Molecular Genetic Characterization Research Articles

Molecular Mechanisms Determining the Role of Bacteria from the Genus Azospirillum in Plant Adaptation to Damaging Environmental Factors.

Agricultural plants are continuously exposed to environmental stressors, which can lead to a significant reduction in yield and even the death of plants. One of the ways to mitigate stress impacts is the inoculation of plant growth-promoting rhizobacteria (PGPR), including bacteria from the genus Azospirillum, into the rhizosphere of plants. Different representatives of this genus have different sensitivities or resistances to osmotic stress, pesticides, heavy metals, hydrocarbons, and perchlorate and also have the ability to mitigate the consequences of such stresses for plants. Bacteria from the genus Azospirillum contribute to the bioremediation of polluted soils and induce systemic resistance and have a positive effect on plants under stress by synthesizing siderophores and polysaccharides and modulating the levels of phytohormones, osmolytes, and volatile organic compounds in plants, as well as altering the efficiency of photosynthesis and the antioxidant defense system. In this review, we focus on molecular genetic features that provide bacterial resistance to various stress factors as well as on Azospirillum-related pathways for increasing plant resistance to unfavorable anthropogenic and natural factors.

Combining morphological and mitochondrial DNA data to describe a new species of Austroniscus Vanhöffen, 1914 (Isopoda, Janiroidea, Nannoniscidae) linking abyssal and hadal depths of the Puerto Rico Trench.

Hadal trenches are perceived as a unique deep-sea ecosystem with fundamentally different communities compared to the nearby abyss. So far, however, scarce information exists about how populations are genetically linked within a trench and about mechanisms for species divergence. The present study presents the morphological and molecular-genetic characterization and description of a new nannoniscid species within the genus Austroniscus Vanhöffen, 1914 obtained from abyssal and hadal depths of the Puerto Rico Trench, NW Atlantic. Samples were collected as part of the Vema-TRANSIT expedition onboard RV Sonne in January 2015. Because of the large depth differences between sampling locations (4,552-8,338 m), we expected to find different species within the genus inhabiting abyssal and hadal sites. Initial morphological examination using traditional light microscopy and Confocal Laser Scanning Microscopy was paired with subsequent molecular analysis based on mtDNA (COI and 16S). Contrary to our assumptions, combined morphological and molecular species delimitation analyses (sGMYC, mPTP, ABGD) revealed the presence of only one species spanning the abyssal and hadal seafloor of the Puerto Rico Trench. In addition, comparison with type material could show that this species belongs to a new species, Austroniscus brandtae n. sp., which is described herein. Incongruence between some species delimitation methods suggesting the presence of multiple species is interpreted as strong genetic population structuring within the trench, which is also supported by the analysis of the haplotype networks. The geographic and bathymetric distribution of Austroniscus species is discussed. The species described herein represents the first in the genus Austroniscus from the Atlantic Ocean and the deepest record of the genus to date, and hence significantly expanding previously known limits of its geographic and bathymetric range.

Exploring HTRA1-Autosomal Dominant Disease: Literature Review of Clinical Features and Molecular Mechanisms

Heterozygous HTRA1-autosomal dominant disease is a gradually recognized hereditary cerebral small vessel disease (cSVD) characterized by debilitating conditions and extensive white matter hyperintensities (WMHs), but doubts remain on the underlying mechanisms of this disease. This review summarizes the clinical, MRI, and molecular genetics features of heterozygous HTRA1-autosomal dominant disease in combination with two better-studied hereditary cSVDs. A total of 31 mutations in HTRA1-autosomal dominant cases documented between 2020 and 2023 were also reviewed, characterizing the mutation features and clinical manifestations. This review aims to gain better insight into the unique characteristics of the disease and its correlations with other hereditary cSVDs.

Hepatitis A virus monitoring in wastewater: A complementary tool to clinical surveillance

Monitoring wastewater is an effective tool for tracking information on trends of enteric viral dissemination. This study aimed to perform molecular detection and genetic characterization of HAV in wastewater and to correlate the results with those obtained from clinical surveillance.Wastewater samples (n=811) of the second most populous city in Argentina were collected from the main wastewater treatment plant (BG-WWTP, n=261), and at 7 local neighborhood collector sewers (LNCS, n=550) during 2017–2022. Clinical samples of acute hepatitis A cases (HA, n=54) were also analyzed. HAV molecular detection was performed by real time RT-PCR, and genetic characterization by RT-Nested PCR, sequencing and phylogenetic analysis.RNA-HAV was detected in sewage samples throughout the entire period studied, and detection frequencies varied according to the location and year (2.9% - 56.5%). In BG-WWTP, 23% of the samples were RNA-HAV+. The highest detection rates were in 2017 (30.0%), 2018 (41.7%) and 2022 (56.5%), which coincides with the highest number of HA cases reported. Twenty-eight (28) sequences were obtained (from clinical and sewage samples), and all were genotype IA. Two monophyletic clusters were identified: one that grouped clinical and wastewater samples from 2017–2018, and another with specimens from 2022, evidencing that environmental surveillance might constitute a replica of viral circulation in the population.These findings evidence that WBE, in a centralized and decentralized sewage monitoring, might be an effective strategy to track HAV circulation trends over time, contributing to the knowledge of HAV in the new post-vaccination epidemiological scenarios in Argentina and in Latin America.

Molecular genetics features of anaplastic thyroid carcinoma

INTRODUCTION: Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid cancer accounting for 12% of all malignancies. Systemic therapy remains the main treatment strategy. Targeted therapy and immunotherapy are prescribed when certain molecular genetic aberrations are detected.
 THE AIM: To investigate the molecular genetic profile of samples of anaplastic thyroid carcinoma.
 MATERIALS AND METHODS: The study included 37 patients with ATC. Mutation V600E BRAF, mutations in the gene NRAS and KRAS were detected by allele-specific polymerase chain reaction (AS-PCR). Microsatellite instability (MSI) was determined by fragment analysis in according to ESMO recommendations. Mutations in the promoter region of the TERT gene were used by Sanger sequencing. NTRK1, EML4-ALK, PAX8/PPARy и RET/PTC translocations were determined in all patients with ATC by real-time polymerase chain reaction (PCR).
 RESULTS: According to the results of the study, the frequency of the V600E mutation in the BRAF gene was 32.4% (12/37). The frequency of aberrations in the NRAS, KRAS genes in anaplastic thyroid carcinoma was 13.5% (n=5). The prevalence of point mutations in the promoter gene TERT in food samples of ATC was 24.3% (n=9). MSI was found in 2.7% (1/37) of cases of anapalastic thyroid carcinoma. NTRK1, EML4-ALK, PAX8/PPARy and RET/PTC translocations were not detected in cases with anaplastic thyroid carcinoma.
 CONCLUSION: The further study of the main specific molecular targets in cancer cells will allow to personalize the tactics of patients with anaplastic thyroid carcinoma.

Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

Clinicopathological and molecular genetic characterization of colorectal angiosarcoma

目的： 探讨结直肠血管肉瘤的临床病理和分子遗传学特点。 方法： 观察和分析4例原发于结直肠的血管肉瘤的临床特征、组织学和免疫表型特点，应用荧光原位杂交（FISH）检测c-MYC基因的扩增状态。 结果： 女性3例，男性1例，年龄56~77岁。3例与盆腹腔恶性肿瘤术后放疗相关。组织学上，2例由交织网状的裂隙样血管腔隙和实性的梭形或上皮样肿瘤细胞组成；1例完全由上皮样肿瘤细胞组成，伴有局灶的单个细胞内原始血管腔隙；1例主要由致密的束状梭形细胞和散在的多形性间变性肿瘤细胞组成，伴有局灶分化好的淋巴管腔隙。免疫表型，4例均弥漫表达ERG和CD31，3例表达c-MYC蛋白，1例共表达细胞角蛋白7和广谱细胞角蛋白。FISH检测示3例与放疗相关的肿瘤均存在c-MYC基因扩增。术后随访3~11个月，3例死于肿瘤，1例带瘤生存。 结论： 结直肠血管肉瘤罕见，侵袭性强，常与恶性肿瘤术后放疗相关。组织学上具有明显的异质性，FISH检测c-MYC扩增可进一步证实与放疗的相关性。.

Molecular genetic features and clinical manifestations in Chinese familial cerebral cavernous malformation: from a novel KRIT1/CCM1 mutation (c.1119dupT) to an overall view.

Cerebral cavernous malformations (CCMs) are common vascular anomaly diseases in the central nervous system associated with seizures, cerebral microbleeds, or asymptomatic mostly. CCMs can be classified as sporadic or familial, with familial cerebral cavernous malformations (fCCMs) being the autosomal dominant manner with incomplete penetrance. Germline mutations of KRIT1, CCM2, and PDCD10 are associated with the pathogenesis of fCCMs. Till now, little is known about the fCCMs mutation spectrum in the Han Chinese population. In this study, we enrolled a large, aggregated family, 11/26 of the family members were diagnosed with CCMs by pathological or neuroradiological examination, with a high percentage (5/9) of focal spinal cord involvement. Genomic DNA sequencing verified a novel duplication mutation (c.1119dupT, p.L374Sfs*9) in exon 9 of the Krev interaction trapped 1 (KRIT1) gene. The mutation causes a frameshift and is predicted to generate a truncated KRIT1/CCM1 protein of 381 amino acids. All our findings confirm that c.1119dupT mutation of KRIT1 is associated with fCCMs, which enriched the CCM genes' mutational spectrum in the Chinese population and will be beneficial for deep insight into the pathogenesis of Chinese fCCMs. Additionally, with a retrospective study, we analyzed the molecular genetic features of Chinese fCCMs, most of the Chinese fCCMs variants are in the KRIT1 gene, and all these variants result in the functional deletion or insufficiency of the C-terminal FERM domain of the KRIT1 protein.

HISTOLOGICAL AND MOLECULAR GENETIC CHARACTERISTICS OF CLINICALLY AGGRESSIVE VARIANTS OF PAPILLARY THYROID CARCINOMA: A LITERATURE REVIEW

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer; this cancer presents relatively low malignancy, good prognosis, and a 10-year survival rate of over 90 %. However, the clinical behaviors of this cancer are complex and varied. PTC is easy to spread via lymphatic ducts, which results in recurrence, metastases, and even death [1]. Differentiated thyroid cancer has a favorable prognosis in terms of relapsefree and overall survival. However, some cases of differentiated thyroid cancer (TC) are characterized by aggressive course with distant metastases. Identification of histological subtypes of papillary thyroid cancer (PTC) and detection of specific mutations may have some prognostic value in the prediction of the disease course and clinical response to treatment. The severity of the disease depends on multiple clinical characteristics, morphological and molecular features of the tumor. Several histological variants of PTC with varying degrees of malignancy have been described so far: from well-differentiated PTC to radioiodine refractory undifferentiated anaplastic carcinoma with a high mitotic index and high probability of relapses. Recurrent PTC mainly refers to localized and distant recurrence, including recurrence of the primary tumor, lymph node metastases, invasion of the esophagus and trachea, invasion of muscles, soft tissues and nerves, and distant metastases. Many factors can affect the recurrence of thyroid cancer, and final conclusion has not yet been reached. Aim: to summarize the available literature data on the molecular genetic characteristics of aggressive variants of papillary thyroid carcinoma. Methods: We conducted a literature review of scientific papers over the past decade using PubMed search engine resources. In addition, a search was conducted using the data of the scientific electronic library eLIBRARY for the above keywords. For this meta-analysis, we used articles containing clinical evidence on the most recent issues regarding aggressive variants of papillary thyroid carcinoma. Results: The highest frequency of BRAF V600E among PTC is shown by the high-cell variant of PTC (90%), also significantly higher with «shoe nail» cells than in classical PTC and varies from 40-90%. In a columnar cell, PTC, mutations in the BRAF V600E gene are much less common than in classic PTC. The diffuse sclerosing type of PTC is characterized by the presence of a RET/PTC translocation, and the percentage of the BRAF V600E mutation is 0-60 in various studies. Conclusion: In the presented work, the morphological and clinical features of patients with prognostic unfavorable variants of PTC are considered. Correct recognition of these variants is an important component in the diagnosis of tumor pathology of the thyroid gland, except for accidentally detected microcarcinomas without signs of invasion into the tissues and into the capsule of the thyroid gland and into the lymphatic vessels. The most aggressive forms of PTC include diffuse sclerosing, tall cell, and insular variants of PTC. Various genetic abnormalities, such as BRAF mutation, can be detected in this case. Thus, in some cases these markers can help to predict the course of TC and to provide adequate and timely treatment.

Spindle Cell Lipoma and Pleomorphic Lipoma: An Update and Review.

Spindle cell lipoma (SCL) is a benign adipocytic tumor that primarily occurs in the subcutis of the posterior neck, upper back, and shoulder, particularly of middle-aged males. SCL and pleomorphic lipoma (PL) represent a morphological spectrum of one disease process. The lesion typically presents as a relatively small (<5 cm), mobile, slow-growing, painless mass. Magnetic resonance imaging reveals the lesion to be a well-defined subcutaneous mass with a mixture of adipose and non-adipose components. Intense enhancement of the non-adipose component is seen after contrast administration. Histologically, SCL is composed of variable distributions of mature adipocytes, bland spindle cells and ropey collagen bundles and PL also contains pleomorphic and multinucleated floret-like giant cells. By immunohistochemistry, the spindle and pleomorphic/floret-like giant cells of SCL/PL are diffusely positive for CD34 and show loss of nuclear RB transcriptional corepressor 1 (RB1) expression. Recent cytogenetic and molecular genetic studies have shown heterozygous deletions of 13q14, including the RB1 gene. SCL/PL can be successfully treated with simple excision, with a very low recurrence rate. Knowledge of these peculiar tumors is important because it can mimic a variety of benign and malignant soft-tissue tumors. This review provides an updated overview of the clinical, radiological, histopathological, cytogenetic, and molecular genetic features of SCL/PL.

Genetic and clinical characteristics of Turkish children with Maturity Onset Diabetes of the Young Type 2 (MODY2): A single center experience

Objective: The aim of the study was to investigate the clinical and molecular genetic characteristics of children with maturity-onset diabetes of the youth-glucokinase (MODY-GCK, MODY type 2). Method: Medical files of 21 patients with suspected MODY-GCK were reviewed retrospectively. The file records of the clinical findings, laboratory results and the suspected clinical diagnoses of MODY were based on (1) asymptomatic fasting hyperglycemia (glucose ≥100mg/dl, HbA1c &amp;lt; 7.5% (at least twice measurement) 2) parents with a history of diabetes without complications or mild fasting hyperglycemia (100-144mg/dL). Results: The mean age at diagnosis was 11.5±4.3 years (min-max, 1.9-17.2). The mean (SD) fasting blood glucose level was 119.1 (9.8) mg/dL. The mean (SD) fasting C-peptide level was 1.3 (0.7) ng/mL, the mean (SD) insulin level was 5.9 (2.3) IU/ml, and the mean (SD) HbA1c level at diagnosis was 6.2 (0.5) %. Among 12 variants detected in the GCK gene, 8 were missense mutation, 2 were non-sense mutation, 1 of them was splice site and 1 of them was frameshift mutation. Eight of them (p. Val227Met, p. Ser282Ala, p.Val183Met, p.Met239Thr, p.Arg304Gln, p.Thr229Met, p.Gly163Asp, p.Cys130Ter) have been previously reported in the literature and 4 variants (c.582+4delA, p.Glu436Ter, p.His106ThrfsTer11, p.Asp133Gly) were novel. Conclusion: We found similar phenotype characteristic of children with GCK-MODY among the children with different variants. The most common mutation type was missense and followed by nonsense, splice site and frameshift mutations. Detection of the molecular defect in patients with MODY is vital for the implementation of appropriate treatment approaches.

Molecular genetic characterization of sudden deaths due to thoracic aortic dissection or rupture

BackgroundSudden deaths due to thoracic aortic dissection or rupture (TADR) are often investigated by forensic pathologists in the United States. Up to a quarter of reported TADR result from a highly penetrant autosomal dominant single gene variant. Testing genes associated with familial TADR provides an underlying etiology for the cause of death and informs effective sudden death prevention for at-risk family members. At the New York City Office of Chief Medical Examiner (NYC-OCME), TADR cases are routinely tested by the in-house, CAP-accredited Molecular Genetics Laboratory. In this retrospective study, TADR and cardiovascular cases were reviewed to understand the burden of TADR in sudden deaths, value of molecular diagnostic testing in TADR, and genotype-phenotype correlations in a demographically diverse TADR cohort. MethodsBetween July 2019 and June 2022, cases with in-house cardiovascular genetic testing at NYC-OCME were retrospectively reviewed. Twenty genes associated with familial TADR were analyzed using high throughput massive parallel sequencing on postmortem tissues or bloodspot cards. Variant interpretation was conducted according to ACMG/AMP guidelines. ResultsA total of 1078 cases were tested for cardiovascular genetic conditions, of which 34 (3%) had TADR. Eight of those TADR cases had a pathogenic or likely pathogenic variant (P/LPV), 4 had a variant of uncertain significance (VUS), and 22 cases were negative for variants in TADR genes. The molecular diagnostic yield using the TADR subpanel was 23.5%. The genes with the greatest prevalence of P/LPV were FBN1 (6), followed by TGFBR2 (2), TGFBR1 (1), and MYLK (1). Highly penetrant P/LPV in TGFBR2, FBN1, and TGFBR1 were found in TADR individuals who died younger than 34 years old. Two P/LPV in FBN1 were secondary findings unrelated to cause of death. P/LPV in FBN1 included five truncating variants located in the N-terminal domains and one missense variant involved in the disulfide bonds of the EGF-like domain. All P/LPV in TGFBR1 and TGFBR2 were missense or in-frame deletion variants located in the protein kinase catalytic domain. Three variants were first reported in this study. ConclusionsMolecular testing of familial TADR-associated genes is a highly effective tool to identify the genetic cause of TADR sudden deaths and benefits surviving at-risk families.

Molecular identification and genetic characterization of Theileria and Anaplasma infection in sheep and goat of North Gujarat, India

Hemoprotozoal diseases are significant health concerns in small ruminants. The present study was conducted to identify and characterize the species of Theileria and Anaplasma in sheep and goats located in different districts of North Gujarat, India. A total of 226 (Banaskantha = 175, Patan = 26, and Bhuj = 25) blood samples were collected from sheep (n = 78) and goats (n = 148), and 46 ticks were collected and identified from sheep and goats. PCR assays were carried out using genus and species-specific primers for Theileria targeting 18S rRNA locus and for Anaplasma targeting the msp5 gene. Overall, 37.2% sheep (29/78) and 10.8% of goats (16/148) were positive for Theileria by PCR, whereas 15.4% of sheep (12/78) and 25.7% goats (38/148) were positive for Anaplasma infection. Moreover, mixed infection was found in 4.4% (10/226) of sheep and goats by PCR. Sanger sequencing of Theileria and Anaplasma positives revealed a high similarity to T. ovis and A. ovis using NCBI blast, respectively. Phylogenetic analyses revealed that the Anaplasma spp. DNA sequences belonged to the A. ovis group and closely associated with the A. ovis nucleotide sequence strain Haibei isolated in China from sheep (GQ483471). The phylogenetic analysis based on the SSU rRNA locus revealed that the Theileria ovis DNA sequences belonged to the T. ovis group and closely related to MW440586 isolated in Kerala, India, from a goat. The majority of ticks (91.3%) were identified as Hyalomma. In conclusion, Theileria ovis and Anaplasma ovis were commonly identified species in sheep and goats and transmitted mainly by Hyalomma ticks in North Gujarat, India, which is important baseline data for future research and control strategies. This is the first report on Theileria and Anaplasma co-infections in sheep and goats from North Gujarat, India.

Clinical and epidemiological characteristic of chronic hepatitis C in the republic of Tyva

Aim: To assess the current epidemiological situation and establish the dominant clinical profile of patients with chronic hepatitis C, taking into account the stage of the disease and the molecular genetic characteristics of the virus in the Republic of Tyva. Materials and methods: Based on the annual reports of the dispensary observation office of the Infectious Hospital of the Republic of Tyva, an electronic database was developed using the licensed Office Excel 2016 program, which included results on the health status of patients, clinical, laboratory and instrumental data, including molecular biological data with the determination of HCV genotypes. The analysis of the data of the state statistical reporting of infectious morbidity in the Russian Federation (form №2 “Information on infectious and parasitic diseases”) was carried out. Analytical tables developed by specialists of the Scientific and Methodological Center of the Saint-Petersburg Pasteur Institute for the period 2012-2022. The data of the Reference Center for Monitoring Viral Hepatitis of the Federal State Budgetary Institution “Central Research Institute of Epidemiology” of Rospotrebnadzor were used. Results. Over the past two years, the decrease in incidence in the Republic of Tyva is likely due to a decrease in the number of examinations, a decrease in patients seeking outpatient care, and insufficient screening in the context of the COVID-19 pandemic. Clinical, laboratory and instrumental examination of patients with HCV revealed the predominance of HCV-infected genotype 1 (49.4%), with F0-F2 fibrosis in 79% of cases. The clinical profile of typical patients with chronic hepatitis C was established; whose share in the total structure of the examined was 38.4%. Conclusion: There is a tendency to reduce the incidence of chronic hepatitis C in the Republic of Tyva. Based on the clinical information base, the most typical characteristics of chronic hepatitis C patients living in the Republic of Tyva have been established.

Clinicopathological and molecular genetic characteristics of primary gastric follicular lymphoma

Primary gastric follicular lymphomas (FLs) have been rarely reported, and little is known about their characteristics. In the present study, we report 5 cases of primary gastric FL and describe their clinicopathological and molecular genetic features. A total of 7 samples from 5 patients were investigated for clinicopathological characteristics and somatic mutations by the targeted sequencing of 50 lymphoma-related genes. Two cases were identified as slightly elevated submucosal tumors and 3 cases as polypoid tumors. Histologically, all cases were low-grade FLs. The immunoprofile was CD20+/CD10+/BCL2+ in 4 cases and CD20+/CD10+/BCL2- in 1 case. The immunostaining pattern for CD21 was similar to that of classic FL. BCL2 rearrangement was not identified in fluorescence in situ hybridization studies in any of the 5 cases. Next-generation sequencing analysis showed mutations in genes involved in epigenetic modifications (KMT2D, ARID1A, EP300, and CREBBP), NK-kB pathway (CARD11), and JAK-STAT pathway as found in classic FL. All cases presented with clinical I without the involvement of regional or systemic lymph nodes. Four patients were well, whereas 1 patient who received endoscopic mucosal resection of tumor without additional chemotherapy or radiotherapy experienced 3 relapses. In conclusion, primary gastric FL is characterized by a low-grade neoplasm with infrequent BCL2 rearrangement. After resection of the lesion, additional treatment such as radiation therapy or chemotherapy is required as there is a possibility of recurrence.

Principles of the differential diagnosis of achondroplasia and pseudoachondroplasia

BACKGROUND: Achondroplasia and pseudoachondroplasia are hereditary systemic skeletal dysplasias characterized by a certain similarity of clinical manifestations; however, they have different etiopathogenetic mechanisms and confirmation methods for molecular genetic diagnosis. Their common phenotypic features often make differential diagnosis difficult during the clinical examination of patients, planning DNA diagnostics, and appropriate time detection of neurosurgical and orthopedic complications.&#x0D; AIM: This study aimed to identify differential diagnostic criteria for achondroplasia and pseudoachondroplasia and optimize the strategy for their molecular genetic diagnosis.&#x0D; MATERIALS AND METHODS: A comprehensive examination of 76 children from 74 unrelated families aged 1 month to 18 years with phenotypic signs of achondroplasia and pseudoachondroplasia was conducted. To clarify the diagnosis through genealogical and amnestic analysis, clinical and neurological examination data according to the standard method and radiographic data were used. Molecular genetic confirmation of diseases was conducted by searching for hotspot mutations in the FGFR3 gene, assessing the number of GAC repeats located in exon 13 of the COMP gene, and new-generation sequencing of the target panel consisting of 166 genes responsible for hereditary skeletal pathology.&#x0D; RESULTS: Based on a comparative analysis of the specific phenotypic characteristics, the criteria for the differential diagnosis of achondroplasia and pseudoachondroplasia were identified. The leading signs of achondroplasia are disproportionate nanism from birth, macrocrania, and facial dysmorphism, which are not specific to pseudoachondroplasia. Certain radiological features are essential in the differential diagnosis of pseudoachondroplasia, which should be considered when referring to patients for molecular genetic analysis. A deletion of the GAC repeat c.1417_1419del in the COMP gene was identified in 27% of patients with pseudoachondroplasia. Thus, the analyses of these two mutations in FGFR3 and COMP were conducted first. In the absence of target mutations, further diagnostic search should be continued with a target panel consisting of 166 genes responsible for hereditary skeletal pathology or whole-exome sequencing.&#x0D; CONCLUSIONS: The analysis of the clinical, radiological, and molecular genetic characteristics of patients with achondroplasia and pseudoachondroplasia, together with the literature data analysis, made it possible to clarify the differential diagnostic criteria for these diseases and optimize the algorithm for their molecular genetic diagnosis.

Abstract 4177: Genetic testing for hereditary colorectal cancer syndromes in Algerian patients: A multicenter study

Abstract Background To date, 5% to 6 % of all colorectal cancers (CRCs) are associated with germline pathogenic variants in cancer predisposition genes that confer inherited predisposition to CRC. The use of genetic testing to identify individuals at risk for hereditary CRC syndromes can help to prevent the development of cancer and in the clinical management of the colorectal patients in the areas of both prevention and treatment. We report here the experience of our research laboratory of genetic testing for hereditary polyposis syndromes and Lynch syndrome (LS), respectively, in 126 patients. Methods Fifty four (54) severe familial adenomatous polyposis (FAP) patients and 72-suspected Lynch syndrome (LS) patients, respectively, were selected from 2851 consecutive colorectal patients at five public hospitals during 2012-2019. Family histories of cancer were obtained from interviews, pedigrees and medical records of patients. Index cases and relatives diagnosed with FAP syndrome or Lynch syndrome have been tested for germline variants in APC, MLH1, MSH2, MSH6 and PMS2 genes, respectively, using PCR-Sanger Sequencing or by NGS using a cancer panel of 30 hereditary cancer genes (Color Genomics). Results We detected 13 germline pathogenic variants in APC gene in 17 unrelated families, one germline pathogenic variant in BMPRA1 gene in juvenile polyposis syndrome (JPS) patient; seven (7) germline pathogenic variants and 2 variants of uncertain clinical significance (VUS) in MMR genes. Interestingly, 4 novel germline pathogenic variants in APC gene and 3 novel germline pathogenic variants in MMR genes, respectively, have been detected in our study. The most occurring germline pathogenic variants in APC gene were c.3927_3931del and c.4728dup that were identified in four and two index cases in 6 unrelated FAP families, respectively. In Lynch syndrome patients, the rare germline pathogenic variant MLH1 c.1546C&amp;gt;T has been found in 21 individuals from 9 LS families, 6 of them related, with two large kindreds. In addition, the recurrent germline pathogenic variant MSH2 c.942+3A&amp;gt;T has been detected in five unrelated index cases with a strong family history of LS syndrome. Moreover, the rare germline VUS PMS2 c.989-107_989-106insA has been detected in 14 unrelated LS patients and could be reclassified as likely benign. Interestingly, our NGS analysis detected the novel BMPR1A pathogenic variant c.1474-1G&amp;gt;C in young JPS patient that has been misdiagnosed as FAP. The in-silico analysis for this novel variant showed an alteration of the wild type acceptor site and an activation of a cryptic acceptor site, respectively, most probably affecting splicing. Conclusions Our current study will contribute to the molecular genetics characterization of hereditary colorectal cancer syndromes in Algerian population that is relevant for clinical management in the areas of genetic testing, early diagnosis, treatment and prevention. Citation Format: Farid Cherbal, Asma-Lamia Boumehdi, Feriel Khider, Karima Landelouci, Abdelwahab Zemam, Sarah Sabri, Adam.Walid Damache, Mohammed Oukkal, Hassen Mahfouf, Ferhat Zebboudj, Mustapha Maaoui. Genetic testing for hereditary colorectal cancer syndromes in Algerian patients: A multicenter study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4177.

Abstract 869: Molecular genetic positioning of small intestine and papilla of Vater carcinomas including clinicopathological classification

Abstract Purpose: Small intestine carcinoma (SIC) cases in Japan have recently been treated with chemotherapy according to colorectal carcinoma classification, while papilla of Vater carcinoma (PVC) cases according to cholangiocarcinoma (CHC) classification. However, few research reports support the molecular genetic validity of these therapeutic choices. Here, we investigated the clinicopathological and molecular genetic factors of SIC and PVC. Patients and Methods: We used the data from the Japanese version of The Cancer Genome Atlas (JCGA). Additionally, molecular genetic data on gastric adenocarcinoma (GAD), colorectal adenocarcinoma (CRAD), pancreatic ductal adenocarcinoma (PDAC), and CHC were also referred to. Results: This study consisted of tumor specimens from 12 cases of SIC and three cases of PVC treated from January 2014 to March 2019. Among them, six cases had pancreatic invasion. t-Distributed Stochastic Neighbor Embedding analysis showed that the gene expression pattern of SIC was similar not only to those of GAD and CRAD, but also to that of PDAC in the pancreatic invasion cases. In addition, PVC resembled the GAD, CRAD, and PDAC, rather than the CHC. The molecular genetic characteristics of the six patients with pancreatic invasion were: one had high microsatellite instability (MSI-High), two had a TP53 driver mutation, and three had tumor mutation burden values &amp;lt; 1 mutation/Mb with no driver mutation. Conclusions: The molecular genetics of SIC and PVC suggested that they may be similar to GAD, CRAD, and PDAC. In addition, the data demonstrate that pancreatic invasive cases may be classified into several subtypes using molecular genetic factors. Citation Format: Yukiyasu Okamura, Masanori Nakamura. Molecular genetic positioning of small intestine and papilla of Vater carcinomas including clinicopathological classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 869.

Genetic diversity and structure of Capparis spinosa L. natural populations using morphological and molecular markers

Capparis spinosa is an orphan shrub widely distributed in various landscapes in the Mediterranean basin and in Asia. The medicinal and culinary value of its products has gained the scientific interest, however due to small-scale efforts for caper cultivation, caper parts are largely collected uncontrollably from wild germplasm, exposing natural populations to bottleneck events. Assessment of the species’ genetic diversity is pivotal for the design of conservation schemes. DNA barcoding of 8 C. spinosa populations, collected from various landscapes in Greece was verified by ITS2, matK and trnL-trnF markers and morphological characterization revealed significant differences regarding the leaf length, width, weight, and the length of internodes (Wilk’s λ = 0.002, p < 0.001), as well as significant correlations between the population’s genotype and the internodal length (R=0.4, p < 0.05). Molecular genetic characterization with 8 SRAP and 8 SCoT markers revealed low diversity indices (Shannon’s I= 0.28, h=0.19). The Analysis of Molecular Variance (AMOVA) suggests that 67% of the variability is detected within each population (p < 0.001), while STRUCTURE analysis inferred the existence of 3 distinct populations, indicating admixtures in 4 populations from southern Greece. The results of this study corroborate the necessity for thorough caper germplasm characterization and deployment of a conservation strategy of Greek wild populations to safeguard the species’ diversity.

102 AI-Based Molecular Classification of Diffuse Gliomas using Rapid, Label-Free Optical Imaging

Molecular classification has transformed the management of brain tumors by enabling more accurate prognostication and personalized treatment. Access to timely molecular diagnostic testing for brain tumor patients is limited, complicating surgical and adjuvant treatment and obstructing clinical trial enrollment. By combining stimulated Raman histology (SRH), a rapid, label-free, non-consumptive, optical imaging method, and deep learning-based image classification, we are able to predict the molecular genetic features used by the World Health Organization (WHO) to define the adult-type diffuse glioma taxonomy, including IDH-1/2, 1p19q-codeletion, and ATRX loss. We developed a multimodal deep neural network training strategy that uses both SRH images and large-scale, public diffuse glioma genomic data (i.e. TCGA, CGGA, etc.) in order to achieve optimal molecular classification performance. One institution was used for model training (University of Michigan) and four institutions (NYU, UCSF, Medical University of Vienna, and University Hospital Cologne) were included for patient enrollment in the prospective testing cohort. Using our system, called DeepGlioma, we achieved an average molecular genetic classification accuracy of 93.2% and identified the correct diffuse glioma molecular subgroup with 91.5% accuracy within 2 minutes in the operating room. DeepGlioma outperformed conventional IDH1-R132H immunohistochemistry (94.2% versus 91.4% accuracy) as a first-line molecular diagnostic screening method for diffuse gliomas and can detect canonical and non-canonical IDH mutations. Our results demonstrate how artificial intelligence and optical histology can be used to provide a rapid and scalable alternative to wet lab methods for the molecular diagnosis of brain tumor patients during surgery.