Expanding the clinical spectrum of biglycan-related Meester-Loeys syndrome.

Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5' untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.

A familial missense variant in the Alzheimer's disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

NOTCH3 C201R variant causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) that can be confused with early-onset Alzheimer's disease.

BACKGROUND: NOTCH3 is the causative gene for autosomal dominant cerebral arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) which is associated with both stroke and dementia. When CADASIL presents primarily as dementia it can be difficult to distinguish from Alzheimer's disease (AD) at both the clinical and neuropathological levels. METHODS: We performed exome sequencing of several affected individuals from a large family affected with AD. PCR amplification and direct Sanger sequencing were used to verify variants detected by exome analysis and to screen family members at-risk to carry those variants. Neuropathologic brain evaluation by immunohistochemistry and MRI were performed for the carriers of the NOTCH3 variant. RESULTS: In a three-generation family with AD, we found a c.601 T > C p.Cys201Arg variant in the NOTCH3 gene that caused clinical and neuropathological manifestations of CADASIL. These features included earlier onset of dementia accompanied by behavioral abnormalities in the father and son and white matter abnormalities in the asymptomatic grandson. The family is one branch of a large pedigree studied by the Alzheimer's Disease Sequencing Project (ADSP). As part of the ADSP linkage analysis and whole genome sequencing endeavor, an ABCA1 variant, p.Ala937Val, was previously found associated with AD in this pedigree. CONCLUSIONS: Our findings, together with other reported pathogenic missense variants of the C201 codon in NOTCH3, support the role of cysteine 201 as a mutation hotspot for CADASIL and highlight the genetic complexity both clinically and pathologically of AD and related dementia.

A familial missense variant in the Alzheimer's Disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset >75 years. All offspring were affected with AD with ages at onset ranging from 53yrs-74yrs. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Novel LMNA mutations in Greek and Myanmar Patients with Progeroid Features and Cardiac Manifestations.

Segmental progeroid syndromes are groups of genetic disorders with multiple features resembling accelerated aging. The International Registry of Werner Syndrome (Seattle, WA) recruits pedigrees of progeroid syndromes from all over the world. We identified two novel LMNA mutations, p.Asp300Gly in a patient from Myanmar, and p.Asn466Lys, in a patient from Greece. Both were referred to our Registry for the genetic diagnosis because of the accelerated aged-appearance and cardiac complications. LMNA mutations are the second most common genetic cause of progeroid syndromes after WRN mutations in our Registry. As the next generation sequencing becomes readily available, we expect to identify more cases of rare genetic diseases in the developing countries.

Alternative splicing in a presenilin 2 variant associated with Alzheimer disease.

OBJECTIVE: Autosomal-dominant familial Alzheimer disease (AD) is caused by by variants in presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Previously, we reported a rare PSEN2 frameshift variant in an early-onset AD case (PSEN2 p.K115Efs*11). In this study, we characterize a second family with the same variant and analyze cellular transcripts from both patient fibroblasts and brain lysates. METHODS: We combined genomic, neuropathological, clinical, and molecular techniques to characterize the PSEN2 K115Efs*11 variant in two families. RESULTS: Neuropathological and clinical evaluation confirmed the AD diagnosis in two individuals carrying the PSEN2 K115Efs*11 variant. A truncated transcript from the variant allele is detectable in patient fibroblasts while levels of wild-type PSEN2 transcript and protein are reduced compared to controls. Functional studies to assess biological consequences of the variant demonstrated that PSEN2 K115Efs*11 fibroblasts secrete less Aß 1-40 compared to controls, indicating abnormal γ-secretase activity. Analysis of PSEN2 transcript levels in brain tissue revealed alternatively spliced PSEN2 products in patient brain as well as in sporadic AD and age-matched control brain. INTERPRETATION: These data suggest that PSEN2 K115Efs*11 is a likely pathogenic variant associated with AD. We uncovered novel PSEN2 alternative transcripts in addition to previously reported PSEN2 splice isoforms associated with sporadic AD. In the context of a frameshift, these alternative transcripts return to the canonical reading frame with potential to generate deleterious protein products. Our findings suggest novel potential mechanisms by which PSEN variants may influence AD pathogenesis, highlighting the complexity underlying genetic contribution to disease risk.

Characterization of Adult Vestibular Organs in 11 CreER Mouse Lines.

Utricles are vestibular sense organs that encode linear head movements. They are composed of a sensory epithelium with type I and type II hair cells and supporting cells, sitting atop connective tissue, through which vestibular nerves project. We characterized utricular Cre expression in 11 murine CreER lines using the ROSA26tdTomato reporter line and tamoxifen induction at 6 weeks of age. This characterization included Calbindin2CreERT2, Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, ParvalbuminCreERT2, Prox1CreERT2, Sox2CreERT2, and Sox9-CreERT2. OtoferlinCreERT2 mice had inducible Cre activity specific to hair cells. GLAST-CreERT2, Id2CreERT2, and Sox9-CreERT2 had inducible Cre activity specific to supporting cells. Sox2CreERT2 had inducible Cre activity in supporting cells and most type II hair cells. ParvalbuminCreERT2 mice had small numbers of labeled vestibular nerve afferents. Calbindin2CreERT2 mice had labeling of most type II hair cells and some type I hair cells and supporting cells. Only rare (or no) tdTomato-positive cells were detected in utricles of Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, and Prox1CreERT2 mice. No Cre leakiness (tdTomato expression in the absence of tamoxifen) was observed in OtoferlinCreERT2 mice. A small degree of leakiness was seen in GLAST-CreERT2, Id2CreERT2, Sox2CreERT2, and Sox9-CreERT2 lines. Calbindin2CreERT2 mice had similar tdTomato expression with or without tamoxifen, indicating lack of inducible control under the conditions tested. In conclusion, 5 lines-GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, Sox2CreERT2, and Sox9-CreERT2-showed cell-selective, inducible Cre activity with little leakiness, providing new genetic tools for researchers studying the vestibular periphery.

Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice.

Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury.