Heterozygous State Research Articles

IFIH1 loss of function predisposes to inflammatory and SARS-CoV-2-related infectious diseases.

The IFIH1 gene, encoding melanoma differentiation-associated protein 5 (MDA5), is an indispensable innate immune regulator involved in the early detection of viral infections. Previous studies described MDA5 dysregulation in weakened immunological responses, and increased susceptibility to microbial infections and autoimmune disorders. Monoallelic gain-of-function of the IFIH1 gene has been associated with multisystem disorders, namely Aicardi-Goutieres and Singleton-Merten syndromes, while biallelic loss causes immunodeficiency. In this study, nine patients suffering from recurrent infections, inflammatory diseases, severe COVID-19 or multisystem inflammatory syndrome in children (MIS-C) were identified with putative loss-of-function IFIH1 variants by whole-exome sequencing. All patients revealed signs of lymphopaenia and an increase in inflammatory markers, including CRP, amyloid A, ferritin and IL-6. One patient with a pathogenic homozygous variant c.2807+1G>A was the most severe case showing immunodeficiency and glomerulonephritis. The c.1641+1G>C variant was identified in the heterozygous state in patients suffering from periodic fever, COVID-19 or MIS-C, while the c.2016delA variant was identified in two patients with inflammatory bowel disease or MIS-C. There was a significant association between IFIH1 monoallelic loss of function and susceptibility to infections in males. Expression analysis showed that PBMCs of one patient with a c.2016delA variant had a significant decrease in ISG15, IFNA and IFNG transcript levels, compared to normal PBMCs, upon stimulation with Poly(I:C), suggesting that MDA5 receptor truncation disrupts the immune response. Our findings accentuate the implication of rare monogenic IFIH1 loss-of-function variants in altering the immune response, and severely predisposing patients to inflammatory and infectious diseases, including SARS-CoV-2-related disorders.

Global Proteomic Analysis Reveals Alterations in Differentially Expressed Proteins between Cardiopathic Lamin A/C Mutations.

Lamin A/C (LMNA) is an important component of nuclear lamina. Mutations cause arrhythmia, heart failure, and sudden cardiac death. While LMNA-associated cardiomyopathy typically has an aggressive course that responds poorly to conventional heart failure therapies, there is variability in severity and age of penetrance between and even within specific mutations, which is poorly understood at the cellular level. Further, this heterogeneity has not previously been captured to mimic the heterozygous state, nor have the hundreds of clinical LMNA mutations been represented. Herein, we have overexpressed cardiopathic LMNA variants in HEK cells and utilized state-of-the-art quantitative proteomics to compare the global proteomic profiles of (1) aggregating Q353 K alone, (2) Q353 K coexpressed with WT, (3) aggregating N195 K coexpressed with WT, and (4) nonaggregating E317 K coexpressed with WT to help capture some of the heterogeneity between mutations. We analyzed each data set to obtain the differentially expressed proteins (DEPs) and applied gene ontology (GO) and KEGG pathway analyses. We found a range of 162 to 324 DEPs from over 6000 total protein IDs with differences in GO terms, KEGG pathways, and DEPs important in cardiac function, further highlighting the complexity of cardiac laminopathies. Pathways disrupted by LMNA mutations were validated with redox, autophagy, and apoptosis functional assays in both HEK 293 cells and in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for LMNA N195 K. These proteomic profiles expand our repertoire for mutation-specific downstream cellular effects that may become useful as druggable targets for personalized medicine approach for cardiac laminopathies.

Wilson’s Disease Caused by Previously Undescribed Homozygous Nucleotide Variant of the <i>ATP7B</i> Gene: Clinical Cases

Wilson’s disease is severe autosomal recessive disease manifested primarily by hepatic, neurological, and psychiatric disorders due to excessive copper deposition in organs and tissues. Clinical case description. The variant with uncertain clinical value of the ATP7B gene, c.2111C>T (p.T704I, chr13:52534294G>A (HG19)), was described in the family where parents are cousins. The eldest daughter out of four children died at the age of 11 due to liver cirrhosis. Wilson’s disease was genetically confirmed in two children (clinically — abdominal form). The younger son was diagnosed heterozygous state of the disease (without any clinical manifestations). The revealed variant of the ATP7B gene was previously identified in 3 more patients with Wilson’s disease, however, in a compound heterozygous state with known pathogenic genetic variant. Conclusion. c.2111C>T (p.T704I) variant of the ATP7B gene can be considered as probably pathogenic. Further research is required to evaluate its functional significance in Wilson’s disease pathogenesis.

Study of the potential role of CASPASE-10 mutations in the development of autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS, FASLG, and FADD, all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute’s genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

Influence of Interferon Gamma Gene rs2430561 Variant on Complicated Course of Pneumonia in Patients with COVID-19

Background: Pneumonia is one of the most common complications of the course of COVID-19. Interferon-γ (IFN-γ) is an essential cytokine for lung defense against intracellular and extracellular pathogens. Since the rs2430561 variant of the IFNG gene, which encodes IFN-γ, affects the production of IFN-γ, it can potentially be associated with a severe course of COVID-19. Objective: The aim of our research was to determine the impact of the rs2430561 variant of the IFNG gene on the propensity of patients to develop and experience a severe course of COVID-19-related pneumonia. Methods: The study included 117 patients with a complicated course of COVID-19- associated pneumonia, who were hospitalized in the intensive care unit. All patients received a standard clinical and laboratory evaluation and course of treatment for COVID- 19. IFNG gene variants were determined by PCR method. For comparisons, we used clinical and laboratory indicators from the inpatients’ medical records, which reflected the course of the patient's disease, taking into account the rs2430561 variants of the IFNG gene. Results: In the group of patients, the following genotype frequencies were found: TT – 18.8%, TA – 52.1%, and AA – 29.1%. The results showed that AA genotype carriers had essentially higher SpO2/FiO2 ratio (p=0.043). High base excess rates were present in patients with the TT genotype (p=0.047). It was found that patients with the T allele (whether homozygous or heterozygous state) had significantly higher concentrations of such electrolytes as potassium and sodium (p=0.016 and p=0.004, respectively). Сonclusion: In patients with COVID-19, the rs2430561 variant of the IFNG gene is linked to a complicated course of pneumonia. Certainly, this finding requires further research.

Identification of a novel GNAS mutation in a family with pseudohypoparathyroidism type 1A

BackgroundPseudohypoparathyroidism (PHP) is caused by loss-of-function mutations at the GNAS gene (as in the PHP type 1A; PHP1A), de novo or inherited at heterozygous state, or by epigenetic alterations at the GNAS locus (as in the PHP1B). The condition of PHP refers to a heterogeneous group of disorders that share common clinical and biological features of PTH resistance. Manifestations related to resistance to other hormones are also reported in many patients with PHP, in association with the phenotypic picture of Albright hereditary osteodystrophy characterized by short stature, round facies, subcutaneous ossifications, brachydactyly, mental retardation and, in some subtypes, obesity. The purpose of our study is to report a new mutation in the GNAS gene and to describe the significant phenotypic variability of three sisters with PHP1A bearing the same mutation.Case presentationWe describe the cases of three sisters with PHP1A bearing the same mutation but characterized by a significantly different phenotypic picture at onset and during follow-up in terms of clinical features, auxological pattern and biochemical changes. Clinical exome sequencing revealed a never before described heterozygote mutation in the GNAS gene (NM_000516.5 c.118_139 + 51del) of autosomal dominant maternal transmission in the three siblings, confirming the diagnosis of PHP1A.ConclusionsThis study reported on a novel mutation of GNAS gene and highlighted the clinical heterogeneity of PHP1A characterized by wide genotype–phenotype variability. The appropriate diagnosis has crucial implications for patient care and long-term multidisciplinary follow-up.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO–K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

COMBINATION OF TWO DIFFERENT C174T AND C235T OF THE ANGIOTENSINOGEN GENE MUTATION AND C677T METHYLENETETRAHYDROFOLATE REDUCTASE GENE MUTATION IN PATIENTS WITH CVD IN THE POPULATION OF AZERBAIJAN

We studied two mutations of C174T (Met174Tre) and C235T (Met235Tre) of the angiotensinogen gene and mutation C677T (Ala677Val) of the methylenetetrahydrofolate reductase gene, as well as their combinations among patients with cardiovascular diseases: coronary heart disease, myocardial infarction and hypertension, using a complex of modern molecular genetic diagnostic methods. A high frequency of these mutations has been established in the group of patients with severe forms of cardiovascular disorders. Homozygous and compound conditions were found in people with severe diseases. In particular, the heterozygous state of the mutation was found in people with moderate hypertension. It was also found that the presence of close blood relationship between the parents of patients with cardiovascular diseases increases the homozygotization of mutations of the genes AGT and MTGFR in probands. Consequently, such persons are susceptible to the disease or in other words, have an increased risk of developing diseases of the cardiovascular system. For the first time, the frequencies of these mutations in the Azerbaijani population have been determined, which are mostly consistent with the frequencies described in other populations of the world. The studied genetic markers are of interest as genes presumably associated with a wide range of cardiovascular diseases and can be used in further population and epidemiological studies. Therefore, the results of molecular genetic studies obtained by us in people with diseases of the cardiovascular system are of great practical importance. Timely prevention by detecting mutations of C174T, C235T of the AGT gene and C677T of the MTGFR gene in patients will allow doctors to carry out qualified treatment of cardiovascular diseases

High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Loss-of-function variants affecting the STAGA complex component SUPT7L cause a developmental disorder with generalized lipodystrophy

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Sudden cardiac death triggered by minimal alcohol consumption in the context of novel PPA2 mutations in 2 unrelated families

Biallelic variants in PPA2 gene cause a rare but lethal mitochondrial disorder. We describe the first four cases reported in Spain of PPA2 disease in two unrelated families. We have conducted a revision of the clinical history, necropsies, and postmortem genetic testing from probands, and clinical evaluation, genetic testing and blood transcript analysis in family members. All the cases harbored biallelic PPA2 variants in compound heterozygous status. Two brothers from family 1 suffered sudden death after a small first intake of alcohol in 2013 and 2022. The sister remains alive but affected with cardiomyopathy, extensive scar on cardiac imaging, and high sensitivity to alcohol intake. The three siblings carried PPA2 c.290A > G (p.Glu97Gly) novel missense variant and PPA2 c.513C > T (p.Cys171 = ) altering splicing site variant, both probably leading to mRNA degradation based on in-silico and transcript analyses. A teenager from family 2 suffered sudden death after a small intake of alcohol in 2018 and carried PPA2 c.683C > T (p.Pro228Leu) missense and PPA2 c.980_983del (p.Gln327fs) novel frameshift variant, both probably leading to abnormal protein structure. All cases were asymptomatic until adolescence. Furthermore, the sister in family 1 has survived as an asymptomatic adult. PPA2 disease can manifest as cardiac arrest in the young, especially after alcohol exposure. Our results show that PPA2 deficiency can be related to different pathogenicity mechanisms such as abnormal protein structure but also mRNA decay caused by synonymous or missense variants. Strict avoidance of alcohol consumption and early defibrillator implantation might prevent lethal arrhythmias in patients at risk.

A novel frameshift mutation in RHAG leads to Rhnull phenotype in a Chinese individual.

We recently encountered a Rhnull phenotype proband within one family in the Chinese population. Rhnull is a rare autosomal recessive disorder characterized by the absence of the Rh antigens on the erythrocyte membrane, resulting in chronic hemolytic anemia. This study described the serological and molecular analysis of a Chinese Rhnull proband and his immediate family. Red blood cells antigen phenotyping and antibody screening/identification were conducted. RHD, RHCE, and RHAG were analyzed using genomic DNA by polymerase chain reaction and sequence analysis. Serologic tests showed a D-C-E-c-e- phenotype in the proband associated with the suspicion of anti-Rh29 (titer 16). Molecular analyses showed a new mutation (c.406dupA) in exon 3 of RHAG. This duplication introduced a reading frameshift (p.Thr136AsnfsTer21). The RHAG mutation was found in the homozygous state for the proband and heterozygous state for his parents. We identified a novel RHAG mutation resulting in the Rhnull phenotype of the regulator type. Inheritance of the novel allele was shown by family study.

A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Поліморфізм клінічних проявів орфанних захворювань у дітей із мутаціями генів ARID1A та ARID1B

The Department of Psychoneurology for Children with Perinatal Pathology and Orphan Diseases at the SI “Institute of Pediatrics, Obstetrics and Gynecology named after academician O.M. Lukyanova of the NAMS of Ukraine” has a multidisciplinary team which provides care to children with orphan diseases from all over Ukraine. Thanks to modern methods of clinical, instrumental and genetic diagnostics, it is possible to establish a definitive diagnosis. Aim - to provide a literature review and a clinical case of a child with pathogenic variants of the ARID family genes, which, in particular, lead to the development of Coffin-Circe syndrome (CCS), autism spectrum disorder and intellectual disability, as well as the development of nervous system tumors in children. The article presents a clinical case of a child with a rare disease - CCS, which is part of the ARID gene mutation spectrum. The general clinical and neurological examination, electroencephalographic sleep monitoring, magnetic resonance imaging, whole-exome sequencing, and pathological examination were performed. The difficulty of diagnosis was that a child with skin manifestations of phacomatosis was diagnosed with subependymal giant cell astrocytoma (SGСA) by magnetic resonance imaging, but there were no mutations characteristic of tuberous sclerosis (TSC1, TSC2). Pathologically, the patient was confirmed to have grade I SGСA according to the World Health Organization classification. Two pathogenic mutations of the ARID1A gene in the compound heterozygous state were detected by whole-exome sequencing. After combining the combined clinical and instrumental signs, the diagnosis of CCS with grade I SGСA was established. Conclusions. The disorders caused by ARID1A and ARID1B mutations belong to a spectrum that includes syndromic and non-syndromic cases of intellectual disability, autism spectrum disorder, and CCS with or without a variety of somatic and neurological symptoms, and are grouped under the general term “ARID-related disorders”. Only a thorough medical history, clinical examination, and modern methods of instrumental and genetic diagnostics can make a diagnosis. The research was carried out in accordance with the principles of the Declaration of Helsinki. Informed consent of the women was obtained for the research. No conflict of interests was declared by the authors.

FAM20A-Associated Amelogenesis Imperfecta: Gene Variants with Functional Verification and Histological Features.

To investigate FAM20A gene variants and histological features of amelogenesis imperfecta and to further explore the functional impact of these variants. Whole-exome sequencing (WES) and Sanger sequencing were used to identify pathogenic gene variants in three Chinese families with amelogenesis imperfecta. Bioinformatics analysis, in vitro histological examinations and experiments were conducted to study the functional impact of gene variants, and the histological features of enamel, keratinised oral mucosa and dental follicle. The authors identified two nonsense variants c. 406C > T (p.Arg136*) and c.826C > T (p.Arg176*) in a compound heterozygous state in family 1, two novel frameshift variants c.936dupC (p.Val313Argfs*67) and c.1483dupC (p.Leu495Profs*44) in a compound heterozygous state in family 2, and a novel homozygous frameshift variant c.530_531insGGTC (p.Ser178Valfs*21) in family 3. The enamel structure was abnormal, and psammomatoid calcifications were identified in both the gingival mucosa and dental follicle. The bioinformatics and subcellular localisation analyses indicated these variants to be pathogenic. The secondary and tertiary structure analysis speculated that these five variants would cause structural damage to FAM20A protein. The present results broaden the variant spectrum and clinical and histological findings of diseases associated with FAM20A, and provide useful information for future genetic counselling and functional investigation.

Hemoglobin Variants in Patients Attending Aden Diagnostic Center by High Performance Liquid Chromatography

Introduction: High performance liquid chromatography (HPLC) is emerging as the method of choice for initial screening and diagnosis of hemoglobinopathies. The use of alkaline and acid gel electrophoresis may result in incorrect diagnosis of hemoglobinopathies. The aim of the study is to investigate the hemoglobin pattern using the HPLC and to correlate the hematological profile with the types of hemoglobin. Methods: A cross-sectional study was conducted in Aden Diagnostic Center from July– December 2022. Over a six-month period, 250 samples of patients aged between six months to thirty years, were evaluated by HPLC for detection of hemoglobinopathies using the Lifotronic Hemoglobin Analyzer H9: β-thalassemia Analysis Mode. Red blood cell and red cell indices were determined using automated hematology analyzer. Results: A total of 152 samples (60.8%) showed different abnormal hemoglobin variants. Sixty-four (25.6%) were diagnosed to have sickle cell anemia, 46 (18.4%) as sickle cell trait, two (0.8) as beta – heterozygous thalassemia based on high level of HbA2 (>3.9%), four (1.6%) as beta – homozygous thalassemia (HbF 25–91%), 18 (7.2%) as compound heterozygous state of sickle - β+ thalassemia, 17 (6.8%) as beta thalassemia trait, and one (0.4%) sample had HbD variant on HPLC was diagnosed as HbD trait. Conclusion: H9-HPLC is suitable for the routine investigation of hemoglobinopatheis because it is very powerful tool in the evaluation of Hb variants, rapid assay time and accurate quantification.

Identification of some genetic markers as productive and reproductive traits in Ukrainian dairy cattle breeding

A Selection based on DNA markers is a breeding technique grounded in the genomic selection value of animals. The aim of the research is to study the genotypic profile of cows of different breeds in Ukrainian breeding in relation to the genes LEP, CSN3, TG5, BLG, and Pit-1 and to identify the probability of using them as markers for reproductive traits in cows. During the study, the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method was used to analyze the genes and determine their polymorphic characteristics. The obtained data indicated that the same gene variants have varying effects in the studied breeds due to their diverse influences on the genomic background. Specifically, the homozygous state of leptin genes (LEPCC) and pituitary-specific transcription factor (PIT-1BB) was observed to impact the reproductive characteristics of cows in the evaluated breeds. Meanwhile, for the genes casein (CSN3AB), thyroglobulin (TG5CT), and beta-lactoglobulin (BLGAВ), the heterozygous state of alleles was found to influence the key reproductive traits of dairy cattle of Holstein origin. The polymorphism of the genes CSN3, βLG, TG, PIT-1, and LEP indicated the presence of genetic potential for the reproductive function of cows and can be utilized as molecular markers in selective breeding, providing significant progress in improving not only the traits of dairy cattle productivity but also reproductive function. Therefore, in the implementation of selective breeding work, it is advisable to consider genotyping for the genes CSN3, βLG, TG, PIT-1, and LEP as an additional criterion for the selection of animals to enhance both their milk and reproductive characteristics.

Prevalence of MTHFR Polymorphisms in Patients With Hypermobile Ehlers-Danlos Syndrome and Hypermobile Spectrum Disorders in a US Hypermobility Clinic.

Hypermobile Ehlers-Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD) are characterized by joint hypermobility, joint subluxations and dislocations, hyperextensible skin, and chronic and progressive multiorgan comorbidities. Diagnosing hEDS and HSD is difficult because of variable phenotypes and unknown genetic etiology. In our clinic, we observed many patients with hEDS and HSD with a high serum level of unmetabolized folate, which suggests that hypermobility may be linked to methylenetetrahydrofolate reductase (MTHFR)-mediated folate metabolism. The present study aims to examine the prevalence of MTHFR polymorphisms, C677T and A1298C, among patients with hEDS and HSD. Clinical and demographic information of patients visiting our hypermobility clinic from January 2023 to July 2023 were retrospectively reviewed. Continuous variables were reported as mean ± SD and range, whereas categorical variables were reported as total count and percentage. Among 157 patients, 93% of patients were female patients, 52.2% were diagnosed with hEDS, and 47.8% were diagnosed with HSD. Interestingly, 85% of the patients had MTHFR C677T and/or A1298C polymorphisms in heterozygous or homozygous state. MTHFR 677CT/TT genotype was present in 52.9% of cases, and 49.7% of patients had 1298AC/CC genotype. In addition,14% of patients with hypermobility exhibited MTHFR 677TT genotype, 10.2% showed 1298CC genotype, and 17.2% displayed combined heterozygosity, collectively representing 41.4% hypermobile patients with two copies of MTHFR variant alleles. There is a high prevalence of MTHFR polymorphisms among patients with hypermobility, which supports the hypothesis that hypermobility may be dependent on folate status.

DISP1 deficiency: Monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations

PurposeDISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog, a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with division transporter dispatched-1 (DISP1) variants. MethodsThis study was based on the identification of at least 1 pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. ResultsA total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor, and congenital nasal pyriform aperture stenosis. These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (ie, oligogenic inheritance). ConclusionThe genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of Sonic hedgehog. We also demonstrated that the very rare solitary median maxillary central incisor and congenital nasal pyriform aperture stenosis combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

Abstract 1410: An effect of Phenylalanine hydroxylase (PAH) deficiency on cancer development in kidney

Abstract Several studies have noted the importance of amino acid metabolism in cancer progression. Preliminary research using public cancer multi-omics data has revealed associations between heterozygous germline mutations in genes associated with amino acid metabolism and cancer development. In particular, the heterozygous status of the PAH gene enhances the risk of developing kidney cancer by more than three times. Phenylalanine hydroxylase (PAH) is an enzyme that converts phenylalanine to tyrosine. It is highly expressed in the liver and kidneys. Analysis of the blood metabolome in prospectively collected PAH heterozygous carriers indicates that heterozygous status causes metabolome fluctuations in the body. By the way, renal cell carcinoma (RCC) is an immunogenic tumor mainly infiltrated by T cells. Accordingly, we suggest that defects in the PAH gene change the metabolites in the body, leading to a reduction in immune function and an increased risk of developing kidney cancer. First, the expression of the PAH protein was confirmed through immunohistochemistry in a tissue microarray from RCC patients, and the results were then compared with clinical information. Next, we simulated the environment of PAH deficiency in vitro by treating the cells with phenylalanine (Phe) and its metabolite, phenylpyruvate (PRA), which accumulates during PAH dysfunction. The cytotoxicity of Phe and PPA was investigated using the WST assay. The Jurkat T cell line was cultured long-term in a medium containing Phe or PPA and then stimulated with PHA/PMA. Cytokine secretion was confirmed in these supernatants. In addition, purified primary human pan T cells were stimulated with Anti-CD3/CD28 in the medium containing either Phe or PPA. Proliferation was evaluated using CFSE staining. The IHC was performed on 467 tissues from RCC patients aged 29 to 101 years. We classified the cases into three scale scores based on the percentage of PAH intensity. Approximately 73% of cases were PAH-negative, and only 27% were positive (score 1+ or 2+). A comparison of IHC results with clinical information showed that lower PAH expression was associated with a lower relapse-free survival rate. Next, Phe and PPA exhibited minimal cytotoxicity. The secretion of representative proinflammatory cytokines IL-2 and TNF- α were dose-dependent and significantly decreased with Phe and PPA treatment. Additionally, cell proliferation, a critical step in acquiring effector functions of T cells, was inhibited by treatment with Phe and PPA, particularly in the CD8+ T cell population compared to CD4+ T cells. These results suggest that metabolite changes within the tissue microenvironment of the kidney, caused by Pah defects, may suppress anti-tumor immunity by impairing the function of immune cells. This novel discovery may contribute to the importance of the PAH gene as a new biomarker for diagnosing kidney cancer and to the development of kidney cancer therapy. Citation Format: Seojeong Kim, Jihyun Park, Jeongmin Park, Chaelin Kang, Kyung Chul Moon, Seung Seok Han, Youngil Koh, Sung-Soo Yoon. An effect of Phenylalanine hydroxylase (PAH) deficiency on cancer development in kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1410.