Introduction

Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS) is a distinctive form of hereditary early onset spastic ataxia that is characterised by spasticity, dysarthria, nystagmus, distal muscle wasting, and finger and foot deformities. Hypermyelination of retinal nerve fibres and atrophy of the superior part of the vermis are the main neuropathological findings. Initially, ARSACS was described among French Canadians in the Charlevoix–Saguenay region of Quebec. After the identification of the SACS gene on proximal chromosome 13q as the causative gene,1 mutations in this gene were detected in patients from other populations, notably Tunisia,2 Turkey,3 Italy,4, 5 Spain,6 Japan7, 8, 9, 10 and Belgium.11 Up to now, approximately 30 different loss-of-function mutations have been reported in the SACS gene.12, 13 We here report a Belgian patient with ARSACS who is a compound heterozygote for an inherited point mutation in the SACS gene on the paternal allele and a de novo microdeletion of maximally 1.47 Mb, comprising the SACS gene, on the maternal chromosome.

Case report

This 28-year-old Belgian male was born as the first child of healthy, non-consanguineous parents, after caesarean section due to breech presentation. Birth weight and length were normal, 3450 g (50th centile) and 51 cm (50th centile), respectively. He started walking at the age of 19 months but never acquired a normal gait, initially showing pelvic instability and progressively declining owing to weakness and contractures of the lower limb muscles. Neurological examination at age 16 revealed muscular hypotrophy of the four limbs, especially of the distal muscles, and spastic hypertonia and paresis of the lower limbs. Muscle strength of the upper limbs was slightly diminished, especially of the proximal muscles. Although patellar reflexes were very intense, ankle jerks were absent and plantar reflexes were bilaterally indifferent. Osteotendon reflexes of the upper limbs were weak. The patient was walking independently with effort, limping with endorotation of the feet. Sensation was not affected. He showed mild dysarthria and was enlisted in special education for mild learning difficulties, with a full scale IQ of 88 on the Wechsler Adult Intelligence Scale (WAIS). Ophthalmologic evaluation was normal. Electromyography showed signs of an axonal sensorimotor polyneuropathy. He had a moderate perceptive bilateral hearing loss. Although brain auditory evoked potentials were normal on the right side, on the left side, waves II and III were absent and there was a prolonged I–V interpeak latency. No scalp response was detected by somatosensory evoked potentials of the nervus tibialis. The peripheral response to somatosensory stimulation of the nervus medianus showed a bilateral minor increase in latency, whereas central conduction was severely prolonged.

At age 26, neurological re-examination revealed progressive spasticity and ataxic gait. At that time, he was walking with crutches. Brain MRI showed atrophy of the vermis superior and the superior cerebellar peduncles; otherwise, brain parenchyma was normal and there were no white matter abnormalities (Figure 1).

Figure 1
figure 1

Brain MRI at the age of 26 years showing atrophy of the vermis superior (black arrows) and the superior cerebellar peduncles. No anomalies of the cerebral hemispheres were detected.

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Materials and methods

Conventional karyotyping with G-banding was performed according to standard techniques. Array-Comparative Genomic Hybridisation (aCGH) was carried out as described.14 We used a microarray containing 3625 BAC/PACs chosen genome wide at 1 Mb intervals and a full tiling array for chromosome 13, with a resolution of 100 kb, containing 1010 BAC/PACs obtained from BACPAC Resources Center (Children's Hospital, Oakland, CA, USA). Fluorescent in situ hybridisation (FISH) using probe RP11-760M1 and real-time quantitative PCR (qPCR) using primers A, B, C, D, E, F and G (Figure 3; Table 1) were performed as described.15 The sequences of the breakpoint-containing regions were investigated using Miropeats (thresholds 400 bp).16 Homology of low copy repeats was investigated using AlignX with standard settings (Vector NTI Advance 10.0.1, Invitrogen). The polymorphic repeat D13S232 was analysed by PCR using specific primers (Table 1) on 50 ng of genomic DNA using Expand Long Template (Roche) and buffer 3 according to the manufacturer's instructions. Mutation analysis of the SACS gene was performed as described.17 This study was approved by the Medical Ethical Committees of University Hospital Leuven and Jichi Medical University.

Figure 3
figure 3

Overview of the deleted region on 13q12.12 comprising six genes, SGCG, SACS, TNFRSF19, MIPEP, SPATA13 and C1QTNF9 (black arrows). PARP4 is not deleted (white arrow). The location of polymorphic marker D13S232 and qPCR primers A, B, C, D, E, F and G is indicated by red vertical lines (primers inside the deleted region are displayed in red). The deleted BAC/PACs (RP11-760M1 and 32k clones RP11-357N10 till RP11-530B16) are displayed in black and the flanking non-deleted BAC/PACs as white boxes. The minimal 1.37 Mb deleted region is shown below as a black box. Grey boxes indicate the breakpoint-containing regions. Red boxes at the bottom line indicate homologous sequences in the breakpoint-containing regions. (See online version for colour figure).

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Table 1 Oligonucleotide sequences of primers used for qPCR analyses
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Results

Conventional karyotyping showed a normal result. Higher-resolution molecular karyotyping by aCGH, using a BAC/PAC microarray with 1 Mb resolution, detected a deletion of a single DNA clone RP11-760M1, located on chromosome 13q12.12, partially overlapping the SACS gene. This microdeletion was confirmed by FISH using probe RP11-760M1 (Figures 2b and 3). Involvement of the SACS gene was demonstrated by qPCR using primer pair A (Figure 3; Table 1). The deleted region was further delineated by means of a chromosome 13 specific tiling array. The deletion spans maximally 1.54 Mb and affects 6 genes (Figures 2a and 3), with breakpoints mapping to a region 22335637-22383193 (proximal) to 23801734-23879339 (distal).

Figure 2
figure 2

(a) Microarray containing BAC/PAC clones covering chromosome 13 with a tiling resolution of 100 kb, showing a maximal deletion of 1.54 Mb on 13q12.12 and spanning clones from RP11-357N10 to RP11-530B16. (b) The microdeletion was confirmed by FISH using probe RP11-760M1. (c) Analysis of the polymorphic marker D13S232 using patient and parental DNA revealed that the 13q12.12 microdeletion occurred de novo and was located on the maternal chromosome. (See online version for colour figure).

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Analysis of the polymorphic marker D13S232 on patient and parental DNA revealed that the 13q12.12 microdeletion occurred de novo and was located on the maternally inherited chromosome 13 (Figure 2c).

The sequences of the breakpoint-containing regions were investigated using Miropeats and AlignX. This revealed the presence of two highly homologous sequences. A first 14 kb sequence starting from 22 384 kb was found to be 94% homologous to a 14 kb sequence starting from 23 796 kb. A second 43 kb sequence starting from 22 400 kb was found to be 91% homologous to a 44 kb sequence starting from 23 822 kb. To fine map the breakpoints, qPCR primers were designed flanking these four regions (primers B, C, D, E, F and G (Figure 3; Table 1)). This demonstrated that the breakpoint-containing regions are located between the primers F and G (proximal) and C and D (distal) and thus coincide with the second 44/43 kb homologous sequences: 22399440–22442680 (proximal) and 23813131–23866606 (distal).

Sequencing of the SACS gene revealed a hemizygous mutation in exon 9 (c.10517T>C) (GeneBank accession number, AL157766) in the SACS gene resulting in an amino-acid change, F3506S (Figure 4). The father was carrier of this mutation, which was not detected in 100 normal Belgian control chromosomes.

Figure 4
figure 4

(a) Partial results of nucleotide sequence analysis of exon 9 of the SACS gene. A T-to-C change in the patient and his father with a heterozygous state, and the normal sequence in his mother are shown. (b) The PCR products after BtsC1 digestion; lane M, 100 bp size markers; lane 1, the father with a heterozygous T-to-C change; lane 2, the patient with a heterozygous T-to-C change; lane 3, the mother with normal sequence. The wild-type BtsC1-digested PCR products gave four bands (260, 125, 122 and 106 bp). The BtsC1-digested PCR products with a heterozygous T-to-C change gave six bands (260, 216, 125, 122, 106 and 44 bp). As the patient had a microdeletion of chromosome 13q in one allele, his BtsC1-digested PCR products gave five bands (216, 125, 122, 106 and 44 bp). The BtsC1-digested band of 44 bp cannot be observed.

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Discussion

In this patient with a complicated form of spastic paraparesis, a de novo microdeletion of maximally 1.54 Mb, encompassing six genes, was detected using aCGH, a high-resolution molecular cytogenetic technique. The SACS gene was located in this region, and the patient's phenotype was typical of ARSACS. In addition, brain imaging revealed the characteristic cerebellar anomalies. This diagnosis was formally confirmed by the demonstration of a paternally inherited point mutation on the other allele. The F3506S variant, caused by c.10517T>C, is predicted by the PolyPhen tool to be probably damaging to the function of the Sacsin protein (PSIC score difference 2.225), as it is situated in a conserved ARM repeat.18

Up to now, all reported mutations in ARSACS patients were point mutations or deletions of a few nucleotides in the SACS gene, resulting in a loss of function. We here report the first case of ARSACS where one of the mutations is a gross chromosomal deletion comprising the SACS gene. Besides SACS, the deleted region contains five other genes, namely SGCG, TNFRSF19, MIPEP, SPATA13 and C1QTNF9. Interestingly, the only feature by which the present patient differs from other cases with ARSACS is the presence of moderate perceptive hearing loss. None of the five genes has a known role in hearing (loss), and therefore it remains uncertain whether the hearing loss is a rare manifestation of ARSACS or is caused by haploinsufficiency of one of the flanking genes (Figure 3).

The aberrant region most likely coincides with 2 formerly described copy number variants, one deletion and one duplication. One 1.4 Mb deletion was described by Pinto et al19 in a cohort of 506 unrelated healthy Caucasian individuals. Two duplications comprising SACS entirely were found by Simon-Sanchez et al20 in DNA extracted from EBV immortalised cell lines, derived from lymphocytes of 276 elderly neurologically normal subjects. In one subject, the duplication was confirmed in DNA extracted directly from the blood.20 The presence of the segmental duplication suggests non-allelic homologous recombination as a recurrent mechanism generating the imbalances found in our patient and the aforementioned subjects. Therefore, ARSACS can be added to the growing list of genomic disorders.

The present observation illustrates that in addition to its usefulness in the detection of candidate genes for autosomal dominant disorders, aCGH may contribute to gene identification for autosomal recessive disorders, as discussed by Coman and Gardner.21

In conclusion, we report on the first case of ARSACS carrying an atypical mutation, that is a de novo chromosomal deletion comprising SACS. This finding underscores the need to include dosage analysis of the SACS gene in mutation analysis in patients with spastic ataxia.