Order Amish Research Articles

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.229Genetics and modeling of TNNT1 genetic variants in nemaline myopathy

TNNT1-related nemaline myopathy (NEM5) comprises a small but increasingly significant cause of nemaline myopathy. Initially described as a severe autosomal recessive condition confined to the Old Order Amish of Pennsylvania in the US, subsequent patients with severe phenotypes have been described throughout the world. The prototypic clinical features include congenital hypotonia and weakness of proximal, bulbar, facial, and neck flexor muscles that may result in death secondary to respiratory insufficiency. This condition appears to exhibit allelic heterogeneity with multiple mutations within the same gene causing the severe recessive form of the disease. We recently described the first autosomal dominant c.311A>T, p.E104V pathogenic variant of TNNT1 gene occurring in an Ashkenazi Jewish family in the United States. This family had an overall milder phenotype than the recessive cases with marked clinical heterogeneity ranging from a Gower's maneuver in childhood to mild difficulty weight training late into adulthood. Recent whole exome sequencing studies in a large cohort of congenital myopathy cases have identified two additional unrelated cases with potentially pathogenic dominant TNNT1 mutations as well as two new instances of possible recessive variants. To assess pathogenicity of these variants we are establishing zebrafish models of both dominant gain of function and recessive loss of function for this gene. Morpholino-based knock down studies lead to significant neuromuscular abnormalities, including poor swimming ability, truncal curvature, and loss of birefringence, all indicative of defective muscle structure and function within the first week of life. Studies to model the dominant variants are in progress.

Association of plasma nitrite levels with obesity and metabolic syndrome in the Old Order Amish.

SummaryObjectivesPlasma nitrite is a metabolite of nitric oxide and reflects endogenous nitric oxide synthase (NOS) activity. Although plasma nitrites were previously linked with obesity and metabolic syndrome (MetS), the direction of association remains inconsistent, possibly due to sample heterogeneity. In a relatively homogeneous population, we hypothesized that nitrite levels will be positively associated with overweight/obesity and MetS.MethodsFasting nitrite levels were measured in 116 Old Order Amish (78% women). We performed age‐and‐sex‐adjusted ancovas to compare nitrite levels between three groups (a) overweight/obese(−)MetS(−), (b) overweight/obese(+)MetS(−) and (c) overweight/obese(+)MetS)(+). Multivariate linear regressions were conducted on nitrite associations with continuous metabolic variables, with successive adjustments for demographics, body mass index, C‐reactive protein and neopterin.ResultsNitrite levels were higher in the obese/overweight(+)MetS(+) group than in the other two groups (p < 0.001). Nitrites were positively associated with levels of triglycerides (p < 0.0001), total cholesterol (p = 0.048), high‐density lipoprotein/cholesterol ratio (p < 0.0001) and fasting glucose (p < 0.0001), and negatively correlated with high‐density lipoprotein‐cholesterol (p < 0.0001). These associations were robust to adjustments for body mass index and inflammatory markers.ConclusionFurther investigation of the connection between obesity/MetS and plasma nitrite levels may lead to novel dietary and pharmacological approaches that ultimately may contribute to reducing the increasing burden of obesity, MetS and cardiovascular morbidity and mortality.

TNNT1 nemaline myopathy: natural history and therapeutic frontier.

We describe the natural history of 'Amish' nemaline myopathy (ANM), an infantile-onset, lethal disease linked to a pathogenic c.505G>T nonsense mutation of TNNT1, which encodes the slow fiber isoform of troponin T (TNNT1; a.k.a. TnT). The TNNT1 c.505G>T allele has a carrier frequency of 6.5% within Old Order Amish settlements of North America. We collected natural history data for 106 ANM patients born between 1923 and 2017. Over the last two decades, mean age of molecular diagnosis was 16 ± 27 days. TNNT1 c.505G>T homozygotes were normal weight at birth but failed to thrive by age 9 months. Presenting neonatal signs were axial hypotonia, hip and shoulder stiffness, and tremors, followed by progressive muscle weakness, atrophy and contractures. Affected children developed thoracic rigidity, pectus carinatum and restrictive lung disease during infancy, and all succumbed to respiratory failure by 6 years of age (median survival 18 months, range 0.2-66 months). Muscle histology from two affected children showed marked fiber size variation owing to both Type 1 myofiber smallness (hypotrophy) and Type 2 fiber hypertrophy, with evidence of nemaline rods, myofibrillar disarray and vacuolar pathology in both fiber types. The truncated slow TNNT1 (TnT) fragment (p.Glu180Ter) was undetectable in ANM muscle, reflecting its rapid proteolysis and clearance from sarcoplasm. Similar functional and histological phenotypes were observed in other human cohorts and two transgenic murine models (Tnnt1-/- and Tnnt1 c.505G>T). These findings have implications for emerging molecular therapies, including the suitably of TNNT1 gene replacement for newborns with ANM or other TNNT1-associated myopathies.

Evaluation of WISP1 as a candidate gene for bone mineral density in the Old Order Amish

Wnt1-inducible signaling pathway protein-1 (WISP1) is a novel target of the Wnt pathway for modulating osteogenesis and improving bone strength. However, it is not clear if genetic variants in the WISP1 region are associated with bone mineral density (BMD) in human. The aim of this study is to investigate the role of genetic variation in WISP1 gene as a determinant of BMD in 1,510 Old Order Amish (OOA). We performed regional association analysis of 58 tag variants within 5 kb upstream and downstream to WISP1 with BMD and found 5 variants that were associated with BMD at multiple skeletal sites (P values from 2.89 × 10−6 to 1.62 × 10−2), with some significant associations even after adjustment for multiple comparisons. To replicate these results in an independent dataset, we performed a look-up of BMD associations with these variants in European ancestry subjects from the large GEFOS Consortium and observed the nominal associations of two of these variants with BMD (P values: 0.031 to 0.048). In conclusion, we have demonstrated that genetic variants surrounding WISP1 are associated with BMD at multiple skeletal sites in the OOA, thus influencing osteoporosis risk. These results support a role for the WISP1 gene on influencing variation in BMD.

0163 Activity And Light Exposure In The Old Order Amish

0163 Activity And Light Exposure In The Old Order Amish

0424 Association between Toxoplasma Gondii Oocyst IgG And Insomnia In The Old Order Amish

0424 Association between Toxoplasma Gondii Oocyst IgG And Insomnia In The Old Order Amish

F181. Association of Plasma Nitrite Levels With Metabolic Syndrome and its Components in the Old Order Amish

Metabolic Syndrome (MetS), a constellation of major cardiovascular risk factors, is highly prevalent in mental illness, thereby increasing morbidity and mortality. NO/nitrite pathways have been previously associated with obesity/metabolic syndrome but the direction of association remains inconsistent, probably due to sample heterogeneity. We now hypothesized that, in the Old Order Amish, a relatively homogeneous population, nitrite levels will be higher in obesity and MetS.

F156. Anhedonia and Hopelessness/Dysphoria Associated With Tooth Loss in the Old Order Amish: Gender Differences and Neopterin Levels-Mediator or Confounder?

Tooth loss, marker of poor dental health, consequence of multiple causes including periodontal, endodontal, and traumatic etiologies, has been previously associated with mental illness. Proposed mediation of this link includes self-neglect secondary to depression and anhedonia. Yet, inflammation, a common consequence of poor dental health, has been previously predictively associated with depression (vicious cycle). As smoking and socioeconomically differences induce marked heterogeneity, we are now examining associations between tooth loss and symptoms of depression in the Old Order Amish (OOA) a more homogeneous adult population, largely nicotine free.

T183. Gender Differences in Seropositivity and Metabolic Associations of Toxoplasma Gondii Oocyst IgG in the Old Order Amish

T183. Gender Differences in Seropositivity and Metabolic Associations of Toxoplasma Gondii Oocyst IgG in the Old Order Amish

F158. Toxoplasma Gondii-Oocyst Seropositivity and Depression in the Old Order Amish

F158. Toxoplasma Gondii-Oocyst Seropositivity and Depression in the Old Order Amish

The effect of congenic myostatin inhibition on a mouse model of osteogenesis imperfecta (OI) type I/IV

Bone is a dynamic organ in part due to its mechanosensitivity. Muscle mass and force are some of the largest physiological loads that bone experience during its life time, with muscle mass being directly proportional to bone mass. One of the factors regulating muscle mass is myostatin, a member of the TGF‐β superfamily, that acts as a negative regulator of muscle growth. Osteogenesis imperfecta (OI), commonly known as brittle bone disease, is a heritable connective tissue disorder characterized by skeletal fragility. A glycine to cysteine substitution in the proα2(I) collagen chain leads to reduced bone mineral density and compromised biomechanical integrity as modeled by the heterozygous G610C (+/G610C) mouse. +/G610C mice model both the genotype and phenotype of 64 affected individuals with mild to moderate type I/IV OI in an Old Order Amish kindred. We investigated the effects of myostatin deficiency on the +/G610C mouse muscle by breeding heterozygote myostatin deficient mice (+/mstn) with +/G610C mice to produce WT, +/mstn, +/G610C, and +/mstn +/G610C mice. After weaning at three weeks of age, mice were weighed weekly until 4 months of age when they were euthanized. After euthanization, 5 hindlimb muscles, the gastrocnemius, quadriceps, tibalis anterior, plantaris, and soleus, were harvested and weighed. Serum was separated from the blood and used to quantify myostatin levels. Serums from +/mstn and +/mstn +/G610C mice demonstrated decreased levels of myostatin as compared to WT and +/G610C mice. +/mstn mice showed increased body weights at four months of age when compared to WT littermates. +/mstn +/G610C mice also had increased body weights as compared to +/G610C littermates, although they did not reach significance. More importantly, however, +/mstn and +/mstn +/G610C mice exhibited significantly increased hindlimb skeletal muscle weights and relative muscle weights as compared to +/G610C mice for the gastrocnemius and quadriceps. These findings demonstrate that myostatin deficiency can lead to increased muscle mass in mice with OI which can potentially lead to increased bone mass and strength.Support or Funding InformationSupported by March of Dimes, National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (RO1 AR055907), the Leda J. Sears Trust, the Kansas City Area Life Sciences Institute Patton Trust Research, Inc., and the University of Missouri Research Board Grant. The project described was also supported by the IMSD EXPRESS Fellows Program (or simply the IMSD EXPRESS Program) via grant number R25GM056901 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 052: Analysis of Platelet Related Traits in the Trans-Omics for Precision Medicine (TOPMed) Whole Genome Sequencing Project

Platelets are critical in inflammation, wound healing, and thrombosis. Platelet count (PLT) and mean platelet volume (MPV) are heritable, frequently assessed measures for which hundreds of genetic associations have been identified, predominantly in European populations. However, causal variants are unknown at most loci, and additional genetic determinants in diverse ancestral populations remain to be discovered. We performed a multi-ancestry whole genome sequencing analysis for PLT in three Phase 1 TOPMed cohorts, the European American Framingham Heart (FHS, n=2259) and Old Order Amish (OOA, n=1095) studies and the African American Jackson Heart Study (JHS, n=3523). MPV was additionally analyzed in JHS and FHS. Heritability in JHS and FHS was 45-73%. Single variant and aggregate burden and sequence kernel association (SKAT) tests [5 and 50 kilobase (kb) sliding windows] were performed using a mixed model approach in GENESIS, with aggregate tests for variants with a minor allele frequency (MAF) &lt; 1% only. Inverse normalized residuals were adjusted for sex, age, study, and an empirical genetic relationship matrix, allowing for heterogeneous variance by study. For PLT, several associations had the same index SNP ( ARHGEF3 , TPM4 ) or were in moderate LD ( TAOK1 , r 2 =0.46) with a previously reported signal; two associations were not in LD (r 2 &lt;0.1) with a known locus. The first locus (rs73022249, p=1.2 x 10 -8 , MAF=16%), an intergenic variant upstream of telomerase reverse transcriptase ( TERT ), is common only in African ancestry populations (~36%, 1% in Europeans) and was significant only in JHS (p=4.7 x 10 -9 , p=0.91 FHS, absent OOA). This variant and an LD proxy (rs10052815) overlap a GATA1 binding site in erythroblasts, the sister lineage of platelet producing megakaryocytes. An intronic TERT variant (rs2736100) 17 kb away (r 2 =0.025, D’=0.418 in included TOPMed cohorts) has been associated with PLT in Europeans, as well as with red blood cell traits, telomere length, pulmonary fibrosis, and multiple cancer subtypes. The second locus (rs73437176, p=1.5 x 10 -8 , MAF=12%), an intronic variant in progestin and adipoQ receptor family member V ( PAQR5 ), is also more frequent in African ancestry populations (~25%, 4% in Europeans) and significant only in JHS (p=2.5 x 10 -8 , FHS p=0.47, OOA p=0.12); the nearest previously identified signal is ~4 Mb away and not in significant LD (D’&lt;0.01). Seven known platelet loci were identified for MPV ( DNM3 , ARHGEF3 , CCDC71L , JMJD1C , RHOF , TAOK1 , TPM4 ), with either the same lead variant as previously reported or a variant in close LD (r 2 &gt;0.75). Aggregated sliding window tests did not identify any additional signals. These results show suggestive evidence for association with PLT for two novel variants common only in African ancestry populations, but larger sample sizes of diverse ancestry will be necessary for replication and further discovery of novel loci.

Interpreting Non-Amish Perceptions of the Old Order Amish Using Cultural Relativism and Human Rights Frameworks

Interpreting Non-Amish Perceptions of the Old Order Amish Using Cultural Relativism and Human Rights Frameworks

Seasonality of blood neopterin levels in the Old Order Amish.

Seasonal changes in non-human animals and seasonal affective disorder (SAD) in humans are associated with immune activation in winter relative to summer. We intended to measure seasonal variation in neopterin, a marker of cellular immunity, and its interactions with gender and seasonality of mood. We studied 320 Amish from Lancaster, PA, USA (men = 128; 40%) with an average age [Standard deviation (SD)] of 56.7 (13.9) years. Blood neopterin level was measured with enzyme-linked immunosorbent assay (ELISA). Seasonality was measured with Seasonal Pattern Assessment Questionnaire (SPAQ). Statistical analysis included analysis of covariance (ANCOVAs) and multivariate linear regression. We also investigated interactions of seasonal differences in neopterin with gender, seasonality scores and estimation of SAD diagnosis. We found a significantly higher neopterin level in winter than in summer (p = 0.006). There were no significant gender or seasonality interactions. Our study confirmed the hypothesized higher neopterin level in winter. A cross sectional design was our major limitation. If this finding will be replicated by longitudinal studies in multiple groups, neopterin could be used to monitor immune status across seasons in demographically diverse samples, even if heterogeneous in gender distribution, and degree of seasonality of mood.

Sleep onset insomnia, daytime sleepiness and sleep duration in relationship to Toxoplasma gondii IgG seropositivity and serointensity.

Toxoplasma gondii (T. gondii) infects central nervous tissue and is kept in relative dormancy by a healthy immune system. Sleep disturbances have been found to precipitate mental illness, suicidal behavior and car accidents, which have been previously linked to T. gondii as well. We speculated that if sleep disruption, particularly insomnia, would mediate, at least partly, the link between T. gondii infection and related behavioral dysregulation, then we would be able to identify significant associations between sleep disruption and T. gondii. The mechanisms for such an association may involve dopamine (DA) production by T. gondii, or collateral effects of immune activation necessary to keep T. gondii in check. Sleep questionnaires from 2031 Old Order Amish were analyzed in relationship to T. gondii-IgG antibodies measured by enzyme-linked immunosorbent assay (ELISA). Toxoplasma gondii seropositivity and serointensity were not associated with any of the sleep latency variables or Epworth Sleepiness Scale (ESS). A secondary analysis identified, after adjustment for age group, a statistical trend toward shorter sleep duration in seropositive men (p = 0.07). In conclusion, it is unlikely that sleep disruption mediates links between T. gondii and mental illness or behavioral dysregulation. Trending gender differences in associations between T. gondii and shorter sleep need further investigation.

Positive association between Toxoplasma gondii IgG serointensity and current dysphoria/hopelessness scores in the Old Order Amish: a preliminary study.

Toxoplasma gondii (T. gondii) IgG seropositivity and serointensity have been previously associated with suicidal self-directed violence (SSDV). Although associations with unipolar depression have also been investigated, the results have been inconsistent, possibly as a consequence of high heterogeneity. We have now studied this association in a more homogeneous population, [that is (i.e.) Old Order Amish (OOA)] with previously reported high T. gondii seroprevalence. In 306 OOA with a mean age of 46.1 ± 16.7 years, including 191 (62.4%) women in the Amish Wellness Study, we obtained both T. gondii IgG titers (by enzyme-linked immunosorbent assay [ELISA]), and depression screening questionnaires (Patient Health Questionnaire [PHQ-9] [n = 280] and PHQ-2 [n = 26]). Associations between T. gondii IgG and dysphoria/hopelessness and anhedonia scores on depression screening questionnaires were analyzed using multivariable linear methods with adjustment for age and sex. Serointensity was associated with both current dysphoria/hopelessness (p = 0.045) and current combined anhedonia and dysphoria/hopelessness (p = 0.043), while associations with simple anhedonia and past/lifelong (rather than current) phenotypes were not significant. These results indicate the need for larger longitudinal studies to corroborate the association between dysphoria/hopelessness and T. gondii IgG-titers. Current hopelessness is a known risk factor for SSDV which responds particularly well to cognitive behavioral therapy, and may be a focused treatment target for T. gondii-positive individuals at high-risk for SSDV.

Abstract 21310: ApoCIII RX19 Mutation Lowers the Production of ApoCIII and Increases the Clearance of VLDL-TG

The APOCIII R19X mutation (rs76353203), has been found in ~5% of the Lancaster Old Order Amish. It is a C -&gt;T substitution at the 55th nucleotide in human aolipoprotein C-III mRNA that prevents translation of mature apoC-III protein. The mutation decreases apoC-III levels by ~50% and confers favorable lipid profiles and less coronary artery calcification, providing evidence that ApoC-III deficiency is cardioprotective. This conclusion is supported by recent data in Danish and U.S. cohorts showing a 40% reduction in myocardial infarction rates in people with this mutation. The mechanisms whereby apoc-III deficiency exerts its effects are not fully understood. The aim of this study was to describe the fractional clearance rates (FCR) and production rates (PR) of apoB containing lipoproteins, apoCIII, and very low density lipoprotein (VLDL) tryglyceride (TG) in five Amish sibling pairs (CT vs. CC) discordant for the APOC3 R19X mutation and matched for age and sex. Methods: Sib pairs completed a 24-hr stable isotope kinetic study using leucine and glycerol. Blood samples were collected over 24-hr period. We measured plasma lipids and apoCIII by Elisa. We determined the FCR and PR of VLDL apoB, apoCIII, and VLDL-TG. Results: The CT subjects had a 38% decrease in baseline TG levels compared to CC. ApoCIII levels were reduced by 42% in the CT vs. CC. We observed reductions in VLDL-Chol (49%), IDL-Chol (52%), VLDL-TG(44%), and IDL-TG(39%) in the CT when compared to CC. The changes in plasma levels were due to an increase in the VLDL apoB FCR (p = 0.001) without significant changes in production. VLDL-TG FCR increased by 76% in the CT when compared to CC (p=0.03). We observed no differences in post-heparin plasma levels of hepatic and lipoprotein lipase activity in the CT vs. CC. The plasma changes in apoCIII were due to and increased FCR in the CT compared to CC. Conclusions: Reductions in plasma TG levels in heterozygous apoC-III deficient individuals is due to enhanced FCRs of VLDL-apoB and VLDL-TG.

Novel polymorphisms associated with hyperalphalipoproteinemia and apparent cardioprotection

Hyperalphalipoproteinemia (HALP) is inversely correlated with coronary heart disease (CHD) although genetic variants associated with high serum levels of high-density lipoprotein cholesterol (HDL-C) have not been shown to be cardioprotective. The objective of the study was to uncover novel genetic variants associated with HALP and possibly with reduced risk of CHD. Exome sequencing data, HDL-C, and triglyceride levels were analyzed in 1645 subjects. They included the University of Maryland outpatients with high HDL-C (n=12), Cardiovascular Health Study (n=210), Jackson Heart Study (n=402), Multi-Ethnic Study of Atherosclerosis (n=404), Framingham Heart Study (n=463), and Old Order Amish (n=154). Novel nonsynonymous single-nucleotide polymorphisms (nsSNPs) were identified in men and women with primary HALP (mean HDL-C, 145 ± 30mg/dL). Using PolyPhen-2 and Combined Annotation Dependent Depletion to estimate the predictive effect of each nsSNP on the gene product, rare, deleterious polymorphisms in UGT1A3, PLLP, PLEKHH1, ANK2, DIS3L, ACACB, and LRP4 were identified in 16 subjects with HALP but not in any tested subject with low HDL-C (<40mg/dL). In addition, a single novel polymorphism, rs376849274, was found in OSBPL1A. The majority of these candidate genes have been implicated in fat and lipid metabolism, and none of these subjects has a history of CHD despite 75% of subjects having risk factors for CHD. Overall, the probability of finding these nsSNPs in a non-high HDL-C population ranges from 1×10-17 to 1×10-25. Novel functional polymorphisms in 8 candidate genes are associated with HALP in the absence of CHD. Future study is required to examine the extent to which these genes may affect HDL function and serve as potential therapeutic targets for CHD risk reduction.

Lipid Metabolism, Abdominal Adiposity, and Cerebral Health in the Amish.

ObjectiveTo assess the association between peripheral lipid/fat profiles and cerebral grey matter (GM) and white matter (WM) in healthy, Old Order Amish (OOA).MethodsBlood lipids, abdominal adiposity, liver lipid contents and cerebral microstructure were assessed in OOA (N=64, 31 Males/33 Females, ages 18–77). Orthogonal factors were extracted from lipid and imaging adiposity measures. GM assessment used the Human Connectome Project protocol to measure whole-brain average cortical thickness. Diffusion weighted imaging derived WM fractional anisotropy and kurtosis anisotropy measurements.ResultsLipid/fat measures were captured by three orthogonal factors explaining 80% of the variance. Factor 1 loaded on cholesterol/LDL-C; Factor 2 on triglyceride/liver measurements; Factor 3 on abdominal fat measurements. A two-stage regression including age/sex (1st stage) and the three factors (2nd stage) examined the peripheral lipid/fat effects. Factors 2 and 3 significantly contributed to WM measures after Bonferroni corrections (p<0.007). No factor significantly contributed to GM. Blood pressure inclusion did not meaningfully alter the lipid/fat-WM relationship.ConclusionsPeripheral lipid/fat indicators significantly and negatively associated with cerebral WM rather than GM, independent of age and blood pressure. Dissecting the fat/lipid components contributing to different brain imaging parameters may open a new understanding of the body-brain connection through lipid metabolism.

Functional analysis of PCSK2 coding variants: A founder effect in the Old Order Amish population.

In humans, noncoding variants of PCSK2, the gene encoding prohormone convertase 2 (PC2), have been previously associated with risk for and age of onset of type 2 diabetes (T2D). The aims of this study were to identify coding variants in PCSK2; to determine their possible association with glucose handling; and to determine functional outcomes for coding variants in biochemical studies. Exome-wide genotyping was performed on 1725 Old Order Amish (OOA) subjects. PCSK2 coding variants were tested for association with diabetes-related phenotypes. In vitro analyses using transfected human PC2-encoding constructs were performed to determine the impact of each mutation on PC2 activity. We identified 10 rare missense coding variants in PCSK2 in various genomic databases. R430W (rs200711626) is greatly enriched in the OOA population (MAF 4.3%). This variant is almost twice as common (MAF 7.4%) in OOA individuals with T2D as in OOA individuals with normal or with normal/impaired glucose tolerance (MAF 3.9% and 2.9%, respectively; p=0.25 and p=0.10). In vitro experiments revealed a broadening of the pH optimum for the R430W variant, which may result in increased activity against PCSK2 substrates. Although the association of the R430W variation with T2D in the OOA population did not reach significance, based upon the broadened pH profile of R430W PC2, we speculate that the presence of this substitution may result in altered processing of PCSK2 substrates, ultimately leading to increased conversion to diabetes.