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Keywords:

  • ataxia-telangiectasia;
  • biopsy;
  • conjunctiva;
  • founder;
  • ocular motor apraxia

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose:  To describe the outcome of ophthalmologic examination of 10 Norwegian children with ataxia-telangiectasia (AT) followed through 5 years.

Methods:  Ten Norwegian patients with AT aged 2–22 years (three females, seven males) were examined. The diagnosis was confirmed clinically as well as with molecular genetic studies. Conventional ophthalmologic examination was performed and supplemented by photographs of the conjunctiva, video recordings and registration of eye motility in five consecutive years. Additionally conjunctival biopsies were performed at the end of the follow-up period.

Results:  General ataxia was usually detected when the child started to walk. All children over the age of 4 years had abnormal saccade movements, a form of ocular motor apraxia. Conjunctival telangiectasias were mostly visible at 4–5 years, primarily within the palpebral fissure. Immunohistochemical examination of conjunctival biopsies showed an increased number of cross-sections of blood vessels and neurons surrounded by glial tissue. There was a tendency to slightly earlier onset of conjunctival telangiectasias in the patients homozygous for a founder mutation compared with the other patients.

Conclusion:  The diagnosis of AT can be supported at preschool age by the onset of ocular motor apraxia and conjunctival telangiectasias. The findings become more prominent with age. The conjunctival telangiectasias seem to appear slightly earlier in the patients who are homozygous for a Norwegian founder mutation than in the rest of the patients.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ataxia telangiectasia (AT) is a rare autosomal recessive disorder characterized by early onset progressive cerebellar ataxia, oculocutaneous telangiectasias, ocular motor apraxia, dysartria and immunodeficiency. Chromosomal instability and hypersensitivity to ionizing radiation are reflected as increased susceptibility to cancer, especially to lymphomas and leukaemia. An increased prevalence of breast cancer has been registered among heterozygous relatives of AT patients (Swift et al. 1987), and similar results have been found in Nordic relatives of AT patients (Olsen et al. 2001). Malignancies or chronic lung failure with pulmonary infections in AT patients may cause death in early adulthood. The responsible gene, ATM (AT Mutated), maps to chromosome 11q22–23 (Gatti et al. 1988) and was identified in 1995 (Savitsky et al. 1995). The ATM protein is a protein kinase involved in DNA double strand breaks response and repair (Shiloh 2003). The disease was previously called Louis-Bar syndrome after the author of the first publication of the condition. (Louis-Bar 1941). The first Norwegian report of AT patients was published by Smeby (1966), who served the Norwegian Eastern Inland; he found five patients with both neurological and ocular characteristics of AT. Systematic prospective registrations of ocular status in AT patients have, to our knowledge, not been published previously. In this study, we performed yearly examination of the ocular symptoms in 10 Norwegian AT patients for 5 years.

Because five patients were homozygous for the Norwegian founder mutation c.3245delATCinsTGAT (Laake et al. 1998), we also looked for a possible phenotype/genotype relationship.

A Danish patient who was compound heterozygous for other mutations in the ATM gene was examined for comparison with the Norwegian patients.

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

Ten patients (three females and seven males) who at the end of the five year's observation time had a mean age of 9.8 (range 2–22) years, represented all but one living and known Norwegian AT patients at that time. These patients were examined yearly for five consecutive years from 1999 to 2003. Four patients did not participate in all examinations because of young age, late diagnosis or death. At the time of diagnosis by paediatrician all had general ataxia, increased serum levels of alfa-fetoprotein and the typical immunological findings such as IgE deficiency, IgA and/or IgG2 deficiency and low numbers of B- and T-lymphocytes (Stray-Pedersen et al. 2004). Six patients had recurrent respiratory infections and four had recurrent pneumonias. Two of the patients had been treated for leukaemia and six had relatives with a history of cancer.

Methods

The clinical ophthalmologic examinations were all performed by the same ophthalmologist (R.R.) and included testing of best corrected visual acuity at 3 m and 30 cm distance. An estimate of accommodation was achieved by measuring distance to blurring of a small object. Refraction was measured by retinoscopy and was expressed as the mean of the spherical equivalent of the two eyes. The anterior segment was inspected and direct ophthalmoscopy was performed.

The presence of conjunctival telangiectasias was documented with photographs taken yearly for 5 years. At the end of the follow-up period, conjunctival biopsies were taken from the palpebral fissure and from the fornix of two of the patients (both homozygous for a Norwegian founder mutation) and were compared with biopsies from a 5-year-old Danish AT patient, compound heterozygous for two other ATM mutations. The biopsies were fixated in 4% formalin, cut into 4 µm sections and subjected to in microscope after immunohistochemical staining against Von Willebrand factor, Actin, Neuron specific enolase, Laminin, Fibronectin,Vitronectin, Vimentin, S-100 protein, Type IV collagen, glial fibrillary acid protein (GFAP) and Synaptofysin according to previously described procedures (Bek 1997).

Ocular motility was analysed for ocular fixation, smooth pursuit movements, saccades and optokinetic nystagmus. The findings were documented by video recordings as well as eye movement recordings performed by the same expert (J.Y.) with the XY-1000 infrared reflection technique (IOTA Inc, Timrå, Sweden).

Mutation analyses of DNA prepared from peripheral blood cells were performed using several techniques including protein truncating test, denaturing gradient gel electrophoresis, heteroduplex analysis and denaturing high-performance liquid chromatography followed by sequencing, as previously described (Laake et al. 1998). The ATM mutation status for seven of the patients has been reported previously (Laake et al. 2000).

The study was approved by the Regional Scientific Ethical Committee of East Norway and was conducted in accordance with the tenets of the Declaration of Helsinki.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The best corrected visual acuity was slightly reduced for the age and was stable in spite of severe motility disturbances. Refraction was, in general, close to emmetropia (Table 1). There were only slight differences between the visual acuity measured at a distance of 3 m and at 30 cm, although a slight tendency to better acuity on the 30 cm distance test was registered. Fusion measured with Worth 4-dot test held at a distance of 1 m was normal except in two patients who had a convergent squint. Accommodation was evaluated in seven of the individuals: it was within normal limits (10 dioptres) in the youngest patients but was reduced to 6–8 dioptres in those older than 12 years of age. Six of the individuals were photophobic and needed filter spectacles.

Table 1.   ATM mutations, onset of ocular telangiectasia and visual acuity with spherical equivalent of final refraction in 10 Norwegian patients with AT for five consecutive years.
Patient ID Born/sex ATM mutation paternal/maternalOnset of OT age (years)VA 1999VA 2000VA 2001VA 2002VA 2003
  1. F, female; M, male; ins, insertion; del, deletion; fs, frameshift; ms, missense; ns, nonsense; OT, ocular telangiectasia; VA, visual acuity; emm, emmetropia.

NOAT 20B2001/Fc.5932G>T(ns) / c.2880delC(fs)    0.5 emm.
NOAT 20A1999/Fc.5932G >T(ns) /c.2880delC(fs)4.5  1.01.01.0 emm.
NOAT 161996/Mc.3245delATCinsTGAT(fs) / c.3245delATCinsTGAT(fs)4.50.50.50.51.01.0 emm.
NOAT 131995/Mc.8432delA(fs) / c.8432delA(fs)51.01.01.01.01.0 emm.
NOAT 181994/Mc.3245delATCinsTGAT(fs) / c.4110delG(ss)6  1.01.00.8 + 2.0
NOAT 111993/Fc.3245delATCinsTGAT(fs) / c.3245delATCinsTGAT(fs)50.61.00.71.01.0 emm.
NOAT 141993/Mc.3245delATCinsTGAT(fs) / c.3245delATCinsTGAT(fs)51.00.60.51.00.5 + 1.0
NOAT 101991/Mc.3245delATCinsTGAT(fs) / c.3245delATCinsTGAT(fs)40.61.00.60.80.8 emm.
NOAT 171989/Mc.6890A>C(ms) / c.3245delATCinsTGAT(fs)100.60.50.50.60.8 + 4.5
NOAT 11980/Mc.3245delATCinsTGAT(fs) / c.3245delATCinsTGAT(fs)40.60.80.7 + 2.5  

The mean age of onset for the telangiectasias was 5 years with a range of 4–10 years, as shown in Table 1. The table also shows a tendency towards earlier onset of conjunctival telangiectasias in the individuals homozygous for the founder mutation (4.5 years) compared with the others.

The telangiectasias were primarily visible in the palpebral fissure, but in the oldest patient they were also seen in the upper and lower conjunctival fornix of conjunctiva. The degree of dilation and tortuosity of the blood vessels increased gradually during the 5 years of observation (Fig. 1). Along with increasing tortuosity and diameter of the vessels in the palpebral fissue, telangiectasias in the skin – for example on the back of the hands and in the ear – were observed (Fig. 2). The telangiectasias in the fornix of the conjunctiva were seen later in the course of the disease.

image

Figure 1.  Photograph of one of the study patients showing the nasal part of conjunctiva through five consecutive years. This shows the progression of dilatation and tortuosity of the vessels.

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image

Figure 2.  Telangiectasias in the ear appeared alongside progression of conjunctival telangiectasias, here shown in one of the study patients.

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The immunohistochemical staining of the conjunctival biopsies showed the same pattern in all the examined cases. The tissue from the rima area contained more cross-sections of blood vessels than the tissue from the fornix. These rimal vessels showed a large variation in calibre, but all had a thin capillary wall consisting of endothelial cells (Von Willebrand factor), a basement membrane (Type IV collagen) and dispersed pericytes externally to the vascular wall (Actin), but no smooth muscle cells located inside the vessel wall. As evidenced by the immunohistochemical staining of peripheral nerves (Neuron specific enolase) and their glial sheathing (Vimentin and S-100 protein), the tissue from the rima area also contained more cross-sections of peripheral nerves of different calibre than did the conjunctival tissue from the fornix. There was no difference in the markers of nerve synapses (Synaptofysin) or extracellular matrix (Laminin, Vitronectin, Fibronectin) between the two tissue areas studied (Fig. 3). There were no differences between the two patients homozygous for the founder mutation and the Danish patient compound heterozygous for other mutations.

image

Figure 3.  Immunohistochemical staining of conjunctival biopsies from a 5-year-old Danish patient with AT. Staining with Type IV collagen presents the cross-sections of the vessels, while S-100 protein brings focus on the nerves. There were no differences in the findings between this patient and two of the Norwegian patients in whom we were allowed to take biopsies.

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None of the patients had cataract or other opacities in the anterior or posterior segment of the eye. The fundus of the eyes appeared normal.

Evaluation of ocular motility was affected by the patients' problems with head stability and tendency to move the head instead of the eyes. These movements were different from the typical head thrusts described by other authors (Baloh et al. 1978; Stell et al. 1989). Our AT patients rather lost their head control if they tried to move their eyes when the head was unsupported. The observed patients older than 12 years kept a stiffer and more stable head position.

Ocular fixation was unstable, with small intruding saccades and tendency to ocular flutter and sometimes nystagmus beats (Fig. 4). The evaluation of ocular fixation for 5 years is shown in Table 2. Normal fixation was lost in all patients over the age of 6 years. It was not possible to obtain reliable information about the time of onset of fixation instability. Nystagmus was noticed exclusively in children over the age of 10 years.

image

Figure 4.  Eye movement recording of fixation in one of the AT subjects. Note the unstable fixation with some nystagmus beats (arrow). Horizontal double arrow, 2 seconds; vertical double arrow, 10 degrees.

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Table 2.   Ocular fixation / Smooth pursuit movements / Saccades in ten Norwegian patients with AT for five consecutive years. (normal:+, subnormal:(+), abnormal:-).
PatientBorn/sexATM19992000200120022003
  1. R/R Homozygous for the Norwegian founder mutation; R/x: Heterozygous for the Norwegian founder mutation; x/x: Other mutation in ATM, founder mutation excluded.

NOAT 20B2001/Fx/x    +/−/+
NOAT 20A1999/Fx/x   +/+/− − / − / −
NOAT 161996/MR/R+/+/++/(+)/−+/−/− − / − / − − / − / −
NOAT 131995/Mx/x − / − / − − / − / − − / − / − − / − / − − / − / −
NOAT 181994/MR /x   − / − / − − / − / − − / − / −
NOAT 111993/FR/R+/−/− − / − / − − / − / − − / − / − − / − / −
NOAT 141993/MR/R − /(+)/− − / − / − − / − / − − / − / − − / − / −
NOAT 101991/MR/R − / − / − − / − / − − / − / − − / − / − − / − / −
NOAT 171989/Mx/R − / − / − − / − / − − / − / − − / − / − − / − / −
NOAT 11980/MR/R − / − / − − / − / − − / − / −   

Smooth pursuit movements were affected from the age of 4 years, when small catch-up saccades were seen (Fig. 5); the problems remained throughout the 5 year observation period (Table 2).

image

Figure 5.  Eye movement recording of horizontal smooth pursuit movements in one of the AT subjects (upper panel). For comparison, a recording of a normal child of comparable age has been added (lower panel). Note the low gain smooth pursuit in the AT child. Horizontal double arrow, 2 seconds; vertical double arrow, 10 degrees.

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There was increased delay time for the initiation of visually and verbally induced saccades. There were many small saccades with low gain both horizontally and vertically (ocular motor apraxia) (Fig. 6, Table 2). None of the Norwegian AT patients had compensating head thrusts.

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Figure 6.  Eye movement recordings of attempts to perform horizontal saccades in one of the AT children (upper panel). Each vertical line represents a stimulus change. For comparison, a recording of a normal child has been added in the lower panel. Horizontal double arrow, 2 seconds; vertical double arrow, 10 degrees.

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We could not elicit any optokinetic nystagmus, except in one patient at 2 years of age.

All four of these components of ocular stability and motility seemed to be irreversibly disturbed from early childhood. However, the vestibulo-ocular reflex (VOR) could be seen in all patients from the earliest age investigated. Ocular motility in these children will be reported more thoroughly elsewhere.

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This prospective cohort study of 10 Norwegian children with AT documents the progression of conjunctival telangiectasias and disturbance in ocular motility. To our knowledge, such a prospective study has not been published before.

By our subjective measurements of accommodation, we confirmed the tendency to reduced accommodation as shown earlier (Farr et al. 2002). The patients with reduced accommodation were provided with bifocal spectacles, but none of these were accepted.

The disturbances in ocular motility, especially in the saccades, appear slightly earlier than the telangiectasias. However, both of these symptoms are important parts of the clinical diagnosis of AT and play a role in the differential diagnosis of other neurodevelopmental disorders (Marr et al. 2005) and of congenital ocular motor apraxia (Cogan 1966). In particular, we looked for the typical head thrusts used to facilitate eye movements; however, we could not demonstrate these in any of the Norwegian patients. We believe that muscle control of the head in our AT patients was too weak to enable the use of compensating head thrusts. This is in contrast to the evidence from other authors (Baloh et al. 1978; Stell et al. 1989), who registered head thrusts in most of the examined AT patients.

Many of the patients suffered from photophobia and needed protecting filter spectacles; these spectacles were used permanently by those who were provided with them. We have not found any explanation for the photophobia.

Although conjunctival telangiectasias are mainly a cosmetic problem, they serve to confirm the diagnosis clinically. Our immunohistochemical examination of the conjunctival biopsies revealed an increased number of cross-sections of vessels and nerves with surrounding glial tissue. The number was greatest in the palpebral rima, which might indicate that sunlight has a role as a causative factor for the development of this vessel abnormality. This assumption is further strengthened by the finding of telangiectasias in the skin on hands and ears. It is unknown whether the increased number of cross-sections of conjunctival vessels and nerves appear simultaneously or whether one of these structures conditions the other. The stainings showed no differences between the two patients homozygous for the Norwegian founder mutation and the Danish patient compound heterozygous for other mutations in the ATM gene. The five consecutive photographs of the nasal part of conjunctiva on each patient indicate that the larger number of cross-sections is related to increasing tortuosity rather than to a greater number of vessels and nerves.

We saw a tendency to somewhat earlier onset of the conjunctival telangiectasias in the patients homozygous for the founder mutations than in the other patients. This is in accordance with the findings of lower concentrations of serum IgG2 in these patients. However, no more infectious episodes were registered in these homozygous patients than in the others (Stray-Pedersen et al. 2004). But the number of patients in our study is small, so these results must be evaluated with caution.

The pathophysiology of both the conjunctival telangiectasias and the motor disturbances is still unclear. The specific ocular ataxia can probably be regarded as a part of the general ataxia. The progression of the disease is a central element in which the atrophy of cerebellum is also involved.

All our AT patients had elevated serum alfa-feto protein. The significance of this finding is not known at present.

It is our hope that this clinical and molecular genetic study of the course of ocular disease in Norwegian AT patients may contribute to the establishment of AT diagnosis at the time when children have their first symptoms of general ataxia (age 1–2 years). Three of our patients were not diagnosed until the age of five or even later, and did not get sufficient medical, social and educational services until they had started school.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by grants from Inger Holms Minnefond and Professor Hjalmar Schiøtz′ Minnefond. We thank Professor Anne-Lise Børresen-Dale for valuable discussion. We thank the AT patients and their families for loyal cooperation during the project period.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References