The families included in our study are from regions with a high prevalence of consanguinity, favouring an increased frequency of genetic homozygosity and diseases with recessive inheritance. From the literature, it is well known that genetic variation within the parkin
gene cause autosomal recessive juvenile parkinsonism (AR-JP) [6
] and is the most common cause of familial parkinsonism known to date [10
], while mutations in the PINK1
gene are recognized as an increasingly important genetic cause of early-onset parkinsonism. A possible effect of heterozygous mutations in both parkin
have been proposed and a digenic effect have been observed [47
]. To this end we identified an exon 4 deletion in parkin
that was homozygous in both affected (figure ) and additional substitutions in PINK1
, which co-segregated with disease (figure ). Some substitutions acknowledged to be common in populations are here discussed according to possible role in disease development.
In our material we identified four non-synonymous substitutions in the kinase domain of PINK1
, of which two are novel and proposed to be putative pathogenic mutations. The protein kinase domain in PINK1
that is located to amino acids 156–509, has a high degree of homology to similar kinases in the Ca2
+/calmodulin family [7
]. Functional studies indicate that mutations in the kinase domain are expected to effect kinase activity as well as substrate binding capability [33
Affected individuals in these families were either homozygous or compound heterozygous and most substitutions were evaluated to be non-pathogenic polymorphisms but could be discussed in relation to impact of a single hit as well as accumulative effect of substitutions. The P416R and S419P substitutions are novel and homozygous in two affected in each of their respective families (figure ). Both are in conserved regions of the gene and predictions using SIFT software for the tolerability of substitutions indicates that especially the P416R substitution is of probable functional importance to the protein. The A340T, E476K and N521T substitutions are assumed to be non-pathogenic polymorphisms [27
] and the D391D synonymous substitution is assumed to have no effect [27
], although the A340T substitution has been proposed to contribute to risk of development of late-onset PD [57
]. Interestingly, in family F, two of these substitutions were found to co-segregate with disease. In the other families with PINK1
polymorphisms, a segregation pattern compatible with recessive inheritance could not be excluded, when taken into account the possibility of reduced penetrance in some offspring due to low age.
In family F we observed an E476K substitution and an N521T substitution segregating with disease. Both affected in family F were compound heterozygous for these putative polymorphisms in the PINK1
gene. The E476K substitution is in the protein serine/threonine kinase domain of the protein, but only to a small degree conserved across species (figure ). The effect of the substitution on the enzymatic activity of PINK1
is not known but the substitution is found as the conserved amino acid in several species [27
] and are common in controls as well. The N521T substitution is located in a conserved region at the 3' of the protein outside the 156–509 residues proposed as the serine/threonine kinase region. This region is conserved across all mammals and has an unknown function. The N521T substitution identified in this region is the most prevalent in our material and thus far one of the most common substitutions found in cases as well as healthy family members with parkinsonism. In previous studies the N521T substitution has been common and recognised merely as a polymorphism [45
] and in studies of early-onset PD homozygous incidences have been confirmed in controls and cases equally [27
The exon 4 deletion in the parkin gene segregates with disease and explains the clinical manifestations. It is curios thou, how additional substitutions are accumulated in the PINK1 gene as revealed in this study, and for this reason the observed substitutions will be discussed.
The A340T substitution introduced into the pedigree of family F was equally found in compound heterozygous state with N521T in family J. Threonine is the conserved amino acid in Rattus norwegicus and Mus musculus (figure ) and this suggests the substitution is most likely a polymorphism. However, we cannot completely exclude a possible effect of the A340T polymorphism in combination with supplementary mutations within PINK1 or in other genes affecting parkinsonism.
is located in the third most common fragile site, FRA6E, proposed to be involved in several types of cancer, acting as a tumor suppressor gene together with other common fragile site genes such as FHIT (FRA3B ; 3p14.2) and WWOX (FRA16D ;16q23) that are also associated with exon deletions [43
]. In some cancer cells there are a high frequency of loss-of-heterozygosity (LOH) observed in D6S1599 (parkin
intron 2) and D6S305 (parkin
intron 6) [43
] as observed in carriers in healthy family members of family F in our study. The mechanism of common fragile sites might indicate some of the mechanism behind the observed parkin
deletions and associate the formation of deletions with a FRA6E site mechanism. The occurrence of parkin
mutations could either be independently recurrent de novo mutational events or a geographical spread through founder effect. In general, exon rearrangements are found to be caused by independent de novo mutational events, while point mutations are spread mainly through founder effect [48
]. The extent of the exon 4 deletion in family F was further mapped and was different from one mapped in a geographically close Turkish family [50
We found a Gln34Arg substitution previously described in India where it might be the result of a geographic founder effect [52
]. In India, the substitution was observed in heterozygous state in affected with parkinsonism, but also in older unaffected family members [52
This might be a dominant negative mutation which contributes to functional variation in the Parkin protein. A functional example may be that the protein still interacts and competes with the wild type protein for the target protein, but lacks functional properties. Therefore, in heterozygous state, this category of mutation might cause a range of phenotypes in response to genetic and environmental influence. Variation in penetrance is discussed for several parkinsonism related mutations. Dominant negative mutations may be a possible explanation of some of the observed heterozygous mutations having a variable "dominant nature". It is different from the loss-of-function mutations generally observed in the parkin gene, where a heterozygous state continues to have a normal phenotype.
Abbas and colleagues [17
] previously detected a mutation in the same location of exon 2 in two British families, in which a 202-203delAG caused "loss-of-function" of the Parkin protein [17
]. The Gln34Arg substitution has not yet been functionally characterized, and its function is therefore still uncertain. The localization to the ubiquitin-like domain suggests it may effect binding to proteasome subunits. The substitution was not observed in the carriers affected sister (patient II.4), who also developed the disease, stating that this substitution is not involved in disease development in both cases. More important, the affected are both carriers of homozygous P416R substitutions which probably cause the clinical symptoms (see discussion on P416R further down).
The Val380Leu mutation has, in previous studies, been found in both healthy and affected individuals. Abbas et al
] reported that the substitution was found in eleven European families and also 16% of the control subjects and it appear to be common across populations [52
] and therefore not considered a main cause of disease development. It might, however, alter the Parkin protein and contribution to the pathogenesis of idiopathic PD, as noted in a study by Lucking et al
], which reported homozygous Val380Leu substitutions to be associated with sporadic PD. Biswas et al [56
] commented on this substitution that it might be an association in some populations when ethnically stratified. In our study the substitution was heterozygous in one affected and absent in the carriers brother who also developed young-onset parkinsonism implying that the substitution was not involved in development of disease in both affected family members (figure ).
A recessive pattern of inheritance should, in a population with high degree of consanguinity, anticipate the observation of homozygous haplotypes identical by decent (IBD) in the parkin
gene, as found in family F where the exon 4 deletion and all four microsatellite markers segregated with disease. Further haplotype assessment did not reveal any haplotype that could associate the parkin
gene with the observed symptoms of young-onset or juvenile parkinsonism across families. Sequencing revealed few substitutions and we were unable to identify specific point mutations in remaining families. Quantitative analysis revealed no exon rearrangements or complete gene deletions/multiplications that could associate the parkin
gene with the observed clinical symptoms. We confirmed less than expected incidences of parkin
caused parkinsonism in the population. Multiple sequence alignment and estimation of the tolerability of the observed parkin
substitutions indicated that they were probably tolerable (Figure ). The reported frequency of parkin
associated parkinsonism was similarly low in populations of India where the Gln34Arg substitution was previously observed [52
, the novel P416R substitution was identified in family G, in a highly conserved motif of the activation segment in the serine/threonine kinase domain [33
]. The substitution was homozygous in both affected members of the family and were the centre amino acid of an APE motif conserved in orthologous sequences and in paralogous protein kinases [33
]. Located in a universally conserved functional motif the substitution is likely to affect kinase activity.
The S419P substitution was identified in family I. Two non-synonymous substitutions and one synonymous substitution, all in homozygous state, were identified in both affected members of the family (figure ). Located in the serine/threonine kinase domain the S419P substitution was the most conserved alteration within the material.
There were no obvious differences between the ages at onset amongst putative parkin and PINK1 mutation carriers as compared to non-carriers (Table ). However, there are similarities regarding age at onset within affected of the same family. For affected carrying P416R, the only substitution to be in a universally highly conserved motif, an earlier age at onset was seen in both affected, as compared to family I carrying three homozygous substitutions, amongst them the S419P substitution. These findings indicate a severe function of the P416R mutation, which could represent a total loss-of-function mutation. Otherwise, the observed variations in age at onset could generally be due to interactions of additional environmental or genetic factors such as accumulation of substitutions causing gradual alterations in protein structure. In light of the SIFT predictions, assessment of the tolerability of the S419P substitution is dependent on further functional analysis.
In some families an accumulation of substitutions are observed. In family F both deletion in parkin and substitutions in PINK1 co-segregate with disease, and a carrier of A340T and N521T is married in. In family G a carrier of N521T is married in to the family through a healthy family member. In family I, the accumulation of substitutions in the two affected, the only two persons available for analysis, are notable. The possibility of a LOH for this region was excluded using the gene and exon dosage SALSA MLPA kit. Accumulation of substitutions should suggest some selective bias based on knowledge of disease in families and therefore suggest a role of these substitutions in disease or in association to disease.