Download Disease clustering: the example of ALS, PD, dementia and

Disease clustering: the example of ALS, PD,
dementia and hereditary ataxias in Italy
Andrea Malaspina*,**
Dario Alimonti*
Tino Emanuele Poloni***
Mauro Ceroni*,***
*Laboratory of Experimental Neurobiology, IRCCS C.
Mondino Institute of Neurology, University of Pavia, Italy,
**Division of Neuroscience and Psychological Medicine,
Imperial College School of Medicine, University of London,
UK,
***Department of Neurology, “Policlinico di Monza”
Hospital, Monza, Italy
Reprint requests to: Prof. Mauro Ceroni,
IRCCS C. Mondino Institute of Neurology
Via Palestro 3, 27100 Pavia, Italy
E-mail: [email protected]
Accepted for publication: November 14, 2002
Summary
The “mini-epidemic” distribution of rare conditions (either sporadic, inherited or due to a transmissible agent)
is frequently described as a cluster. Genetic abnormalities and environmental factors are usually investigated
to explain the presence of a disease cluster. We have reported a cluster of amyotrophic lateral sclerosis (ALS)
cases in a small area of central Italy, where an identical
SOD1 gene mutation was found both in familial ALS
(FALS) cases and in one apparently sporadic ALS individuals. Along with this cluster of ALS patients, we review important clusters of neurological disorders in Italy
and discuss the importance of an accurate estimation of
their regional/local prevalence. This approach is likely to
facilitate molecular investigations, the search for environmental agents and the analysis of gene-environment
interaction in disease presentation and development.
The organisation of national registers that record, in particular, the geographical distribution of neurological disorders, might represent a good research strategy.
KEY WORDS: Cluster, environmental factors, Italy, mutations, neuro logical disorders.
Introduction
Epidemiological studies constitute a powerful tool for
the identification of clues to disease aetiology and for
the evaluation of risk factors (e.g., environmental or ocFunctional Neurology 2002; 17(4): 177-182
cupational hazards) in public health management (1,2).
Geographical epidemiology has become a distinct
branch of epidemiology, with increasing attention being
devoted to describing the health status of small, geographically-circumscribed populations (3).
In medical terms, a cluster is a “mini-epidemic” distribution of a pathological condition within a well-defined region, where it accounts for a higher-than-expected disease prevalence (i.e., above the estimated national
prevalence). When, in some families, more “disease
cases” occur than would be expected on the basis of
the occurrence rate in the general population, we also
talk of familial clustering. The identification of a cluster
depends on the precise evaluation of several factors,
including the expected prevalence of the disease in the
given area and the geographical distribution of the patients under investigation. However, it can be argued
that an “aggregation” of patients may also occur by
chance or as a consequence of a statistical bias in the
process of patient selection (2).
A genetic mutation with a recessive or dominant pattern
of inheritance is often responsible for the appearance of
a disease cluster, within a family or a geographical
area. Exposure to environmental factors is another important cause of disease clustering. As postulated in
Parkinson’s disease (PD), when the disease is not
clearly related to a single gene mutation (4), the combined action of several genes together with exposure to
environmental factors may be important pathogenically.
In a situation where there is genetic predisposition to
the disease and exposure to environmental precipitating factors (for members of some families but not for
members of other families), a monogenic inheritance
may be mimicked (5).
The importance of identifying disease clusters is increasingly debated. Systematic surveillance systems
have been developed to identify foci of increased disease occurrence (together with possible risk factors),
but despite this, their reliability remains a matter of discussion (6).
Social and cultural background can also play a part in
the uneven distribution of several medical conditions (2).
In addition to the aetiological factors, such as genetic
abnormalities and/or environmental factors, socio-cultural habits and traditions appear to contribute to regional
differences in general health status and in the development of several pathological disorders. A strong association between a population’s health status and social
and economic characteristics has been shown by many
methodological approaches, such as the “deprivation indices” developed in the United Kingdom (7). A significant association between antibodies against CMV and
specific socio-demographic/geographical factors has also been found. The prevalence of these antibodies was
significantly higher in females and in subjects residing in
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A. Malaspina et al.
the South of Italy (8). CMV infection may be a critical
determinant for the appearance of neurological disorders, particularly in immune-compromised patients.
To emphasise the importance of identifying regionallyconfined clusters of patients suffering from a neurological condition, we describe our experience with a cluster
of patients suffering from ALS. We have studied both
the genetic and the environmental factors that may
have played a role in the high occurrence of ALS in the
area studied (9). The major clusters of neurological disorders in Italy are also reviewed, paying particular attention to analysis of genetic mutations, environmental
hazards and gene-environment interaction. We discuss
the importance of studying the geographical distribution
of patients affected by a specific neurological disorder
and the possible use of disease registers that would allow collection of and easy access to all the information
regarding a patient population.
Amyotrophic lateral sclerosis
A 41-year-old man presenting with rapidly progressive
weakness of the lower limbs was admitted to our clinic.
The patient had a family history of ALS, and detailed in vestigation of the pedigree led to the identification of a
large kindred with an autosomal dominant inheritance
for ALS (FALS). The proband’s mother died of ALS at
36 after 4 years of illness and his grandfather died at 55
after a 12-year disease course. In this particular family,
we identified a total of 13 cases spanning back 6 generations to the beginning of the 19th century. Data were
collected from interviews with family members, medical
records, and church/civic records of births and deaths.
With the cooperation of local general practitioners (GPs),
we also analysed the distribution of other ALS cases in
the area where the proband was identified: an area of
approximately 400 km2 located in the north of the central
Italian province of Ascoli Piceno, including the valleys
around Fermo (the area). A total of 12 different ALS cases (2 familial and 10 sporadic) were identified in a population numbering 166,451 people (1997). The prevalence
of the disease according to our findings was 7.8/100,000,
higher than the national frequency (which ranges between 1.56 (10) and 5.4 (11) per 100,000).
The SOD1 gene molecular analysis of the FALS cases
(including our proband) yielded a missense point mutation causing the substitution of Leucine with Phenylalanine in exon 4 (L84F). We extended the molecular investigation to the sporadic ALS cases and to those
members of the pedigrees at risk of inheriting the disease. The L84F SOD1 gene mutation was also detected in a 60-year-old sporadic ALS patient with unknown
father and with clinical features that overlapped those
of the FALS cases. Furthermore, the L84F mutation
was also found in a 65-year-old unaffected woman belonging to the proband’s family.
One of the two new FALS patients was linked to the
family lineage of our proband. In this pedigree a case of
conjugal ALS was present in a previous generation, but
no subject homozygous for the L84F mutation was detected. Due to the low population prevalence of ALS,
this event may suggest that the disease had been inherited by two apparently unrelated individuals in a
small community or that they had been exposed to the
178
same environmental agent. A total of nine cases of conjugal ALS have been previously described (12), two of
these from Italy (southern Italy and Sardinia). All these
couples, including our family, originated from small, inbred rural communities.
The presence of a significant number of individuals sharing the same rare molecular abnormality in a small isolated population may suggest a common founder for the
SOD1 gene mutation and a high level of inbreeding within the population in the area. The presence of ALS cases
bearing a SOD1 gene mutation increased the total number of affected individuals in the area, and ultimately, the
local disease prevalence to 7.8/100,000. The prevalence
of sporadic ALS in the area was 4.8/100,000, which is
within national estimated range. In this small population
the SOD1 gene mutation spreads vertically through apparently distant lineages and distinct family pedigrees.
Environmental factors were also investigated in search of
other elements explaining the “apparently” high occurrence of ALS cases in this area. A putative external
agent was unlikely to explain the appearance of the disease in subsequent generations (transmitted in an autosomal dominant fashion). Therefore, if an exogenous
agent had been responsible for the occurrence of the
disease throughout the generations, a “subtle” element,
perhaps linked to a well-established local practice, could
be postulated. We discovered that shoe manufacturing
spread rapidly within a well-defined period in the area
and that many individuals, including our FALS cases,
were employed in this activity (whereas in the past, agriculture had been the most important form of occupation
for the area’s population). Nevertheless, no evidence of
increased exposure to toxicants in these individuals or in
the general population of the area emerged. Furthermore, analysis of local aqueduct water did not reveal
high concentrations of manganese or magnesium, as reported in the previous investigations on environmental
hazards in ALS (13,14). Finally, we were unable to find
environmental factors that could explain the trend of generational anticipation that was present in these families.
Few other studies of the regional distribution of the disease are available in the literature. As for other neurological disorders, Sardinia has been a focus of epidemiological studies because it is isolated from foreign influence and has communities, throughout the island, that
have maintained close kinship ties. Giagheddu et al.
(15) analysed the occurrence of ALS cases in various
areas of the island, and noted a significantly heterogeneous distribution of the disease.
Maurelli et al. (16) reported 3 families in the province of
Pavia, an area in northern Italy. Reconstruction of the
pedigree was difficult and the SOD1 gene analysis was
not carried out. Prior to our study, only one family harbouring a SOD1 gene mutation had been reported in
Italy (17), while more recently, six other mutations have
been identified in seven ALS families (18). The authors
do not comment on the pattern of territorial distribution
of these families.
Parkinson’s disease (PD)
Unlike ALS, Parkinson’s disease (PD) shows a much
higher prevalence, varying in Italy from 74 to 257 cases
per 100,000 members of the population (19). The crude
Functional Neurology 2002; 17(4): 177-182
Disease clustering: ALS, PD, dementia and hereditary ataxias in Italy
prevalence of idiopathic PD in Italy varies considerably,
depending on the method of study utilised (20-22). An
important “aggregation” of PD cases was detected in
Sicily, where a door-to-door method of investigation
was adopted (23).
Despite the presence in the southern Italian Campania
and Molise regions (including the Salerno province) of
an overall prevalence that is comparable to that of other
western populations, a large kindred of autosomal dominant PD was found in a small village in the Salerno
province (24). The procedures for identification and
characterisation of the families living in this area were
similar to those used in our study on the ALS familial
cluster. Initially, two large kindreds were found in the
village of Contursi (Salerno province) where the rate of
occurrence of the illness was significantly high. The disease in the two kindreds showed a phenotypic homogeneity. Further investigation revealed a common origin
of the two families and an autosomal dominant pattern
of inheritance of the disease. Molecular studies confirmed the presence of a mutation in exon 4 in the αsynuclein gene with Ala to Thr substitution at position
53 (25,26). As in our experience with the ALS cluster,
molecular investigations confirmed a genetic mutation
to be responsible for the significant increase in the
number of PD cases in the above PD familial cluster.
Prior to the publication of these results, De Michele et
al. analysed PD-related environmental and genetic risk
factors in these regions of southern Italy. Family history
was found to be the major risk factor for the disease,
with 33% of the patients having had at least one affected relative (27). No environmental risk factor has been
reported to act on the genetic background described in
the Contursi families. The search for an external toxic
agent in PD has focused in the past on the study of
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine),
which shows a selective toxicity for the neurones of the
substantia nigra (28). However, no PD clusters caused
by MPTP-related environmental toxins have, to date,
been described (29). Several studies have also
analysed susceptibility markers within important metabolic pathways, to identify genetic polymorphisms
which might aggregate with the disease. Of these, the
“pharmacogenetic” marker P4502D6 debrisoquine 4hydroxylase (a cytochrome P450-dependent enzyme
which metabolises over 25 drugs) can be present in the
“extensive” and “poor” metaboliser aplotype conformations. No clear association between PD and any of
these allelic variants has been found (4).
Dementia
Alzheimer’s disease (AD) is the most common of the
various forms of dementia. In the last few years, large
Italian pedigrees harbouring different genetic abnormalities and showing a cluster-like distribution of early-onset familial AD (FAD) have been found in the southern
part of the Italian peninsula. Initially, two unrelated families from the Calabria region had been found to carry a
mutation in exon 17 of the amyloid precursor protein
gene (APP717) on chromosome 21, which segregates
with an early-onset form of AD (30). The study of other
large pedigrees based in the same area has led to the
identification of another genetic abnormality on chromoFunctional Neurology 2002; 17(4): 177-182
some 14 (31). These pedigrees also contained cases of
early-onset FAD and were finally merged into two larger
kindreds through an extensive epidemiological and genealogical reconstruction.The molecular analysis identified a Met 146 Leu missense mutation in the presenilin
1 (PS-1) gene.
Migration from southern Italy to other Italian regions or
abroad explains the molecular findings of other families
with early-onset FAD harbouring the same genetic mutations. A large South American pedigree from Argentina with early-onset FAD was recently found to bear the
presenilin 1 gene mutation (32), but no clear genealogical link with the above Calabrian families could be identified. In Turin (northern Italy), a pedigree with autosomal dominant early-onset FAD was found to contain
1,950 members, spanning eight generations with at
least thirty living affected individuals (33). The ancestors of the proband were from Calabria (southern Italy)
from where they had emigrated to the northern part of
Italy, France and United States. The “Turin family”
shows several genealogical and clinical similarities with
other large FAD pedigrees originating from Calabria.
An association of apolipoprotein E (ApoE) epsilon 4 allele (chromosome 19) with late-onset familial and sporadic AD was also sought in a number of Italian patients, but a geographical clustering of AD cases and of
this allele was not identified (34).
Several foci of high incidence of Creutzfeldt-Jacob disease (CJD) have been reported in France, Chile, Slovakia, Israel and among Libyan Jews (3,35,36). All of
these resulted from high incidence of familial CJD in a
restricted area. In Italy, a large familial cluster of CJD
was found in the Calabria region, where, as mentioned
above, the prevalence of dementia-related disorders
(including the prevalence of early-onset FAD) linked to
different genetic mutations, has been found to be significantly high. The CJD cluster in this region was later
found to bear the E200K Prp gene mutation (37,38).
Autosomal dominant spinocerebellar ataxias
(ADCAs)
Autosomal dominant spinocerebellar ataxia type 1
(ADCA1), the most common form of dominant ataxia,
is another inherited neurological condition described
mainly in the southern part of Italy, particularly in Sicily
(39). Genetic heterogeneity is a specific hallmark of
this condition where different loci have been found to
bear a pathological expansion of a trinucleotide repeat.
Molecular analysis in 51 families from these regions
harbouring this disorder identified an SCA2 expansion
in 30 families (39). In particular, molecular studies performed in 12 of 15 families with ADCA type I originating from mid-eastern Sicily identified the SCA2 mutation in 11 of them (91.6%) (40). Despite the lack of a
clear analysis of the territorial distribution of these families in this part of the island, the presence of a substantial number of ADCA1 pedigrees with the same
molecular abnormality in a small territory would strongly support the idea of a clustering distribution, where
the disease and the mutation have spread through apparently unrelated families. Another pedigree with mutations in the SCA2 gene has been reported to have a
southern Italian ancestry (41).
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A. Malaspina et al.
Concluding remarks
We have emphasised the importance of an accurate
analysis of the territorial distribution of patients afflicted
by neurological disorders. For a number of reasons, including socio-cultural factors, historical traditions and
the particular regional/geographical conformation of the
country, the Italian peninsula emerges as an extraordinary pool of inherited disorders. In certain regions of
southern Italy, the rural populations are geographically
isolated, which explains the significantly increased
presence of several genetic disorders, most of them
neurological. These diseases can appear in familial
clusters and in some cases, a genetic abnormality has
been found to cause the illness.
Epidemiological searches in confined regions, where
patients with a family history of a neurological disorder
can be identified, appear to be of primary importance.
The validity of this approach is confirmed by our experience with an ALS cluster in central Italy. In our study
we identified a concentration of familial and sporadic
ALS patients confined within a restricted area, i.e., a
cluster of the disease. Subsequent studies discovered
an underlying genetic defect (a SOD1 gene mutation)
causing the spread of the pathological condition within
“apparently” non-related pedigrees (9). In a previous
molecular investigation of several individuals suffering
from sporadic and familial ALS and originating from different Italian regions, we failed to identify SOD1 gene
mutations (42). These findings support the idea that the
evaluation of patients within well-characterised clusters
may represent a priority in the quest to find either
known molecular defects or new aetiological clues.
Clusters of patients suffering from a specific neurological
disorder may differ as regards the clinical features of the
illness and the severity of its evolution. In the reported
ALS pedigrees and in the clusters of familial CJD, individuals carrying the same genetic mutation show extreme variability in age at onset and disease duration
(3,9,26,35,43,44). A comparative study of these clusters
may provide further insight into genetic and/or environmental factors influencing the disease phenotype. The
geographical location of a disease cluster can also be
highly informative with regard to the role of toxic agents
in the disease aetiology. To date, no significant relationship between a specific neurological disorder and a welldefined neurotoxic element has been identified, with the
exception of some toxic polyneuropathies (45). An increased concentration of different elements, including
metals in certain areas of the CNS of patients affected by
different neurological disorders, has been suspected
(46,47). Studies of the basal ganglia, which included
measurement of the levels of iron, potassium, silica,
sodium, sulphur, zinc and aluminium, as well as assessment of exposure to other environmental agents (particularly pesticides, industrial solvents and MPTP), did not
reveal any pathological accumulation (48).
Single gene abnormalities or environmental factors may
cover the entire range of possible aetiologies of a neurological disorder. However, a complex interplay between
various genes and environmental xenobiotics is likely to
explain, at least in some cases, both the heterogeneous
distribution of a neurological disorder and the occurrence
of areas (i.e., clusters) with a higher-than-expected
prevalence of the disorder. The islands of Sardinia and
180
Sicily and the southern part of the Italian peninsula show
an undoubtedly high prevalence of various neurological
disorders and a cluster-like distribution of some of these
conditions. In Sardinia, for example, diseases like myasthenia gravis, multiple sclerosis and insulin-dependent diabetes mellitus show frequencies up to 3-5 times higher
than those found in the rest of Italy (49). The social context in these areas, characterised by large family groups
and people traditionally not inclined to emigrate, may
have favoured the spread of gene aplotypes involved in
the disease presentation and development. As we have
pointed out, subsequent migration from these areas with
high rates of inherited disorders has, as reported above
for AD and PD, resulted in the appearance of the same
hereditary diseases (and possibly of associated genetic
mutations) in other recipient regions and countries.
The relationship between genetic determinants and environmental factors is currently the focus of study through
the project “Colombo 2000”, an analysis of the phenomenon of massive migration from Italy to Argentina. The project represents a “natural experiment” into gene-environment interactions in disease pathogenesis. Data on incidence, prevalence and mortality rates in the original and
in the migrant population (exposed to different environmental factors) may be compared to elucidate risk factors, as well as protective influences, for different neurological disorders. Preliminary data from this study have
shown differences in mortality rates for brain tumours,
Parkinson’s disease, Alzheimer’s disease, motor neuron
diseases, stroke and alcohol-related diseases, between
Italy and Argentina (50,51).
In conclusion, we suggest that the observations outlined above underline the importance of careful analysis
of the “territorial distribution” of patients afflicted by various neurological disorders. The implementation of regional and national disease registers, which report the
origins of cases, would greatly favour such investigations and represent a crucial step towards the elucidation of the role of genetic mutations, environmental factors and/or gene-environment interaction in the pathogenesis of specific neurological disorders.
Acknowledgments
We are grateful to the patients and their families who
kindly agreed to participate in the study. We wish to acknowledge Prof. de Belleroche (Department of Neuromuscular Diseases, Division of Neuroscience and Psychological Medicine, ICSM), the UK Motor Neuron Disease Association, the financial support of the “C.
Mondino Institute of Neurology” Foundation (Pavia),
AISLA (the Italian Amyotrophic Lateral Sclerosis Association), the Hayward Foundation, Smith’s Charities and
Charing Cross Hospital Trustees. Dr A. Malaspina is
grateful for the support of the Riquier family, from whom
he received an award.
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