Background and aim: Duplication of the lamin B1 gene (LMNB1) has recently been described in a rare form of autosomal dominant adult-onset leucoencephalopathy. The aim of the study was to evaluate the presence of LMNB1 gene defects in a series of eight patients with diffuse adult-onset hereditary leucoencephalopathy.
Methods: Clinical features of tested patients included a variable combination of pyramidal, cerebellar, cognitive and autonomic dysfunction. Neuroradiological data (MRI) showed symmetrical and diffuse white-matter lesions in six cases, and multifocal confluent lesions in two. LMNB1 full gene deletion/duplication and point mutations were searched using a TaqMan real-time PCR assay and direct sequencing of all coding exons.
Results: One patient carried a 140–190 kb duplication involving the entire LMNB1 gene, the AX748201 transcript and the 3′ end of the MARCH3 gene. Clinical and neuroimaging data of this proband and an affected relative overlapped with the features already described in patients with LMNB1 duplication. Lamin B1 expression was found increased in lymphoblasts. No LMNB1 gene defect was identified in the remaining seven probands.
Conclusions: LMNB1 gene duplication appears characteristic of a subset of adult-onset autosomal dominant leucoencephalopathies, sharing autonomic dysfunction at onset, diffuse T2-hyperintensity of supra- and infratentorial white matter, sparing of U-fibres and optic radiations. The variable phenotypes in the remaining cases lacking LMNB1 defects (five with autosomal dominant transmission) suggest that adult-onset leucoencephalopathies are genetically heterogeneous.
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Hereditary leucoencephalopathies are rare disorders characterised by progressive degeneration of myelin in the central nervous system, with variable degree of involvement in the peripheral nervous system. The most common forms include recessive autosomal or X linked patterns, and onset in infancy or childhood.1 Autosomal dominant forms include Alexander disease, due to mutations in the GFAP gene, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leucoencephalopathy (CADASIL), and adult-onset Autosomal Dominant Leucodystrophy (ADLD, OMIM#169500), a very rare disease due to duplication of the LMNB1 gene.2 ADLD is characterised by onset in the fourth to fifth decade with autonomic dysfunction and diffuse white-matter disorder.3 A limited number of families with or without autonomic dysfunctions and autosomal dominant inheritance are reported, in which the involvement of the LMNB1 gene is unknown.4–10
The aim of our work was to evaluate the presence of full LMNB1 gene deletion/duplication and point mutations in the coding region, in a selected group of eight unrelated cases with an adult-onset leucoencephalopathy. One of these patients had a phenotype completely overlapping ADLD.
MATERIALS AND METHODS
Clinical and neuroradiological features of ADLD patients
We collected eight unrelated Italian cases with familial adult-onset leucoencephalopathy (table 1). All onset above 30 years, with a slow progression of neurological symptoms; MRI was suggestive for white-matter pathology, and at least one first-degree relative was affected by a similar disorder. At least six were compatible with an autosomal dominant transmission pattern. Seven patients (28, 41, 43, 69, 89, 94 and 109) were referred after a molecular test for exclusion of CADASIL (DHPLC analysis of exons 2–23 of the NOTCH3 gene). Patient G769 had a phenotype overlapping ADLD, and was sent by the Medical Genetics Unit in Ferrara with a diagnosis of “ADLD compatible with LMNB1 duplication.”
Analysis of LMNB1 gene mutations and lamin B1 expression in lymphoblasts
The number of LMNB1 copies was determined by a quantitative duplex PCR assay, based on the relative amplification of the target sequence (LMNB1) and the internal standard RNaseP. TaqMan real-time RT-PCR analysis was used to measure expression levels of LMNB1 vs TBP or G6PDH reference genes in one affected individual (III-4) and three healthy controls (see supplementary data, and supplementary fig 2).
The LMNB1 gene (5q23.2, 11 exons; chr5: 126 140–126 200 kb, NM_005573.2) was screened for point mutations in the coding region, using primers designed to flank each exon, in the eight affected index cases (conditions available upon request).
To define the boundary of the duplicated region, we compared the sequencing profiles of heterozygous Single Nucleotide Polymoprhisms (SNP) or heterozygous Short Tandem Repeats (STRs) in patient III-4 vs normal subjects, as described by Padiath et al2 (see supplementary fig 3).
We found one patient with LMNB1 duplication belonging to a three-generation autosomal dominant pedigree (G769, fig 1B). The duplication was confirmed in a second affected member of the family (III-4).
Using the analysis of relative peak height ratio of heterozygous microsatellite markers and SNPs, we defined the duplicated segment to a region of 140–190 kb, which contains the LMNB1 gene, the AX748201 transcript and the 3′ end of the MARCH3 gene (fig 1C, and supplementary fig 3). The characterisation of the breakpoints was not possible, but, using published primers, we excluded that this was one of the two large duplications, whose breakpoints had already been identified (families K4233, K2685 and K50069).2 However, this duplication is similar to that described in family K4975 of Japanese origin (>150 kb), whose boundaries were not defined.2
According to published data, LMNB1 gene expression is increased in the brain of patients with LMNB1 duplication.2 We found that LMNB1 gene expression was fivefold higher than controls also in the lymphoblastoid cell line of patient III-4 (supplementary fig 2).
In the remaining series of seven cases, no point mutation was detected, screening the coding exons and flanking introns of the LMNB1 gene.
Clinical and neuroradiological phenotype of patients with LMNB1 duplication
The disease onset at 35 years with autonomic dysfunctions (urinary retention, fecal incontinence and sexual disturbances gradually evolving to impotence). A diagnosis of “possible MS” was suggested at a first neurological and neuroradiological exam. Four years later, impairment in walking due to gait imbalance and weakness of the legs appeared. Spastic crying and laughing were also present. The patient underwent an extensive workup to exclude a demyelinating disorder due to a known metabolic defect. Lysosomal enzyme activities were normal (exosoaminidase A and B, arylsulfatase A, galactosylcerebrosidase, α and β mannosidase). CSF exam was unremarkable, and oligoclonal bands were not detected. No anomalies were evident at the electroneurographic exam, and a neuropsychological evaluation failed to detect any deficit in cognitive functions.
The disease progressed slowly, and 15 years from the onset, the patient was wheel-chaired and presented a mild cognitive impairment with deficit in frontal functions (Wisconsin Card Sorting Test, Stroop test) and the involvement of orthosympathetic functions (Tilt test, Valsalva test and deep breathing test).
Cerebral MRI a few years after the onset of symptoms showed a diffuse T2 hyperintensity of the white matter in the cerebellar hemispheres, posterior limbs of the internal capsulae, centrum semiovalis and corpus callosum with sparing of optic radiations, subcortical U-fibres and a thin rim of periventricular white matter (see fig 1D). Pons, medulla oblongata and vermis appeared mildly hypotrophic. A slight supratentorial atrophy was present. No cystic degeneration or calcifications were detectable.
The patient had a positive family history of neurological diseases. His father died at 57 years, unable to walk; his brother (III-5) developed primary progressive multiple sclerosis (MS); a paternal uncle (II-6) had been diagnosed in mid 1980 with pseudobulbar syndrome and “vascular subcortical encephalopathy.”
Neurological symptoms were represented by micturition disorder at 45 years, followed, a few years later, by slight disturbances of the gait.
The CSF exam was unremarkable; MRI demonstrated a diffuse and symmetrical involvement of the white matter comparable with patient III-4, except that corpus callosum was less prominently involved.
Patient II-6 received a diagnosis of “pseudobulbar syndrome” at 61 years. Clinical features included micturition disorder followed by pyramidal (motor impairment) and later by bulbar signs (dysphagia and dysarthria). No deficit in cognitive function or psychiatric symptoms were reported. A CT scan revealed diffuse hypodensity of periventricular and subcortical white matter, interpreted as being of ischaemic origin. DNA was not available for molecular confirmation of the LMNB1 gene duplication.
Duplication of the LMNB1 gene has recently been identified in four families of Irish-American or Japanese origin with ADLD.2
Among eight unrelated probands with adult-onset hereditary leucoencephalopathy, we found one patient carrying LMNB1 duplication (G769), belonging to a large autosomal dominant pedigree. Clinical and MRI data for this patient and an affected relative showed that the onset and progression of the disease overlap with those reported for patients with LMNB1 duplication. In all ADLD cases, onset is in the 4th–5th decade of life with involvement of the autonomic nervous system (bowel and bladder dysfunction, impotence, orthostatic hypotension and decreased sweating). Pyramidal and cerebellar signs followed later.
MRI shows a diffuse alteration of the supra- and infratentorial white matter, in particular of the cerebellum, the corticospinal tracts and the corpus callosum, with relative preservation of periventricular white matter and sparing of U-fibres without a significant atrophy.2 3 11 12 The neuroimaging was slightly different in the youngest patient (III-6), with a relative sparing of the corpus callosum.
LMNB1 is the only functional gene within the duplicated segment, whose breakpoints are different from the two characterised so far,2 suggesting that duplications of this gene originated from independent mutational events.
We have also shown that LMNB1 expression levels are increased in lymphoblasts, implying that expression analysis may be a useful tool as first-line screening for LMNB1 involvement in patients with leucoencephalopathy.
It has not yet been clarified if point mutations may mimic the phenotype of the duplication. Although we did not find any point mutation in the seven probands negative for LMNB1 duplication, the study of a larger series of patients is needed to rule out the possibility that ADLD is caused by point mutations in this gene, and to verify whether this phenotype is invariably associated with the duplication. Moreover, five of these patients had an autosomal dominant leucoencephalopathy, suggesting that these rare diseases are clinically/genetically heterogeneous.
Finally, due to the late age at onset, the slow progression and the paucity of initial symptoms of the disease associated with LMNB1 duplication, we suggest considering the diagnosis of ADLD in all patients with autonomic dysfunction and a mild non-specific white-matter alteration of the central nervous system, also known as “leucoaraiosis.”
This work was supported by the “Associazione EE Rulfo per la Genetica Medica,” MURST 60%, Telethon grant GGP07110. We thank the technical assistance of P Pappi and V Giangarrà. We are gratefully indebted to the families and patients who participated in this study.
▸ Supplementary materials, methods and figures are published online only at http://jnnp.bmj.com/content/vol80/issue2
Competing interests: None.
Ethics approval: Ethics approval was provided by the Department of Genetics Biology and Biochemistry internal ethics committee.
Patient consent: Obtained.
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