The linkage analysis included 69 multiplex families with 174 affected individuals genotyped at <5 cM resolution. The maximum lod score on chromosome 1 was 5.7 from multipoint nonparametric linkage analysis. The lod-1 interval spanned 7 MB from D1S199 to D1S2749 which corresponds to the physical position of the PINK1 locus. To determine if the linkage signal was due to mutations within PINK1, the gene was successfully sequenced in 89 familial index cases. Fourteen nonsynonymous coding changes were identified and assessed within other family members from 92 families (76 multiplex families and an additional 16 singleton families), 240 nonfamilial patients with PD and 368 control subjects (). Eight substitutions have been identified previously, including Q115L, P196L, A340T, A383T, G411S, E476K, Q456X, and N521T. Novel variants identified in this study included Q129X, Q129fsX157, T145M, R152W, G227R, and G440E. Of these changes, Q129X, Q129fsX157, G440E, and Q456X appear to be pathogenic as they were found in a homozygous state in 46 out of 50 (92%) of the affected individuals within 14 families (). Excluding these kindreds from linkage analysis abolished the chromosome 1p35-36 signal.
Table 1 Frequency of PINK1 mutations in Parkinson disease family probands, nonfamilial patients, and control subjects
Figure Four pedigrees are shown representative of families with homozygous PINK1 Q129X, Q129fsX157, G440E, or Q456X mutations
Evidence for pathogenicity of the other 10 PINK1 mutations was equivocal. One coding substitution N521T was common (heterozygous frequency of 24.8%, homozygous frequency 4.1% in controls) and did not segregate with parkinsonism in the families. This was excluded from further analyses of the demographic and clinical characteristics of PINK1 carriers. Other substitutions were uncommon (<5% frequency) in controls and generally found in a heterozygous state with the exception of homozygous inheritance of Q115L, A383T, and E476K in three control subjects aged 64, 49, and 49 years at examination. Nonfamilial patients were also identified with homozygous substitutions Q129X (n = 1, AAO 38 years) and Q456X (n = 5, average AAO 38 ± 9) which are putatively pathogenic.
Demographic characteristics are reported for the affected and unaffected members of families with parkinsonism, nonfamilial patients with PD, and control subjects, stratified by PINK1 status (). All participants were of Arab-Berber ethnicity with the exception of one family which originated from Turkey and two from Southern Europe. All PINK1 mutations were in individuals of Arab-Berber descent. Within families, unaffected individuals with homozygous PINK1 mutations were marginally younger at examination (34 ± 5 years) than the onset age of affected homozygous PINK1 individuals (36 ± 12 years); therefore some may yet develop parkinsonism.
Table 2 Demographic characteristics of subjects focused on four putatively pathogenic PINK1 mutations
Subsequent analyses focus on subjects with pathogenic mutations, Q129X, Q129fsX157, G440E, and Q456X. Clinical characteristics of patients with parkinsonism with PINK1 homozygous and heterozygous mutations were compared to those without PINK1 variants (). The mean age at onset of parkinsonism in familial PINK1 homozygous patients (36 ± 12) was younger than in familial PD with heterozygous PINK1 mutations (69 ± 8 years, Wilcoxon rank sum test p < 0.005) or affected family members without any of the four potentially pathogenic PINK1 mutations (57 ± 15 years, Wilcoxon rank sum test p < 0.0001). The mean age at onset of PD in six individuals with nonfamilial PD and PINK1 homozygous mutations (38 ± 9 years) was younger than in nonfamilial PD without any of the four mutations (58 ± 12 years, Wilcoxon rank sum test p < 0.001).
Table 3 Characteristics of patients with Parkinson disease (PD) with PINK1 homozygous and heterozygous mutations compared to those without PINK1 mutations
In familial patients with homozygous PINK1
mutations, in contrast to patients without any PINK1
mutation, the duration of parkinsonism was longer (Wilcoxon rank sum test p
< 0.0001) and Hoehn and Yahr scores were not significantly different (). The notable exception were higher mean Hoehn & Yahr scores in nonfamilial patients with homozygous PINK1
mutations and with a very long duration of disease, although the sample size was too small for significance (n = 6, p
= 0.1). The majority of familial patients without PINK1
mutations recalled tremor as a first feature (83%) whereas gait or balance problems were infrequent (14%). In contrast, familial PINK1
homozygotes noted their first symptom as either tremor (54%) or deterioration in gait or balance (37%). At examination, the type of parkinsonism in most affected individuals were classified as mixed, with both rigidity and tremor present, but PINK1
homozygotes more often had akinetic-rigid parkinsonism (31%), compared to heterozygotes (0%) or individuals without PINK1
mutations (13%). Clinical differences on first presentation and subtype at examination between PINK1
homozygotes and PINK1
heterozygote or patients without PINK1
mutations were statistically significant (p
< 0.0002). Data on cognition and dementia are limited to neurologist observations as more than 60% of the study population was unable to read and therefore their MMSE scores were not valid.18
Some families carried more than one PINK1 sequence variation. One familial affected individual (onset age 74) was a compound heterozygote for both Q129X and A340T substitutions; another familial patient (onset age 60) was a compound heterozygote for Q456X and A383T, whereas two unaffected individuals (ages 44 and 69) from two families were compound heterozygous for both Q456X and A340T. In two of the eight families with the Q456X mutation, the proband did not screen positive for the mutation but it was identified in other family members.
Within the 14 families with PINK1 mutations, PRKN sequencing and dosage analysis was performed and LRRK2 point mutations including 6055G>A (G2019S) were assayed. There are seven heterozygous PINK1 carriers who also inherited either M192L (n = 2), P153R (n = 4), or D394N (n = 1) heterozygous PRKN substitutions. However, all were unaffected with a mean age at examination of 54 ± 19, range 26–73 years. Of three homozygous PINK1 carriers with heterozygous P153R PRKN substitutions, two were affected at 30 and 36 years while the other remains unaffected at examination at 35 years. In one family, one affected (age 56 years at onset) and one unaffected individual (age 62 years at examination) also carried a heterozygous Lrrk2 G2019S substitution.