cells. With short duration flashes, such as li- ght-adapted cone responses, the activities of both bipolar cell types merge into a single positive b-wave. With long duration stimu- li, as used for the recording of the ON-OFF responses, two positive waves occur, the b- -waves and the d-waves.18,25 The first one of these, the ON-response b-wave, indicates the activity of the ON-bipolar cells, while the second one, the OFF-response d-wave, measures the contributions from the OFF- -bipolar cells.26,27 ON-OFF ERGs have pre- viously been shown to be useful for the se- lective evaluation of ON- and OFF-pathway abnormalities of the retinal cone system.26 More recent studies have shown that this response is highly indicative for detection of the preferential ON-pathway abnormali- ty in retinal pathologies, such as cCSNB or melanoma-associated retinopathy.24,26,28 On the other hand, in icCSNB, where both the ON- and OFF-responses are reduced, this functional abnormality indicates dysfuncti- on of both the ON- and OFF-bipolar cells. This difference between cCSNB and icCSNB was seen in all of our pediatric patients here, and it appears to be highly characteristic for differential diagnosis between those two cli- nical entities, as previously described.5,10,21 However, the ON-OFF ERG abnormalities alone do not provide information for the diagnosis of cCSNB and icCSNB, as the de- fective night vision has to be confirmed by there was evident nystagmus in six of these 12 children. The present study compared children with cCSNB and icCSNB from the age of 5 years, when they were old enough to coope- rate with the recordings with ocular contact electrodes. The ERG features have been pre- viously reported in adults and mixed popu- lations, including children.5,9,23 One study investigated a pediatric population but did not specify the ages of the patients.24 In our study, the ERG characteristics in the chil- dren with cCSNB and icCSNB were consi- stent with previous descriptions.21 As previ- ously shown, the electronegative waveform of the dark-adapted combined rod–cone response indicates post-photoreceptor dys- function.5 In the present study, the combi- ned rod–cone responses were reduced with cCSNB and icCSNB to similar extents, and therefore the electronegative waveform did not provide information for differential di- agnosis between cCSNB and icCSNB. The most significant differentiation between the children with cCSNB and icCSNB in the present study was seen in the amplitude and implicit time of the rod response, cone response, 30-Hz flicker response, and OFF- -response. The cone response a-wave was di- stinctly broadened in children with cCSNB, as also indicated previously.21 This broade- ning is a consequence of a missing positive component of the cone ON-bipolar cells. However, this functional anomaly can be more easily detected with ON-OFF ERGs. ON-OFF ERGs do not represent a stan- dardized electrophysiological procedure, I-26 Zdrav Vestn Supl | junij 2012 | Letnik 81 IZVIrnI čLaneK/OrIgInaL artIcLe ght be observed. These can be seen as signifi- cant reductions in the sum of the oscillatory potential amplitude associated with the rod activity or with a reduced light-adapted b- -wave and 30-Hz flicker amplitude.34 Con- sistently with this description, we found re- duced light-adapted ERG amplitudes in the mother of a boy with icCSNB (child 12), who was a possible carrier, but this finding has not yet been confirmed by genetic analysis. However, even when genetic testing is acces- sible, ERGs still have an important role in the diagnosis of cCSNB from icCSNB, even though they cannot discriminate between X-linked and autosomal recessive mutations that cause these types of retinal disorder. To conclude, cCSNB and icCSNB are ge- netically heterogeneous and their clinical features are not specific enough to establish diagnosis. However, children can be diagno- sed and differentiated by the use of standard full-field ERGs. ON-OFF ERG recordings are also relevant in children, because they define ON- versus OFF-bipolar cell dysfunc- tion, and therefore these also differ between cCSNB and icCSNB. acknowledgement The authors wish to thank Mrs. Marija Jesenšek and Mrs. Ana Jeršin, who were in- volved in the clinical ERG recording, and to Mrs. Barbara Klemenc, who prepared some of the figures. This study was supported by the Slovene Research Agency, Grant No. J3– 6167 and P3–0333, and was in part presented at the anual meeting of the European Pae- diatric Ophthalmological Society (EPOS), in Portorož, Slovenia, in 2007. abnormal dark-adapted ERGs, as well as for the light-adapted ERG characteristics. The ISCEV does not currently have a standard for ERG recording in younger children (less than seven years old), as these children do not always cooperate in the re- cordings with ocular contact electrodes and Ganzfield stimuli. Indeed, some laboratories sedate younger children for their ERG recor- dings.29 Other laboratories use a noninvasi- ve Great Ormond Street Hospital (GOSH) protocol that uses skin electrodes, while the children are alert and sit in a parent’s lap.30,31 According to our experience, the noninvasi- ve GOSH protocol is already sensitive eno- ugh for the detection of CSNB features in babies and young children from the age of a few months. An example of the sensitivity of detection of CSNB abnormalities is shown in Figure 7, where the ERG abnormalities that were detected following the GOSH pro- tocol at a younger age (child of 6 years) were subsequently confirmed using the standard full-field ERG protocol one year later. In recent years, genetic testing has beco- me fundamental for the diagnosis of patients with CSNB, as ophthalmological and ERG findings are similar for all the genetic forms of cCSNB or icCSNB. To our knowledge, the only phenotypic differences have been detected using dark-adapted 15-Hz-flicker ERGs in cCSNB patients with different gene mutations.32 Patients with NYX and TRPM1 gene mutations showed similar 15-Hz-flicker ERG responses, while differences were fo- und for those with GRM6 gene mutations, which suggested some differences in the rod pathways.33 For all of the children inclu- ded in the present study, the blood samples were sent for genetic screening, although to date we have received the result of only one of the children with icCSNB, in whom the CACNA1F mutation was confirmed. In five of these children, X-linked inheritance is suspected, according to their family histori- es. In the other children, none of the family members have shown signs of CSNB, and therefore the manner of inheritance and the mutation cannot be revealed without gene- tic testing. 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