Non-classical Research Articles

Non-classical (NC), heptagon-containing fullerenes obtained via chlorination-promoted cage transformations: C76(NC2a)Cl24 and C76(NC2b)Cl28.

High-temperature chlorination of C76 fullerene with SbCl5 proceeds via five Stone-Wales rearrangements, resulting in non-classical (NC) C76(NC1a)Cl24 with two heptagons and 14 pentagons partically fused in pairs and triples. C76(NC2b)Cl28 with isomeric carbon cage was obtained by chlorination-promoted cage shrinkage of C80via two C2 losses. The pathways of skeletal cage trasformations, the chlorination patterns, and formation energies are discussed in detail.

The injured monocyte: The link to chronic critical illness and mortality following injury.

This study aimed to understand the altered innate immune response in severely injured patients leading to chronic critical illness (CCI). Specifically, it focused on characterizing the monocyte populations and their correlation with CCI development and long-term complications. Over a 3-year period, we monitored patients with severe injuries for up to 1-year postinjury. Chronic critical illness was defined as an ICU stay exceeding 14 days with persistent organ failure. Blood samples were collected on Days 1 and 5 for monocyte phenotypic expression analysis using cytometry by time flight. The monocyte subpopulations studied were classical (CL), intermediate (INT), and nonclassical (NC), along with cell surface receptor expression and activation. Out of 80 enrolled patients, 26 (32.5%) developed CCI. Patients with CCI had more severe injuries (Injury Severity Score, 32.4 + 5.2 vs. 29.6 + 4.1, p = 0.01) and received a higher number of red blood cells (8.9 + 4.1 vs. 4.7 + 3.8 units, p < 0.01) compared with those without CCI. In patients with CCI, the NC monocytes were significantly reduced by over twofold early, and significantly increased later, compared with those without CCI. Moreover, significant changes in intracellular cytokine expression and cell receptors were observed within each monocyte subpopulation in patients with CCI, indicating an increased proinflammatory phenotype but decreased phagocytic capacity and antigen presentation. The development of CCI and the presence of this unique monocyte phenotype were associated with a significantly increased risk of infection, discharge to a long-term care facility, and 1-year mortality of 27%. Development of CCI following severe injury is associated with significant long-term morbidity and unacceptably high mortality. The altered NC phenotype with reduced phagocytic capacity and antigen presentation in patients developing CCI after severe injury is appears partially responsible. Early identification of this unique phenotype may help predict and treat patients at risk for CCI, leading to improved outcomes. Prognostic and Epidemiological; Level III.

Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~20-60% residual activity) exhibited NC and SV phenotypes, respectively. This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.

Imbalance of Circulating Monocyte Subsets in Subjects with Newly Emerged and Recurrent Hospital-Acquired Pneumonia.

Hospital-acquired pneumonia (HAP) is one of the most common diseases in the intensive care unit, where the development of disease is closely related with the host immune response. Monocytes play an important role in both innate and adaptive immune system. We aimed to investigate the changes of circulating monocyte subsets in subjects with HAP to explore its value in monitoring HAP. In total, 60 HAP patients and 18 healthy individuals were enrolled in this study. Human monocyte subsets are classified into 3 groups: nonclassical (NC), intermediate (ITM), and classical (CL). Also, programmed death ligand 1 (PD-L1) expression on circulating monocyte subsets was measured by flow cytometry. Data showed that the ratio of NC, ITM, and CL among monocytes was comparable between HAP patients and healthy controls (P > .05). There was a remarkable imbalance of NC and CL in newly emerged HAP compared to healthy controls (P < .05), subsequently reaching normalization in recurrent HAP (P > .05). Furthermore, although PD-L1 was seemly constitutively expressed by NC, ITM, and CL groups regardless of disease status, it was noted that PD-L1 was dominantly expressed in the CL group (P < .05). Given distinct PD-L1 expression, a shift of CL/NC in newly emerged HAP would constitute an inhibitory anti-pathogen immune response. Normalization of circulating monocyte subsets on recurrence of HAP might be the consequence of immune memory of bacterial infection.

Genetic analysis and novel variation identification in Chinese patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency is an autosomal-recessive disorder caused by mutations in the CYP21A2 gene. The aim of the study was to analyze the molecular data of 155 21-OHD patients and retrospectively investigated the common allelic mutations of CYP21A2 in 1442 Chinese 21-OHD patients. Clinical features and mutations of CYP21A2 gene in 155 unrelated 21-OHD patients were examined. Of the 155 patients, 103 cases were salt-wasting (SW) forms, 38 were simple virilizing (SV) forms and 14 were non-classical (NC) forms. In general, two types of mutations including common allelic mutations (281/310, 90.6%) and rare mutations (29/310, 9.4%) were detected, among them four novel variants c.835G>T, c.1081C>T, c.1423C>T and c.651 + 2 T > G were identified. In 1442 Chinese 21-OHD patients, the most frequently mutations were I2G (36.2%), large deletion/conversion (20.7%) and p.I173N (17.8%), while p.V282L has the lowest frequency. In this study, we provided detailed clinical data and mutation spectrum in Chinese 21-OHD patients. Moreover, four novel CYP21A2 variants (c.835G>T, c.1081C>T, c.1423C>T and c.651 +2 T > G) were identified and computational structural modeling indicated that these novel variations probably affect structural stability. Our findings improve the understanding of CYP21A2 mutational spectrum and contribute to the precise diagnosis and prenatal counseling.

Single-cell expression quantitative trait loci (eQTL) analysis of SLE-risk loci in lupus patient monocytes

BackgroundWe performed expression quantitative trait locus (eQTL) analysis in single classical (CL) and non-classical (NCL) monocytes from patients with systemic lupus erythematosus (SLE) to quantify the impact of well-established genetic risk alleles on transcription at single-cell resolution.MethodsSingle-cell gene expression was quantified using qPCR in purified monocyte subpopulations (CD14++CD16− CL and CD14dimCD16+ NCL) from SLE patients. Novel analysis methods were used to control for the within-person correlations observed, and eQTLs were compared between cell types and risk alleles.ResultsThe SLE-risk alleles demonstrated significantly more eQTLs in NCLs as compared to CLs (p = 0.0004). There were 18 eQTLs exclusive to NCL cells, 5 eQTLs exclusive to CL cells, and only one shared eQTL, supporting large differences in the impact of the risk alleles between these monocyte subsets. The SPP1 and TNFAIP3 loci were associated with the greatest number of transcripts. Patterns of shared influence in which different SNPs impacted the same transcript also differed between monocyte subsets, with greater evidence for synergy in NCL cells. IRF1 expression demonstrated an on/off pattern, in which expression was zero in all of the monocytes studied from some individuals, and this pattern was associated with a number of SLE risk alleles. We observed corroborating evidence of this IRF1 expression pattern in public data sets.ConclusionsWe document multiple SLE-risk allele eQTLs in single monocytes which differ greatly between CL and NCL subsets. These data support the importance of the SPP1 and TNFAIP3 risk variants and the IRF1 transcript in SLE patient monocyte function.

Impact of Newborn Screening on Clinical Presentation of Congenital Adrenal Hyperplasia.

Background and Objectives: The main reason for Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) is to prevent adrenal insufficiency that can lead to life-threatening conditions. On the other hand, screening programs are not always sensitive and effective enough to detect the disease. We aimed to evaluate impact of the national NBS on the clinical presentation of patients with CAH in Lithuania. Materials and Methods: A retrospective study was performed on data of 88 patients with CAH from 1989 to 2020. Patients with confirmed CAH were divided into two groups: (1) 75 patients diagnosed before NBS: 52 cases with salt-wasting (SW), 21 with simple virilising (SV) and two with non-classical (NC) form; (2) 13 patients diagnosed with NBS: 12 cases with SW and 1 case with SV form. For the evaluation of NBS effectiveness, data of only male infants with salt-wasting CAH were analysed (n = 36, 25 unscreened and nine screened). Data on gestational age, birth weight, weight, symptoms, and laboratory tests (serum potassium and sodium levels) on the day of diagnosis, were analysed. Results: A total of 158,486 neonates were screened for CAH from 2015 to 2020 in Lithuania and CAH was confirmed in 13 patients (12 SW, one–SV form), no false negative cases were found. The sensitivity and specificity of NBS program for classical CAH forms were 100%; however, positive predictive value was only 4%. There were no significant differences between unscreened and screened male infant groups in terms of age at diagnosis, serum potassium, and serum sodium levels. Significant differences were found in weight at diagnosis between the groups (−1.67 ± 1.12 SDS versus 0.046 ± 1.01 SDS of unscreened and screened patients respectively, p = 0.001). Conclusions: The sensitivity and specificity of NBS for CAH program were 100%, but positive predictive value—only 4%. Weight loss was significantly lower and the weight SDS at diagnosis was significantly higher in the group of screened patients.

Comprehensive and Cost-Effective Strategy for Genetic Screening of Congenital Adrenal Hyperplasia (CAH) in India

Background: With substantial challenges in molecular analysis of 21 hydroxylase deficiency and lack of studies in extended panel of genes implicated in CAH, genetic diagnosis is largely unavailable and unaffordable in India. Therefore, we aim to develop a cost-effective screening strategy in CAH using Allele Specific PCR and Targeted Next-Generation Sequencing (NGS). Methods: Long range PCR and restriction digestion were utilized to specifically amplify the CYP21A2 gene whereas multiplex PCR was used to amplify CYP11B1, CYP17A1, CYP19A1 and POR genes. In house developed Allele Specific PCR (ASPCR) for 8 hotspot mutations in CYP21A2 gene and targeted NGS for five genes was carried out. The results were validated using Sanger sequencing and MLPA. Results: Of the 50 patients suspected for 21 hydroxylase deficiency, 64% (n=32) were of Salt Wasting phenotype (SW), 30% (n=15) with Simple Virilizing (SV) phenotype and 6% (n=3) of the study population were suspected for non-classical (NC) CAH. The mutation positive rate of ASPCR was 86% (n=43). Seven patients carried more than two biallelic mutations indicating smaller gene conversions. The predominant mutation identified among the study subjects was I2G splice variant in SW phenotype (38%) and I172N in the SV phenotype (41%). Based on the Long range PCR amplification and restriction digestion we identified one patient with large gene conversion and one patient with large 30kb deletion. These results were confirmed with MLPA.Additionally, utilizing the Targeted NGS we identified five patients with CYP21A2 variants (two patients with novel variants c.1274G>T, c.17_18delTG and two other variants in three patients - c.1451G>C, c.143A>G). We also identified a CYP19A1:c.1142A>T gene variant in a patient who was initially suspected for 21 hydroxylase deficiency. Out of six patients with 11 beta hydroxylase deficiency four patients were positive for homozygous CYP11B1 variants (c.1201-1G>A, c.1200 + 1delG, c.412C>T) and two patients with compound heterozygous variants (c.1024C>T and c.1012dupC, c.623G>A and c.412C>T). Discussion: Utilizing the novel allele specific PCR followed by NGS we identified a total of 96% (48/50) of 21 hydroxylase deficiency patients with homozygous or compound heterozygous mutations and 2% (2/50) were positive for single heterozygous variant in CYP21A2 gene. ASPCR followed by multigene targeted NGS assay for genetic screening in CAH has shown to be a sensitive and specific strategy established in a clinical setting. To best of our knowledge this is the most cost-effective and comprehensive multigene screening carried out in India.

Rapid fall in circulating non-classical monocytes in ST elevation myocardial infarction patients correlates with cardiac injury.

Myocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. We retrospectively analyzed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical, and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand, circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (P<.05) and correlated inversely with left ventricular function (r=0.41, P=.04). The early fall in NC monocytes post-reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. In conclusion, rapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature.

Challenges of CYP21A2 genotyping in children with 21-hydroxylase deficiency: determination of genotype-phenotype correlation using next generation sequencing in Southeastern Anatolia.

Although it is known that there is generally a good correlation between genotypes and phenotypes, the number of studies reporting discrepancies has recently increased, exclusively between milder genotypes and their phenotypes due to the complex nature of the CYP21A2 gene and methodological pitfalls. This study aimed to assess CYP21A2 genotyping in children with 21-hydroxylase deficiency (21-OHD) and establish their predictive genotype-phenotype correlation features using a large cohort in Southeastern Anatolia's ethnically diverse population. The patients were classified into three groups: salt-wasting (SW), simple virilizing (SV) and non-classical (NC). The genotypes were categorized into six groups due to residual enzyme activity: null-A-B-C-D-E. CYP21A2 genotyping was performed by sequence-specific primer and sequenced with next generation sequencing (NGS), and the expected phenotypes were compared to the observed phenotypes. A total of 118 unrelated children with 21-OHD were included in this study (61% SW, 24.5% SV and 14.5% NC). The pathogenic variants were found in 79.5% of 171 mutated alleles (60.2%, 22.2%, and 17.6% in SW, SV and NC, respectively). Patient distribution based on genotype groups was as follows: null-16.1%, A-41.4%, B-6.0%, C-14.4%, E-22%). In2G was the most common pathogenic variant (33.9% of all alleles) and the most common variant in the three phenotype groups (SW-38.8%, SV-22.2% and NC-23.3%). The total genotype-phenotype correlation was 81.5%. The correlations of the null and A groups were 100% and 76.1%, respectively, while it was lower in group B and poor in group C (71.4% and 23.5%, respectively). This study revealed that the concordance rates of the severe genotypes with their phenotypes were good, while those of the milder genotypes were poor. The discrepancies could have resulted from the complex characteristics of 21-OHD genotyping and the limitations of using NGS alone without integrating with other comprehensive methods.

Deciphering the fate of slan+ -monocytes in human tonsils by gene expression profiling.

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan+ -monocytes. In recent studies, we and other groups have reported that, in tonsils, slan marks dendritic cell (DC)-like cells, as defined by morphological, phenotypical, and functional criteria. However, subsequent investigations in lymphomas have uncovered a significant heterogeneity of tumor-infiltrating slan+ -cells, including a macrophage-like state. Based on their emerging role in tissue inflammation and cancer, herein we investigated slan+ -cell fate in tonsils by using a molecular-based approach. Hence, RNA from tonsil slan+ -cells, conventional CD1c+ DCs (cDC2) and CD11b+ CD14+ -macrophages was subjected to gene expression analysis. For comparison, transcriptomes were also obtained from blood cDC2, classical (CL), intermediate (INT), NC, and slan+ -monocytes. Data demonstrate that the main trajectory of human slan+ -monocytes infiltrating the tonsil tissue is toward a macrophage-like population, displaying molecular features distinct from those of tonsil CD11b+ CD14+ -macrophages and cDC2. These findings provide a novel view on the terminal differentiation path of slan+ -monocytes, which is relevant for inflammatory diseases and lymphomas.

Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency.

21-hydroxylase deficiency is the most common cause of Congenital Adrenal Hyperplasia. It presents as severe or classical forms-salt wasting and simple virilizing-and a mild or nonclassical (NC). Several studies have reported the frequency of pathogenic variants in different populations, although few of them included a large number of NC patients. To analyse the CYP21A2 gene defects in a large cohort of Argentine patients. Molecular characterization of 628 patients (168 classical, 460 nonclassical, representing 1203 nonrelated alleles), 398 relatives, 126 partners. Genetic variants were assessed by allele-specific PCR, PCR-RFLP or direct sequencing. Deletions, duplications and large gene conversions (LGC) were studied by Southern blot/MLPA or long-range PCR. Biological implications of novel variants were analysed by structure-based in silico studies. The most frequent pathogenic variants were p.V282L (58%) in NC alleles and c.293-13C>G (31.8%) and p.I173N (21.1%) in classical. Deletions and LGC were found at low frequency (6.2%), 57 alleles had rare pathogenic variants, and 3 had novel variants: p.(S166F); p.(P189R), p.(R436L). Genotype-phenotype correlation was observed in 98.6% of the cases, 11 asymptomatic first-degree relatives had pathogenic variants in both alleles, and 21/126 partners were carriers. We conducted a comprehensive genetic characterization of the largest cohort of 21-hydroxylase patients from the region. In particular, we add to the molecular characterization of a large number of NC patients and to the estimation of the disease carrier's frequency in our population.

3521 Distinct single cell gene expression in peripheral blood monocytes correlates with treatment response groups to TNF-alpha inhibition in rheumatoid arthritis

OBJECTIVES/SPECIFIC AIMS: The cellular mechanisms that underlie the IFNβ/α ratio that predicts response are not known. Effects of IFN on single immune cells may be masked in whole blood or mixed cell populations. By studying the effect of IFNβ/α activity ratio on individual monocytes, we can determine the functional impact of the IFN ratio and suggest the cellular mechanisms that underlie response/non-response to TNFi therapy in RA. METHODS/STUDY POPULATION: We used single cell analysis to investigate whether monocyte gene expression differs significantly between RA patients according to their pre-TNFi serum IFN-β/α ratio. Single classical (CL) and non-classical (NC) blood-derived monocytes were isolated from 15 seropositive RA subjects prior to biologic therapy. Subjects were grouped by pre-TNFi serum IFN-β/α ratio into two groups, those with a high IFN-β/α ratio (≥1.3, n = 6) and those with a low IFN-β/α ratio (&lt;1.3, n = 9). 87 target genes were analyzed. Genes that varied significantly between the groups by categorical analyses were tested in multivariate logistic regression models. RESULTS/ANTICIPATED RESULTS: Every participant was seropositive for rheumatoid factor and antibodies to cyclic citrullinated peptide. Among the participants in the groups, there were no significant differences in age or DAS scores (P&gt;0.05). The treatments were comparable and none were being treated with biologic therapy. There were striking differences in monocyte gene expression between patients with pre-treatment blood IFNβ/α activity &lt;1.3 and ≥1.3. Expression of (1) key type I IFN pathway genes (JAK1, STAT2, IFIT2, IFIH1, PRDM1); (2) IL12; (3) CD36; and (4) CTLA4 were the strongest differentiators between groups (p&lt;0.0001 for each, corrected for multiple comparisons). DISCUSSION/SIGNIFICANCE OF IMPACT: In this study we were able to measure gene expression in single monocytes from seropositive RA patients prior to biologic treatment. Within-cell co-expression patterns demonstrate biological differences in monocytes of RA patients with an IFNβ/α ≥1.3, the ratio of type I IFNs which predicts non-response to TNFi. The data suggest that there may be differential IFN production and pathway activation in patients who do not respond to TNFi. The increased expression of CD36 in monocytes from RA patients with high IFN β/α activity may be a reflection of increased “foam cells” in the inflamed tissue of patients who do not respond to TNFi. Enrichment of CTLA4 in those with high serum IFNβ/α suggests that CTLA4-Ig may be less likely to be an effective alternative for someone who is not likely to respond to TNFi. Current work includes determining whether the peripheral blood findings reflect altered cellular composition, type I IFN production and signaling in the synovium. Significance: This work will help to develop a more individualized approach to therapy in RA and determine an immunological basis of response/non-response to TNFi.

Chlorination-promoted Transformation of Isolated Pentagon Rule C78 into Fused-pentagons- and Heptagons-containing Fullerenes.

Cage transformations in fullerenes are rare phenomena which are still not fully understood. We report the first skeletal transformation of an Isolated-Pentagon-Rule (IPR) isomer of C78 fullerene upon high-temperature chlorination which proceeds by six-step Stone-Wales rearrangements affording non-IPR, non-classical (NC) C78 (NC2)Cl24 with two cage heptagons, six pairs of fused pentagons, and an unprecedented loop-like chlorination pattern. The following loss of a C2 unit results in C76 (NC3)Cl24 containing three cage heptagons.

Single-cell gene expression patterns in lupus monocytes independently indicate disease activity, interferon and therapy

ObjectivesImportant findings can be masked in gene expression studies of mixed cell populations. We examined single-cell gene expression in SLE patient monocytes in the context of clinical and immunological features.MethodsMonocytes...

Linking the degree of virilization in females with congenital adrenal hyperplasia to genotype.

Mutations of CYP21A2 variably decrease 21-hydroxylase activity and result in a spectrum of disease expressions in patients with congenital adrenal hyperplasia (CAH). We examined the association between CYP21A2 mutations and virilization (Prader score) in females with CAH. The study population included 187 CAH females with fully characterized CYP21A2 mutations. One hundred fifty-eight patients were sorted into groups by expected enzyme activity (percent of normal activity) of the less severely affected allele: (A) null, 0%; (B) I2G, 1%; (C) I172N, 2%; and (D) V281L, >2%. We observed an inverse relationship between virilization and residual enzyme activity (P < 0.001). Subjects in group A or B had a significantly higher likelihood (unadjusted odds ratio: 16; P < 0.001) of developing severe virilization compared with those in group C. Surprisingly, 24% of group D patients, whose mutation is usually associated with nonclassical (NC) CAH, had severe virilization. Among subjects with the NC P30L mutation, 66% expressed unexpected virilization. Virilization, usually leading to extensive reconstructive surgery, is highly likely in patients with null or I2G mutations; however, NC mutations (P30L/V281L) may also lead to unexpected virilization. These findings have implications for prenatal counseling and highlight the need for additional investigations into other factors that influence virilization in CAH.

Skeletal Transformation of a Classical Fullerene C88 into a Nonclassical Fullerene Chloride C84Cl30 Bearing Quaternary Sequentially Fused Pentagons.

A classical fullerene is composed of hexagons and pentagons only, and its stability is generally determined by the Isolated-Pentagon-Rule (IPR). Herein, high-temperature chlorination of a mixture containing a classical IPR-obeying fullerene C88 resulted in isolation and X-ray crystallographic characterization of non-IPR, nonclassical (NC) fullerene chloride C84(NC2)Cl30 (1) containing two heptagons. The carbon cage in C84(NC2)Cl30 contains 14 pentagons, 12 of which form two pairs of fused pentagons and two groups of quaternary sequentially fused pentagons, which have never been observed in reported carbon cages. All 30 Cl atoms form an unprecedented single chain of ortho attachments on the C84 cage. A reconstruction of the pathway of the chlorination-promoted skeletal transformation revealed that the previously unknown IPR isomer C88(3) is converted into 1 by two losses of C2 fragments followed by two Stone-Wales rearrangements, resulting in the formation of very stable chloride with rather short C-Cl bonds.

Molecular diagnosis of Chinese patients with 21-hydroxylase deficiency and analysis of genotype-phenotype correlations.

ObjectiveThe spectrum of molecular defects in Chinese patients with 21-hydroxylase deficiency (21-OHD), and genotype–phenotype relationships are unknown.MethodsWe screened eight patients with non-classical (NC) 21-OHD and 35 with classical 21-OHD, and detected nine known mutations.ResultsThe most frequent mutation among the 43 21-OHD cases was p.Ile172Asn (allele frequency, 36.0%), followed by c.290-13A/C > G (20.9%), Del (8.6%), p.Pro30Leu (7.0%), p.Gln318Ter (7.0%), p.Val281Leu (4.7%), p.Arg356Trp (2.3%), p.[Ile236Asn; Val237Glu; Met239Lys] (2.3%), and E3Δ8 bp (1.2%). The frequency spectrum of CYP21A2 mutations in the Chinese population was similar to that in the Japanese population, except that p.Val281Leu was identified in Chinese NC21-OHD patients at a frequency of 25.0%, whereas it was absent in Japanese patients. We found that genotype could predict phenotype in 88.3% of patients.ConclusionSome characteristics appear to be unique to the Chinese population, but genotype was strongly predictive of phenotype.

Chloro Derivatives of Azafullerenes C59 NCl5 and Non-Classical C97 NCl21.

High-temperature chlorination of HPLC fullerene fractions containing mainly C92 /C94 and C102 /C104 resulted in the isolation and X-ray structural characterization of chloro derivatives of azafullerenes, C59 NCl5 and C97 NCl21 . It was assumed that formation of azafullerenes in the arc-discharge synthesis of fullerenes occurred due to air leakage into the reactor. The molecule of C59 NCl5 contains an isolated aromatic pyrrole ring on the fullerene cage and possesses C5v symmetrical shape typical of other known C59 NR5 derivatives. The molecule of non-classical (NC) C97 N(NC)Cl21 contains an NC6 heptagon on the azafullerene cage that assumes its formation by a C2 loss from C99 N in the course of chlorination. The chlorination pattern is characterized by the presence of stabilizing isolated aromatic systems and isolated double C=C bonds on the C97 N(NC) azafullerene cage.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population.

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA-A, -B, -DRB1 alleles/haplotypes, cause deficiency of the 21-hydroxylase enzyme (21-OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non-classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA-A, -B, and -DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype, 23 had the HLA-A*33:01-B*14:02-DRB1*03:01 haplotype, 8 had the B*14:02-DRB1*01:02 combination and 5 were carrying the HLA-A*68:02-B*14:02-DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA-B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02-DRB1*01:02 (P < 0.001) and HLA-A*68:02-B*14:02-DRB1*13:03 (P < 0.001) as well as HLA-A*33:01-B*14:02-DRB1*01:02 and HLA-A*33:01-B*14:02-DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA-A*33:01-B*14:02-DRB1*03:01 and HLA-A*68:02-B*14:02-DRB1*13:03 haplotypes.