The proband, patient II-4, presented at 13 years of age along with his 18 year old brother, patient II-2.The two brothers were the sons of first cousin Arab-Moslem parents of Palestinian origin (figure 3). The parents and three siblings were healthy. Pregnancy, delivery and early psychomotor development of the two patients was normal. On physical examination at 11 years, patient II-4 was first noted to have debilitating bradykinesia, rigidity, postural instability, hypomimia, and asymmetric tremor at rest. Therapeutic trials with Amantadine, Pramipexole, and L-Dopa did not provide any relief and the patient became wheel-chair bound at age 13. Despite being cognitively normal he could not attend school due to his physical impairment. Patient II-2 was reported to suffer from identical symptoms during childhood and had deteriorated to a dependent state by age 18. The course of his disease was more insidious with bradykinesia noted at 7 years and later appearance of rigidity, tremor and postural instability. The physical examination of both patients revealed hypomimia, slow and dysarthric speech, bradykinesis, pill-rolling tremor at rest, and postural instability with inability to walk. Glabellar tap was unextinguishable and no gaze paresis was elicited. In patient II-2 hypometric saccades were noted. Tone was increased in limbs with no spasticity, pyramidal signs or dystonia. Deep tendon reflexes were symmetric and normal. Plantar reflexes were downgoing. Fine alternating movements, finger and foot tapping were slow and reduced in amplitude. No cerebellar or sensory deficits were found. Brain Magnetic Resonance Imaging (MRI) was unremarkable in both patients. The parents, another brother and two sisters were healthy at ages 11 to 21 and had normal neurologic examinations.
The study was approved by the Hadassah Institutional Review Board and the Ministry of Health. The parents consented to participate.
Single nucleotide polymorphism (SNP) genotyping was performed in the DNA samples of the two patients and an unaffected brother, with the Affymetrix GeneChip Human Mapping 250 K Nsp Array as previously described. Homozygous regions larger than 2 Mb were manually searched. Selected SNP markers were used for genotyping the remaining family members. The carrier rate of the pathogenic mutation was determined by Sanger sequencing of the relevant exon in DNA samples of 133 anonymous ethnic matched controls.
The DNA sample of patient II-2 was enriched for exonic sequences with the SureSelect Human All Exon v.2 Kit, which targets 44 Mb (Agilent, Santa Clara, CA, USA). Sequencing was carried out on a GAIIx (Illumina, San Diego, CA, USA) with 100-bp paired-end runs. Image analysis and base calling were performed with the Genome Analyzer Pipeline version 1.5 using default parameters. The sequence reads were aligned to human genome assembly hg18 (GRCh36) with DNAnexus software (Palo Alto, CA) using default parameters.
Total RNA was isolated from fibroblasts of patient II-4 and from five normal unrelated adult controls, using Tri Reagent (Sigma). DNA traces were removed by treatment with TURBO DNAse kit (Ambion). RNA was reversed transcribed using Improm II kit (Ambion) and random hexamers primers. DNA concentration of two plasmids, one with an ACTB insert and the other with a cDNA insert encompassing exon 4 and 5 of DNAJC6, was determined by spectrophotometer. Serial dilution of the plasmids' DNA was performed in order to create a calibration curve on real time PCR instrument (ABI 7900). These calibration curves were used to determine the copy number of the respective transcripts in RNA samples from the patient and the controls. We used the concentration of the ACTB cDNA to normalize the concentration of the DNAJC6 cDNA and each sample was PCR four times.
In order to study the endosomal system, transferrin uptake was measured in fibroblasts of patient II-2 as previously described with some modifications. Cells were incubated for 1 h at 37 C in labeling medium (F12 containing 10 mM HEPES, pH 7.3 and 0.2% w/v BSA) to remove unlabeled transferrin. They were then labeled for 1 h on ice with 50 microg/ml Alexa fluor 488-transferrin (Invitrogen) in labeling medium. After rinsing twice with warm labeling medium, they were incubated at 37 C for either 20 or 40 min to allow transferrin uptake. The cells were then fixed with 2% formaldehyde in PBS (RT, 30 min) and examined with a Zeiss Axiovert 200 microscope equipped with a 100x oil immersion objective.
Online Mendelian Inheritance in Man (OMIM) http://www.omim.org/ SeattleSeq Annotation website - http://snp.gs.washington.edu/SeattleSeqAnnotation/ PolyPhen-2 prediction of functional effects of human nsSNPs http://coot.embl.de/PolyPhen/ SIFT - Sorting Tolerant From Intolerant http://sift.jcvi.org/ Mutation taster - http://www.mutationtaster.org/ NHLBI Exome Sequencing Project Exome Variant Server - http://evs.gs.washington.edu/EVS/.