Exome sequencing in the pediatric neuromuscular clinic leads to more frequent diagnosis of both neuromuscular and neurodevelopmental conditions.

INTRODUCTION/AIMS: Exome sequencing (ES) has proven to be a valuable diagnostic tool for neuromuscular disorders, which often pose a diagnostic challenge. The aims of this study were to investigate the clinical outcomes associated with utilization of ES in the pediatric neuromuscular clinic and to determine if specific phenotypic features or abnormal neurodiagnostic tests were predictive of a diagnostic result. METHODS: This was a retrospective medical record review of 76 pediatric neuromuscular clinic patients who underwent ES. Based upon clinical assessment prior to ES, patients were divided into two groups: affected by neuromuscular (n = 53) or non-neuromuscular (n = 23) syndromes. RESULTS: A diagnosis was made in 28/76 (36.8%), with 29 unique disorders identified. In the neuromuscular group, a neuromuscular condition was confirmed in 78% of those receiving a genetic diagnosis. Early age of symptom onset was associated with a significantly higher diagnostic yield. The most common reason neuromuscular diagnoses were not detected on prior testing was due to causative genes not being present on disease-specific panels. Changes to medical care were made in 57% of individuals receiving a diagnosis on ES. DISCUSSION: These data further support ES as a powerful diagnostic tool in the pediatric neuromuscular clinic and highlight the advantages of ES over gene panels, including the ability to identify diagnoses regardless of etiology, identify genes newly associated with disease, and identify multiple confounding diagnoses. Rapid and accurate diagnosis by ES can not only end the patient's diagnostic odyssey, but often impacts patients' medical management and genetic counseling of families.

Pediatric Myelodysplastic Syndrome With Germline RRAS Mutation: Expanding the Phenotype of RASopathies.

The RAS/mitogen-activated protein kinase pathway plays a significant role in cell cycle regulation. Germline mutation of this pathway leads to overlapping genetic disorders, RASopathies, and is also an important component of tumorigenesis. Here we describe a rare case of myelodysplastic syndrome with monosomy 7 in a pediatric patient with a germline RRAS mutation. RRAS mutations have been implicated in the development of juvenile myelomonocytic leukemia, but our case suggests RRAS mutations display a broader malignant potential. Our case supports the recommendation that genetic testing should include RRAS in suspected RASopathy patients and if identified, these patients undergo surveillance for hematologic malignancy.

CAGI SickKids challenges: Assessment of phenotype and variant predictions derived from clinical and genomic data of children with undiagnosed diseases.

Whole-genome sequencing (WGS) holds great potential as a diagnostic test. However, the majority of patients currently undergoing WGS lack a molecular diagnosis, largely due to the vast number of undiscovered disease genes and our inability to assess the pathogenicity of most genomic variants. The CAGI SickKids challenges attempted to address this knowledge gap by assessing state-of-the-art methods for clinical phenotype prediction from genomes. CAGI4 and CAGI5 participants were provided with WGS data and clinical descriptions of 25 and 24 undiagnosed patients from the SickKids Genome Clinic Project, respectively. Predictors were asked to identify primary and secondary causal variants. In addition, for CAGI5, groups had to match each genome to one of three disorder categories (neurologic, ophthalmologic, and connective), and separately to each patient. The performance of matching genomes to categories was no better than random but two groups performed significantly better than chance in matching genomes to patients. Two of the ten variants proposed by two groups in CAGI4 were deemed to be diagnostic, and several proposed pathogenic variants in CAGI5 are good candidates for phenotype expansion. We discuss implications for improving in silico assessment of genomic variants and identifying new disease genes.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

A retrospective review of multiple findings in diagnostic exome sequencing: half are distinct and half are overlapping diagnoses.

PURPOSE: We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). METHODS: Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. RESULTS: Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. CONCLUSION: Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

Exome sequencing in neonates: diagnostic rates, characteristics, and time to diagnosis.

PURPOSE: Neonatal patients are particularly appropriate for utilization of diagnostic exome sequencing (DES), as many Mendelian diseases are known to present in this period of life but often with complex, heterogeneous features. We attempted to determine the diagnostic rates and features of neonatal patients undergoing DES. METHODS: The clinical histories and results of 66 neonatal patients undergoing DES were retrospectively reviewed. RESULTS: Clinical DES identified potentially relevant findings in 25 patients (37.9%). The majority of patients had structural anomalies such as birth defects, dysmorphic features, cardiac, craniofacial, and skeletal defects. The average time for clinical rapid testing was 8 days. CONCLUSION: Our observations demonstrate the utility of family-based exome sequencing in neonatal patients, including familial cosegregation analysis and comprehensive medical review.

Review of X-linked syndromes with arthrogryposis or early contractures-aid to diagnosis and pathway identification.

The following is a review of 50 X-linked syndromes and conditions associated with either arthrogryposis or other types of early contractures. These entities are categorized as those with known responsible gene mutations, those which are definitely X-linked, but the responsible gene has not been identified, and those suspected from family history to be X-linked. Several important ontology pathways for known disease genes have been identified and are discussed in relevance to clinical characteristics. Tables are included which help to identify distinguishing clinical features of each of the conditions.

Clinical Cytogenetics: Current Practices and Beyond.

BACKGROUND: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics. CONTENT: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements. SUMMARY: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria mitochondrial disorder: case report and literature review.

Background: Leigh syndrome is a rare, genetic, and severe mitochondrial disorder characterized by neuromuscular issues (ataxia, seizure, hypotonia, developmental delay, dystonia) and ocular abnormalities (nystagmus, atrophy, strabismus, ptosis). It is caused by pathogenic variants in either mitochondrial or nuclear DNA genes, with an estimated incidence rate of 1 per 40,000 live births. Case presentation: Herein, we present an infant male with nystagmus, hypotonia, and developmental delay who carried a clinical diagnosis of Leigh-like syndrome. Cerebral magnetic resonance imaging changes further supported the clinical evidence of an underlying mitochondrial disorder, but extensive diagnostic testing was negative. Trio exome sequencing under a research protocol uncovered compound-heterozygous missense variants in the HTRA2 gene (MIM: #606441): NM_013247.5:c.1037A>T:(p.Glu346Val) (maternal) and NM_013247.5:c.1172T>A:(p.Val391Glu) (paternal). Both variants are absent from public databases, making them extremely rare in the population. The maternal variant is adjacent to an exon-intron boundary and predicted to disrupt splicing, while the paternal variant alters a highly conserved amino acid and is predicted to be damaging by nearly all in silico tools. Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria, type VIII (MGCA8), an extremely rare autosomal recessive disorder with fewer than ten families reported to date. Variant interpretation is challenging given the paucity of known disease-causing variants, and indeed we assess both paternal and maternal variants as Variants of Uncertain Significance under current American College of Medical Genetics guidelines. However, based on the inheritance pattern, suggestive evidence of pathogenicity, and significant clinical correlation with other reported MGCA8 patients, the clinical care team considers this a diagnostic result. Conclusion: Our findings ended the diagnostic odyssey for this family and provide further insights into the genetic and clinical spectrum of this critically under-studied disorder.

Chemical characterization of pro-inflammatory amyloid-beta peptides in human atherosclerotic lesions and platelets.

Amyloid-ß (Aß) peptides are intimately involved in the inflammatory pathology of atherosclerotic vascular disease (AVD) and Alzheimer's disease (AD). Although substantial amounts of these peptides are produced in the periphery, their role and significance to vascular disease outside the brain requires further investigation. Amyloid-ß peptides present in the walls of human aorta atherosclerotic lesions as well as activated and non-activated human platelets were isolated using sequential size-exclusion columns and HPLC reverse-phase methods. The Aß peptide isolates were quantified by ELISA and structurally analyzed using MALDI-TOF mass spectrometry procedures. Our experiments revealed that both aorta and platelets contained Aß peptides, predominately Aß40. The source of the Aß pool in aortic atherosclerosis lesions is probably the activated platelets and/or vascular wall cells expressing APP/PN2. Significant levels of Aß42 are present in the plasma, suggesting that this reservoir makes a minor contribution to atherosclerotic plaques. Our data reveal that although aortic atherosclerosis and AD cerebrovascular amyloidosis exhibit clearly divergent end-stage manifestations, both vascular diseases share some key pathophysiological promoting elements and pathways. Whether they happen to be deposited in vessels of the central nervous system or atherosclerotic plaques in the periphery, Aß peptides may promote and perhaps synergize chronic inflammatory processes which culminate in the degeneration, malfunction and ultimate destruction of arterial walls.

Early autophagic response in a novel knock-in model of Huntington disease.

The aggregation of mutant polyglutamine (polyQ) proteins has sparked interest in the role of protein quality-control pathways in Huntington's disease (HD) and related polyQ disorders. Employing a novel knock-in HD mouse model, we provide in vivo evidence of early, sustained alterations of autophagy in response to mutant huntingtin (mhtt). The HdhQ200 knock-in model, derived from the selective breeding of HdhQ150 knock-in mice, manifests an accelerated and more robust phenotype than the parent line. Heterozygous HdhQ200 mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age and striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Striatal NIIs accumulate earlier in HdhQ200 mice than in HdhQ150 mice. The earlier appearance of aggregate pathology in HdhQ200 mice is paralleled by earlier and more rapidly progressive motor deficits: progressive imbalance and decreased motor coordination by 50 weeks, gait deficits by 60 weeks and gross motor impairment by 80 weeks of age. At 80 weeks, heterozygous HdhQ200 mice exhibit striatal and cortical astrogliosis and a approximately 50% reduction in striatal dopamine receptor binding. Increased LC3-II protein expression, which is noted early and sustained throughout the disease course, is paralleled by increased expression of the autophagy-related protein, p62. Early and sustained expression of autophagy-related proteins in this genetically precise mouse model of HD suggests that the alteration of autophagic flux is an important and early component of the neuronal response to mhtt.

Inherited and de novo variants extend the etiology of TAOK1-associated neurodevelopmental disorder.

Alterations in the TAOK1 gene have recently emerged as the cause of developmental delay with or without intellectual impairment or behavioral abnormalities (MIM # 619575). The 32 cases currently described in the literature have predominantly de novo alterations in TAOK1 and a wide spectrum of neurodevelopmental abnormalities. Here, we report four patients with novel pathogenic TAOK1 variants identified by research genome sequencing, clinical exome sequencing, and international matchmaking. The overlapping clinical features of our patients are consistent with the emerging core phenotype of TAOK1-associated syndrome: facial dysmorphism, feeding difficulties, global developmental delay, joint laxity, and hypotonia. However, behavioral abnormalities and gastrointestinal issues are more common in our cohort than previously reported. Two patients have de novo TAOK1 variants (one missense, one splice site) consistent with most known alterations in this gene. However, we also report the first sibling pair who both inherited a TAOK1 frameshift variant from a mildly affected mother. Our findings suggest that incomplete penetrance and variable expressivity are relatively common in TAOK1-associated syndrome, which holds important implications for clinical genetic testing.

A Decade's Experience in Pediatric Chromosomal Microarray Reveals Distinct Characteristics Across Ordering Specialties.

Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

Macrophage-mediated degradation of beta-amyloid via an apolipoprotein E isoform-dependent mechanism.

Recent studies suggest that bone marrow-derived macrophages can effectively reduce beta-amyloid (Abeta) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Abeta, we cultured murine macrophages on top of Abeta plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP(717V>F) mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Abeta in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Abeta degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Abeta than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Abeta was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Abeta-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Abeta degradation by macrophages. These apoE isoform-dependent effects of macrophages on Abeta degradation suggest a novel "peripheral" mechanism for Abeta clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimer's disease.

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy.

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1's enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners.  These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations' biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

Verification of somatic CAG repeat expansion by pre-PCR fractionation.

The inheritance of a long CAG repeat causes several late onset neurological disorders including Huntington's disease (HD). Longer CAG repeats correlate with earlier onset of HD suggesting an increased toxicity for the products of long repeat alleles. PCR based data has been used to show that HD CAG repeat expansion beyond the inherited length occurs in affected tissues indicating a possible role for somatic instability in the disease process. PCR, however, is prone to artifacts resulting from expansion of repeat sequences during amplification. We describe a method to distinguish between CAG repeat expansions that exist in vivo and those that potentially occur during PCR. The method involves size fractionation of genomic restriction fragments containing the expanded repeats followed by PCR amplification. The application of this method confirms the presence of somatic expansions in the brains of a knock-in mouse model of HD.

Novel pathogenic variants and genes for myopathies identified by whole exome sequencing.

Neuromuscular diseases (NMD) account for a significant proportion of infant and childhood mortality and devastating chronic disease. Determining the specific diagnosis of NMD is challenging due to thousands of unique or rare genetic variants that result in overlapping phenotypes. We present four unique childhood myopathy cases characterized by relatively mild muscle weakness, slowly progressing course, mildly elevated creatine phosphokinase (CPK), and contractures. We also present two additional cases characterized by severe prenatal/neonatal myopathy. Prior extensive genetic testing and histology of these cases did not reveal the genetic etiology of disease. Here, we applied whole exome sequencing (WES) and bioinformatics to identify likely causal pathogenic variants in each pedigree. In two cases, we identified novel pathogenic variants in COL6A3. In a third case, we identified novel likely pathogenic variants in COL6A6 and COL6A3. We identified a novel splice variant in EMD in a fourth case. Finally, we classify two cases as calcium channelopathies with identification of novel pathogenic variants in RYR1 and CACNA1S. These are the first cases of myopathies reported to be caused by variants in COL6A6 and CACNA1S. Our results demonstrate the utility and genetic diagnostic value of WES in the broad class of NMD phenotypes.

Mouse Huntington's disease homolog mRNA levels: variation and allele effects.

Huntington's disease homolog (Hdh) mRNA levels in mice with different Hdh alleles were measured. Brain Hdh mRNA levels varied up to threefold in genetically identical wild-type mice, indicating nongenetic factors influence Hdh expression. Striatal Hdh mRNA levels from an allele with a repeat expanded to 150 CAGs were diminished compared with wild-type and showed variation that might contribute to phenotypic variability in the Hdh(CAG)150 knock-in mouse model. To determine whether Hdh mRNA levels are tightly regulated, we assessed these levels in mice heterozygous for a deletion of the Hdh promoter. The loss of one allele reduced Hdh mRNA levels in most tissues, suggesting mechanisms to maintain Hdh mRNA levels are not in effect and should not impede therapies designed to destroy mutant huntingtin mRNA. Finally, we found a correlation between tissue mRNA levels and the susceptibility of the Hdh locus to Cre-mediated deletion. The two tissues with the highest levels of Hdh mRNA, testes and brain, were the only tissues susceptible to Cre-mediated recombination between loxP sites at Hdh locus. In contrast, the same Cre-expressing line caused recombination in every tissue for loxP sites at another genomic location. The pattern of Cre susceptibility at Hdh suggests a correlation between chromatin accessibility and high levels of Hdh expression in testes and brain.

Neurochemical profile of dementia pugilistica.

Dementia pugilistica (DP), a suite of neuropathological and cognitive function declines after chronic traumatic brain injury (TBI), is present in approximately 20% of retired boxers. Epidemiological studies indicate TBI is a risk factor for neurodegenerative disorders including Alzheimer disease (AD) and Parkinson disease (PD). Some biochemical alterations observed in AD and PD may be recapitulated in DP and other TBI persons. In this report, we investigate long-term biochemical changes in the brains of former boxers with neuropathologically confirmed DP. Our experiments revealed biochemical and cellular alterations in DP that are complementary to and extend information already provided by histological methods. ELISA and one-dimensional and two dimensional Western blot techniques revealed differential expression of select molecules between three patients with DP and three age-matched non-demented control (NDC) persons without a history of TBI. Structural changes such as disturbances in the expression and processing of glial fibrillary acidic protein, tau, and α-synuclein were evident. The levels of the Aß-degrading enzyme neprilysin were reduced in the patients with DP. Amyloid-ß levels were elevated in the DP participant with the concomitant diagnosis of AD. In addition, the levels of brain-derived neurotrophic factor and the axonal transport proteins kinesin and dynein were substantially decreased in DP relative to NDC participants. Traumatic brain injury is a risk factor for dementia development, and our findings are consistent with permanent structural and functional damage in the cerebral cortex and white matter of boxers. Understanding the precise threshold of damage needed for the induction of pathology in DP and TBI is vital.

Morphological and pathological evolution of the brain microcirculation in aging and Alzheimer's disease.

Key pathological hallmarks of Alzheimer's disease (AD), including amyloid plaques, cerebral amyloid angiopathy (CAA) and neurofibrillary tangles do not completely account for cognitive impairment, therefore other factors such as cardiovascular and cerebrovascular pathologies, may contribute to AD. In order to elucidate the microvascular changes that contribute to aging and disease, direct neuropathological staining and immunohistochemistry, were used to quantify the structural integrity of the microvasculature and its innervation in three oldest-old cohorts: 1) nonagenarians with AD and a high amyloid plaque load; 2) nonagenarians with no dementia and a high amyloid plaque load; 3) nonagenarians without dementia or amyloid plaques. In addition, a non-demented (ND) group (average age 71 years) with no amyloid plaques was included for comparison. While gray matter thickness and overall brain mass were reduced in AD compared to ND control groups, overall capillary density was not different. However, degenerated string capillaries were elevated in AD, potentially suggesting greater microvascular "dysfunction" compared to ND groups. Intriguingly, apolipoprotein ε4 carriers had significantly higher string vessel counts relative to non-ε4 carriers. Taken together, these data suggest a concomitant loss of functional capillaries and brain volume in AD subjects. We also demonstrated a trend of decreasing vesicular acetylcholine transporter staining, a marker of cortical cholinergic afferents that contribute to arteriolar vasoregulation, in AD compared to ND control groups, suggesting impaired control of vasodilation in AD subjects. In addition, tyrosine hydroxylase, a marker of noradrenergic vascular innervation, was reduced which may also contribute to a loss of control of vasoconstriction. The data highlight the importance of the brain microcirculation in the pathogenesis and evolution of AD.