Two hundred sixty nine unrelated Iranian subjects of ≥ 60 years of age, consisting of late-onset NCD patients (n = 115) and controls (n = 154) were recruited from the provinces of Tehran, Qazvin, and Rasht. In each NCD case, the Persian version of the Abbreviated Mental Test Score (AMTS)8,9 was implemented (AMTS < 7 was an inclusion criterion for NCD), medical records were reviewed in all participants, and CT-scans were taken where possible (approximately 40% of instances). Furthermore, in a number of subjects, the Mini-Mental State Exam (MMSE) Test10 was implemented in addition to the AMTS. A score of < 24 was an inclusion criterion for NCD.
The AMTS is currently one of the most accurate primary screening instruments to increase the probability of NCD11. The Persian version of the AMTS is a valid cognitive assessment tool for older Iranian adults, and can be used for NCD screening in Iran8.
The control group was selected based on cognitive AMTS of > 7 and MMSE > 24, lack of major medical history, and normal CT-scan where possible. The cases and controls were matched based on age, gender, and residential district. The subjects' informed consent was obtained (from their guardians where necessary) and their identities remained confidential throughout the study. The research was approved by the Ethics Committee of the Social Welfare and Rehabilitation Sciences, Tehran, Iran, and was consistent with the principles outlined in an internationally recognized standard for the ethical conduct of human research. All methods were performed in accordance with the relevant guidelines and regulations.
Allele and genotype analysis of the RASGEF1C (GGC)-repeat.
Genomic DNA was obtained from peripheral blood using a standard salting out method. PCR reactions for the amplification of the RASGEF1C (GGC)-repeat were set up with the following primers
PCR reactions were carried out with a GC-TEMPase 2x master mix (Amplicon) in a thermocycler (Peqlab-PEQStar) under the following conditions: touchdown PCR: 95 ◦C for 5 min, 20 cycles of denaturation at 95 ◦C for 45 s, annealing for 45 s at 67◦C (-0.5 decrease for each cycle) and extension at 72 ◦C for 1 min, and 30cycles of denaturation at 95 ◦C for 40 s, annealing at 57 ◦C for 45 s and extension at 72 ◦C for 1 min, and a final extension at 72 ◦C for 10 min. All samples included in this study were sequenced by the forward primer, using an ABI PRISM 377 DNA sequencer.
Analysis of the RASGEF1C (GGC)-repeat across vertebrates.
The interval between + 1 and + 100 of the TSS of the RASGEF1C was searched across all the available orders of vertebrates based on Ensembl 103. The Ensembl alignment program and CodonCode Aligner were implemented for the sequence alignments across the selected species.