Novel ALG12 variants and hydronephrosis in siblings with impaired N-glycosylation

BackgroundALG12-CDG is a rare autosomal recessive type I congenital disorder of glycosylation (CDG) due to pathogenic variants in ALG12 which encodes the dolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-alpha-6-mannosyltransferase. Thirteen patients from unrelated 11 families have been reported, most of them result in broad multisystem manifestations with clinical variability. It is important to validate abnormal glycosylation to establish causal relationship.Case reportHere, we report two siblings with novel compound heterozygous variants in ALG12: c.443T>C, p.(Leu148Pro) and c.412_413insCGT, p.(Gln137_Phe138insSer). Both patients showed global developmental delay, microcephaly, hypotonia, failure to thrive, facial dysmorphism, skeletal malformations and coagulation abnormalities, which are common in ALG12-CDG. In addition, one of our patients showed left hydronephrosis, which is a novel clinical feature in ALG12-CDG. Brain MRI showed hypoplasia of cerebrum, brain stem and cerebellar vermis in both patients. N-glycosylation defects of trypsin digested transferrin peptides were revealed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and electrospray ionization MS verified the lack of N-glycans in transferrin.ConclusionsThe present study can add hydronephrosis to phenotypic spectrum of ALG12-CDG. Since the symptoms of ALG12-CDG are quite diverse, the combination of whole-exome sequencing and transferrin glycopeptide analysis with MS, can help diagnosis of ALG12-CDG.     

0.5 Mb Array as a First‐Line Prenatal Cytogenetic Test in Cases without Ultrasound Abnormalities and Its Implementation in Clinical Practice

Using whole‐genome array testing instead of karyotyping in prenatal diagnosis for all indications may be desirable because of the higher diagnostic yield and shorter reporting time. The goal of this research was finding the optimal array resolution that could replace routine prenatal karyotyping in cases without ultrasound abnormalities, for example, referred for advanced maternal age or abnormal first trimester screening. As variants of unknown clinical significance (VOUS), if reported, might complicate decision‐making about continuation of pregnancy, such an optimal array resolution should have a high abnormality detection rate and reveal a minimal amount of VOUS. The array data of 465 fetuses were retrospectively evaluated with several resolution levels, and the Decipher microdeletion/microduplication syndrome list was reviewed to assess what could be theoretically missed with a lower resolution. A 0.5‐Mb resolution showed a high diagnostic yield potential and significantly minimized the number of VOUS. Based on our experience, we recommend genomic SNP array as a first‐tier test in prenatal diagnosis. The resolution should be chosen based on the indication. In cases of fetal ultrasound abnormalities or intrauterine fetal death (IUFD), high‐resolution analysis should be done. In other cases, we advise replacing karyotyping by SNP array analysis with 0.5 Mb resolution.     

海洋细胞毒药物与抗体偶联物的制备及生物活性评价

目的 以抗体N-糖链为偶联位点,制备曲妥珠单抗与海洋细胞毒药物单甲基澳瑞他汀E(MMAE)偶联物,并研究其对乳腺癌细胞BT474的杀伤作用。 方法 合成含叠氮和炔基修饰的唾液酸化寡糖,采用化学酶法制备含叠氮或炔基修饰的曲妥珠单抗,利用生物正交反应分别制备相应的抗体药物偶联物。利用SDS-PAGE 对转糖基及偶联过程进行监测,表面等离子共振技术(SPR)对叠氮或炔基修饰前后抗体与FcγRIIIa亲和力进行检测。利用CCK-8法评价两种抗体药物偶联物对乳腺癌细胞株BT474的杀伤作用。结果 成功制备了含叠氮或炔基修饰的曲妥珠单抗及N-糖链糖基位点抗体药物偶联物,修饰抗体与FcγRIIIa的亲和力较曲妥珠单抗增加,相比曲妥珠单抗,两种抗体MMAE偶联物对BT474有更明显的生长抑制作用。结论 基于抗体糖链末端唾液酸位点偶联的方法可有效应用于海洋细胞毒抗体偶联药物的开发。     

Polycythaemia associated with homozygosity for the abnormal haemoglobin Sherwood Forest (β104 (G6) Arg → Thr)

Summary. We describe a 22-year-old Pakistani male with polycythaemia associated with homozygosity for a highaffinity haemoglobin mutant, Hb Sherwood Forest. This haemoglobin variant has an amino acid substitution in the β globin chain at position 104, Arg → Thr. In the two previously reported instances of this haemoglobin mutant the individuals were heterozygotes and were haematologically normal. We show here that the homozygous state for the mutation is associated with a compensatory erythrocytosis resulting from decreased delivery of oxygen to the tissues. A family study showed that both parents and two siblings are heterozygotes for the haemoglobin mutant and are haematologically normal. To our knowledge, this represents the first example of a β-globin mutation producing polycythaemia in homozygotes but not in heterozygotes.     

Skin Advanced Glycation End Product Accumulation Is Poorly Reflected by Glycemic Control in Type 2 Diabetic Patients (ZODIAC-9)

Background

Glycemic memory can be reflected by tissue accumulation of advanced glycation end products (AGEs). In type 1 diabetes mellitus (T1DM) patients, hemoglobin A1c (HbA1c) levels over various time periods poorly predicted the accumulation of different AGEs in skin biopsies. Our aim was to investigate whether HbA1c assessments can predict the change in skin AGEs during time in type 2 diabetes mellitus (T2DM).

Methods

We included 452 T2DM patients participating in a shared-care setting, who are screened annually for HbA1c and diabetic complications. Baseline and follow-up levels of skin AGEs were assessed with a validated noninvasive autofluorescence (AF) method, which is based on the fluorescence characteristics of certain AGEs.

Results

Our study population had a mean age of 65 years and 54% were female. After a mean follow-up duration of 3.3 years, linear regression analyses showed weak relationships among different assessments of HbA1c (baseline, maximum, mean, and variance of HbA1c) and skin AF at follow-up. Baseline skin AF and age were predictors of skin AF at follow-up, but diabetes duration, smoking, and creatinine were of less or no predictive value for skin AF at follow-up.

Conclusions

In our T2DM population, integrated HbA1c assessments over years poorly predict the change in skin AGE level measured by skin AF. These findings agree with results in patients with T1DM. This suggests either the need for longer exposure to glucose disturbances to change tissue AGEs or other mechanisms, such as oxidative stress, leading to AGE accumulation.     

Comparison of N-glycan pattern of recombinant human coagulation factors II and IX expressed in Chinese hamster ovary (CHO) and African green monkey (Vero) cells

The N-glycan patterns of recombinant human coagulation factors II (rF-II) and IX (rF-IX), derived from both transfected Chinese hamster ovary (CHO) cells and African green monkay (Vero) cells produced at industrial scale, were analyzed by binding to carbohydrate-specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. Human plasma-derived coagulation factors II (hpF-II) and IX (hpF-IX) exhibited complex-type glycan structures with carbohydrate chains capped with (2–6)-sialic acid. Terminal galactose-(1–4)-N-acetylglucosamine units were detected in hpF-IX. Both CHO cell-derived rF-II and rF-IX exhibited complex-type glycosylation and contained (2–3)-sialic acid in addition to terminal galactose-(1–4)-N-acetylglucosamine. Vero cell-derived rF-IX exhibited a complex-type glycan structure similar to that of CHO cell-derived rF-IX. In contrast, rF-II produced by Vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing high-mannose structures and complex-type glycosylation containing (2–3)-sialic acid. Galactose-(1–4)-N-acetylglucosamine structures and a low concentration of (2–6)-sialic acid were detected in both microheterogeneity fractions of Vero cell-derived rF-II. Although different in their carbohydrate structures, coagulation factors II and IX obtained recombinantly from both transformed CHO cells and Vero cells exhibited coagulation activities comparable with the plasma-derived proteins.     

Non-enzymatic glycosylation of serum proteins as an indicator of diabetic control

Summary In 44 diabetic patients treated either with insulin or with oral antidiabetic agents, non-enzymatic glycosylation of serum proteins (GSP), expressed as nmol of 5-hydroxymethylfurfural/mg protein, showed higher values than those found in control subjects. In the diabetics GSP was more closely correlated to the degree of glycometabolic control during the 15 days preceding the assay, than to the glycosylated hemoglobin (HbAl). However, GSP values did not correlate significantly with fasting blood glucose levels. It may be concluded that GSP can be used as an index of the medium-term control of diabetes mellitus.     

重型颅脑外伤术后颅内感染患者脑脊液高迁移率族蛋白-1和RAGE水平的变化及意义

目的:观察重型颅脑外伤(sever traumatic brain injury,s TBI)术后发生颅内感染的患者高迁移率族蛋白-1(High mobility group box 1,HMGB-1)和晚期糖基化终产物受体(receptor for advanced glycosylation end products,RAGE)的脑脊液(cerebrospinal fluid,CSF)水平,探讨其对诊断颅内感染的价值。方法:54例s TBI术后拟诊发生颅内感染的患者根据最后诊断分为颅内感染组(n=12)和非颅内感染组(n=42)。酶联免疫法吸附法(enzyme-linked immunosorbent assay,ELISA)检测拟诊颅内感染时CSF中的HMGB-1和RAGE蛋白水平。受试者工作曲线(receiver operating characteristic,ROC)分析其对颅内感染的诊断意义。结果:脑脊液的HMGB-1和RAGE水平在颅内染组和非颅内感染组之间比较差异具有统计学意义(t=6.711,P=0.000;t=2.683,P=0.008)。脑脊液HMGB-1和RAGE水平诊断颅内感染的ROC的曲线下面积分别为:0.899(95%CI:0.822~0.975)、0.682(95%CI:0.555~0.809)。HMGB-1的cut-off值分别为209.50 ng/m L(敏感度:0.75,特异性:0.93);RAGE的cut-off值为108.50 ng/m L(敏感度:1.00,特异性:0.43)。结论:联合检测脑脊液HMGB-1和RAGE水平对s TBI术后是否发生颅内感染具有重要的诊断价值。     

糖耐量异常患者骨密度水平测定及其与血糖代谢指标的关系探究

目的 研究糖耐量异常(IGT)患者的骨密度水平及其与血糖代谢指标的关系.方法 将河北医科大学第一医院门诊体检发现的IGT患者290例纳入研究的观察组,同期体检的健康者290例纳入研究的对照组,检测两组受试者的血糖代谢指标空腹血糖、空腹血浆胰岛素、胰岛素样生长因子、糖基化终末产物、糖化血红蛋白水平以及L3、L4椎体、股骨颈、大转子、Ward's区的骨密度,分析骨密度水平与血糖代谢指标的关系.结果 两组受试者空腹血糖水平无明显差异(P＞0.05).观察组患者的晚期糖基化终末产物、糖化血红蛋白水平高于对照组,胰岛素、胰岛素样生长因子水平以及L3、L4腰椎、股骨颈、Ward's三角区、大转子的骨密度水平均低于对照组.糖基化终末产物、糖化血红蛋白水平与骨密度水平呈负相关,胰岛素、胰岛素样生长因子水平与骨密度水平呈正相关.结论 IGT患者的骨密度显著下降,且骨密度与血糖代谢指标胰岛素、胰岛素样生长因子、糖基化终末产物、糖化血红蛋白水平密切相关.     

Quantitative analysis of β1,6GlcNAc-branched N-glycans on β4 integrin in cutaneous squamous cell carcinoma

α6β4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased β1,6GlcNAc-branched N-glycans on β4 integrin suppress β4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of β1,6GlcNAc-branched N-glycans with β4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that β1,6GlcNAc-branched N-glycans are upregulated on β4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of β1,6GlcNAc-branched N-glycans on β4 integrins as well as β4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of β1,6GlcNAc-branched N-glycans to β4 integrin compared with normal skin samples. These findings suggest that the addition of β1,6GlcNAc-branched N-glycans onto β4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of β1,6GlcNAc-branched N-glycans on β4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression.