Association of Alleles of Human Leukocyte Antigen Class II Genes and Severity of COVID-19 in Patients of the 'Red Zone' of the Endocrinology Research Center, Moscow, Russia.

The aim of this study was to assess the correlations of clinical features of patients with moderate and severe courses of COVID-19, comorbidity (endocrine, autoimmune, cardiovascular, oncological, and pulmonary diseases), and alleles of the HLA class II system genes. One hundred COVID-19 patients hospitalized in the Endocrinology Research Centre, Moscow, Russia, were analyzed for age, gender, smoking, comorbidity, and invasive mechanical ventilation. Computer tomography was used to assess the severity of the disease. HLA-DRB1, HLA-DQA1, and HLA-DQB1 alleles were identified in samples from 100 patients and samples from 327 randomly selected individuals collected in the prepandemic period (control group). There was no association of gender, age, weight, body mass index, smoking, and comorbidity with the severity of COVID-19. Allele DQB1*06:02-8 was more common in patients (p < 0.00005), and DQB1*06:01 and DQB1*05:03 were more common in the control group (p < 0.00005, and p = 0.0011, respectively). DQB1*06:02-8 can probably be considered as predisposing to moderate and severe COVID-19, and DQB1*06:01 can be considered as protective. No association of these alleles with comorbidity was found. Our results suggest that carriers of predisposing alleles, with cardiovascular and non-autoimmune endocrine diseases, should take more stringent preventive measures, and if infected, a more aggressive COVID-19 treatment strategy should be used.

A Founder Mutation in the POMC 5'-UTR Causes Proopiomelanocortin Deficiency Through Splicing-Mediated Decrease of mRNA.

CONTEXT: The syndrome of adrenal insufficiency, obesity, and red hair is a rare autosomal recessive disorder. The majority of disease-causing variants associated with the syndrome are located in the coding region of the POMC gene. OBJECTIVE: This work describes 7 unrelated patients who shared a novel homozygous mutation in the 5'-untranslated region (UTR) of the POMC gene and functionally characterize this novel variant. METHODS: Whole-exome sequencing (WES) with autozygosity mapping, Sanger sequencing, model expression system studies, and RNA sequencing were used for identification of the disease-causing variant and its subsequent functional characterization. Seven unrelated patients of the Perm Tatar ethnic group presented with hypoglycemia and excessive weight gain, low plasma adrenocorticotropin, and cortisol. Five of 7 children had red hair; 6 of 7 patients also showed signs of bronchial obstruction. RESULTS: WES showed shared autozygosity regions overlapping the POMC gene. Sanger sequencing of the POMC 5'-UTR detected a homozygous variant chr2:25391366C > T (hg19) at the splice donor site of intron 1. As demonstrated by the model expression system, the variant led to a significant decrease in the POMC messenger RNA level. Analyses of the patients' haplotypes were suggestive of the founder effect. We estimate that the mutation must have occurred at least 4.27 generations ago (95% CI, 0.86-7.67). CONCLUSION: This report presents a new molecular mechanism of POMC deficiency and contributes to the information on phenotypic variability in patients with this disorder.

Circular RNAs and thyroid cancer: Closed molecules, open possibilities.

Thyroid neoplasms requiring differential diagnosis between thyroid cancer and benign tumors can be detected in more than half of the healthy population. A generally accepted method that allows assessing the risk of malignant potential and determining the indications for surgical treatment of thyroid tumor is a fine-needle aspiration biopsy followed by a cytological examination. Nevertheless, in patients with indeterminate categories of cytological conclusions according to Bethesda system, the positive predictive value of the cytology result is significantly lower than desired and often leads to unjustified surgical treatment. In this regard, the search for alternative diagnostic solutions continues. Circular RNAs are a group of non-coding RNAs distinguished by a closed structure formed by covalent bonding of the nucleotide chain ends. Recent studies allow us to conclude that many different circular RNAs are involved in processes mediating oncogenesis in the thyroid gland, and their altered expression in tissue, blood, and exosomes of plasma may be a characteristic sign of thyroid cancer and certain clinicopathological features of its course. The purpose of this review is to analyze the accumulated data on the association of various circular RNAs with thyroid cancer and to discuss possible ways to improve the diagnosis and treatment of the disease based on the assessment of the expression of these molecules.

Familial Acromegaly and Bilateral Asynchronous Pheochromocytomas in a Female Patient With a MAX Mutation: A Case Report.

Background: There are very few cases of co-occurring pituitary adenoma (PA) and pheochromocytomas (PCC)/paragangliomas caused by MAX mutations. No cases of familial PA in patients with MAX mutations have been described to date. Case Presentation: We describe a 38-year-old female patient, presenting with clinical and biochemical features of acromegaly and PCC of the left adrenal gland. Whole-exome sequencing was performed [NextSeq550 (Illumina, San Diego, CA, USA)] identifying a nonsense mutation in the MAX gene (NM_002382) [c.223C>T (p.R75X)]. The patient had a medical history of PCC of the right adrenal gland diagnosed aged 21 years and prolactinoma diagnosed aged 25 years. Cabergoline treatment was effective in achieving remission of prolactinoma at age 33 years. The patient's father who died at age 56 years of a heart attack had a medical history of PA and prominent acromegalic features, which supports the familial presentation of the disease. Conclusion: This clinical case gives an insight into the clinical presentation of familial PA and PCC probably associated with a MAX mutation.

Search for Genetic Predictors of Adult Autoimmune Polyendocrine Syndrome in Monozygotic Twins.

Autoimmune polyendocrine syndromes (APS) are a heterogeneous group of diseases characterized by the presence of autoimmune dysfunction of 2 or more endocrine glands and other non-endocrine organs. The components of the syndrome can manifest throughout life: in childhood-APS type 1 (the juvenile type) and in adulthood-APS type 2, 3, and 4 (the adult types). Adult types of APS are more common in clinical practice. It is a polygenic disease associated with abnormalities in genes encoding key regulatory proteins of the major histocompatibility complex (MHC). The search of for candidate genes responsible for mutations in adult APS is continuing. Genetic predisposition is insufficient for the manifestation of the APS of adults, since the penetrance of the disease, even among monozygotic twins, does not approach 100% (30-70%). The article presents the case of isolated Addison's disease and APS type 2 in monozygotic twins with a revealed compound heterozygosity in the candidate gene VTCN1.

Successful Use of Denosumab for Life-Threatening Hypercalcemia in a Pediatric Patient with Primary Hyperparathyroidism.

INTRODUCTION: Primary hyperparathyroidism (PHPT) is rare and usually symptomatic in children. There is no approved medication to lower serum calcium levels in this patient group. Denosumab is used in adult patients with osteoporosis and hyperparathyroidism. To our knowledge, only 1 case of denosumab treatment in a child with severe PHPT has been reported to date. CASE PRESENTATION: A 16-year-old female was referred to our clinic with symptoms including pathologic fractures, nausea, emesis, and progressive weight loss. At admission, her serum total calcium was 4.17 mmol/L (reference range 2.15-2.55), parathyroid hormone 2,151 pg/mL (15-65), and phosphate 1.07 mmol/L (1.45-1.78). Due to potentially life-threatening hypercalcemia, denosumab 60 mg subcutaneously was administered after obtaining informed consent. Serum calcium levels were reduced within 12 h of injection and the patient's condition rapidly improved, which allowed genetic testing to be done prior to surgery. A heterozygous mutation in the CDC73 gene was revealed, and a parathyroidectomy was performed on day 22 after denosumab administration. Morphological examination revealed solitary parathyroid adenoma. After surgery, hypocalcemia developed requiring high doses of alfacalcidol and calcium supplements. CONCLUSION: Our case supports the previous observations in adults that denosumab can be safely and effectively used as a preoperative treatment in patients with PHPT and severe hypercalcemia and shows that it may be used in pediatric patients.

Insulin autoimmune syndrome.

SUMMARY: Insulin autoimmune syndrome (Hirata's disease) is a disorder caused by development of autoantibodies to insulin and manifested by hypoglycaemic syndrome. The overwhelming majority of physicians do not include it in the differential diagnosis of hypoglycaemic states because of a misconception of an extremely low prevalence of this condition. This results in unnecessary drug therapy and unjustified surgical interventions in patients that otherwise would be successfully treated conservatively. This disease is strongly associated with certain alleles of the HLA gene. In most cases, this condition develops in predisposed individuals taking drugs containing sulfhydryl groups. Formation of autoantibodies to insulin may be observed in patients with other autoimmune disorders, as well as in those with multiple myeloma or monoclonal gammopathy of undetermined significance. This paper presents the first Russian case report of insulin autoimmune syndrome in an adult patient. LEARNING POINTS: Insulin autoimmune syndrome, Hirata's disease, anti-insulin antibodies, and hypoglycaemia.

Minigene splicing assessment of 20 novel synonymous and intronic glucokinase gene variants identified in patients with maturity-onset diabetes of the young.

The next-generation sequencing (NGS) has become a routine method for diagnostics of inherited disorders. However, assessment of the discovered variants may be challenging, especially when they are not predicted to change the protein sequence. Here we performed a functional analysis of 20 novel or rare intronic and synonymous glucokinase (GCK) gene variants identified by targeted NGS in 1,130 patients with maturity-onset diabetes of the young. Human Splicing Finder, ver 3.1 and a precomputed index of splicing variants (SPIDEX) were used for in silico prediction. In vitro effects of GCK gene variants on splicing were tested using a minigene expression approach. In vitro effect on splicing was shown for 9 of 20 variants, including two synonymous substitutions. In silico and in vitro results matched in about 50% of cases. The results demonstrate that novel or rare apparently benign GCK gene variants should be regarded as potential splicing mutations.

Non-lethal Raine Syndrome in a Middle-Aged Woman Caused by a Novel FAM20C Mutation.

Raine syndrome is a rare hereditary disease caused by mutations in the FAM20C gene. Only 18 non-lethal cases have been reported, the majority of them being children and young adults aged up to 30. Due to the rarity of the disease, genotype-phenotype correlations are not available and patient life expectancy is unknown, thus making descriptions of each novel case of particular importance. In this article, we describe a case of an Armenian woman, living in Russia, who was followed-up from age 36 to 39, presenting with pain in the extremities, osteosclerosis with periosteal bone formation, multiple calcifications in solid organs, midface hypoplasia, exophthalmos, amelogenesis imperfecta, shortening of distal phalanges, pectus excavatum, and hypophosphatemia due to renal phosphate wasting. Whole exome sequencing was performed on NextSeq 550 (Illumina, USA) and compound heterozygous variants were identified in the FAM20C gene (reference sequence NM_020223): a frameshift insertion c.1107_1108insTACTG (p.Tyr369fs) and a missense substitution c.1375C > G (p.Arg459Gly). This is the first reported case of a middle-aged patient presenting classical symptoms of Raine syndrome caused by novel compound heterozygous mutations in the conserved C-terminal domain of FAM20C gene.

High frequency of pathogenic and rare sequence variants in diabetes-related genes among Russian patients with diabetes in pregnancy.

AIMS: Diabetes in pregnancy may be associated with monogenic defects of beta-cell function, frequency of which depends on ethnicity, clinical criteria for selection of patients as well as methods used for genetic analysis. The aim was to evaluate the contribution and molecular spectrum of mutations among genes associated with monogenic diabetes in non-obese Russian patients with diabetes in pregnancy using the next-generation sequencing (NGS). METHODS: 188 non-obese pregnant women with diabetes during pregnancy were included in the study; among them 57 subjects (30.3%) met the American Diabetes Association (ADA) criteria of preexisting pregestational diabetes (pre-GDM), whereas 131 women (69.7%) fulfilled criteria of gestational diabetes mellitus (GDM). A custom NGS panel targeting 28 diabetes causative genes was used for sequencing. The sequence variants were rated according to the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: In total, 23 pathogenic, 18 likely pathogenic and 16 variants of uncertain significance were identified in 59/188 patients (31.4%). The majority of variants (38/59) were found in GCK gene. No significant differences in the number of variants among the two study groups (pre-GDM and GDM) were observed. CONCLUSIONS: The study suggests that frequency of monogenic variants of diabetes might be underestimated, which warrants a broader use of genetic testing, especially in pregnancy.

High frequency of mutations in 'dyshormonogenesis genes' in severe congenital hypothyroidism.

OBJECTIVE: Results of the screening of disease causative mutations in congenital hypothyroidism (CH) vary significantly, depending on the sequence strategy, patients' inclusion criteria and bioinformatics. The objective was to study the molecular basis of severe congenital hypothyroidism, using the next generation sequencing (NGS) and the recent guidelines for assessment of sequence variants. DESIGN: 243 patients with CH (TSH levels at neonatal screening or retesting greater than 90 mU/l) and 56 control subjects were included in the study. METHODS: A custom NGS panel targeting 12 CH causative genes was used for sequencing. The sequence variants were rated according to American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: In total, 48 pathogenic, 7 likely pathogenic and 57 variants of uncertain significance were identified in 92/243 patients (37.9%), while 4 variants of uncertain significance were found in 4/56 control subjects (7.1%). 13.1% (12/92) of the cases showed variants in 'thyroid dysgenesis' (TD) genes: TSHR, n = 6; NKX2-1, n = 2; NKX2-5, n = 1; PAX8, n = 3. The variants in 'dyshormonogenesis' (DH) genes were found in 84.8% (78/92) of cases: TPO, n = 30; DUOX2, n = 24; TG, n = 8; SLC5A5, n = 3; SLC26A4, n = 6; IYD, n = 1. 8 patients showed oligonenic variants. The majority of variants identified in DH genes were monoallelic. CONCLUSIONS: In contrast to earlier studies demonstrating the predominance of TD in severe CH, the majority of variants identified in our study were in DH genes. A large proportion of monoallelic variants detected among DH genes suggests that non-mendelian mechanisms may play a role in the development of CH.

Preoperative planning for advanced modelling of anterolateral thigh flaps in the treatment of severe haemifacial atrophy in Parry-Romberg and Goldenhar syndrome.

BACKGROUND: Technological advancement in medical science is constantly innovating solutions to the varied and complex challenges of surgery. Digital diagnostics and prospective microsurgery are rapidly evolving. Three-dimensional (3-D) imagery and computed tomography (CT) scanning can determine accurate dimensions of many defects. Subsequently, a thorough understanding of micro-vasculature and application of microsurgical techniques allows modelling of flaps to obtain an accurate transplant resulting in an aesthetic outcome following the very first operation. METHODS: Two patients with Parry-Romberg syndrome and one patient with haemifacial microsomia (Goldenhar syndrome) were treated with anterolateral thigh (ALT) flaps to restore facial volume, contour, and symmetry. In each case, a different approach in planning and performing the intervention was applied:The patient in the first case had a full-thickness ALT flap transplant with significant overcorrection.The patient in the second case had reconstruction with a partially thinned ALT flap guided by a clinically formed template made per manual measurements.The patient in the third case had reconstruction with a precise primary thinned ALT flap with a template made according to data obtained from superimposed 3-D photographs and CT scans. RESULTS: All flaps survived. In cases 1 and 2, a corrective intervention was required to achieve acceptable facial symmetry. In case 3, a very good aesthetic result was achieved immediately after the first operation. CONCLUSIONS: Digital methods of 3-D analysis offer great opportunities in creating a precise operative plan, and modern surgical techniques make it feasible to implement it intra-operatively. Overall, these methods shortened the rehabilitation time by avoiding further revision surgeries.

A Novel Mutation in the Critical P-Box Residue of Steroidogenic Factor-1 Presenting with XY Sex Reversal and Transient Adrenal Failure.

BACKGROUND: Although the importance of steroidogenic factor-1 (SF1, NR5A1) for adrenal development is supported by numerous in vitro and in vivo studies, cases of SF1 deficiency associated with adrenal failure are exceptionally rare. The first human NR5A1 mutation was a heterozygous de novo p.G35E variant identified in a patient with disorder of sex development (DSD) 46,XY and primary adrenal insufficiency. Here we describe another association of the "classic" SF1 phenotype with a novel NR5A1 mutation affecting G35 residue. METHODS: We describe the clinical characteristics of a phenotypically female patient presenting at 2 months with signs of adrenal insufficiency. DSD 46,XY was diagnosed at 4 years. The NR5A1 gene was analyzed by Sanger sequencing. Minigene splicing and dual luciferase reporter assays were used to characterize effects of the novel mutation on splicing and transcription, respectively. RESULTS: Sequencing of the NR5A1 gene revealed a de novo heterozygous c.104G>A:p.G35D substitution. The minigene experiments demonstrated that c.104G>A substitution did not affect splicing. However, transactivation activity of the p.G35D mutant was clearly impaired, which was comparable with the effect of the p.G35E mutation. CONCLUSIONS: The findings stress the importance of G35 residue for adrenal development. The current observation also suggests that some patients with SF1 deficiency may present with transient adrenal failure.

Primary hyperparathyroidism in young patients in Russia: high frequency of hyperparathyroidism-jaw tumor syndrome.

BACKGROUND: Primary hyperparathyroidism (PHPT) is a relatively rare disorder among children, adolescents and young adults. Its development at an early age is suspicious for hereditary causes, though the need for routine genetic testing remains controversial. OBJECTIVE: To identify and describe hereditary forms of PHPT in patients with manifestation of the disease under 40 years of age. DESIGN: We enrolled 65 patients with PHPT diagnosed before 40 years of age. Ten of them had MEN1 mutation, and PHPT in them was the first manifestation of multiple endocrine neoplasia type 1 syndrome. METHODS: The other fifty-five patients underwent next-generation sequencing (NGS) of a custom-designed panel of genes, associated with PHPT (MEN1, CASR, CDC73, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2C, CDKN2D). In cases suspicious for gross CDC73 deletions multiplex ligation-dependent probe amplification was performed. RESULTS: NGS revealed six pathogenic or likely pathogenic germline sequence variants: four in CDC73 c.271C>T (p.Arg91*), c.496C>T (p.Gln166*), c.685A>T (p.Arg229*) and c.787C>T (p.Arg263Cys); one in CASR c.3145G>T (p.Glu1049*) and one in MEN1 c.784-9G>A. In two patients, MLPA confirmed gross CDC73 deletions. In total, 44 sporadic and 21 hereditary PHPT cases were identified. Parathyroid carcinomas and atypical parathyroid adenomas were present in 8/65 of young patients, in whom CDC73 mutations were found in 5/8. CONCLUSIONS: Hereditary forms of PHPT can be identified in up to 1/3 of young patients with manifestation of the disease at <40 years of age. Parathyroid carcinomas or atypical parathyroid adenomas in young patients are frequently associated with CDC73 mutations.

Progressive Generalized Lipodystrophy as a Manifestation of Autoimmune Polyglandular Syndrome Type 1.

We describe APS1 in a boy with generalized lipodystrophy, oral candidiasis, autoimmune hepatitis and adrenal insufficiency. It is the first time when generalized lipodystrophy is associated with APS1.

Torque Teno Virus (TTV) distribution in healthy Russian population.

BACKGROUND: Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. There is a lot of data on the prevalence and genetic heterogeneity of TTV in healthy populations and in patients with various diseases now available. However, little is known about TTV load among healthy human population. In this study we analyzed TTV load in the group of 512 Russian elite athletes, who are supposed to be, by some standards, the healthiest part of the human population. RESULTS: The prevalence rate of TTV among the Russian Olympic Reserve members was 94% (for test sensitivity about 1000 genome equivalents per 1 ml of blood). Quantities varied from 103 (which corresponded to detection limit) to 1010 copies per 1 ml of blood, with median at 2.7 x 106 copies. CONCLUSION: About 94% of healthy individuals in Russian population have more than 1000 TTV genome copies per 1 ml of blood. This result exceeds the previously published data, and can be explained by either more sensitive PCR test system or by higher TTV distribution in Russian population or both. TTV viral load neither depends on gender, nor age.