CAG/CTG repeat expansions at the Huntington’s disease–like 2 locus are rare in Huntington’s disease patients

Patients and methods.

We screened for the HDL2 expansion in 39 index patients with HD-like phenotype (chorea and dementia) and in patients with chorea without dementia (n = 13) or with subcortical dementia without chorea (n = 22), but with abnormal movements such as myoclonus (n = 2), dystonia (n = 2), rigidity (n = 5), or cerebellar ataxia (n = 13). Blood samples were taken from all patients after informed consent, and DNA was extracted using standard procedures. All patients had CAG repeats in the normal size range in the IT15 and DRPLA genes. In addition, CAG repeat expansions at the SCA-1, -2, -3, -6, -7, -8, -12, and -17 loci were excluded in the 13 index patients presenting with ataxia. In 27 patients, including 11 with typical HD, the transmission of the disease was autosomal dominant. Forty-seven had no known family history of chorea or dementia. Most families originated in France (n = 60) or continental Europe (n = 6) and five were of North African origin. The three other families were from Guinea, French West Indies, and Colombia. Mean age at onset was 43 ± 15 years and ranged from 1 to 83 years.

The distribution of the normal allele was determined in 95 healthy unrelated controls: 44 French and 51 North Africans.

We determined the CAG/CTG repeat sizes at the HDL2 locus by PCR with 200 ng genomic DNA, 0.8 μmol/L of primers L237–1 and L237–2,⁵ 0.2 mmol/L each dNTP, 2% deionized formamide, and 2.5 U of Taq DNA polymerase in the PCR buffer supplied by the manufacturer (Invitrogen, Carlsbad, CA) in a final volume of 25 μL. Samples then underwent 5 minutes of denaturation at 96 °C followed by 35 cycles of denaturation at 95 °C-1mn, annealing 59 °C-1mn, and extension at 72 °C-1mn, in a GeneAmp 9600 thermocycler (PE Biosystems, Foster City, CA). The last extension step was prolonged by 10 minutes. Aliquots of the PCR products then were diluted to 1:5 in formamide loading buffer and run in a 4% acrylamide gel on an ABI Prism 377 automated sequencer. GENSCAN and GENOTYPER software were used to genotype all individuals. Both alleles of the patient carrying an expansion were agarose-purified and subcloned in PGEM-T (Promega) before sequencing by Big Dye terminator chemistry (PE Biosystems).

Results.

A single patient (SAL-2289-001), without a known family history of the disease, carried a pure uninterrupted 50 CAG/CTG repeat at the HDL2 locus. All other 73 patients had repeats ranging from 10 to 17 units and did not differ significantly from the controls (8–28 units). The distribution was similar (p > 0.05) in the control populations, although it was broader in the North Africans (8–28) than in the French (9–18). Eighty-five percent of the control chromosomes carried 13 to 16 repeats. Heterozygosity was 62%.

The patient, a 44-year-old Moroccan woman, working as a cleaning agent, had mild choreic movements of the face and extremities for 2 years. She also had slight memory loss and a bedside Mini-Mental Status score of 22/30. Detailed neuropsychological evaluation revealed subcortical dementia similar to that found in HD caused by expansion in the IT15 gene: abnormal attention, global inertia, and memory deficit. Interestingly, memory impairment was atypical. Visual retention and logical memory were affected but performance on the paired associated tests of the Wechsler Memory Scale⁶ was normal, whereas it has been reported to be specifically impaired in HD,⁷ even during the presymptomatic stage of the disease.⁸ Enlargement of the anterior horn of the lateral ventricles was observed on cerebral MRI, suggesting atrophy of the head of the caudate nucleus (figure). Furthermore, atrophy of the cerebral cortex was particularly marked after only 2 years of disease duration. The patient’s father had died accidentally at age 54, but her mother (61 years old) was considered healthy. There were no other known affected individuals in the paternal family.

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Figure. Axial plane T2-weighted MRI (A) of patient SAL-2289–001 showing enlargement of the lateral ventricles and cortical atrophy. Gradient echo T2-weighted MRI coronal plane (B) showing enlargement of frontal horns indirectly reflecting atrophy of the caudate nucleus.

Discussion.

Andrew et al.⁹ found that 1% of patients diagnosed with HD did not have expanded CAG repeats in the IT15 gene. In our experience, only 88% (542/618) of families with suspected HD and 93% (542/583) of those with typical HD—i.e., chorea associated with dementia—were found to have CAG repeat expansions in the IT15 gene. Clinically diagnosed HD therefore is genetically heterogeneous.

Our report demonstrates further genetic heterogeneity of HD, because the CAG/CTG repeat expansion at the HDL2 locus is responsible for only 2% of our series of unexplained typical HD cases and 0.2% of all the HD families tested. Therefore, one or more genes must account for the other cases. Some of them might result from the recently identified mutation in the gene encoding the prion protein found in a family with HD.¹⁰ However, the phenotype of these patients was not typical of HD.

HD is also clinically heterogeneous. Age at onset and progression rate vary widely among patients, as do movement abnormalities and psychiatric involvement. The clinical and radiologic profile of patient SAL-2289–001, however, cannot be distinguished from that of patients with HD with an expansion in the IT15 gene. Our patient’s phenotype also is similar to other HDL-2 patients reported by Margolis et al.,⁴ who all show atrophy of the cerebral cortex that is particularly prominent on MRI scans of our patient.

Interestingly, all HDL2 patients described so far have been of African or African American descent.⁵ This could be because of a founder effect or because of recurrent mutations on a predisposing haplotype in this population. However, the parents of our North African patient were not black.

The size distribution of normal alleles in the French and North African controls was similar to that of white and African Americal controls,⁵ except that the upper range extended up to 28 repeats in the North African group. We did not find large alleles (30–40 repeats) as in the study of Holmes et al.⁵

The finding of several HD-like patients with CAG/CTG expansion in the junctophilin-3 gene demonstrates that HD is genetically heterogeneous. However, greater heterogeneity is suspected because HDL2 accounts only for approximately 2% of clinically typical HD cases without expansions in the IT15 gene. The existence of genetic heterogeneity in HD has major implications for presymptomatic testing of families in which HD has been diagnosed on clinical grounds. Exclusion of repeats in the IT15 and junctophilin-3 genes in these families does not exclude the presence of another mutation in them.