Diagnostic Approach to Children with Unexplained Global Developmental Delay in Pediatric Neurology Outpatient Clinic.

BACKGROUND: Global developmental delay (GDD) is a common pediatric disorder that affects up to 3% of children. Due to the heterogeneous etiology of GDD, diagnostic procedures and algorithms are complex and diverse. The aim of our study was to investigate the diagnostic yield of genetic, metabolic, and imaging studies in establishing the etiology of unexplained GDD (UGDD). METHODS: In this retrospectively observational study, we examined the medical records of all children diagnosed with UGDD at the Department of Pediatric Neurology, University Medical Centre Ljubljana, Slovenia, between January and December 2019. We evaluated the effectiveness of various genetic, metabolic, and magnetic resonance imaging (MRI) tests in identifying the underlying cause of GDD. Additionally, we assessed subgroups of patients to determine whether any of the studied tests were particularly beneficial based on their clinical symptoms. RESULTS: A total of 123 patients met the inclusion criteria, with a median age of 4.3 years (range, 0-16 years), of which 71 (57.7%) were males. Genetic diagnosis was established in 47.1% (58/123) of patients. Metabolic laboratory testing did not identify a metabolic disease in any of the tested participants (114/123) and MRI was critical for diagnosis in only 1/81 (1.2%) patient. CONCLUSION: Our findings strongly suggest that genetic testing surpasses MRI and metabolic testing in establishing the etiology of UGDD in a pediatric neurology outpatient setting. This information will help guide the diagnostic evaluation of these children.

Further exploration of cardiac channelopathy and cardiomyopathy genes in stillbirth.

OBJECTIVE: To explore genetic variation including whole genome copy number variation and sequence analysis of 98 genes associated with pediatric or adult cardiomyopathies, cardiac channelopathies, and sudden death in an unexplained intrauterine fetal death cohort. METHODS: The study population included 55 stillbirth cases that remained unexplained after thorough postmortem examination, excluding maternal, fetal, and placental causes of stillbirth. Molecular karyotyping was performed in 55 cases and the trio exome sequencing approach was applied in 19 cases. RESULTS: The analysis revealed six rare variants with predicted effects on protein function in six genes (CASQ2, DSC2, KCNE1, LDB3, MYH6, and SCN5A) previously reported in cases of stillbirth or severe early onset pediatric cardiac related phenotypes. When applying strict American College of Genetics and Genomics classification guidelines, these are still variants of uncertain significance. CONCLUSIONS: Several potentially stillbirth-related genetic variants were detected in our cohort, adding to the growing literature on cardiac phenotype gene variation in stillbirth. However, the mechanisms of action, gene-gene interaction, and contribution of the uterine environment are still to be deciphered. In order to advance our knowledge of the genetics of unexplained fetal death, there is an evident need for international collaboration and field standardization.

The genetic approach to stillbirth: A ¼systematic review«.

Unexplained stillbirth is defined as a stillbirth with no known cause after the exclusion of common causes, including obstetric complications, infections, placental insufficiency or abruption, umbilical cord complications, and congenital abnormalities with or without known genetic cause. More than 60% of stillbirth cases remain unexplained. The aim of this systematic review was to investigate the known genetic causes of unexplained stillbirth cases and to evaluate the current position and future directions for the use of genetic and genomic testing in expanding the knowledge in this field. A systematic search through several databases was performed using the keywords genetics and stillbirths in humans. Different methods to detect various types of causal genetic aberrations have been used in the past decades, from standard karyotyping to novel methods such as chromosomal microarray analysis and next generation sequencing technologies. Apart from common chromosomal aneuploidies, a promising hypothesis about genetic causes included genes related to cardiomyopathies and channelopathies. However, these were tested in the research settings, since molecular karyotyping is currently the standard approach in the routine evaluation of genetic causes of stillbirth. Hereby, we provide evidence that expanding knowledge using novel genetic and genomic testing might uncover new genetic causes of unexplained stillbirth.

A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6, GRHL3, and TBX22.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Identification of women at risk for hereditary breast and ovarian cancer in a sample of 1000 Slovenian women: a comparison of guidelines.

BACKGROUND: An important number of breast and ovarian cancer cases is due to a strong genetic predisposition. The main tool for identifying individuals at risk is recognizing a suggestive family history of cancer. We present a prospective study on applying three selected clinical guidelines to a cohort of 1000 Slovenian women to determine the prevalence of at-risk women according to each of the guidelines and analyze the differences amongst the guidelines. METHODS: Personal and family history of cancer was collected for 1000 Slovenian women. Guidelines by three organizations: National Comprehensive Cancer Network (NCCN), American College of Medical Genetics in cooperation with National Society of Genetic Counselors (ACMG/NSGC), and Society of Gynecologic Oncology (SGO) were applied to the cohort. The number of women identified, the characteristics of the high-risk population, and the agreement between the guidelines were explored. RESULTS: NCCN guidelines identify 13.2% of women, ACMG/NSGC guidelines identify 7.1% of women, and SGO guidelines identify 7.0% of women from the Slovenian population, while 6.2% of women are identified by all three guidelines as having high-risk for hereditary breast and ovarian cancer. CONCLUSIONS: We identified 13.7% of women from the Slovenian population as being at an increased risk for breast and ovarian cancer based on their personal and family history of cancer using all of the guidelines. There are important differences between the guidelines. NCCN guidelines are the most inclusive, identifying nearly twice the amount of women as high-risk for hereditary breast and ovarian cancer as compared to the AGMG/NSCG and SGO guidelines in the Slovenian population.

Comprehensive use of extended exome analysis improves diagnostic yield in rare disease: a retrospective survey in 1,059 cases.

PurposeWe sought to determine the analytical sensitivity of several extended exome variation analysis approaches in terms of their contribution to diagnostic yield and their clinical feasibility.MethodsWe retrospectively analyzed the results of genetic testing in 1,059 distinct cases referred for exome sequencing to our institution. In these, we routinely employed extended exome analysis approaches in addition to basic variant analysis, including (i) copy-number variation (CNV) detection, (ii) nonconsensus splice defect detection, (ii) genomic breakpoint detection, (iv) homozygosity mapping, and (v) mitochondrial variant analysis.ResultsExtended exome analysis approaches assisted in identification of causative genetic variant in 44 cases, which represented a 4.2% increase in diagnostic yield. The greatest contribution was associated with CNV analysis (1.8%) and splice variant prediction (1.2%), and the remaining approaches contributed an additional 1.2%. Analysis of workload has shown that on average nine additional variants per case had to be interpreted in the extended analysis.ConclusionWe show that extended exome analysis approaches improve the diagnostic yield of heterogeneous genetic disorders and result in considerable increase of diagnostic yield of exome sequencing with a minor increase of interpretative workload.

Angiotensin-converting enzyme insertion/deletion gene polymorphism and interferon-ß treatment response in multiple sclerosis patients: a preliminary report.

We investigated the effect of the functional insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene on the response to interferon-ß (IFN-ß) therapy in Croatian and Slovenian patients with multiple sclerosis (MS). A total of 275 IFN-ß treated MS patients [162 responders (Rs) and 113 nonresponders (NRs)] were genotyped by PCR. The ACE I/D genotype distribution and allele frequencies did not differ between female Rs and NRs. However, male NRs tended to have a greater prevalence of the DD genotype (P=0.073; odds ratio: 2.64; 95% confidence interval: 0.91-7.60) and a significantly higher frequency of the D allele (P=0.022; odds ratio: 2.43; 95% confidence interval: 1.13-5.20) than male Rs. Multiple forward stepwise regression analysis indicated that the negative response to IFN-ß therapy was associated with the ACE-DD genotype in men (ß=0.371; multiple R change: 0.132; P=0.009) and a higher pretreatment relapse rate in both men (ß=-0.438; multiple R change: 0.135; P=0.015) and women (ß=-0.208; multiple R change: 0.042; P=0.034). The ACE I/D polymorphism and pretreatment relapse rate accounted for ∼26.7% of the IFN-ß response variability among the men in the sample. Further studies of a larger number of MS patients from different populations are necessary to evaluate these preliminary findings.

Phenotype-driven gene target definition in clinical genome-wide sequencing data interpretation.

PURPOSE: Genome-wide sequencing approaches are increasingly being used in place of disease gene panel sequencing approaches. Despite the well-recognized benefits of these approaches, they also carry with them an increased burden of analyzing overwhelmingly large gene targets and an increased possibility of detecting incidental findings. METHODS: We propose a novel approach for design of individualized phenotype gene panels using the set of signs and symptoms observed and selecting relevant genes on the basis of known phenotype-gene associations. RESULTS: We used results of diagnostic exome sequencing in 405 cases submitted to our institution to show retrospectively that using the phenotype gene panel increases the sensitivity of masked exome analysis (increase from 25.4 to 29.7% in overall diagnostic yield). We also show that such a strategy enables the possibility of masked analysis of genome-wide sequencing data in patients with poorly defined and multifaceted clinical presentations. Ultimately, we show that this approach enables control over the incidental findings rate (0.25% in phenotype gene panels). Finally, we provide a Web tool for customized phenotype panel creation (available at http://www.kimg.eu/generator). CONCLUSION: In conclusion, we present a novel approach to a phenotype-driven diagnostic process of genome scale sequencing data that harnesses the sensitivity of these approaches while restricting the analysis to genes relevant to clinical presentation in patient.Genet Med 18 11, 1102-1110.

Clinical utility of array comparative genomic hybridisation in prenatal setting.

BACKGROUND: The objective of reported study was to evaluate the clinical utility of prenatal microarray testing for submicroscopic genomic imbalances in routine prenatal settings and to stratify the findings according to the type of fetal ultrasound anomaly. METHODS: From July 2012 to October 2015 chromosomal microarray testing was performed in 218 fetuses with varying indications for invasive prenatal diagnosis: abnormal karyotype, ultrasound anomalies, pathogenic variant in previous pregnancy or carriership in a parent. RESULTS: The detection rate in the group of fetuses with ultrasound anomalies was 10,0% for pathogenic copy number variants (CNVs), five of them being larger than 8 Mb and expected to be seen on prenatal karyotype. If only those pathogenic CNVs below the classical karyotype resolution are considered, chromosomal microarray testing provided an additional 7,7% diagnostic yield in here reported series. When stratified according to the ultrasound anomalies, the highest percentage of pathogenic CNVs were detected in the group of fetuses with multiple congenital anomalies (16,7%) and lowest in the group of isolated in utero growth restriction (6,3%). In the group of cases with isolated increased nuchal translucency we identified a small interstitial deletion of 16p24.1 involving FOXF1 gene. Prenatal aCGH also provided important insights into cases with seemingly balanced chromosomal rearrangements found on prenatal karyotype, where additional pathogenic CNV were discovered. CONCLUSION: Prenatal chromosomal microarray testing significantly increases the diagnostic yield when compared with conventional karyotyping. The highest added value is shown in prenatal diagnostics in fetuses with abnormal ultrasound results. Variants of unknown significance and risk factor CNVs present important challenges and should be discussed with parents in advance, therefore pretest counseling prior to prenatal testing is very important.

11q terminal deletion and combined immunodeficiency (Jacobsen syndrome): Case report and literature review on immunodeficiency in Jacobsen syndrome.

Antibody deficiency is common finding in patients with Jacobsen syndrome (JS). In addition, there have been few reports of T-cell defects in this condition, possibly because most of the reported patients have not been specifically evaluated for T-cell function. In this article, we present a child with an 11q deletion and combined immunodeficiency and we perform a literature overview on immunodeficiency in JS. Our patient presented with recurrent bacterial and prolonged viral infections involving the respiratory system, as well as other classic features of the syndrome. In addition to low IgM, IgG4, and B-cells, also low recent thymic emigrants, helper and naïve T-cells were found. We propose that patients with Jacobsen syndrome need thorough immunological evaluations as T-cell dysfunction might be more prevalent than previously reported. Patients with infections consistent with T-cell defects should be classified as having combined immunodeficiency. © 2016 Wiley Periodicals, Inc.

Nutriepigenomics: the role of nutrition in epigenetic control of human diseases.

PURPOSE OF REVIEW: Nutrients or even diets affect the epigenome by lifelong remodeling. Nutritional imbalances are associated with noncommunicable diseases. Thus, nutriepigenomics is a promising field in the treatment of complex human diseases. RECENT FINDINGS: The epigenome is susceptible to changes and can be shaped by nutritional states, especially in prenatal period through transgenerational mechanisms and in early postnatal life when critical developmental processes are taking place. Although more stable, the epigenetic marks in adulthood are also dynamic and modifiable by environmental factors including diet. SUMMARY: The present review is focused on the most recent knowledge of epigenetically active nutrients/diets including transgenerational inheritance and prenatal predispositions related to increased risk for cancer, metabolic syndrome, and neurodegenerative diseases.

Specific gene expression differences in cumulus cells as potential biomarkers of pregnancy.

The development of an objective and accurate test that could help select embryos with the highest chance of achieving pregnancy in IVF procedures is an important goal of reproductive medicine. For this purpose, cumulus cell gene expression is being studied to find biomarkers of pregnancy. Several recent studies have proposed potential biomarkers of pregnancy expressed in cumulus cells; however, these have mostly not been validated on an independent set of samples. The aim of this study was to analyse the expression of EFNB2, RGS2 and VCAN genes proposed as biomarkers of pregnancy in cumulus cells by quantitative polymerase chain reaction. Gene expression was evaluated in 43 individual cumulus cell samples, derived from a highly homogenous group of 43 women. The same protocol for ovarian stimulation was used for all women, and elective single embryo transfer was performed. Expression levels of RGS2 and VCAN did not differ between cumulus cells of implanted and non-implanted embryos. EFNB2 showed borderline higher expression in cumulus cells of non-implanted embryos, which is contradictory to previous studies. Altogether, the results of previous studies in which EFNB2, RGS2 and VCAN were proposed as biomarkers of pregnancy could not be replicated in our set of cumulus cell samples.

The Genetic Architecture of Congenital Heart Disease in Neonatal Intensive Care Unit Patients-The Experience of University Medical Centre, Ljubljana.

Congenital heart disease (CHD) is the most commonly detected congenital anomaly and affects up to 1% of all live-born neonates. Current guidelines support the use of chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) as diagnostic approaches to identify genetic causes. The aim of our study was to evaluate the diagnostic yield of CMA and NGS in a cohort of neonates with both isolated and syndromic CHD. The present study included 188 infants under 28 days of age with abnormal echocardiography findings hospitalized at the Department of Neonatology, UMC Ljubljana, between January 2014 and December 2023. Phenotypic data were obtained for each infant via retrospective medical chart review. We established the genetic diagnosis of 22 distinct syndromes in 17% (32/188) of neonates. The most frequent genetic diagnoses in diagnosed cases were 22q11.2 microdeletion and CHARGE syndromes, followed by Noonan syndrome and Williams syndrome. In addition, we detected variants of uncertain significance in 4.8% (9/188) of neonates. Timely genetic diagnosis is important for the detection of syndrome-related comorbidities, prognosis, reproductive genetic risks and, when appropriate, genetic testing of other family members.

Assessment of pathogenic variation in gynecologic cancer genes in a national cohort.

Population-based estimates of pathogenic variation burden in gynecologic cancer predisposition genes are a prerequisite for the development of effective precision public health strategies. This study aims to reveal the burden of pathogenic variants in a comprehensive set of clinically relevant breast, ovarian, and endometrial cancer genes in a large population-based study. We performed a rigorous manual classification procedure to identify pathogenic variants in a panel of 17 gynecologic cancer predisposition genes in a cohort of 7091 individuals, representing 0.35% of the general population. The population burden of pathogenic variants in hereditary gynecologic cancer-related genes in our study was 2.14%. Pathogenic variants in genes ATM, BRCA1, and CDH1 are significantly enriched and the burden of pathogenic variants in CHEK2 is decreased in our population compared to the control population. We have identified a high burden of pathogenic variants in several gynecologic cancer-related genes in the Slovenian population, most importantly in the BRCA1 gene.

Optical genome mapping in an atypical Pelizaeus-Merzbacher prenatal challenge.

Pathogenic genetic variants represent a challenge in prenatal counseling, especially when clinical presentation in familial carriers is atypical. We describe a prenatal case involving a microarray-detected duplication of PLP1 which causes X-linked Pelizaeus-Merzbacher disease, a progressive hypomyelinating leukodystrophy. Because of atypical clinical presentation in an older male child, the duplication was examined using a novel technology, optical genome mapping, and was found to be an inverted duplication, which has not been previously described. Simultaneously, segregation analysis identified another healthy adult male carrier of this unique structural rearrangement. The novel PLP1 structural variant was reclassified, and a healthy boy was delivered. In conclusion, we suggest that examining structural variants with novel methods is warranted especially in cases with atypical clinical presentation and may in these cases lead to improved prenatal and postnatal genetic counseling.

Evaluation of Optical Genome Mapping in Clinical Genetic Testing of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, resulting in the faulty expression of the DUX4 gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.

The Endometrial Transcriptome of Metabolic and Inflammatory Pathways During the Window of Implantation Is Deranged in Infertile Obese Polycystic Ovarian Syndrome Women.

Introduction and Aim: Obese women with polycystic ovarian syndrome (PCOS) have a reduced rate of spontaneous conception even when their cycles are ovulatory. Endometrial receptivity is an important factor for poor implantation and increased miscarriage rates. Mechanisms in which both pathologies modify the endometrium are not fully clarified. The aim of our study was to compare the endometrial transcriptomic profiles between infertile obese PCOS (O-PCOS) women and infertile normal weight subjects during the window of implantation in ovulatory menstrual cycles. Methods: We conducted a prospective transcriptomic analysis of the endometrium using RNA sequencing. In this way, potential endometrial mechanisms leading to the poor reproductive outcome in O-PCOS patients could be characterized. Endometrial samples during days 21-23 of the menstrual cycle were collected from infertile O-PCOS women (n = 11) and normal weight controls (n = 10). Subgroups were defined according to the ovulatory/anovulatory status in the natural cycles, and O-PCOS women were grouped into the O-PCOS ovulatory (O-PCOS-ovul) subgroup. RNA isolation, sequencing with library reparation, and subsequent RNAseq data analysis were performed. Results: Infertile O-PCOS patients had 610 differentially expressed genes (DEGs), after adjustment for multiple comparisons with normal weight infertile controls, related to obesity (MXRA5 and ECM1), PCOS (ADAMTS19 and SLC18A2), and metabolism (VNN1 and PC). In the ovulatory subgroup, no DEGs were found, but significant differences in canonical pathways and the upstream regulator were revealed. According to functional and upstream analyses of ovulatory subgroup comparisons, the most important biological processes were related to inflammation (TNFR1 signaling), insulin signaling (insulin receptor signaling and PI3/AKT), fatty acid metabolism (stearate biosynthesis I and palmitate biosynthesis I), and lipotoxicity (unfolded protein response pathway). Conclusions: We demonstrated that endometrial transcription in ovulatory O-PCOS patients is deranged in comparison with the control ovulatory endometrium. The most important pathways of differentiation include metabolism and inflammation. These processes could also represent potential mechanisms for poor embryo implantation, which prevent the development of a successful pregnancy. ClinicalTrials.gov ID: NCT03353948.

Recommendations for reporting results of diagnostic genomic testing.

Results of clinical genomic testing must be reported in a clear, concise format to ensure they are understandable and interpretable. It is important laboratories are aware of the information which is essential to make sure the results are not open to misinterpretation. As genomic testing has continued to evolve over the past decade, the European Society of Human Genetics (ESHG) recommendations for reporting results of diagnostic genetic testing (biochemical, cytogenetic and molecular genetic) published in 2014 have been reviewed and updated to provide the genomic community with guidance on reporting unambiguous results.

Potential protective role of a NOD2 polymorphism in the susceptibility to multiple sclerosis is not associated with interferon therapy.

Pattern recognition receptors, such as specific nucleotide-binding oligomerization domain protein 2, and their polymorphisms may be involved in the pathogenesis of multiple sclerosis (MS). They may also play a role in the formation of neutralizing antibodies against interferon-ß (INF-ß), and may exhibit lowered efficacy. Identification of these polymorphisms may be useful for early identification of potential non-responders and to allow for modification of treatment regimens earlier. The differences in genotype distribution and allele frequency of the rs3135499 and rs2066842 NOD2 polymorphisms between patients with MS and healthy controls were analysed in the present study. The group of patients were divided into responders and non-responders to INF-ß therapy to evaluate the association of both polymorphisms with response to therapy. No differences in the genotype frequencies between the responder and non-responder groups were observed. However, a statistically significant difference in genotype frequencies of TT homozygotes for rs2066842 between patients with MS and healthy controls was observed (χ2=11.8; P=0.003). A recessive genotype model and allele distribution in rs2066842 suggest that the genotype TT and allele T itself are protective against MS. The odds ratio of 0.12 represents an 8.33x lower risk for MS if an individual has a TT genotype. The significantly lower incidence of the TT genotype of rs2066842 in patients with MS suggests that the TT genotype and T allele may be a protective genetic factor against MS.