Transmission Disequilibrium Test Research Articles

Association between variants around IRF6 and non-syndromic orofacial cleft in Western Han Chinese.

Considering limitations of previous studies and differences across populations and subtypes, this study aimed to identify new potential SNPs around IRF6 associated with non-syndromic orofacial cleft (NSOC) in Western Han Chinese. We recruited 376NSOC case-parent trios, including 125 non-syndromic cleft lip only (NSCLO) trios, 151 non-syndromic cleft lip and palate (NSCLP) trios, and 100 non-syndromic cleft palate only (NSCPO) trios. Twenty-two single-nucleotide polymorphisms (SNPs) were genotyped using MassARRAY method.Hardy-Weinberg equilibrium test, allelic transmission disequilibrium test (TDT) analysis, sliding-window haplotype TDT analysis, and tests for parent-of-origin effect were performed using the PLINK software.Pairwise linkage disequilibrium (LD) was computed using the Haploview program. In TDT analysis, allele A at rs17015217 (p=0.00011, OR=0.61 and 95% CI: 0.47-0.78) and allele T at rs12080691 (p=0.00011, OR=0.61 and 95% CI: 0.47-0.78) were under-transmitted among NSCLO trios but over-transmitted among NSCPO trios. Haplotypes showing evidence of under-transmission in NSCLO trios were over-transmitted in NSCPO trios. In tests for parent-of-origin effects, T allele at rs12080691 presented paternal under-transmission among NSCLO trios but over-transmission among NSCPO trios. Allele A at rs17015217 and allele T at rs12080691 are associated with NSCLO and NSCPO with potential to have opposite effects on two subtypes in this sample from Western Han Chinese.

Linkage and association of PAX7 polymorphisms (rs742071, rs766325, and rs4920520) with the risk of non-syndromic cleft lip with/without cleft palate: A systematic review and meta-analysis

The studies have reported several additional non-syndromic cleft lip/palate (NSCL/P) susceptibility loci. This systematic review and meta-analysis aimed to evaluate the linkage and association of PAX7 polymorphisms with the NSCL/P risk. A comprehensive search was conducted in the PubMed, Cochrane Library, Web of Science, and Scopus databases until May 15, 2021. The association between PAX7 polymorphisms and NSCL/P susceptibility was analyzed by calculation of the odds ratios (ORs) and 95% confidence intervals (CIs), and the linkage was assessed by the allelic transmission disequilibrium test. Some articles included more than one study (reporting more than one polymorphism). Therefore, nine articles including 13 studies were entered into the meta-analysis. With regard to the association of rs742071 polymorphism with the risk of NSCL/P, the pooled OR was 1.33 for the allelic (P < 0.0001), 1.98 for the homozygous (P = 0.0007), 1.41 for the heterozygous (P = 0.0267), and 1.15 for the dominant (P = 0.2542) models. For the association of PAX7 rs766325 and rs4920520 polymorphisms with the risk of NSCL/P, the pooled OR was 0.96 (P = 0.5571) and 1.14 (P = 0.1020), respectively. In addition, the pooled allelic transmission disequilibrium test for s742071 and rs766325 polymorphisms did not show any allelic linkage between these polymorphisms and susceptibility to NSCL/P. The main results of the present systematic review and meta-analysis showed an association between PAX7 rs742071 polymorphism and NSCL/P susceptibility; but there was no linkage or association with rs766325 and rs4920520 polymorphisms.

A Comprehensive Investigation on Potential Risk Factors for NSCL/P in a Rural District of Hebei Province, China.

Non syndromic cleft lip with or without palate (NSCL/P), one of the most common birth defects, is closely related to various risk factors. However, information regarding risk factors for NSCL/P in rural districts in China is very limited thus far. The objective of this study was designed to identify the potential risk factors for NSCL/P in rural districts. A comprehensive retrospective investigation including 435 NSCL/P patients and 402 healthy children was carried out in Hebei Province, China. Multiple logistic regression analysis and transmission disequilibrium test (TDT) were respectively used to identify non-genetic and genetic risk factors for NSCL/P, and then PLINK was used to explore the relationship between non-genetic and genetic risk factors. The results showed that maternal periconceptional exposure to pesticides and herbicides, as well as low parental education level were involved in the increased risk of NSCL/P, whereas maternal folic acid and multivitamin supplementation use during preconception period were associated with the reduced risk of NSCL/P. TDT analysis identified 2 single nucleotide polymorphisms (SNPs) (rs7078160 and rs4752028) in VAX1 and one SNP (rs17563) in BMP4 as the genetic risk factors for NSCL/P. Further analysis showed that the genetic risk factors were closely related with the negative non-genetic risk factors. Our study identified the potential risk factors for NSCL/P in rural districts, thus providing a theoretical basis for the prevention of NSCL/P occurrence.

LGR4 Gene Polymorphisms Are Associated With Bone and Obesity Phenotypes in Chinese Female Nuclear Families.

ObjectiveThe current study was conducted to determine whether peak bone mineral density (BMD) and obesity phenotypes are associated with certain LGR4 gene polymorphisms found in Chinese nuclear families with female children.MethodsA total of 22 single nucleotide polymorphisms (SNPs) located in and around the LGR4 gene were identified in 1,300 subjects who were members of 390 Chinese nuclear families with female children. Then, BMD readings of the femoral neck, total hip, and lumbar spine as well as measurements of the total lean mass (TLM), total fat mass (TFM), and trunk fat mass were obtained via dual-energy X-ray absorptiometry. The quantitative transmission disequilibrium test was used to analyze the associations between specific SNPs and LGR4 haplotypes and peak BMD as well as between LGR4 haplotypes and TLM, percent lean mass, TFM, percent fat mass, trunk fat mass, and body mass index (BMI).ResultsHere, rs7936621 was significantly associated with the BMD values for the total hip and lumbar spine, while rs10835171 and rs6484295 were associated with the trunk fat mass and BMI, respectively. Regarding the haplotypes, we found significant associations between GAA in block 2 and trunk fat mass and BMI, between AGCGT in block 3 and total hip BMD, between TGCTCC in block 5 and femoral neck BMD, and between TACTTC in block 5 and both lumbar spine and femoral neck BMD (all P-values < 0.05).ConclusionGenetic variations of the LGR4 gene are related to peak BMD, BMI, and trunk fat mass.

Genome-wide family-based study in torus palatinus affected individuals

ObjectiveTori or exostoses are bony growths that appear in different oral regions. Torus palatinus, more specifically, develop in the palate midline and can impair proper word pronunciation and hinder the fabrication and use of dentures. Even though a multifactorial inheritance model has been suggested for torus palatinus appearance, precise genetic factors involved in its etiology remain unclear. Hence, in this study we aimed to identify variants across the genome of individuals from 46 Filipino families that associate with torus palatinus. DesignAll families were composed of fishermen or landless rural dwellers who provided blood samples for DNA extraction and genotyping. A total of 3519 single nucleotide polymorphisms (SNPs) were analyzed through a transmission disequilibrium test in individuals affected by torus palatinus and their unaffected family members. ResultsFourteen SNPs showed trends for associations to the level of p < .005 threshold and several others were nominally (p < .05) associated with torus palatinus. We highlight SNP rs6582285, which is located in the CAPS2 gene, being the C allele less transmitted than the T allele in our sample. The C allele of CAPS2 rs6582285 protects from having torus palatinus whereas the other associations found were linked to an increased risk of developing the condition. ConclusionsTrends for associations were identified for several markers across the genome, supporting the hypothesis that torus palatinus has a multifactorial mode of inheritance. We hope that our study contributes to a better understanding of torus palatinus etiology and helps guide future research in examining genes for this often-overlooked condition in different populations.

The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3h after infection of mouse middle ear.

Myopia in African Americans Is Significantly Linked to Chromosome 7p15.2-14.2.

PurposeThe purpose of this study was to perform genetic linkage analysis and association analysis on exome genotyping from highly aggregated African American families with nonpathogenic myopia. African Americans are a particularly understudied population with respect to myopia.MethodsOne hundred six African American families from the Philadelphia area with a family history of myopia were genotyped using an Illumina ExomePlus array and merged with previous microsatellite data. Myopia was initially measured in mean spherical equivalent (MSE) and converted to a binary phenotype where individuals were identified as affected, unaffected, or unknown. Parametric linkage analysis was performed on both individual variants (single-nucleotide polymorphisms [SNPs] and microsatellites) as well as gene-based markers. Family-based association analysis and transmission disequilibrium test (TDT) analysis modified for rare variants was also performed.ResultsGenetic linkage analysis identified 2 genomewide significant variants at 7p15.2 and 7p14.2 (in the intergenic region between MIR148A and NFE2L3 and in the noncoding RNA LOC401324) and 2 genomewide significant genes (CRHR2 and AVL9) both at 7p14.3. No genomewide results were found in the association analyses.ConclusionsThis study identified a significant linkage peak in African American families for myopia at 7p15.2 to 7p14.2, the first potential risk locus for myopia in African Americans. Interesting candidate genes are located in the region, including PDE1C, which is highly expressed in the eyes, and known to be involved in retinal development. Further identification of the causal variants at this linkage peak will help elucidate the genetics of myopia in this understudied population.

Abstract 874: Predicted leukocyte telomere length and risk of germ cell tumors

Abstract Genetically predicted leukocyte telomere length (LTL) has been evaluated in a number of studies of childhood and adult cancer, with evidence that longer predicted LTL is associated with increased cancer risk. We use Mendelian Randomization (MR) to test whether genetically predicted LTL is associated with germ cell tumors (GCT) in pediatric, adolescent, and adult cases using six SNPs that are validated predictors of LTL. A seventh SNP in the validated genetic predictor of LTL, rs2736100, a variant on TERT, is associated with adult testicular GCT (TGCT); therefore, in our overall analysis, we removed this SNP to avoid violating MR assumptions. Pediatric GCT samples were obtained from a Children's Oncology Group study and state biobank programs in Michigan and California. Genotyping array data were generated using the Illumina HumanCoreExome Beadchip. For each SNP, we estimated main effects for pediatric GCT within 610 complete case-parent trios using the transmission disequilibrium test. We did the same in an independent case-control dataset of 803 pediatric cases and 1,022 controls stratified by ancestry using multivariable logistic regression adjusted for sex, ancestry-specific principal components (PCs), and study site. Effect estimates were meta-analyzed and used to perform the MR analyses. In a separate sample of 396 cases and 1,589 matched controls from the UK Biobank, genotyping array data was used to evaluate the association between a polygenic risk score capturing genetically predicted LTL and TGCT in men &amp;lt;50 years old using multivariable logistic regression adjusted for global ancestry PCs (PC1-10). We observed a positive association between LTL and overall risk of GCT in both the pediatric cases (OR [95% CI] = 2.52 [1.19, 5.34] per kb of LTL; p=0.016) and the adult TGCT cases (OR [95% CI] = 1.94 [0.60, 6.28] per kb of LTL; p=0.266). Subgroup analyses in our pediatric GCT cases show consistent findings of longer LTL and risk across subgroups, with the exception of teratomas. Additionally, we evaluated the individual SNP effect of TERT/rs2736100-A and observed a significant association in pediatric GCT (OR [95% CI] = 1.21 [1.09, 1.35]; p =0.0003) which is driven by male cases diagnosed in adolescence (age at dx 11-19 years). A significant association was also observed between TERT/rs2736100-A and adult TGCT (OR [95% CI] = 1.25 [1.07, 1.46]; p=0.005). Similar to analyses of other childhood and adult cancers, our results indicate that longer telomere length is associated with an increased risk of GCT in pediatric, adolescent, and adult GCT. We also observed a strong association between pediatric GCT and TERT and confirmed previous findings with adult TGCT. These results indicate further analyses of variants in genes relevant for telomere function and maintenance are warranted. Citation Format: Shannon S. Cigan, Ava C. Kelley, John J. Meredith, Erica K. Langer, Anthony J. Hooten, John A. Lane, Benjamin R. Cole, Nathan Pankratz, Jenny Poynter. Predicted leukocyte telomere length and risk of germ cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 874.

Estimating genetic nurture with summary statistics of multigenerational genome-wide association studies

Marginal effect estimates in genome-wide association studies (GWAS) are mixtures of direct and indirect genetic effects. Existing methods to dissect these effects require family-based, individual-level genetic, and phenotypic data with large samples, which is difficult to obtain in practice. Here, we propose a statistical framework to estimate direct and indirect genetic effects using summary statistics from GWAS conducted on own and offspring phenotypes. Applied to birth weight, our method showed nearly identical results with those obtained using individual-level data. We also decomposed direct and indirect genetic effects of educational attainment (EA), which showed distinct patterns of genetic correlations with 45 complex traits. The known genetic correlations between EA and higher height, lower body mass index, less-active smoking behavior, and better health outcomes were mostly explained by the indirect genetic component of EA. In contrast, the consistently identified genetic correlation of autism spectrum disorder (ASD) with higher EA resides in the direct genetic component. A polygenic transmission disequilibrium test showed a significant overtransmission of the direct component of EA from healthy parents to ASD probands. Taken together, we demonstrate that traditional GWAS approaches, in conjunction with offspring phenotypic data collection in existing cohorts, could greatly benefit studies on genetic nurture and shed important light on the interpretation of genetic associations for human complex traits.

Association of Novel Locus With Rheumatic Heart Disease in Black African Individuals

Rheumatic heart disease (RHD), a sequela of rheumatic fever characterized by permanent heart valve damage, is the leading cause of cardiac surgery in Africa. However, its pathophysiologic characteristics and genetics are poorly understood. Understanding genetic susceptibility may aid in prevention, control, and interventions to eliminate RHD. To identify common genetic loci associated with RHD susceptibility in Black African individuals. This multicenter case-control genome-wide association study (GWAS), the Genetics of Rheumatic Heart Disease, examined more than 7 million genotyped and imputed single-nucleotide variations. The 4809 GWAS participants and 116 independent trio families were enrolled from 8 African countries between December 31, 2012, and March 31, 2018. All GWAS participants and trio probands were screened by use of echocardiography. Data analyses took place from May 15, 2017, until March 14, 2021. Genetic associations with RHD. This study included 4809 African participants (2548 RHD cases and 2261 controls; 3301 women [69%]; mean [SD] age, 36.5 [16.3] years). The GWAS identified a single RHD risk locus, 11q24.1 (rs1219406 [odds ratio, 1.65; 95% CI, 1.48-1.82; P = 4.36 × 10-8]), which reached genome-wide significance in Black African individuals. Our meta-analysis of Black (n = 3179) and admixed (n = 1055) African individuals revealed several suggestive loci. The study also replicated a previously reported association in Pacific Islander individuals (rs11846409) at the immunoglobulin heavy chain locus, in the meta-analysis of Black and admixed African individuals (odds ratio, 1.16; 95% CI, 1.06-1.27; P = 1.19 × 10-3). The HLA (rs9272622) associations reported in Aboriginal Australian individuals could not be replicated. In support of the known polygenic architecture for RHD, overtransmission of a polygenic risk score from unaffected parents to affected probands was observed (polygenic transmission disequilibrium testing mean [SE], 0.27 [0.16] SDs; P = .04996), and the chip-based heritability was estimated to be high at 0.49 (SE = 0.12; P = 3.28 × 10-5) in Black African individuals. This study revealed a novel candidate susceptibility locus exclusive to Black African individuals and an important heritable component to RHD susceptibility in African individuals.

The rs1634330 Polymorphisms in the SOST Gene Are Associated with Body Composition in Chinese Nuclear Families with Male Offspring.

Objective The purpose of this study was to explore the effect of the SOST gene polymorphisms on body composition in Chinese nuclear families with male offspring. Methods 1,016 individuals were recruited from 335 Chinese nuclear families with male offspring. The nuclear families consist of at least one male offspring aged 18 to 44. We genotyped the 10 tagged single-nucleotide polymorphisms (SNPs) in SOST gene (rs7220711, rs865429, rs851057, rs1708635, rs2023794, rs1234612, rs74252774, rs1634330, rs851058, and rs1513670) in all the above people. We used dual-energy X-ray absorptiometry to measure the composition of the human body. The quantitative transmission disequilibrium test (QTDT) was used to analyze the associations of the SNPs with the body composition. Results QTDT analysis showed that rs1634330 was significantly associated with trunk LM (P < 0.05). However, haplotypes were not found to be significantly associated with the body composition in the within-family association. The 1000 permutations were consistent with these within-family association results. Conclusions Our results showed that the genetic variation in the SOST gene may contribute to variations in the body composition of Chinese male offspring.

Genetic variations at 10q26 regions near FGFR2 gene and its association with non-syndromic cleft lip with or without cleft palate

ObjectivesIn our study, we focussed on three SNPs in the non-coding regions near FGFR2 gene, as studies on non-coding variants in the genome are the novel trends to identify the susceptible loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). FGFR2 gene is selected as a candidate gene based on knock out animal models and the role played in syndromic forms of clefting. FGFR2 gene also plays an important role in FGF signaling pathway during craniofacial development. MethodsIn the present study 148 case-parent triads were assessed for three SNPs rs10749408, rs11199874 and rs10788165 near FGFR2 gene by using TaqMan allelic discrimination method. Transmission disequilibrium test (TDT) was used to find the allelic association. Linkage disequilibrium (LD) between the markers was analysed using Haploview program 4.2. Haplotype transmission effects were estimated using FAMHAP package. The possible parent-of-origin effects were assessed by likelihood based approach. ResultsTDT analysis of three SNPs failed to show significant transmission disortion from heterozygous parents to the affected child and are not associated with NSCL/P. Linkage disequilibrium analysis showed strong LD between rs11199874 and rs10788165 SNPs. In the haplotype TDT analysis, GG haplotype of rs11199874-rs10788165 showed significant undertransmission to affected child. No significant parent-of-origin effects were observed. ConclusionThe present study on noncoding variants near FGFR2 gene is not associated with NSCL/P. As the numbers of triads included in the study are less, further studies are needed including large sample size to find association with NSCL/P.

Two tSNPs in BRIP1 are associated with breast cancer during TDT analysis.

ObjectivesThis study aimed to investigate and confirm the association between 15 single nucleotide polymorphisms of four susceptibility genes (NBS1, TP53, PTEN, and BRIP1) and the susceptibility of breast cancer.MethodsThe genome DNA was extracted from peripheral blood and tumor tissues from one hundred and seventeen core families. 15 SNPs were detected by PCR. The transmission disequilibrium test (TDT) and the Hardy–Weinberg equilibrium (HWE) are used to verify the association between these SNPs and breast cancer. Further correlation between SNPs and certain pathological features of the tumor, including tumor size, location of lymph nodes, pathologic classification, and the stage and subtype of breast cancer, are analyzed by the chi‐square test and logistic regression analysis.ResultsBased on TDTs, two SNPs of rs7220719 and rs11871753 in BRIP1 showed a significant association with breast cancer, while the other 13 selected SNPs did not. However, further statistical analysis demonstrated no obvious differentiation in the clinical characteristics of breast cancer between 37 patients with rs7220719 and 80 patients with wild types. Similar results were also found for rs11871753.ConclusionsThe data provided the evidence for the association between two SNPs of BRIP1 and breast cancer, but did not affect certain clinical phenotypes.

Transmission Based Conditional Logistic Model for Testing Main and Interaction Effects

Transmission disequilibrium test (TDT) is a popular family based genetic association method. Under multiplicative assumption, a conditional logistic regression for matched pair, affected offspring with allele transmitted from parents and pseudo-offspring (control) with allele non-transmitted from parents, was built to detect the main effects of genes and gene-covariate interactions. When there exist genotype uncertainties, expectation-maximization (EM) algorithm was adopted to estimate the coefficients. The transmission model was applied to detect the association between M235T polymorphism in AGT gene and essential hypertension (ESH). Most of parents are not available in the 126 families from HongKong Chinese population. The results showed M235T is associated with hypertension and there is interaction between M235T and the case’s sex. The allele T is higher risk for male than female.

Association of MICA and HLA‐B alleles with leprosy in two endemic populations in Brazil

Leprosy is a prevalent disease in Brazil, which ranks as the country with the second highest number of cases in the world. The disease manifests in a spectrum of forms, and genetic differences in the host can help to elucidate the immunopathogenesis. For a better understanding of MICA association with leprosy, we performed a case-control and a family-based study in two endemic populations in Brazil. MICA and HLA-B alleles were evaluated in 409 leprosy patients and in 419 healthy contacts by PCR-SSOP-Luminex-based technology. In the familial study, analysis of 46 families was completed by direct sequencing of all exons and 3'/5'untranslated regions, using the Ilumina MiSeq platform. All data were collected between 2006 and 2009. Statistical analysis was performed using the Chi-square or Fisher's exact test together with a multivariate analysis. Family-based association was assessed by transmission disequilibrium test (TDT) software FBAT 2.0.4. We found associations between the haplotype MICA*002-HLA-B*35 with leprosy in both the per se and the multibacillary (MB) forms when compared to healthy contacts. The MICA allele *008 was associated with the clinical forms of paucibacillary (PB). Additionally, MICA*029 was associated with the clinical forms of MB. The association of MICA*029 allele (MICA-A4 variant) with the susceptibility to the MB form suggests this variant for the transmembrane domain of the MICA molecule may be a risk factor for leprosy. Two MICA and nine HLA-B variants were found associated with leprosy per se in the Colônia do Prata population. Linkage disequilibrium analysis revealed perfect linkage disequilibrium (LD) between HLA-B markers rs2596498 and rs2507992, and high LD (R2 =.92) between these and the marker rs2442718. This familial study demonstrates that MICA association signals are not independent from those observed for HLA-B. Our findings contribute the knowledge pool of the immunogenetics of Hansen's disease and reveals a new association of the MICA*029 allele.

Exploring parent-of-origin effects for non-syndromic cleft lip with or without cleft palate on PTCH1, PTCH2, SHH, SMO genes in Chinese case-parent trios

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect, affecting 1.4 per 1 000 live births, and multiple genetic and environmental risk factors influencing its risk. All the known genetic risk factors accounted for a small proportion of the heritability. Several authors have suggested parent-of-origin effects (PoO) may play an important role in the etiology of this complex and heterogeneous malformation. To clarify the genetic association between PTCH1, PTCH2, SHH and SMO in hedgehog (HH) pathway and NSCL/P, as well as testing for potential PoO effects in Chinese case-parent trios. We tested for transmission disequilibrium tests (TDT) and PoO effects using 83 common single nucleotide polymorphic (SNP) markers of HH pathway genes from 806 NSCL/P case-parent trios. These trios were drawn from an international consortium established for a genome-wide association studies (GWAS) of non-syndromic oral clefts of multiple ethnicities. DNA samples were collected from each trio. Single marker and haplotype based analysis were performed both in TDT tests and PoO effects. SNPs were excluded if they (ⅰ) had a call rate of &#60; 95%, (ⅱ) had a minor allele frequency (MAF) of &#60; 0.05, (ⅲ) had Mendelian errors over all trios of >5%, (ⅳ) had a genotype distribution in the parents that deviated from the Hardy-Weinberg equilibrium (HWE) (<i>P</i> &#60; 0.000 1). The process was done using Plink (version 1.07, <a href="http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml" target="_blank">http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml</a>). TDT test was performed in Plink v1.07. A log-linear model was used to explore PoO effects using Haplin v6.2.1 as implemented in R package v3.4.2. Significance level was assessed using the Bonferroni correction. A total of 18 SNPs were dropped due to low MAF, thus leaving 65 SNPs available for the analysis. Thus the Bonferroni threshold was 7.7×10-4 (0.05/65). Nominal significant association with NSCL/P was found at a SNP (rs4448343 in PTCH1, P=0.023) and six haplotypes (rs10512249-rs4448343, rs1461208-rs7786445, rs10512249-rs4448343, rs16909865-rs10512249-rs4448343, rs1461208-rs7786445-rs12698335, and rs288756-rs288758-rs1151790, P < 0.05). A total of six haplotypes (rs288765-rs1233563, rs12537550-rs11765352, rs872723-rs288765-rs1233563, rs288765-rs1233563-rs288756, rs6459952-rs12537550-rs11765352, and rs12537550-rs11765352-rs6971211) showed PoO effect (P < 0.05). None of the results remained significant after the Bonferroni correction (P>7.7×10-4). Neither significant association between SNPs within HH pathway and the risk of NSCL/P nor PoO effects was seen in this study.

Causal inference in genetic trio studies

We introduce a method to draw causal inferences-inferences immune to all possible confounding-from genetic data that include parents and offspring. Causal conclusions are possible with these data because the natural randomness in meiosis can be viewed as a high-dimensional randomized experiment. We make this observation actionable by developing a conditional independence test that identifies regions of the genome containing distinct causal variants. The proposed digital twin test compares an observed offspring to carefully constructed synthetic offspring from the same parents to determine statistical significance, and it can leverage any black-box multivariate model and additional nontrio genetic data to increase power. Crucially, our inferences are based only on a well-established mathematical model of recombination and make no assumptions about the relationship between the genotypes and phenotypes. We compare our method to the widely used transmission disequilibrium test and demonstrate enhanced power and localization.

Multi-omic studies on missense PLG variants in families with otitis media

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

Candidate-gene association analysis for a continuous phenotype with a spike at zero using parent-offspring trios

In this paper, we propose the class of generalized additive models for location, scale and shape in a test for the association of genetic markers with non-normally distributed phenotypes comprising a spike at zero. The resulting statistical test is a generalization of the quantitative transmission disequilibrium test with mating type indicator, which was originally designed for normally distributed quantitative traits and parent-offspring data. As a motivational example, we consider coronary artery calcification (CAC), which can accurately be identified by electron beam tomography. In the investigated regions, individuals will have a continuous measure of the extent of calcium found or they will be calcium-free. Hence, the resulting distribution is a mixed discrete-continuous distribution with spike at zero. We carry out parent-offspring simulations motivated by such CAC measurement values in a screening population to study statistical properties of the proposed test for genetic association. Furthermore, we apply the approach to data of the Genetic Analysis Workshop 16 that are based on real genotype and family data of the Framingham Heart Study, and test the association of selected genetic markers with simulated coronary artery calcification.

Association of Genetic Variation in the 3'UTR of LHX6, IMMP2L, and AADAC With Tourette Syndrome.

Background: Tourette Syndrome (TS) is a neurodevelopmental disorder that presents with motor and vocal tics early in childhood. The aim of this study was to investigate genetic variants in the 3' untranslated region (3'UTR) of TS candidate genes with a putative link to microRNA (miRNA) mediated regulation or gene expression.Methods: We used an in silico approach to identify 32 variants in the 3'UTR of 18 candidate genes putatively changing the binding site for miRNAs. In a sample composed of TS cases and controls (n = 290), as well as TS family trios (n = 148), we performed transmission disequilibrium test (TDT) and meta-analysis.Results: We found positive association of rs3750486 in the LIM homeobox 6 (LHX6) gene (p = 0.021) and rs7795011 in the inner mitochondrial membrane peptidase subunit 2 (IMMP2L) gene (p = 0.029) with TS in our meta-analysis. The TDT showed an over-transmission of the A allele of rs1042201 in the arylacetamide deacetylase (AADAC) gene in TS patients (p = 0.029).Conclusion: This preliminary study provides further support for the involvement of LHX6, IMMP2L, and AADAC genes, as well as epigenetic mechanisms, such as altered miRNA mediated gene expression regulation in the etiology of TS.