Alessandro Brussino

Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28

Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA–deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration.

An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene

Several diagnostic strategies have been applied to the detection of FMR1 gene repeat expansions in fragile X syndrome. Here, we report a novel polymerase chain reaction-based strategy using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany) and the osmolyte betaine. Repeat expansions up to approximately 330 CGGs in males and up to at least approximately 160 CGGs in carrier women could be easily visualized on ethidium bromide agarose gels. We also demonstrated that fluorescence analysis of polymerase chain reaction products was a reliable tool to verify the presence of premutation and full mutation alleles both in males and in females. This technique, primarily designed to detect premutation alleles, can be used as a routine first screen for expanded FMR1 alleles.

FMR1 gene premutation is a frequent genetic cause of late-onset sporadic cerebellar ataxia

In an Italian population of 275 unrelated men affected by adult-onset sporadic progressive cerebellar ataxia, the authors found six patients carrying an FMR1 gene premutation. Age at onset (range, 53 to 69 years) and clinical-neuropathologic findings were consistent with the fragile-X tremor ataxia syndrome (FXTAS), although tremor was not as common as previously described. FXTAS accounted for 4.2% of the cases diagnosed at >50 years, suggesting that it is a frequent genetic cause of late-onset sporadic ataxia.

Modulation of the age at onset in spinocerebellar ataxia by CAG tracts in various genes

Polyglutamine-coding (CAG)n repeat expansions in seven different genes cause spinocerebellar ataxias. Although the size of the expansion is negatively correlated with age at onset, it accounts for only 50-70% of its variability. To find other factors involved in this variability, we performed a regression analysis in 1255 affected individuals with identified expansions (spinocerebellar ataxia types 1, 2, 3, 6 and 7), recruited through the European Consortium on Spinocerebellar Ataxias, to determine whether age at onset is influenced by the size of the normal allele in eight causal (CAG)n-containing genes (ATXN1-3, 6-7, 17, ATN1 and HTT). We confirmed the negative effect of the expanded allele and detected threshold effects reflected by a quadratic association between age at onset and CAG size in spinocerebellar ataxia types 1, 3 and 6. We also evidenced an interaction between the expanded and normal alleles in trans in individuals with spinocerebellar ataxia types 1, 6 and 7. Except for individuals with spinocerebellar ataxia type 1, age at onset was also influenced by other (CAG)n-containing genes: ATXN7 in spinocerebellar ataxia type 2; ATXN2, ATN1 and HTT in spinocerebellar ataxia type 3; ATXN1 and ATXN3 in spinocerebellar ataxia type 6; and ATXN3 and TBP in spinocerebellar ataxia type 7. This suggests that there are biological relationships among these genes. The results were partially replicated in four independent populations representing 460 Caucasians and 216 Asian samples; the differences are possibly explained by ethnic or geographical differences. As the variability in age at onset is not completely explained by the effects of the causative and modifier sister genes, other genetic or environmental factors must also play a role in these diseases.

Missense mutations in the AFG3L2 proteolytic domain account for ∼1.5% of European autosomal dominant cerebellar ataxias

Spinocerebellar ataxia type 28 is an autosomal dominant form of cerebellar ataxia (ADCA) caused by mutations in AFG3L2, a gene that encodes a subunit of the mitochondrial m‐AAA protease. We screened 366 primarily Caucasian ADCA families, negative for the most common triplet expansions, for point mutations in AFG3L2 using DHPLC. Whole‐gene deletions were excluded in 300 of the patients, and duplications were excluded in 129 patients. We found six missense mutations in nine unrelated index cases (9/366, 2.6%): c.1961C>T (p.Thr654Ile) in exon 15, c.1996A>G (p.Met666Val), c.1997T>G (p.Met666Arg), c.1997T>C (p.Met666Thr), c.2011G>A (p.Gly671Arg), and c.2012G>A (p.Gly671Glu) in exon 16. All mutated amino acids were located in the C‐terminal proteolytic domain. In available cases, we demonstrated the mutations segregated with the disease. Mutated amino acids are highly conserved, and bioinformatic analysis indicates the substitutions are likely deleterious. This investigation demonstrates that SCA28 accounts for ∼3% of ADCA Caucasian cases negative for triplet expansions and, in extenso, to ∼1.5% of all ADCA. We further confirm both the involvement of AFG3L2 gene in SCA28 and the presence of a mutational hotspot in exons 15–16. Screening for SCA28, is warranted in patients who test negative for more common SCAs and present with a slowly progressive cerebellar ataxia accompanied by oculomotor signs. Hum Mutat 31:1–8, 2010.

A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

A family with autosomal dominant leukodystrophy linked to 5q23.2–q23.3 without lamin B1 mutations

Background and purpose:  Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult‐onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD.

A novel family with Lamin B1 duplication associated with adult-onset leucoencephalopathy.

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.

Two Italian Families with ITPR1 Gene Deletion Presenting a Broader Phenotype of SCA15

Spinocerebellar ataxia type15 (SCA15) is a pure ataxia characterized by very slow progression. Only seven families have been identified worldwide, in which partial deletions and a missense mutation of the inositol triphosphate receptor type I gene (ITPR1) have been reported. We examined a four-generation Italian family segregating an autosomal dominant cerebellar ataxia, in which linkage analysis was positive for the SCA15 locus. We performed a genomic real-time polymerase chain reaction to search for ITPR1 gene deletions in this family and in 60 SCA index cases negative for mutations in the SCA1–3, 6–8, 10, 12, and dentatorubral-pallidoluysian atrophy genes. The deleted segments were characterized using a custom array comparative genomic hybridization analysis. We have identified two families with an ITPR1 gene deletion: in one, the deletion involved ITPR1 only, while in the other both sulfatase-modifying factor 1 and ITPR1. Clinical data of ten patients and brain MRI (available for six) showed that the phenotype substantially overlapped known SCA15 cases, but we also noted buccolingual dyskinesias, facial myokymias, and pyramidal signs never reported in SCA15. ITPR1 expression analysis of two deleted cases showed a half dose. Our results further support ITPR1 gene as causative of SCA15. The families reported show that SCA15 is present in Italy and has a greater variability in the age at onset and clinical features than previously reported. We propose that the search for ITPR1 deletions is mandatory in the clinical hypothesis of SCA15 and that ITPR1-reduced expression in blood may be a useful marker to identify SCA15 patients harboring genomic deletions and possibly point mutations causing reduction of mRNA level.

Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication‐based mechanisms such fork stalling and template switching or microhomology‐mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele‐specific LMNB1 expression levels.