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Abstract
Objective To classify familial amyotrophic lateral sclerosis (FALS) on the base of family history, and to determine whether frequency of mutations in major amyotrophic lateral sclerosis (ALS) genes varies in different FALS categories.
Methods Included in the study are 53 FALS families. Patients were classified as definite, probable and possible FALS, according to recently proposed criteria. Seven ALS-associated genes, including SOD1, TARDBP, FUS, ANG, ATXN2, OPTN and C9ORF72, were analysed.
Results Thirteen patients (24.5%) were included in the definite group. The great majority of our FALS cases (40/53, 75.5%) were families with only two affected relatives; of these, 31 (58.5%) were included in the probable, and 9 (17%) in the possible FALS categories. The percentage of mutations was 61.5% in definite, 41.9% in probable and 11.1% in possible FALS. With respect to probable FALS, if cases with parent-to-child transmission of the disease were considered separately, the mutational load increased to 61.5%, as observed in definite FALS.
Conclusions Our findings provide evidence that frequency of mutations in currently known ALS genes varies widely among different FALS categories. Families with only two affected relatives have heterogeneous genetic components, the chance to detect mutations being higher in cases with parent-to-child transmission.
- Familial amyotrophic lateral sclerosis
- motor neuron disease
- genetics
- neuropathy
- neurophysiol
- clinical
- vasculitis
- neuromuscular
- evoked potentials
- somatosensory
- neurophysiology
- ALS
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- Familial amyotrophic lateral sclerosis
- motor neuron disease
- genetics
- neuropathy
- neurophysiol
- clinical
- vasculitis
- neuromuscular
- evoked potentials
- somatosensory
- neurophysiology
- ALS
Introduction
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder involving upper and lower motor neurons. The disease occurs sporadically (SALS) in the majority of cases, while nearly 5% of patients have a positive family history.1 An increasing number of genes have been identified over the last 20 years, accounting for about 50%–60% of familial ALS (FALS) and a small proportion of SALS.2 ,3 Though the distinction between familial and sporadic ALS is widely accepted, several studies suggest that it should be considered artificial.4–6 Furthermore, the definition of FALS has not been clearly established, and criteria for FALS were recently proposed, incorporating family history and genetic analysis.7
In the present work, we classified 53 families with FALS into different categories on the basis of family history, and evaluated whether the prevalence of mutations in seven genes (SOD1, TARDBP, C9ORF72, FUS, OPTN, ANG and ATXN2) differed in each category. We focused primarly on kindred with only two affected individuals, since heritability in this FALS subgroup is uncertain.
Methods
From 1987 to February 2012, a cohort of 980 ALS patients was evaluated in the ALS Clinic Center of the Catholic University of Rome. All patients were from the central or southern regions of Italy.
Fifty-seven patients (5.8%) had one or more affected relatives and were diagnosed as FALS, while 923 (94.2%) had no family history and, therefore, diagnosed as SALS. DNA samples were available from 53 of the 57 patients with FALS. All patients met the El–Escorial diagnostic criteria for ALS.8 We classified the 53 patients into definite, probable and possible FALS categories by using previously proposed family history criteria.7 Included in the definite group were patients with at least two first- or second-degree relatives with ALS; the probable group comprised patients with one first- or second-degree-affected relative; the possible group included patients with one distant affected relative. First-degree relatives included parents, offspring and siblings; second-degree relatives included grandparent and aunts/uncles.
Based on clinical patterns in the fully developed state of the disease, each patient was classified as having one of the following phenotypes: classic, upper motor neuron dominant (UMN–D), flail arm or pure lower motor neuron, as we have previously described.9 Survival was defined as the period from disease onset to last follow-up, or death or tracheostomy, and it was analysed using the Kaplan–Meier method.
Coding exons and flanking intronic regions of SOD1 (MIM:147450), ANG (MIM:105850), TARDBP (MIM: 605078), FUS (MIM: 137070) and OPTN(MIM:602432) genes were amplified and screened by direct sequencing on genomic DNA. ATXN-2 (MIM: 601517) and C9ORF72 genes (MIM: 614260) were analysed using a fluorescent-PCR, as described.10 All participants gave written informed consent.
Results
Definite FALS
Thirteen patients (24.5%) had at least two affected relatives and were included in this group. The number of affected members in individual families varied from three to eight, with a total of 59 patients (figure 1). Thirty-one patients were males and 28 females. Clinical data were available from 44 patients: age of onset ranged from 24 to 77 years (mean 50.6); median survival was 33 months. Familial segregation was in accordance with an autosomal-dominant pattern of inheritance in 12 families. The disease was maternally transmitted in nine of them (69.2%) and paternally transmitted in the remaining three. In one relative with three affected siblings, the pattern of inheritance was unclear, since the parents had died at 64 and 68 years of age, without signs of motor neuron disease or fronto-temporal dementia. Of the 13 patients, eight (61.5%) had mutations in one of the analysed ALS genes. SOD1 mutations were detected in three patients (A4V, G93D, L84F), FUS mutations in one (R521C), and TARDBP mutations in another one (G376D). The remaining three patients, including the one with unclear inheritance pattern, harboured the hexanucleotide expansion of C9ORF72. (see Web table).
Number of affected members in families with familial amyotrophic lateral sclerosis. Seventy-five per cent of our 53 families comprised only two affected relatives.
Probable FALS
Thirty-one patients (58.5%) had one first- or second-degree relative with ALS. Of the total 62 patients, including index patients and relatives, 35 were males and 27 were females. Clinical data were available from 53 of them: age of onset ranged from 34 to 77 years (mean 57.7), median survival was 30 months.
Thirteen of the 31 probable FALS patients (41.9%) had mutations in one ALS gene: four in SOD1 (D101G, L144F, L38R, S134G), seven in C9ORF72 and two in ATXN2 (33 CAG repeats).
The 31 patients with probable FALS were divided into subgroups A and B on the basis of degree of kinship of the affected pair of relatives. In probable FALS-A were included 13 patients with a direct-line first-degree-affected relative, who was the father in eight instances and the mother in 5. In this subgroup, the percentage of index patients with mutations was 61.5%. Probable FALS-B included 18 patients, whose affected relatives were siblings (11 families) or second-degree relatives (uncle/aunt in 6; grandmother in 1). In this subgroup, the percentage of index patients with mutations was 27.7%. No differences were observed between patients with one affected sibling (27.3%), and those with one second-degree-affected relative (28.6%).
Possible FALS
Nine patients (17%) had one relative with ALS who was more distant than first or second degree. Of them, 12 were males and six were females. The second affected relative was the proband's first cousin in four families, first cousin once removed in 4, and great-aunt in one. Data could be collected from only 12 patients: age of onset ranged from 33 to 70 years (mean 52.4) and median survival was 49 months. Only one of the nine patients with possible FALS (11.1%) had a mutation in the screened genes, consisting of an intermediate-length CAG expansion (32 repeats) in ATXN2.
Discussion
ALS is considered to be familial if one or more relatives are reported as affected by the same disease, but no consensus exists on standard definition criteria for FALS.7 ,11 The number of affected relatives and degree of relationship vary greatly among kindred, and in several instances, the pattern of inheritance is unclear.12
In the present work, we classified 53 families with FALS into four groups, mainly according to recently suggested clinical criteria,7 and found that frequency of mutation in seven ALS-associated genes varied considerably among different categories.
Thirteen of our 53 families (24.5%) had at least three ascertained patients and were classified as definite FALS. A high percentage of index patients with mutations (61.5%) were observed in this category, as expected.
The great majority of our FALS cases (40/53, 75.5%) were families with only two affected relatives (figure 1), a frequency significantly higher than that previously reported in a cohort of FALS families from France and Canada (50%).12 Different opinions exist as to whether these patients should be classified as FALS.11 In fact, the possibility that a second person among relatives is affected by chance if one person already has ALS may not be excluded.12
We found that if the 40 patients in this category of FALS are stratified according to family history, rates of mutations vary from 61.5% to 11.1% in different subgroups, suggesting heterogeneous genetic components (figure 2). In thirty-one of these 40 families, the two affected members were first- or second-degree relatives, and they were included in the probable FALS category. In this group, 41.9% of index patients had mutations. However, when cases of FALS with parent-to-child transmission of the disease (probable-A) were considered separately, the chance of detecting mutations was the same as that found in definite FALS, suggesting a Mendelian-like heritability. The smaller rate of mutations observed in families where the other affected member is a more distant relative, raises the hypothesis that a polygenic contribution of small-effect variants or low-penetrance variants is the underlying cause of the disease in these cases.
Genetic component in different amyotrophic lateral sclerosis (ALS) groups. Rates of mutation vary widely in different ALS categories. Classification is described in the text.
Accordingly, in the nine patients who were defined as possible FALS, only one (11.1%) harboured a gene mutation, consisting of intermediate length (32 repeats) ATXN2 expansion. Although the limited number of patients belonging to this group does not allow definite conclusions, the genetic component in possible FALS seems comparable with that of our cohort of sporadic cases, in which mutations in the same genes were detected at a frequency of 11%.10
Several studies suggest that the dichotomy between familial and sporadic ALS is less clear than previously assumed, including the observation that relatives of patients with ALS have an increased risk of being affected.5 ,13
The results of this study provide an empirical confirmation of this hypothesis, showing that genetic components due to the currently known major ALS genes exist on a continuum, ranging from fully penetrant phenotypes to apparently sporadic cases.
The discovery of new large- and low-effect genes in the near future will reasonably increase the percentage of patients with definite genetic aetiology, and hopefully, will elucidate missing hereditability of ALS.
Acknowledgments
We are indebted to the individuals and their families who participated in this project. We thank Federazione Italiana Gioco Calcio and I.CO.M.M. onlus, Association for ALS research.
References
Footnotes
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Linked article 303127.
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Competing interests None.
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Patient consent Obtained.
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Ethics approval The study was approved by the local ethical committee.
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Provenance and peer review Not commissioned; externally peer reviewed.
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