VPS13B Gene Research Articles

Cohen syndrome: Can early-onset recurrent infections and hypotonia provide early diagnosis and intervention for intellectual disability?

Cohen syndrome is a rare disease associated with neurodevelopmental disorders, especially intellectual disability (ID), neutropenia and recurrent infections are consistently reported in cases. Neutropenia is an important part of the syndrome, as well as ID. Homozygous variants in the VPS13B gene, located on chromosome 8q22 and containing 62 exons, have been found to cause Cohen syndrome. Cohen syndrome is commonly diagnosed when dysmorphological findings and developmental delay become more apparent. However, the identification of some findings with increasing age has caused the diagnosis of Cohen syndrome to be delayed. Cases diagnosed with ID were evaluated using whole-exome sequencing/clinical exome sequencing method. Family segregation analysis was performed using Sanger sequencing. We presented the clinical and genetic findings of three cases diagnosed with Cohen syndrome and their parents in detail. In this study, we presented the occurrence of symptoms in different age groups, and the prognosis of three cases carrying the VPS13B gene variants, including three different variant types: missense, frameshift and nonsense. Although our cases had different variant types, they shared important similarities on the onset period and prognosis of the symptoms. All cases presented hypotonia, difficulties in swallowing, recurrent respiratory tract infections, neutropenia, delay in motor development, ID and hyperactivity. Our cases did not have a diagnosis of autism spectrum disorder. All cases had increased willingness to engage in social communication. We emphasize the importance of early-onset recurrent infections and hypotonia for early diagnosis and preventive genetic counselling in Cohen syndrome.

Exploring the pathological mechanisms underlying Cohen syndrome.

Cohen Syndrome (CS) is a rare autosomal recessive disorder caused by biallelic mutations in the VPS13B gene. It is characterized by multiple clinical features, including acquired microcephaly, developmental delay, intellectual disability, neutropenia, and retinal degeneration. VPS13B is part of the bridge-like lipid transport (BLTP) protein family, which in mammals also includes VPS13A, -C, and -D. The proteins of this family are peripheral membrane proteins with different sub-cellular localization, but all share similar structural features and have been proposed to act as lipid transport proteins at organellar membrane contact sites. VPS13B is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. Here we present a review of the experimental data on the function of the protein at the cellular level, discussing the potential link with disease phenotype and review the studies on animal models recapitulating features of the human disease.

Clinical findings, treatment and outcome of trapped neutrophil syndrome in Border Collies: 12 cases (2011-2022).

This study aimed to evaluate clinical signs, diagnostic findings, treatment administered and short- (survival to 28 days) and long-term prognosis (survival >6 months) in dogs diagnosed with trapped neutrophil syndrome. Medical records of 12 dogs (10 Border Collies and two Border Collie Crossbreeds) homozygous for VPS13B gene mutation causing trapped neutrophil syndrome from seven veterinary institutions between January 2011 and June 2022 were evaluated retrospectively. The most common clinical signs at the time of diagnosis were pyrexia, abnormal gait and gastrointestinal signs. Concurrent metaphyseal osteopathy and immune-mediated polyarthritis were common. Seven dogs had a segmented neutrophil count below, four dogs within and one dog above the analyser reference interval at presentation. Two dogs had a septic source identified and both were additionally identified to be homozygous mutant positive on DNA testing by polymerase chain reaction (PCR)for canine cyclic neutropenia. All dogs received at least one antimicrobial agent and 10 dogs received treatment with prednisone or prednisolone (median starting dose 1 mg/kg/day; range 0.5 to 2.5 mg/kg/day). Nine dogs were alive at 28 days and six dogs were alive at 6 months post-diagnosis. Trapped neutrophil syndrome should be suspected in young Border Collies with pyrexia, lameness and gastrointestinal signs. Neutropenia may not always be present and long-term survival is possible. A septic focus was not commonly identified in our population; however, our results suggest that if identified, testing for concurrent canine cyclic neutropenia should be considered.

Cohen syndrome combined with psychiatric symptoms: a case report

BackgroundCohen syndrome (CS) is a rare autosomal recessive inherited condition characterized by pathological changes affecting multiple systems. The extensive clinical variability associated with CS poses a significant diagnostic challenge. Additionally, there is limited documentation on the co-occurrence of CS with psychiatric symptoms.Case reportWe report a case of a 30-year-old patient exhibiting characteristic physical features and psychiatric symptoms. Whole exome sequencing identified two heterozygous variants, a nonsense variation c.4336 C > T and a missense mutation c.4729G > A. Integrating clinical manifestations with genetic test results, we established the diagnosis of CS combined with psychiatric symptoms.ConclusionsThis case introduces a novel missense variant as a candidate in the expanding array of VPS13B pathogenic variants. Its clinical significance remains unknown, and further investigation may broaden the spectrum of pathogenic variants associated with the VPS13B gene. Early diagnosis of CS is crucial for the prognosis of young children and holds significant importance for their families.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Ophthalmic findings in Cohen syndrome patient without subjective ophthalmic complaints: A case report.

Cohen syndrome is a rare genetic disorder that can cause various symptoms, including ophthalmic manifestations that can significantly impact a patient's visual health and quality of life. We present the case of a 12-year-old boy diagnosed with Cohen syndrome who exhibited retinal degeneration and macular edema but could not express ophthalmic symptoms due to a developmental disability. The patient was diagnosed with Cohen syndrome by a heterozygous mutation in the VPS13B gene by whole exome sequencing and referred to ophthalmology for an ophthalmic examination. Ophthalmologic tests, including visual acuity, intraocular pressure, slit lamp examination, fundus photography, and optical coherence tomography, were performed. Visual acuity and intraocular pressure were not measured due to poor cooperation, and no abnormal findings were observed on the slit lamp examination. However, peripheral retinal degeneration was observed in the fundus examination, and cystoid macular edema was observed in both eyes on optical coherence tomography. Regular ophthalmologic examination is important for patients with Cohen syndrome, especially those with developmental disabilities who may not be able to express their symptoms. Clinicians should be aware of the potential ophthalmologic manifestations of Cohen syndrome and the importance of timely diagnosis and management.

Quantitative trait locus analysis for endophenotypes reveals genetic substrates of core symptom domains and neurocognitive function in autism spectrum disorder

Autism spectrum disorder (ASD) represents a heterogeneous group of neurodevelopmental disorders and is largely attributable to genetic risk factors. Phenotypic and genetic heterogeneity of ASD have been well-recognized; however, genetic substrates for endophenotypes that constitute phenotypic heterogeneity are not yet known. In the present study, we compiled data from the Autism Genetic Resource Exchange, which contains the demographic and detailed phenotype information of 11,961 individuals. Notably, the whole-genome sequencing data available from MSSNG and iHART for 3833 individuals in this dataset was used to perform an endophenotype-wide association study. Using a linear mixed model, genome-wide association analyses were performed for 29 endophenotype scores and 0.58 million common variants with variant allele frequency ≥ 5%. We discovered significant associations between 9 genetic variants and 6 endophenotype scores comprising neurocognitive development and severity scores for core symptoms of ASD at a significance threshold of p < 5 × 10–7. Of note, the Stereotyped Behaviors and Restricted Interests total score in Autism Diagnostic Observation Schedule Module 3 was significantly associated with multiple variants in the VPS13B gene, a causal gene for Cohen syndrome and a candidate gene for syndromic ASD. Our findings yielded loci with small effect sizes due to the moderate sample size and, thus, require validation in another cohort. Nonetheless, our endophenotype-wide association analysis extends previous candidate gene discovery in the context of genotype and endophenotype association. As a result, these candidate genes may be responsible for specific traits that constitute core symptoms and neurocognitive function of ASD rather than the disorder itself.

Generation of two iPSC lines from healthy donor with a heterozygous mutation in the VPS13B gene

The human induced pluripotent stem cell (iPSC) lines, iCS-MAF1-1 and iCS-MAF1-11, were generated from fibroblasts. The donor has a heterozygous mutation in the VPS13B gene, which manifests in her child as Cohen syndrome. It is a Golgi pathology, characterized by postnatal microcephaly and delayed growth and mental development. However, the process underlying pathological changes leading to the onset of the disease is still unknown. The use of iPSC will allow describing the early stages of neurogenesis, which is undoubtedly relevant for identifying key stages of development, at which phenotypic manifestations of mutations in the VPS13B gene are found.

A Novel Homozygous VPS13B Splice-Site Mutation Causing the Skipping of Exon 38 in a Chinese Family With Cohen Syndrome.

Background: Cohen syndrome (CS) is a clinically heterogeneous disorder characterized by extensive phenotypic variation with autosomal recessive inheritance. VPS13B was identified to be the disease-causing gene for CS. The objectives of the present study were to screen likely pathogenic mutations of the patient with developmental delay and mental retardation, and to determinate the effect of this splice-site mutation by reverse transcription analysis.Methods: Whole exome sequencing (WES) in combination with Sanger sequencing were performed to identify the causative mutations of this CS family. Subsequently, the impact of the intronic variant on splicing was analyzed by reverse transcription and the construction of expression vector.Results: A novel homozygous splice-site mutation (c.6940+1G>T) in the VPS13B gene was identified in this proband. Sanger sequencing analysis of the cDNA demonstrated that the c.6940+1G>T variant could cause the skipping of entire exon 38, resulting in the loss of 208 nucleotides and further give rise to the generation of a premature in-frame stop codon at code 2,247.Conclusions: The homozygous VPS13B splicing variant c.6940+1G>T was co-segregated with the CS phenotypes in this family and was identified to be the cause of CS after comprehensive consideration of the clinical manifestations, genetic analysis and cDNA sequencing result.

A GWAS in Latin Americans identifies novel face shape loci, implicating VPS13B and a Denisovan introgressed region in facial variation.

To characterize the genetic basis of facial features in Latin Americans, we performed a genome-wide association study (GWAS) of more than 6000 individuals using 59 landmark-based measurements from two-dimensional profile photographs and ~9,000,000 genotyped or imputed single-nucleotide polymorphisms. We detected significant association of 32 traits with at least 1 (and up to 6) of 32 different genomic regions, more than doubling the number of robustly associated face morphology loci reported until now (from 11 to 23). These GWAS hits are strongly enriched in regulatory sequences active specifically during craniofacial development. The associated region in 1p12 includes a tract of archaic adaptive introgression, with a Denisovan haplotype common in Native Americans affecting particularly lip thickness. Among the nine previously unidentified face morphology loci we identified is the VPS13B gene region, and we show that variants in this region also affect midfacial morphology in mice.

Cohen Syndrome-Associated Cataract Is Explained by VPS13B Functions in Lens Homeostasis and Is Modified by Additional Genetic Factors.

PurposeCohen syndrome (CS) is a rare genetic disorder caused by variants of the VPS13B gene. CS patients are affected with a severe form of retinal dystrophy, and in several cases cataracts also develop. The purpose of this study was to investigate the mechanisms and risk factors for cataract in CS, as well as to report on cataract surgeries in CS patients.MethodsTo understand how VPS13B is associated with visual impairments in CS, we generated the Vps13b∆Ex3/∆Ex3 mouse model. Mice from 1 to 3 months of age were followed by ophthalmoscopy and slit-lamp examinations. Phenotypes were investigated by histology, immunohistochemistry, and western blot. Literature analysis was performed to determine specific characteristic features of cataract in CS and to identify potential genotype–phenotype correlations.ResultsCataracts rapidly developed in 2-month-old knockout mice and were present in almost all lenses at 3 months. Eye fundi appeared normal until cataract development. Lens immunostaining revealed that cataract formation was associated with the appearance of large vacuoles in the cortical area, epithelial–mesenchymal transition, and fibrosis. In later stages, cataracts became hypermature, leading to profound retinal remodeling due to inflammatory events. Literature analysis showed that CS-related cataracts display specific features compared to other forms of retinitis pigmentosa-related cataracts, and their onset is modified by additional genetic factors. Corroboratively, we were able to isolate a subline of the Vps13b∆Ex3/∆Ex3 model with delayed cataract onset.ConclusionsVPS13B participates in lens homeostasis, and the CS-related cataract development dynamic is linked to additional genetic factors.

Cohen Syndrome Patient iPSC-Derived Neurospheres and Forebrain-Like Glutamatergic Neurons Reveal Reduced Proliferation of Neural Progenitor Cells and Altered Expression of Synapse Genes.

Cohen syndrome (CS), a rare autosomal recessive disorder, has been associated with genetic mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. However, the cellular mechanism underlying CS pathogenesis in patient-derived human neurons remains unknown. We identified a novel compound heterozygous mutation, due to homozygous variation of biparental origin and heterozygous variation inherited from the father, in the VPS13B gene in a 20-month-old female patient. To understand the cellular pathogenic mechanisms, we generated induced pluripotent stem cells (iPSCs) from the fibroblasts of the CS patient. The iPSCs were differentiated into forebrain-like functional glutamatergic neurons or neurospheres. Functional annotation from transcriptomic analysis using CS iPSC-derived neurons revealed that synapse-related functions were enriched among the upregulated and downregulated genes in the CS neurons, whereas processes associated with neurodevelopment were enriched in the downregulated genes. The developing CS neurospheres were small in size compared to control neurospheres, likely due to the reduced proliferation of SOX2-positive neural stem cells. Moreover, the number of SV2B-positive puncta and spine-like structures was significantly reduced in the CS neurons, suggesting synaptic dysfunction. Taking these findings together, for the first time, we report a potential cellular pathogenic mechanism which reveals the alteration of neurodevelopment-related genes and the dysregulation of synaptic function in the human induced neurons differentiated from iPSCs and neurospheres of a CS patient.

Homozygosity mapping and whole exome sequencing provide exact diagnosis of Cohen syndrome in a Saudi family

BackgroundCohen syndrome (CS) is a rare multi-system autosomal recessive disorder with a high prevalence in the Finnish population. Clinical features of Finnish-type CS are homogeneous, however, in non-Finnish populations, CS diagnosis is challenging due to broad phenotypic variability. MethodsWe studied a consanguineous family having three affected individuals with clinical features of severe intellectual disability and global developmental delay. Clinical diagnosis of the phenotype could not be established based on the features. Therefore, whole genome SNP genotyping and whole exome sequencing (WES) were performed on DNA samples from affected and unaffected family members. ResultsHomozygosity mapping identified a shared loss of heterozygosity region on chromosome 8q22.1-q22.3 and WES data analysis revealed an insertion-deletion (indel) mutation (c.11519_11521delCAAinsT) in the VPS13B gene. The indel is predicted to cause a frameshift resulting in a premature termination of the VPS13B protein (NP_060360.3:p.Pro3840Leufs*2). ConclusionVPS13B encodes a giant transmembrane protein called vacuolar protein sorting 13 homolog B. VPS13B is known to play a role in the glycosylation of Golgi proteins and in endosomal-lysosomal trafficking. Moreover, it is thought to function in vesicle mediated transport and sorting of proteins within the cell. The mechanism by which abnormalities of the VPS13B protein lead to the phenotype of CS is currently unknown. Here, in this study, we successfully established a clinical diagnosis of CS cases from a family using genomic analyses. Clinical re-examination of the patients revealed characteristic ocular abnormalities.

Serpin B1 defect and increased apoptosis of neutrophils in Cohen syndrome neutropenia.

Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS. KEY MESSAGES: Cohen syndrome patients' neutrophils display normal morphology and functions. Cohen syndrome patients' neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors' neutrophils. No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients' neutrophils. SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.

First case report of Cohen syndrome in the Tunisian population caused by VPS13B mutations

BackgroundCohen syndrome is a rare autosomal recessive developmental disorder that comprises variable clinical features counting developmental delay, pigmentary retinopathy, myopia, acquired microcephaly, truncal obesity, joint hypermobility, friendly disposition and intermittent neutropenia. VPS13B (vacuolar protein sorting 13, yeast, homologue of B) gene is the only gene responsible for Cohen Syndrome, causative mutations include nonsense, missense, indel and splice-site variants. The integrity of the Golgi apparatus requires the presence of the peripheral membrane protein VPS13B that have an essential function in intracellular protein transport and vesicle-mediated sorting.Case presentationIn this study, we performed whole exome sequencing (WES) in a Tunisian family with two young cases having developmental delay, hypotonia, autism spectrum disorder, ptosis and thick hair and eyebrows. The proposita presented also pigmentory retinopathy. Compound heterozygous mutation in VPS13B gene was detected by WES. This mutation inherited from healthy heterozygous parents, supports an unpredictable clinical diagnosis of Cohen Syndrome. The proband’s phenotype is explained by the presence of compound heterozygous mutations in the VPS13B gene. This finding refined the understanding of genotype-phenotype correlation.ConclusionsThis is the first report of a Tunisian family with Cohen syndrome mutated in the VPS13B gene.

Síndrome de Cohen diagnosticado con hibridación genómica comparativa por microarreglos

Cohen syndrome (CS) is an uncommon autosomal recessive genetic disorder attributed to damage on VPS13B gene, locus 8q22-q23. Characteristic phenotype consists of intellectual disability, microcephaly, facial dysmorphism, ophthalmic abnormalities, truncal obesity and hipotony. Worldwide, around 150 cases have been published, mostly in Finish patients. We report the case of a 3 year-old male, with short height, craniosynostosis, facial dysmorphism, hipotony, and developmental delay. He was diagnosed with Cohen syndrome using Microarray Comparative Genomic Hibridization (aCGH) that showed homozygous deletion of 0.153 Mb on 8q22.2 including VPS13B gene, OMIM #216550. With this report we contribute to enlarge epidemiological databases on an uncommon genetic disorder. Besides, we illustrate on the contribution of aCGH to the etiological diagnosis of patients with unexplained intellectual disability, delayed psychomotor development, language difficulties, autism and multiple congenital anomalies.

Insulin response dysregulation explains abnormal fat storage and increased risk of diabetes mellitus type 2 in Cohen Syndrome

Cohen Syndrome (CS) is a rare autosomal recessive disorder, with defective glycosylation secondary to mutations in the VPS13B gene, which encodes a protein of the Golgi apparatus. Besides congenital neutropenia, retinopathy and intellectual deficiency, CS patients are faced with truncal obesity. Metabolism investigations showed abnormal glucose tolerance tests and low HDL values in some patients, and these could be risk factors for the development of diabetes mellitus and/or cardiovascular complications. To understand the mechanisms involved in CS fat storage, we used two models of adipogenesis differentiation: (i) SGBS pre-adipocytes with VPS13B invalidation thanks to siRNA delivery and (ii) CS primary fibroblasts. In both models, VPS13B invalidation led to accelerated differentiation into fat cells, which was confirmed by the earlier and increased expression of specific adipogenic genes, consequent to the increased response of cells to insulin stimulation. At the end of the differentiation protocol, these fat cells exhibited decreased AKT2 phosphorylation after insulin stimulation, which suggests insulin resistance. This study, in association with the in-depth analysis of the metabolic status of the patients, thus allowed us to recommend appropriate nutritional education to prevent the occurrence of diabetes mellitus and to put forward recommendations for the follow-up of CS patients, in particular with regard to the development of metabolic syndrome. We also suggest replacing the term obesity by abnormal fat distribution in CS, which should reduce the number of inappropriate diagnoses in patients who are referred only on the basis of intellectual deficiency associated with obesity.

Cohen syndrome is associated with major glycosylation defects

Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

Changing facial phenotype in Cohen syndrome: towards clues for an earlier diagnosis

Cohen syndrome (CS) is a rare autosomal recessive condition caused by mutations and/or large rearrangements in the VPS13B gene. CS clinical features, including developmental delay, the typical facial gestalt, chorioretinal dystrophy (CRD) and neutropenia, are well described. CS diagnosis is generally raised after school age, when visual disturbances lead to CRD diagnosis and to VPS13B gene testing. This relatively late diagnosis precludes accurate genetic counselling. The aim of this study was to analyse the evolution of CS facial features in the early period of life, particularly before school age (6 years), to find clues for an earlier diagnosis. Photographs of 17 patients with molecularly confirmed CS were analysed, from birth to preschool age. By comparing their facial phenotype when growing, we show that there are no special facial characteristics before 1 year. However, between 2 and 6 years, CS children already share common facial features such as a short neck, a square face with micrognathia and full cheeks, a hypotonic facial appearance, epicanthic folds, long ears with an everted upper part of the auricle and/or a prominent lobe, a relatively short philtrum, a small and open mouth with downturned corners, a thick lower lip and abnormal eye shapes. These early transient facial features evolve to typical CS facial features with aging. These observations emphasize the importance of ophthalmological tests and neutrophil count in children in preschool age presenting with developmental delay, hypotonia and the facial features we described here, for an earlier CS diagnosis.

Real-time PCR genotyping assay for canine trapped neutrophil syndrome and high frequency of the mutant allele in Border collies

Trapped neutrophil syndrome is an autosomal recessive inherited neutropenia in Border collies. The causative mutation is a 4base pair deletion in exon 19 of the canine VPS13B gene. In this study, a real-time PCR assay was developed and a genotyping survey was carried out in Border collies in Japan. The carrier frequency was 11.1%, suggesting that the mutant allele frequency is high enough to warrant measures to control and prevent the disease.