Genome-wide association and functional genomic analyses for various hoof health traits in North American Holstein cattle.

Hoof diseases are a major welfare and economic issue in the global dairy cattle production industry, which can be minimized through improved management and breeding practices. Optimal genetic improvement of hoof health could benefit from a deep understanding of the genetic background and biological underpinning of indicators of hoof health. Therefore, the primary objectives of this study were to perform genome-wide association studies, using imputed high-density genetic markers data from North American Holstein cattle, for 8 hoof-related traits: digital dermatitis, sole ulcer, sole hemorrhage, white line lesion, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, and toe ulcer, and a hoof health index. De-regressed estimated breeding values from 25,580 Holstein animals were used as pseudo-phenotypes for the association analyses. The genomic quality control, genotype phasing, and genotype imputation were performed using the PLINK (version 1.9), Eagle (version 2.4.1), and Minimac4 software, respectively. The functional genomic analyses were performed using the GALLO R package and the DAVID platform. We identified 22, 34, 14, 22, 28, 33, 24, 43, and 15 significant markers for digital dermatitis, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, sole hemorrhage, sole ulcer, toe ulcer, white line lesion disease, and the hoof health index, respectively. The significant markers were located across all autosomes, except BTA10, BTA12, BTA20, BTA26, BTA27, and BTA28. Moreover, the genomic regions identified overlap with various previously reported quantitative trait loci for exterior, health, meat and carcass, milk, production, and reproduction traits. The enrichment analyses identified 44 significant gene ontology terms. These enriched genomic regions harbor various candidate genes previously associated with bone development, metabolism, and infectious and immunological diseases. These findings indicate that hoof health traits are highly polygenic and influenced by a wide range of biological processes.

Association between a genetic index for lameness resistance and the incidence of claw horn lesions in Holstein cows.

BACKGROUND: This study aimed to determine the association between the lameness advantage genetic index and four outcomes: sole haemorrhage (SH), sole ulcers (SU), white line lesions (WL), and lameness during mobility scoring. METHODS: We enrolled 2352 Holstein cows from four predominantly housed dairy herds in the UK. Cows were mobility scored and foot lesions recorded at four time points from before calving to late lactation. Cows were genotyped and genetic indexes were assigned to each cow following national genetic evaluations. Lameness records and genetic indexes were matched for 2107 cows. Four separate multivariable logistic regression models, which included farm and parity as covariables, were used to quantify the association between the lameness advantage index and whether animals were affected by SH, SU, WL, or lameness. RESULTS: The odds ratios (95% confidence intervals) for one-point increases in the lameness advantage index were 0.79 (0.72-0.86), 0.68 (0.59-0.78), 0.94 (0.84-1.04), and 0.82 (0.74-0.91) for SH, SU, WL, and lameness, respectively. The same trends were present when the sire's lameness advantage index was evaluated in place of the animal's own, although the strength of this association was generally weaker. CONCLUSION: The lameness advantage index is associated with SH, SU, and lameness, therefore selection on the lameness advantage index could be considered in herds aiming to reduce lameness. Where genomic testing of heifers is not conducted, sire lameness advantage index may still be effective to reduce SH and SU incidence.

Transcriptome diversity and differential expression in supporting limb laminitis.

Laminitis results in impaired tissue integrity and Inflammation of the epidermal and dermal lamellae connecting the hoof capsule to the underlying distal phalanx and causes loss-of-use, poor quality of life and euthanasia in horses. Historically, studies to better understand the etiology of laminitis by documenting changes in gene expression were hampered by the paucity of gene annotation specific to hoof tissues. Next-generation sequencing enables improvements to annotation by incorporating equine- and hoof-specific transcripts. Here we characterize the hoof lamellar tissue transcriptome of naturally occurring supporting limb laminitis (SLL) using archived lamellar tissue from Thoroughbred racehorses consisting of 13 SLL hospital cases and seven age-matched control horses. This was achieved using: 1) Applied transcriptome annotation by long-read sequencing to document transcript diversity and 2) short-read RNA sequencing to document changes in gene expression correlating to the developmental and acute stages of naturally occurring SLL. 1.99Gbp of long-read transcriptome sequencing deeply documented 5067 unique loci, while short read RNA-seq under very stringent quality filters described 66 differentially expressed loci. Functional analysis of these loci revealed alterations in cell replication and growth, stress response and leukocyte recruitment and activation pathways. Differential expression of the Ezrin and TIMP3 genes suggests they may have utility as biomarkers for laminitis disease, while NR1D1 and genes relevant to the inflammasome are promising targets for novel pharmacological treatments.

Genome-wide association study between copy number variants and hoof health traits in Holstein dairy cattle.

Genome-wide association studies based on SNP have been completed for multiple traits in dairy cattle; however, copy number variants (CNV) could add genomic information that has yet to be harnessed. The objectives of this study were to identify CNV in genotyped Holstein animals and assess their association with hoof health traits using deregressed estimated breeding values as pseudophenotypes. A total of 23,256 CNV comprising 1,645 genomic regions were identified in 5,845 animals. Fourteen genomic regions harboring structural variations, including 9 deletions and 5 duplications, were associated with at least 1 of the studied hoof health traits. This group of traits included digital dermatitis, interdigital dermatitis, heel horn erosion, sole ulcer, white line lesion, sole hemorrhage, and interdigital hyperplasia; no regions were associated with toe ulcer. Twenty candidate genes overlapped with the regions associated with these traits including SCART1, NRXN2, KIF26A, GPHN, and OR7A17. In this study, an effect on infectious hoof lesions could be attributed to the PRAME (Preferentially Expressed Antigen in Melanoma) gene. Almost all genes detected in association with noninfectious hoof lesions could be linked to known metabolic disorders. The knowledge obtained considering information of associated CNV to the traits of interest in this study could improve the accuracy of estimated breeding values. This may further increase the genetic gain for these traits in the Canadian Holstein population, thus reducing the involuntary animal losses due to lameness.

Paraneoplastic Secretion of Multiple Phosphatonins From a Deep Fibrous Histiocytoma Causing Oncogenic Osteomalacia.

CONTEXT: Literature suggests that oncogenic osteomalacia is usually caused by a benign mesenchymal tumor secreting fibroblast growth factor subtype-23 (FGF-23), but the involvement of other phosphatonins has only been scarcely reported. We have previously published a seemingly typical case of oncogenic osteomalacia. Following curative neoplasm resection, we now report unique molecular characteristics and biology of this tumor. CASE DESCRIPTION: A 25-year-old man had been diagnosed with severe oncogenic osteomalacia that gradually crippled him over 6 years. 68Ga-DOTA-TATE positron emission tomography/computed tomography scan localized the culprit tumor to his left sole, which on resection revealed a deep fibrous histiocytoma displaying a proliferation of spindle cells with storiform pattern associated with multinucleated giant cells resembling osteoclasts. Circulating FGF-23, which was elevated more than 2-fold, declined to undetectable levels 24 h after surgery. Microarray analysis revealed increased tumor gene expression of the phosphatonins FGF-23, matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein subtype 4, with elevated levels of all 3 proteins confirmed through immunoblot analysis. Differential expression of genes involved in bone formation and bone mineralization were further identified. The patient made an astonishing recovery from being wheelchair bound to fully self-ambulant 2 months postoperatively. CONCLUSION: This report describes oncogenic osteomalacia due to a deep fibrous histiocytoma, which coincidentally has been found to induce profound muscle weakness via the overexpression of 3 phosphatonins, which resolved fully upon radical resection of the tumor. Additionally, genes involved in bone formation and bone remodeling contribute to the molecular signature of oncogenic osteomalacia.

Pediatric fibromyxoid soft tissue tumor with PLAG1 fusion: A novel entity?

The classification of undifferentiated soft tissue tumors continues to evolve with the expanded application of molecular analysis in clinical practice. We report three cases of a unique soft tissue tumor in young children (5 months to 2 years old) displaying a purely fibromyxoid histology, with positive staining for desmin and CD34. In two cases, RNA sequencing detected a YWHAZ-PLAG1 gene fusion, while in the third case, a previously unreported EEF1A1-PLAG1 fusion was identified. PLAG1 fusions have been reported in several pathologic entities including pleomorphic adenoma, myoepithelial tumors of skin and soft tissue, and lipoblastoma, the latter occurring preferentially in young children. In these tumors, expression of a full length PLAG1 protein comes under the control of the constitutively active promoter of the partner gene in the fusion, and the current cases conform to that model. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1 in all three cases. Our findings raise the possibility of a novel fibromyxoid neoplasm in childhood associated with these rare PLAG1 fusion variants. The only other report of a PLAG1-YWHAZ fusion occurred in a pediatric tumor diagnosed as a "fibroblastic lipoblastoma." This finding raises the possibility of a relationship with our three cases, even though our cases lacked any fat component. Further studies with regard to a shared pathogenesis are required.

Genetics and Signaling Pathways of Laminitis.

Laminitis is a devastating disease with diverse etiologies and few, if any, effective treatments. Gene expression and hypothesis-generating genomic studies have provided a fresh look at the key molecular players at crucial timepoints in diverse experimental and naturally affected tissues. We summarize findings to date, and propose a unifying model of the laminitis disease process that includes several pathogenesis concepts shared with other diseases of epidermal and epithelial tissues. The value of these new pathways as potential therapeutic targets is exciting but will require careful future work to validate new methods and launch systematic clinical trials.

Assessment of the FAM174A 11G allele as a risk allele for equine metabolic syndrome.

An 11G nucleotide repeat in the 3' UTR of FAM174A was recently postulated as a risk allele with a dominant mode of inheritance for equine metabolic syndrome (EMS) and laminitis status in Arabian horses. The objective of this project was to evaluate this hypothesis in a large and diverse across-breed population. A total of 301 ponies, 292 Morgans, 64 Arabians, 49 Tennessee Walking Horses and 59 Quarter Horses were genotyped for six observed G repeat alleles in the FAM174A 3' UTR. Phenotype data included laminitis status, baseline insulin, glucose, non-esterified fatty acids, triglycerides, adiponectin, leptin, ACTH, insulin and glucose post oral sugar test, and two proxies for insulin resistance. The 11G allele frequencies were 18.8, 6.9, 1.8, 0.2 and 0.0% in the Arabians, Tennessee Walkers, ponies, Morgans and Quarter Horses respectively. Association analyses between FAM174A genotype and EMS phenotypes, and between allele count and EMS phenotypes, identified no statistically significant associations. When a dominant effect for the 11G allele was evaluated, a statistically significant association with adiponectin levels was identified in the ponies, and pairwise comparisons revealed that the estimated marginal means were higher in ponies with the 11G allele vs. alternative alleles (i.e. the allele had a protective effect). In conclusion, our data do not support the FAM174A 11G allele as a risk allele for EMS in our studied breeds.

Symposium review: Multiple-trait single-step genomic evaluation for hoof health.

Hoof lesions represent an important issue in modern dairy herds, with reported prevalence in different countries ranging from 40 to 70%. This high prevalence of hoof lesions has both economic and social consequences, resulting in increased labor expenses and decreasing animal production, longevity, reproduction, health, and welfare. Therefore, a key goal of dairy herds is to reduce the incidence of hoof lesions, which can be achieved both by improving management practices and through genetic selection. The Canadian dairy industry has recently released a hoof health sub-index. This national genetic evaluation program for hoof health was achieved by creating a centralized data collection system that routinely transfers data recorded by hoof trimmers into a coherent and sustainable national database. The 8 most prevalent lesions (digital dermatitis, interdigital dermatitis, interdigital hyperplasia, heel horn erosion, sole hemorrhage, sole ulcer, toe ulcer, and white line lesion) in Canada are analyzed with a multiple-trait model using a single-step genomic BLUP method. Estimated genomic breeding values for each lesion are combined into a sub-index according to their economic value and prevalence. In addition, data recorded within this system were used to create an interactive management report for dairy producers by Canadian DHI, including the prevalence of lesions on farm, their trends over time, and benchmarks with provincial and national averages.

Two-stage genome-wide association study for the identification of causal variants underlying hoof disorders in cattle.

Feet and legs disorders influence dairy cattle breeding by their effect on animal welfare, economic losses due to lower production and fertility, costs of treatment, and problems with herd management. In our study, we estimated heritabilities and performed a 2-step GWAS for 3 traits describing hoof health: hoof health status defined by a veterinarian (HSV), hoof health status defined by a claw trimmer (HSC), and the total number of hoof disorders (NHD), scored in 1,998 Fleckvieh and 979 Braunvieh cows. The individuals were genotyped with a high-density (HD) panel consisting of 76,934 SNP. For significant genomic regions, the SNP information was enhanced by SNP imputed from the whole-genome sequence of Fleckvieh and Braunvieh bulls from the 1000 Bulls Genome project. The heritabilities were estimated to be 0.035 for HSV, 0.249 for HSC, and 0.279 for NHD. Based on the first-stage GWAS with SNP from the HD panel, 7 significant genomic regions on 6 chromosomes were defined: (1) 120 SNP spanning 15,522 bp on BTA1, including the TOPBP1 gene; (2) 4,139 SNP spanning 1,426,046 bp on BTA7, including the RIOK2 and RGMB genes; (3) 167 SNP spanning 167,352 bp on BTA13, including the C13H20orf194 gene; (4) 2 regions on BTA14, one harboring 1,071 SNP spanning 380,024 bp, including RRM2B and NCALD, and the other comprising 632 SNP spanning 385,111 bp, including STK3; (5) 328 SNP on BTA15, spanning 235,567 bp between FAM168A and PLEKHB1; and (6) 1,549 SNP on BTA22, spanning 596,101 bp in the neighborhood of PTPRG. Then, we conducted a second-stage GWAS based on SNP from whole-genome sequences within the significant regions obtained in the first stage of the analysis. For HSV, the highest additive effect was estimated for 23 SNP located within a region on BTA15, close to FAM168A, corresponding to a predicted gene sequence. For HSC, the highest additive effect was attributed to 44 SNP located within a region of BTA22 corresponding to 4 predicted gene sequences, with rs135082893 within a sequence encoding a microRNA. Another potential causal mutation for HSC was rs134142607 on BTA13, within the exon of C13H20orf194. For NHD, 33 SNP with the highest estimated effect were located on BTA7 within a region of a predicted gene positioned between RIOK2 and RGMB. On BTA14, all significant SNP were located in introns of STK3, which is responsible for the "abnormal gait" phenotype in mice.

Congenital spindle cell rhabdomyosarcoma.

Spindle cell and sclerosing rhabdomyosarcoma (ssRMS) is a rare variant of rhabdomyosarcoma, which includes three distinct subtypes. In infants, these tumors are commonly associated with recurring fusions involving VGLL2 or NCOA2 and have a favorable prognosis. We present four cases of ssRMS and 16 additional cases from the literature, which show that these patients present with localized disease and have an excellent prognosis regardless of surgical margin or lack of radiation therapy. Molecularly defined spindle cell rhabdomyosarcoma in infants is likely a biologically distinct entity which may not require the aggressive multimodal treatment used for other subtypes of rhabdomyosarcoma.

Genomic analysis of claw lesions in Holstein cows: Opportunities for genomic selection, quantitative trait locus detection, and gene identification.

Claw lesions are the third most important health issue in dairy cattle, after mastitis and reproductive disorders, and genomic selection is a key component for long-term improvement of claw health. The objectives of this study were to assess the feasibility of a genomic evaluation for claw health in French Holstein cows, explore possibilities to increase evaluation accuracy, and gain a better understanding of the genetic determinism of claw health traits. The data set consisted of 48,685 trimmed Holstein cows, including 9,646 that were genotyped; 478 genotyped sires were also used. Seven claw lesion traits were evaluated using BLUP, genomic BLUP, BayesC, and single-step genomic BLUP, and the accuracies obtained using these approaches were measured through a validation study. The BayesC approach was used to detect quantitative trait locus (QTL) regions associated with the 7 individual traits (digital dermatitis, heel horn erosion, interdigital hyperplasia, sole hemorrhage circumscribed, sole hemorrhage diffused, sole ulcer, and white line fissure) based on their Bayes factor. Annotated genes on these regions were reported. Genomic evaluation approaches generally did not allow for greater accuracies than BLUP, except for single-step genomic BLUP. Accuracies were moderate, but best and worst validation animals were correctly discriminated and showed significant differences in lesion frequencies. A total of 192 QTL regions were identified, including 13 with major evidence or involved for 2 of the traits. A high number of genes were present on these regions, and several had functions associated with the immune system. In particular, the EPYC gene is located close to a major evidence QTL for resistance to digital dermatitis that is also a QTL for interdigital hyperplasia (on chromosome 5, around 20.9 MB) and has been associated with Ehlers-Danlos syndrome in cattle. Genomic selection can be used to improve resistance to individual claw lesions, and several possibilities exist to improve accuracies of genomic evaluations.

Short communication: Use of lameness scoring to genetically improve claw health in Austrian Fleckvieh, Brown Swiss, and Holstein cattle.

The specific objective of this study was to evaluate the use of lameness scoring to genetically improve claw health in Austrian Fleckvieh, Brown Swiss, and Holstein cows based on data from the "Efficient Cow" project. In 2014, a 1-yr data collection was carried out. Data from 6,519 cows kept on 161 farms were recorded. At each time of milk recording, lameness scores were assessed by trained staff of the milk recording organizations. Hoof trimming on these farms was documented and recorded as well. Veterinarian diagnoses and culling due to foot and leg problems from these farms were available from the routine recording system. As repeated lameness records per cow and lactation were available, an overall lactation lameness score was calculated. Estimated heritabilities for lameness were 0.11, 0.05, and 0.09 for Fleckvieh, Brown Swiss, and Holstein, respectively; however, only heritability estimates for Fleckvieh were significantly different from zero. Breeding values for lameness were obtained, reversed in sign, and cows were ranked according to their breeding value. A low breeding value for lameness resistance (the bottom 10% of the cows) was associated with a significantly higher frequency of trimmed cows, which indicates that the cows selected by the farmer to be trimmed are not completely random. Additionally, a high breeding value for lameness resistance (the top 10% of the cows) was associated with lower frequencies of claw diseases recorded at trimming, claw and leg diagnoses, and culling due to foot and leg problems, which highlights the usefulness of lameness scoring for genetic improvement of claw health. Overall, selecting for a better lameness score has the potential to reduce claw diseases, especially the frequency of severe claw diseases that lead to culling.

Invited review: Genetics and claw health: Opportunities to enhance claw health by genetic selection.

Routine recording of claw health status at claw trimming of dairy cattle has been established in several countries, providing valuable data for genetic evaluation. In this review, we examine issues related to genetic evaluation of claw health; discuss data sources, trait definitions, and data validation procedures; and present a review of genetic parameters, possible indicator traits, and status of genetic and genomic evaluations for claw disorders. Different sources of data and traits can be used to describe claw health. Severe cases of claw disorders can be identified by veterinary diagnoses. Data from lameness and locomotion scoring, activity information from sensors, and feet and leg conformation traits are used as auxiliary traits. The most reliable and comprehensive information is data from regular hoof trimming. In genetic evaluation, claw disorders are usually defined as binary traits, based on whether or not the claw disorder was present (recorded) at least once during a defined time period. The traits can be specific disorders, composite traits, or overall claw health. Data validation and editing criteria are needed to ensure reliable data at the trimmer, herd, animal, and record levels. Different strategies have been chosen, reflecting differences in herd sizes, data structures, management practices, and recording systems among countries. Heritabilities of the most commonly analyzed claw disorders based on data from routine claw trimming were generally low, with ranges of linear model estimates from 0.01 to 0.14, and threshold model estimates from 0.06 to 0.39. Estimated genetic correlations among claw disorders varied from -0.40 to 0.98. The strongest genetic correlations were found among sole hemorrhage (SH), sole ulcer (SU), and white line disease (WL), and between digital/interdigital dermatitis (DD/ID) and heel horn erosion (HHE). Genetic correlations between DD/ID and HHE on the one hand and SH, SU, or WL on the other hand were, in most cases, low. Although some of the studies were based on relatively few records and the estimated genetic parameters had large standard errors, there was, with some exceptions, consistency among studies. Various studies evaluate the potential of various data soureces for use in breeding. The use of hoof trimming data is recommended for maximization of genetic gain, although auxiliary traits, such as locomotion score and some conformation traits, may be valuable for increasing the reliability of genetic evaluations. Routine genetic evaluation of direct claw health has been implemented in the Netherlands (2010); Denmark, Finland, and Sweden (joint Nordic evaluation; 2011); and Norway (2014), and other countries plan to implement evaluations in the near future.

BAP1: case report and insight into a novel tumor suppressor.

BACKGROUND: BRCA1-Associated-Protein 1 (BAP1) is a dynamic tumor suppressor which, when mutated, has been associated with an increased risk of uveal melanoma, cutaneous melanoma, mesothelioma, and several other cancers. Germline BAP1 mutations have been extensively studied, where they have been found to cause hereditary cancer susceptibility. However, their sporadic counterparts, tumors that display a loss of BAP1 expression due to somatically arising mutations in the BAP1 gene, remain a poorly described entity. CASE PRESENTATION: Here we present the case of a 49-year-old female who presented with an asymptomatic dome-shaped pink papule on the dorsal foot which was found on biopsy to be deficient in the BAP1 tumor suppressor. While the patient's family history did not suggest the presence of a familial cancer syndrome, germline genetic testing was performed and was negative. The patient underwent surgical excision of this sporadically appearing "BAPoma" by Mohs surgery. CONCLUSIONS: Given the relatively banal clinical appearance of these dome-shaped neoplasms, sporadic BAPomas may often be overlooked by clinicians and dermatologists. In addition to providing a representative case, here we also provide a synopsis of the current understanding of these neoplasms, both in terms of the histopathological features, as well as the molecular mechanisms underlying BAP1 function and its ability to prevent tumorigenesis.

Acral melanoma foot lesions. Part 1: epidemiology, aetiology, and molecular pathology.

Acral melanoma (AM) is a rare subtype of cutaneous malignant melanoma (MM) found on acral skin, primarily on the soles of the feet. Although rare, it is the most common subtype of MM found in patients of African or East Asian ethnicity and has a poor prognosis, often because of the more advanced stage of presentation at diagnosis. The pathogenesis of AM is unclear, but genetic alterations, including mutations in BRAF, NRAS, and KIT have been implicated. Early diagnosis of AM is important for a better prognosis, but its identification is often challenging, leading to easy misdiagnosis. In the first of this two-part review, we review the history, epidemiology, aetiology and molecular pathology of AM; in part 2 we will review diagnosis and management.

Podiatric Physicians and Genomic Medicine.

It has been more than 14 years since identification of the human genome. This phenomenon is creating a revolution in all components of the health-care world. To date, little has been included in the podiatric medical literature despite the fact that so many of the conditions affecting the pedal extremity have genomic implications. Genomics will have a major effect on prevention, diagnosis, and patient management and needs to be included in podiatric medical practice as well as in the curriculum of podiatric medical schools.

Corkscrew Claw.

Corkscrew claw (CSC) is a conformational abnormality of the digit and affecting mostly but not exclusively the claws of the back leg, first reported during the 1950s in Dutch black and white cattle. The affected claws are longer and narrower than the claw and have an inward and upward spiral rotation of the toe. Similarly, the bearing surface of the wall is displaced inward. The animal starts to bear weight on the abaxial wall surface, particularly the caudal segment, and the sole may become completely non-weight bearing. The axial wall is displaced dorsomedially and a fold develops in the wall.

Genetic Influence on Accessory Navicular Bone in the Foot: A Korean Twin and Family Study.

An accessory navicular bone (AN) is the most common accessory ossicle in the foot. The presence of an AN bone can trigger various foot problems, such as posterior tibial tendon pathology, flattening of the medial longitudinal arch, and medial foot pain. Despite the clinical influence of presence of an AN in foot disease, the research regarding its inheritance is still insufficient. A total of 135 pairs of monozygotic (MZ) twins, 25 pairs of dizygotic (DZ) twins, and 676 singletons from families were enrolled in order to estimate genetic influences on AN. After confirmation of zygosity and family relationship with a tandem repeat marker kit and questionnaires, the presence and type of the AN was classified through bilateral feet radiographic examination. The heritability of an AN was estimated using quantitative genetic analysis based on a variance decomposition model considering various types of family relationships: father-offspring pair, mother-offspring pair, and pooled DZ twin and sibling pairs. As a result, approximately 40.96% of the participants in this study had an AN in either foot, with type II being the most common type. The heritability for the presence of any type of an AN in any foot was estimated as 0.88 (95% CI [0.82, 0.94]) after adjusting for age and sex. Specifically, type II AN showed the highest heritability of 0.82 (95% CI [0.71-0.93]). The high heritability of an AN found in this large twin and family study suggests that an AN is determined by the substantial influence of genetic factor.