Supplementary
Methods
Genetic analyses
Probes for custom targeted panel were designed with DesignStudio (Illumina), amplicon length averaged 250 bp (for 2 x 150 bp read lengths on the MiSeq). Samples were run to reach a depth of coverage of >150x. Data were automatically analyzed by MiSeq Reporter software (Illumina) on the instrument. Alignment was done against the human reference sequence build GRCh37/Hg19. Based on the hypothesis that the causative mutations underlying these rare diseases are almost absent in the general population, the variants with allele frequency >0.01 (Minor allele frequency, MAF) identified in the ExAc databases ( were filtered out. Synonymous, intronic, and UTR variants not predicted to have an effect on splice site were also removed. Variants of interest were validated by Sanger sequencing as previously described [23]. Primer sequences are available on request.
Statistical analysis
In order to assess whether the observed frequency of patientscarrying multiple variants was in excess of what one might expect on the basis of chance, we performed a binomial test using the statistical analysis program R (CRAN; R-project.org). The numbers of variants in the control population for each gene other than the C9orf72 RE we found in our cohort were identified in the ExAC database. Both the total number and the number of rare (AF < 0.001) variants observed in the ExAC population were noted and used to calculate the expected frequency of co-occurring variants, using the following formula: (observed number of patients carrying a variant/the total number of patients) (the observed number of controls carrying a variant/the total number of controls).The following formula was usedto perform the binomial test: (pbinom ([number of patients with multiple mutations], [total number of patients], [expected frequency], lower.tail = FALSE, log.p = FALSE).If the binomial p value is smaller than 0.05 this means that the observed frequency of co-occurring variants is higher than expected based on chance.
Results
Clinical presentation of patients with C9orf72 RE in association with other rare variants
Family A
The proband is a 54-year-old woman with a 5-year history of progressive cognitive impairment. Her brother was diagnosed with ALS at the age of 51 and her mother presented at age 67 years with progressive weakness and died at age 69 with ALS. The patient’s personal history had been unremarkable until the age of 49, when relatives reported memory deficits. From age 52 years she experienced progressive gait impairment with daily falls and hallucinations. At the neurological examination (NE) she was disoriented, hypomimic, hypophonic and dysarthric. Pout and palmomontal reflexes were present. There was marked axial and bilateral limb rigidity with moderately severe bradykinesia in the arms. Deep tendon reflexes (DTR) were brisk. Gait was slow and broad based, and there was postural instability. The patient scored 23/30 at the Mini-Mental State Evaluation (MMSE) and a more extensive evaluation confirmed the presence of multi-domain dementia. The CSF analysis showed slightly reduced levels of A-beta1-42 (441 pg/ml; normal values 562-1018) protein. The EMG showed only chronic multisegmental denervation signs and the transcranial magnetic stimulation (TMS) was normal. A brain MRI was unremarkable but a SPECT I123-Ioflupane showedbilateral hypocaptation in the basal ganglia.
Family B
The proband is a 54-year-old woman presenting with a year history of emotional lability followed by a rapidly evolving cognitive impairment with aphasia nominum and memory deficits. Her previous history was unremarkable. A sister was reported to have developed a speech disorder some years before but no further information could be obtained. On the NE she appeared disoriented and unable to maintain the instructions of the examiner. Memory was impaired with deficit in 3 words short- and long-term retrieval. Her face was astonished and she displayed vertical gaze palsy. Her speech was dysarthric. A mild postural and resting tremor was observed along with distal parcellar myoclonus of the hands. Muscle strength was unimpaired but amyotrophy and sporadic fasciculations were noted in the hands. DTR were diffusely brisk. She scored 21/30 at the MMSE. A more detailed neuropsychological assessment showed multi-domain deficit with a relatively preserved memory function.
A brain CT scan showed mild frontal atrophy and a PET scan confirmed the presence of a bilateral frontal and temporal hypofixation of the radiotracer and the presence of the same finding on thalami and basal ganglia.
Family C
The proband presented at age 72 with a 2-year history of progressive weakness of the right leg. Her brother died at age 55 years with a diagnosis of ALS, and her mother died at age 70 years for unknown causes, affected by depression and cognitive impairment. The NE showed a patient fatuous and slightly disinhibited with pout and palmomontal reflexes. Signs of UMN and LMN involvement were observed in the lower limbs. EMG showed subacute neurogenic signs in the muscles explored in the upper and lower limbs. TMS showed signs of corticospinal tract involvement in the lower limbs. She scored 24.4/30 at MMSE and a more detailed neuropsychological assessment revealed deficit of verbal memory, verbal logic, attention and executive functions. Cortical atrophy, prevalent on the temporal regions, was present at the brain MRI.CSF analysis showed normal levels of t-tau, p-tau and A-beta1-42 proteins.
Family D
The proband presented at age 65 with progressive cognitive decline with prominent memory impairment, apathy and self-neglect followed by gait impairment. Her mother died at age 72 years after an 8-year history of dementia. On the NE she appeared apathetic and disoriented, with severe non-fluent aphasia. She also displayed vertical gaze palsy, lower limb dystonia and pyramidal signs. The brain MRI showed cortical atrophy prevalent in the left frontotemporal lobes. The SPECT confirmed a marked hypo perfusion of the left fronto-temporo-parietal lobes associated with less severe hypoperfusion of the other brain areas and of the cerebellum. She scored 24/30 at MMSE and a neuropsychological assessment revealed deficit of praxis and executive functions. After 9 months the MMSE score was 12/30. The CSF routine analysis and total tau protein levels were normal. Her cognitive status progressively worsened and she became bedridden in 2 years time. She could not eat and was fed by gastrostomy. After 2 more years she started suffering from epileptic seizures that were treated with phenytoin.The patient eventually died after 5 years from clinical onset in a nursing home.
Genetic analyses
Statistical analysis demonstrated that the frequency of patients with multiple mutations is higher than one might expect on the basis of chance both considering all (P = 1.33675e-06) and only rare variants (P = 2.607644e-08).