From Amish farm dust to bacterial lysates: The long and winding road to protection from allergic disease.

Allergic diseases typically begin in early life and can impose a heavy burden on children and their families. Effective preventive measures are currently unavailable but may be ushered in by studies on the "farm effect", the strong protection from asthma and allergy found in children born and raised on traditional farms. Two decades of epidemiologic and immunologic research have demonstrated that this protection is provided by early and intense exposure to farm-associated microbes that target primarily innate immune pathways. Farm exposure also promotes timely maturation of the gut microbiome, which mediates a proportion of the protection conferred by the farm effect. Current research seeks to identify allergy-protective compounds from traditional farm environments, but standardization and regulation of such substances will likely prove challenging. On the other hand, studies in mouse models show that administration of standardized, pharmacological-grade lysates of human airway bacteria abrogates allergic lung inflammation by acting on multiple innate immune targets, including the airway epithelium/IL-33/ILC2 axis and dendritic cells whose Myd88/Trif-dependent tolerogenic reprogramming is sufficient for asthma protection in adoptive transfer models. To the extent that these bacterial lysates mimic the protective effects of natural exposure to microbe-rich environments, these agents might provide an effective tool for prevention of allergic disease.

In-Depth Mass Spectrometry Analysis Reveals the Plasma Proteomic and N-Glycoproteomic Impact of an Amish-Enriched Cardioprotective Variant in B4GALT1.

B4GALT1 encodes ß-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.

A biallelic SNIP1 Amish founder variant causes a recognizable neurodevelopmental disorder.

SNIP1 (Smad nuclear interacting protein 1) is a widely expressed transcriptional suppressor of the TGF-ß signal-transduction pathway which plays a key role in human spliceosome function. Here, we describe extensive genetic studies and clinical findings of a complex inherited neurodevelopmental disorder in 35 individuals associated with a SNIP1 NM_024700.4:c.1097A>G, p.(Glu366Gly) variant, present at high frequency in the Amish community. The cardinal clinical features of the condition include hypotonia, global developmental delay, intellectual disability, seizures, and a characteristic craniofacial appearance. Our gene transcript studies in affected individuals define altered gene expression profiles of a number of molecules with well-defined neurodevelopmental and neuropathological roles, potentially explaining clinical outcomes. Together these data confirm this SNIP1 gene variant as a cause of an autosomal recessive complex neurodevelopmental disorder and provide important insight into the molecular roles of SNIP1, which likely explain the cardinal clinical outcomes in affected individuals, defining potential therapeutic avenues for future research.

"The Graves Cannot Be Dug Fast Enough": Excess Deaths Among US Amish and Mennonites During the 1918 Flu Pandemic.

Estimating the lethal impact of a pandemic on a religious community with significant barriers to outsiders can be exceedingly difficult. Nevertheless, Stein and colleagues (2021) developed an innovative means of arriving at such an estimate for the lethal impact of COVID-19 on the Amish community in 2020 by counting user-generated death reports in the widely circulated Amish periodical The Budget. By comparing monthly averages of reported deaths before and during the COVID-19 pandemic, Stein and colleagues were able to arrive at a rough estimate of "excess deaths" during the first year of the pandemic. Our research extends the same research method, applying it to the years during and immediately preceding the global influenza pandemic of 1918. Results show similarly robust findings, including three notable "waves" of excess deaths among Amish and conservative Mennonites in the USA in 1918, 1919, and 1920. Such results point to the promise of utilizing religious periodicals like The Budget as a relatively untapped trove of user-generated data on public health outcomes among religious minorities more than a century in the past.

Missense variants in SORT1 are associated with LDL-C in an Amish population.

Common noncoding variants at the human 1p13.3 locus associated with SORT1 expression are among those most strongly associated with low-density lipoprotein cholesterol (LDL-C) in human genome-wide association studies. However, validation studies in mice and cell lines have produced variable results regarding the directionality of the effect of SORT1 on LDL-C. This, together with the fact that the 1p13.3 variants are associated with expression of several genes, has raised the question of whether SORT1 is the causal gene at this locus. Using whole exome sequencing in members of an Amish population, we identified coding variants in SORT1 that are associated with increased (rs141749679, K302E) and decreased (rs149456022, Q225H) LDL-C. Further, analysis of plasma lipoprotein particle subclasses by ion mobility in a subset of rs141749679 (K302E) carriers revealed higher levels of large LDL particles compared to noncarriers. In contrast to the effect of these variants in the Amish, the sortilin K302E mutation introduced into a C57BL/6J mouse via CRISPR/Cas9 resulted in decreased non-high-density lipoprotein cholesterol, and the sortilin Q225H mutation did not alter cholesterol levels in mice. This is indicative of different effects of these mutations on cholesterol metabolism in the two species. To our knowledge, this is the first evidence that naturally occurring coding variants in SORT1 are associated with LDL-C, thus supporting SORT1 as the gene responsible for the association of the 1p13.3 locus with LDL-C.

Associations of genome-wide and regional autozygosity with 96 complex traits in old order Amish.

BACKGROUND: Autozygosity, the proportion of the genome that is homozygous by descent, has been associated with variation in multiple health-related traits impacting evolutionary fitness. Autozygosity (FROH) is typically measured from runs of homozygosity (ROHs) that arise when identical-by-descent (IBD) haplotypes are inherited from each parent. Population isolates with a small set of common founders have elevated autozygosity relative to outbred populations. METHODS: In this study, we examined whether degree of autozygosity was associated with variation in 96 cardiometabolic traits among 7221 Old Order Amish individuals residing in Lancaster County, PA. We estimated the average length of an ROH segment to be 6350 KB, with each individual having on average 17.2 segments 1.5 KB or larger. Measurements of genome-wide and regional FROH were used as the primary predictors of trait variation in association analysis. RESULTS: In genome-wide FROH analysis, we did not identify any associations that withstood Bonferroni-correction (p = 0.0005). However, on regional FROH analysis, we identified associations exceeding genome-wide thresholds for two traits: serum bilirubin levels, which were significantly associated with a region on chromosome 2 localized to a region surrounding UGT1A10 (p = 1 × 10- 43), and HbA1c levels, which were significantly associated with a region on chromosome 8 localized near CHRNB3 (p = 8 × 10- 10). CONCLUSIONS: These analyses highlight the potential value of autozygosity mapping in founder populations.

Visuospatial and Verbal Memory Differences in Amish Individuals With Alzheimer Disease and Related Dementias.

BACKGROUND: Verbal and visuospatial memory impairments are common to Alzheimer disease and Related Dementias (ADRD), but the patterns of decline in these domains may reflect genetic and lifestyle influences. The latter may be pertinent to populations such as the Amish who have unique lifestyle experiences. METHODS: Our data set included 420 Amish and 401 CERAD individuals. Sex-adjusted, age-adjusted, and education-adjusted Z-scores were calculated for the recall portions of the Constructional Praxis Delay (CPD) and Word List Delay (WLD). ANOVAs were then used to examine the main and interaction effects of cohort (Amish, CERAD), cognitive status (case, control), and sex on CPD and WLD Z-scores. RESULTS: The Amish performed better on the CPD than the CERAD cohort. In addition, the difference between cases and controls on the CPD and WLD were smaller in the Amish and Amish female cases performed better on the WLD than the CERAD female cases. DISCUSSION: The Amish performed better on the CPD task, and ADRD-related declines in CPD and WLD were less severe in the Amish. In addition, Amish females with ADRD may have preferential preservation of WLD. This study provides evidence that the Amish exhibit distinct patterns of verbal and visuospatial memory loss associated with aging and ADRD.

Psychometric approaches to defining cognitive phenotypes in the Old Order Amish.

OBJECTIVE: Memory and cognitive problems are central to the diagnosis of Alzheimer's disease (AD). Psychometric approaches to defining phenotypes can aid in identify genetic variants associated with AD. However, these approaches have mostly been limited to affected individuals. Defining phenotypes of both affected and unaffected individuals may help identify genetic variants associated with both AD and healthy aging. This study compares psychometric methods for developing cognitive phenotypes that are more granular than clinical classifications. METHODS: 682 older Old Order Amish individuals were included in the analysis. Adjusted Z-scores of cognitive tests were used to create four models including (1) global threshold scores or (2) memory threshold scores, and (3) global clusters and (4) memory clusters. An ordinal regression examined the coherence of the models with clinical classifications (cognitively impaired [CI], mildly impaired [MI], cognitively unimpaired), APOE-e4, sex, and age. An ANOVA examined the best model phenotypes for differences in clinical classification, APOE-e4, domain Z-scores (memory, language, executive function, and processing speed), sex, and age. RESULTS: The memory cluster identified four phenotypes and had the best fit (χ2  = 491.66). Individuals in the worse performing phenotypes were more likely to be classified as CI or MI and to have APOE-e4. Additionally, all four phenotypes performed significantly differently from one another on the domains of memory, language, and executive functioning. CONCLUSIONS: Memory cluster stratification identified the cognitive phenotypes that best aligned with clinical classifications, APOE-e4, and cognitive performance We predict these phenotypes will prove useful in searching for protective genetic variants.

De novo mutations across 1,465 diverse genomes reveal mutational insights and reductions in the Amish founder population.

De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains <1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability (h2), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

No association between SCN9A and monogenic human epilepsy disorders.

Many studies have demonstrated the clinical utility and importance of epilepsy gene panel testing to confirm the specific aetiology of disease, enable appropriate therapeutic interventions, and inform accurate family counselling. Previously, SCN9A gene variants, in particular a c.1921A>T p.(Asn641Tyr) substitution, have been identified as a likely autosomal dominant cause of febrile seizures/febrile seizures plus and other monogenic seizure phenotypes indistinguishable from those associated with SCN1A, leading to inclusion of SCN9A on epilepsy gene testing panels. Here we present serendipitous findings of genetic studies that identify the SCN9A c.1921A>T p.(Asn641Tyr) variant at high frequency in the Amish community in the absence of such seizure phenotypes. Together with findings in UK Biobank these data refute an association of SCN9A with epilepsy, which has important clinical diagnostic implications.

Pathways to Immunity: Patterns of Excess Death Across the United States and Within Closed Religious Communities.

Public health officials promoted COVID-19 vaccines to limit burdens placed on the U.S. healthcare system and end the pandemic. People in some closed religious communities refused to vaccinate and likely acquired temporary immunity through infection. This paper compares the death rates in Amish, Old Order Mennonites, and conservative Mennonite groups to a rate estimated for the U.S. population. Approximately two-thirds of the U.S. population was immunized against COVID-19, while few in the Amish/Mennonite community were. We find divergent patterns. Once vaccines became available, excess deaths declined in the general population and remained elevated among Amish and Mennonites. Vaccination campaigns must consider and value the cultural beliefs of closed religious communities to be effective.

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Impact of parental relatedness on reproductive outcomes among the Old Order Amish of Lancaster County.

Genetically isolated populations that arise due to recent bottleneck events have reduced genetic variation reflecting the common set of founders. Increased genetic relatedness among members of isolated populations puts them at increased risk for some recessive disorders that are rare in outbred populations. To assess the burden on reproductive health, we compared frequencies of adverse reproductive outcomes between Amish couples who were both heterozygous carriers of a highly penetrant pathogenic or likely pathogenic variant and noncarrier couples from the same Amish community. In addition, we evaluated whether overall genetic relatedness of parents was associated with reproductive outcomes. Of the 1824 couples included in our study, 11.1% were at risk of producing a child with an autosomal recessive disorder. Carrier couples reported a lower number of miscarriages compared to noncarrier couples (p = 0.02), although the number of stillbirths (p = 0.3), live births (p = 0.9), and number of pregnancies (p = 0.9) did not differ significantly between groups. In contrast, higher overall relatedness between spouses was positively correlated with number of live births (p < 0.0001), pregnancies (p < 0.0001), and stillbirths (p = 0.03), although not with the number of miscarriages (p = 0.4). These results highlight a complex association between relatedness of parents and reproductive health outcomes in this community.

Clinical characterization of familial hypercholesterolemia due to an amish founder mutation in Apolipoprotein B.

BACKGROUND: Familial hypercholesterolemia (FH) due to a founder variant in Apolipoprotein B (ApoBR3500Q) is reported in 12% of the Pennsylvania Amish community. By studying a cohort of ApoBR3500Q heterozygotes and homozygotes, we aimed to characterize the biochemical and cardiac imaging features in children and young adults with a common genetic background and similar lifestyle. METHODS: We employed advanced lipid profile testing, carotid intima media thickness (CIMT), pulse wave velocity (PWV), and peripheral artery tonometry (PAT) to assess atherosclerosis in a cohort of Amish ApoBR3500Q heterozygotes (n = 13), homozygotes (n = 3), and their unaffected, age-matched siblings (n = 9). ApoBR3500Q homozygotes were not included in statistical comparisons. RESULTS: LDL cholesterol (LDL-C) was significantly elevated among ApoBR3500Q heterozygotes compared to sibling controls, though several ApoBR3500Q heterozygotes had LDL-C levels in the normal range. LDL particles (LDL-P), small, dense LDL particles, and ApoB were also significantly elevated among subjects with ApoBR3500Q. Despite these differences in serum lipids and particles, CIMT and PWV were not significantly different between ApoBR3500Q heterozygotes and controls in age-adjusted analysis. CONCLUSIONS: We provide a detailed description of the serum lipids, atherosclerotic plaque burden, vascular stiffness, and endothelial function among children and young adults with FH due to heterozygous ApoBR3500Q. Fasting LDL-C was lower than what is seen with other forms of FH, and even normal in several ApoBR3500Q heterozygotes, emphasizing the importance of cascade genetic testing among related individuals for diagnosis. We found increased number of LDL particles among ApoBR3500Q heterozygotes but an absence of detectable atherosclerosis.

Heterozygosity for a Pathogenic Variant in SLC12A3 That Causes Autosomal Recessive Gitelman Syndrome Is Associated with Lower Serum Potassium.

BACKGROUND: Potassium levels regulate multiple physiologic processes. The heritability of serum potassium level is moderate, with published estimates varying from 17% to 60%, suggesting genetic influences. However, the genetic determinants of potassium levels are not generally known. METHODS: A whole-exome sequencing association study of serum potassium levels in 5812 subjects of the Old Order Amish was performed. A dietary salt intervention in 533 Amish subjects estimated interaction between p.R642G and sodium intake. RESULTS: A cluster of variants, spanning approximately 537 kb on chromosome 16q13, was significantly associated with serum potassium levels. Among the associated variants, a known pathogenic variant of autosomal recessive Gitelman syndrome (p.R642G SLC12A3) was most likely causal; there were no homozygotes in our sample. Heterozygosity for p.R642G was also associated with lower chloride levels, but not with sodium levels. Notably, p.R642G showed a novel association with lower serum BUN levels. Heterozygotes for p.R642G had a two-fold higher rate of self-reported bone fractures and had higher resting heart rates on a low-salt diet compared with noncarriers. CONCLUSIONS: This study provides evidence that heterozygosity for a pathogenic variant in SLC12A3 causing Gitelman syndrome, a canonically recessive disorder, contributes to serum potassium concentration. The findings provide insights into SLC12A3 biology and the effects of heterozygosity on electrolyte homeostasis and related subclinical phenotypes that may have implications for personalized medicine and nutrition.

Rituals of Contagion in Closed Religious Communities: A Case Study of Amish and Mennonite Communities in the USA During the Beginning of the COVID-19 Pandemic.

At the onset of the COVID-19 pandemic, government and medical guidelines emphasized social distancing to limit exposure. These guidelines significantly impacted closed religious communities, particularly those opposed to modern technologies, such as Amish and Mennonite communities. How did these religious communities respond to COVID-19 policies in the USA? We draw data from Ohio and Pennsylvania scribe entries published in an Amish/Mennonite correspondence newspaper. While some of these communities altered church rituals to comply with government directives, others maintained communal worship without disruption. Mennonite communities were more likely to conform to guidelines.

The burden of pathogenic variants in clinically actionable genes in a founder population.

Founder populations may be enriched with certain genetic variants of high clinical impact compared to nonfounder populations due to bottleneck events and genetic drift. Using exome sequencing (ES), we quantified the load of pathogenic variants that may be clinically actionable in 6136 apparently healthy adults living in the Lancaster, PA Old Order Amish settlement. We focused on variants in 78 genes deemed clinically actionable by the American College of Medical Genetics and Genomics (ACMG) or Geisinger's MyCode Health Initiative. ES revealed 3191 total variants among these genes including 480 nonsynonymous variants. After quality control and filtering, we applied the ACMG/AMP guidelines for variant interpretation and classified seven variants, across seven genes, as either pathogenic or likely pathogenic. Through genetic drift, all seven variants, are highly enriched in the Amish compared to nonfounder populations. In total, 14.7% of Lancaster Amish individuals carry at least one of these variants, largely explained by the 13% who harbor a copy of a single variant in APOB. Other studies report combined frequencies of pathogenic/likely pathogenic (P/LP) variants in actionable genes between 2.0% and 6.2% in outbred populations. The Amish population harbors fewer actionable variants compared to similarly characterized nonfounder populations but have a higher frequency of each variant identified, offering opportunities for efficient and cost-effective targeted precision medicine.

Mendelian disease research in the Plain populations of Lancaster County, Pennsylvania.

Founder populations have long contributed to our knowledge of rare disease genes and phenotypes. From the pioneering work of Dr. Victor McKusick to today, research in these groups has shed light on rare recessive phenotypes, expanded the clinical spectrum of disease, and facilitated disease gene identification. Current clinical and research studies in these special groups augment the wealth of knowledge already gained, provide new insights into emerging problems such as variant interpretation and reduced penetrance, and contribute to the development of novel therapies for rare genetic diseases. Clinical developments over the past 30 years have altered the fundamental relationship with the Lancaster Plain communities: research has become more collaborative, and the knowledge imparted by these studies is now being harnessed to provide cutting-edge translational medicine to the very community of vulnerable individuals who need it most.

Identifying barriers and considerations for cochlear implantation in Amish children.

OBJECTIVE: To determine if barriers to cochlear implantation (CI) exist with respect to Amish children and to describe unique considerations associated with CI and subsequent otologic care in the Amish population. METHODS: Out of all patients who underwent CI at a tertiary care pediatric hospital from 2008 to 2019, Amish children were age-matched to the remainder of the cohort to compare demographics and care-related metrics including etiology of hearing loss, age at time of initial hearing-related appointment and at CI, total number of pre- and post-operative audiologic and otologic appointments, and post-operative complications. Social considerations that may pose barriers to care were collected for descriptive analysis. RESULTS: Since 2008, 232 children underwent CI, of which 8 implants were performed on Amish children. Six (75%) Amish children underwent newborn hearing screening and 3(38%) were found to have syndromic etiology for hearing loss. While Amish patients had a lower number of both audiologic (15 vs 33.5, p<.001) and otologic (4.5 vs 8.5, p=.028) appointments when compared to age-matched controls, median age at the time of implantation for the whole sample was not different between groups (2.5 vs 2.0 years, p=.211). From a social standpoint, limitations in transportation, telephone communication, and ability to recharge processor batteries must be considered in the Amish population. CONCLUSION: Amish children undergoing CI face unique barriers to care including transportation and technologic limitations, leading to overall fewer hearing-related appointments when compared to an age-matched cohort. Understanding societal differences is important to facilitate optimal care for Amish children with hearing loss.

The persistently high fertility of a North American population: A 25-year restudy of parity among the Ohio Amish.

Why do the North American Amish maintain high fertility when surrounding populations have nearly all completed the demographic transition? Using the same theoretical predictors and methods as a 1996 Population Studies paper, we explore fertility changes, specifically changes in mean parity, between 1988 and 2015 among one sizeable Amish population in Ohio. Findings suggest that wealth flow shifts (as measured by a decline in farming families) and institutional changes (reflected in Amish denominational gradations) help to explain a decline in mean parity from 5.3 to 4.85, while ideological pronatalism (represented by higher fertility among church leaders) helps to explain why fertility has not been more responsive to structural incentives to limit family size. While this restudy confirms the trend of a slow decline in Amish fertility, it also invites a more methodologically expansive inquiry into Amish fertility patterns.