Andrey Goltsov

Genomic identification in the historical case of the Nicholas II royal family

Accurate unambiguous identification of ancient or historical specimens can potentially be achieved by DNA analysis. The controversy surrounding the fate of the last Russian Emperor, Nicholas II, and his family has persisted, in part, because the bodies of 2 children, Prince Alexei and 1 of his sisters, have not been found. A grave discovered in 1991 contained remains putatively identified as those of the Russian Royal family. However, not all family members were represented. Here, we report the results of genomic analyses of new specimens, the human remains of 2 burned skeletons exhumed from a grave discovered in July 2007, and the results of a comprehensive genomic analysis of remains from the 1991 discovery. Additionally, ≈117 years old archival blood specimens from Nicholas II were obtained and genotyped, which provided critical material for the specific determination of individual identities and kinship identifications. Results of genotypic analyses of damaged historical specimens were evaluated alongside samples from descendants of both paternal and maternal lineages of the European Royal families, and the results conclusively demonstrate that the recently found remains belong to children of Nicholas II: Prince Alexei and his sister. The results of our studies provide unequivocal evidence that the remains of Nicholas II and his entire family, including all 5 children, have been identified. We demonstrate that convergent analysis of complete mitochondrial genome sequences combined with nuclear DNA profiles is an efficient and conclusive method for individual and kinship identification of specimens obtained from old historic relics.

Age- and genotype-related neurophysiologic reactivity to oxidative stress in healthy adults.

The epsilon4 allele of the apolipoprotein E gene (ApoE), as well as aging increase the risk of Alzheimers and vascular diseases. Electroencephalogram (EEG) reactivity to hyperventilation (HV) depends on hypocapnia-induced cerebral vasoconstriction, which may be impaired in subjects with subclinical cerebrovascular disease. Quantitative EEG at rest and under 3-minute HV was examined in 125 healthy subjects divided into younger (age range 28-50) and older (age range 51-82) cohorts and stratified by ApoE genotype. The younger ApoE-epsilon4 carriers had excessive EEG reactivity to HV characterized by the manifestation of high-voltage delta, theta activity and sharp waves, and larger HV-induced changes in EEG relative powers than in the younger ApoE-epsilon4 noncarriers. EEG reactivity to HV decreased with aging, and in the ApoE-epsilon4 carriers the decrease was more pronounced than in the ApoE-epsilon4 noncarriers. The older ApoE-epsilon4 carriers had smaller HV-induced changes in EEG relative powers than the older ApoE-epsilon4 noncarriers. A marked decline of EEG reactivity to HV in the older ApoE-epsilon4 carriers suggests the possible impact of vascular factors on the pathogenesis of ApoE-induced Alzheimer disease.

Whole-genome sequencing identifies a novel ABCB7 gene mutation for X-linked congenital cerebellar ataxia in a large family of Mongolian ancestry

X-linked congenital cerebellar ataxia is a heterogeneous nonprogressive neurodevelopmental disorder with onset in early childhood. We searched for a genetic cause of this condition, previously reported in a Buryat pedigree of Mongolian ancestry from southeastern Russia. Using whole-genome sequencing on Illumina HiSeq 2000 platform, we found a missense mutation in the ABCB7 (ABC-binding cassette transporter B7) gene, encoding a mitochondrial transporter, involved in heme synthesis and previously associated with sideroblastic anemia and ataxia. The mutation resulting in a substitution of a highly conserved glycine to serine in position 682 is apparently a major causative factor of the cerebellar hypoplasia/atrophy found in affected individuals of a Buryat family who had no evidence of sideroblastic anemia. Moreover, in these affected men we also found the genetic defects in two other genes closely linked to ABCB7 on chromosome X: a deletion of a genomic region harboring the second exon of copper-transporter gene (ATP7A) and a complete deletion of PGAM4 (phosphoglycerate mutase family member 4) retrogene located in the intronic region of the ATP7A gene. Despite the deletion, eliminating the first of six metal-binding domains in ATP7A, no signs for Menkes disease or occipital horn syndrome associated with ATP7A mutations were found in male carriers. The role of the PGAM4 gene has been previously implicated in human reproduction, but our data indicate that its complete loss does not disrupt male fertility. Our finding links cerebellar pathology to the genetic defect in ABCB7 and ATP7A structural variant inherited as X-linked trait, and further reveals the genetic heterogeneity of X-linked cerebellar disorders.

Mutational re-modeling of di-aspartyl intramembrane proteases: uncoupling physiologically-relevant activities from those associated with Alzheimer's disease

The intramembrane proteolytic activities of presenilins (PSEN1/PS1 and PSEN2/PS2) underlie production of β-amyloid, the key process in Alzheimer’s disease (AD). Dysregulation of presenilin-mediated signaling is linked to cancers. Inhibition of the γ-cleavage activities of PSENs that produce Aβ, but not the ε-like cleavage activity that release physiologically essential transcription activators, is a potential approach for the development of rational therapies for AD. In order to identify whether different activities of PSEN1 can be dissociated, we designed multiple mutations in the evolutionary conserved sites of PSEN1. We tested them in vitro and in vivo assays and compared their activities with mutant isoforms of presenilin-related intramembrane di-aspartyl protease (IMPAS1 (IMP1)/signal peptide peptidase (SPP)). PSEN1 auto-cleavage was more resistant to the mutation remodeling than the ε-like proteolysis. PSEN1 with a G382A or a P433A mutation in evolutionary invariant sites retains functionally important APP ε- and Notch S3- cleavage activities, but G382A inhibits APP γ-cleavage and Aβ production and a P433A elevates Aβ. The G382A variant cannot restore the normal cellular ER Ca2+ leak in PSEN1/PSEN2 double knockout cells, but efficiently rescues the loss-of-function (Egl) phenotype of presenilin in C. elegans. We found that, unlike in PSEN1 knockout cells, endoplasmic reticulum (ER) Ca2+ leak is not changed in the absence of IMP1/SPP. IMP1/SPP with the analogous mutations retained efficiency in cleavage of transmembrane substrates and rescued the lethality of Ce-imp-2 knockouts. In summary, our data show that mutations near the active catalytic sites of intramembrane di-aspartyl proteases have different consequences on proteolytic and signaling functions.The intramembrane proteolytic activities of presenilins (PSEN1/PS1 and PSEN2/PS2) underlie production of β-amyloid, the key process in Alzheimers disease (AD). Dysregulation of presenilin-mediated signaling is linked to cancers. Inhibition of the γ-cleavage activities of PSENs that produce Aβ, but not the ε-like cleavage activity that release physiologically essential transcription activators, is a potential approach for the development of rational therapies for AD. In order to identify whether different activities of PSEN1 can be dissociated, we designed multiple mutations in the evolutionary conserved sites of PSEN1. We tested them in vitro and in vivo assays and compared their activities with mutant isoforms of presenilin-related intramembrane di-aspartyl protease (IMPAS1 (IMP1)/signal peptide peptidase (SPP)). PSEN1 auto-cleavage was more resistant to the mutation remodeling than the ε-like proteolysis. PSEN1 with a G382A or a P433A mutation in evolutionary invariant sites retains functionally important APP ε- and Notch S3- cleavage activities, but G382A inhibits APP γ-cleavage and Aβ production and a P433A elevates Aβ. The G382A variant cannot restore the normal cellular ER Ca2+ leak in PSEN1/PSEN2 double knockout cells, but efficiently rescues the loss-of-function (Egl) phenotype of presenilin in C. elegans. We found that, unlike in PSEN1 knockout cells, endoplasmic reticulum (ER) Ca2+ leak is not changed in the absence of IMP1/SPP. IMP1/SPP with the analogous mutations retained efficiency in cleavage of transmembrane substrates and rescued the lethality of Ce-imp-2 knockouts. In summary, our data show that mutations near the active catalytic sites of intramembrane di-aspartyl proteases have different consequences on proteolytic and signaling functions.

DECLINE OF FUNCTIONAL INTERHEMISPHERIC CONNECTIVITY IN AGING: ASSOCIATION WITH PICALM GENOTYPE

Background: Type 2 diabetes (T2D) is associated with poorer cognitive function, accelerated cognitive decline, and higher risk for dementia, accompanied by atrophy in different brain structures. The association of the Transcription factor 7-like 2 (TCF7L2) gene with T2D is one of the most reproducible findings in T2D genetics. Since TCF7L2 is expressed in the brain, we investigated the association between volumes of the hippocampus, amygdala and frontal cortex of cognitively normal T2D elderly. Our hypothesis was that T2D genetic risk variants within TCF7L2will be associated with greater brain atrophy among diabetic patients. Methods:T2D subjects (n1⁄4177) from the Israel Diabetes and Cognitive Decline study who were both genotyped for the TCF7L2single nucleotide polymorphisms (SNPs) rs7903146, rs11196205, rs12255372 and underwent brain MRI, were included in this analysis. Images were processed and regional volumes were calculated with SPM software. For each SNP, we used ANCOVA to investigate the association of the T2D risk allele with volume of hippocampus, amygdala and frontal cortex under a recessive model of inheritance controlling for relevant demographic and clinical variables (sex, age, years of education, duration of T2D, HBA1C, total cholesterol, systolic and diastolic blood pressure, BMI, and ICV [intra-cranial volume]). Results: In all three TCF7L2 SNPs, amygdalar volume was 9-12% lower among carriers of the TD2 risk allele homozygous genotype: rs7903146: TT genotype vs. CT+TT genotypes (p1⁄40.003); rs11196205 CC genotype vs. CG+GG genotypes (p1⁄40.001), and rs12255372TT genotype vs. GT+GG genotypes (p1⁄40.003). These SNPs were not associated with hippocampal or frontal cortex volumes. Conclusions: TCF7L2 genetic variants are associated with smaller amygdalar volume in T2D elderly, suggesting an atrophic process in the amygdala in those with the risk variants. In addition, the amygdala has been implicated in supporting memory processes affected in dementia. Further study of TCF7L2 role in this region is needed, as amygdalar atrophy has been reported in T2D and the amygdala has been recently reported as a glucose sensing region.

Effect of Alzheimer's risk variant CLU on EEG alterations in normal aging

Phenotype APOE alone 30 Soares, Potter, et al. 2012, Arch Neurol Plasma Biomarkers Associated With the Apolipoprotein E Genotype and Alzheimer Disease Copy number variations (CNVs) 3 Swaminathan, Huentelman, et al. 2012, PLoS ONE Analysis of Copy Number Variation in Alzheimer’s Disease in a Cohort of Clinically Characterized and Neuropathologically Verified Individuals Candidate SNPs 26 Kauwe, Cruchaga, et al. 2011, PLoS ONE Fine mapping of genetic variants in BIN1, CLU, CR1 and PICALM for association with cerebrospinal fluid biomarkers for Alzheimer’s disease Candidate genes/ pathways 7 Swaminathan, Shen, et al. 2012, Brain Imaging Behav Amyloid pathway-based candidate gene analysis of [(11)C]PiB-PET in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort