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Aim of the Study: Osteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts., Materials and Methods: Fibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14 C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content., Results and Conclusions: Osteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Early diagnosis and monitoring of disease activity are essential in cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). We aimed to establish exhaled molecular profiles as the first step in assessing the potential of breath analysis., Methods: Exhaled breath was analyzed by electronic nose in 25 children with CF, 25 with PCD and 23 controls. Principle component reduction and canonical discriminant analysis were used to construct internally cross-validated ROC curves., Results: CF and PCD patients had significantly different breath profiles when compared to healthy controls (CF: sensitivity 84%, specificity 65%; PCD: sensitivity 88%, specificity 52%) and from each other (sensitivity 84%, specificity 60%). Patients with and without exacerbations had significantly different breath profiles (CF: sensitivity 89%, specificity 56%; PCD: sensitivity 100%, specificity 90%)., Conclusion: Exhaled molecular profiles significantly differ between patients with CF, PCD and controls. The eNose may have potential in disease monitoring based on the influence of exacerbations on the VOC-profile., (Copyright © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

We report an Indonesian patient with bone fragility and congenital joint contractures. The initial diagnosis was Osteogenesis Imperfecta type III (OI type III) based on clinical and radiological findings. Because of (i) absence of COL1A1/2 mutations, (ii) a consanguineous pedigree with a similarly affected sibling and (iii) the existence of congenital joint contractures with absence of recessive variants in PLOD2, mutation analysis was performed of the FKBP10 gene, recently associated with Bruck syndrome and/or recessive OI. A novel homozygous deletion in FKBP10 was discovered. Our report of the first Indonesian patient with clinically Bruck syndrome, confirms the role of causative recessive FKBP10 mutations in this syndrome., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Purpose: To identify a molecular genetic cause in patients with a clinical diagnosis of osteogenesis imperfecta (OI) type I/IV., Methods: The authors performed multiplex ligation-dependent probe amplification analysis of the COL1A1 gene in a group of 106 index patients., Results: In four families with mild osteogenesis imperfecta and no other phenotypic abnormalities, a deletion of the complete COL1A1 gene on one allele was detected, a molecular finding that to our knowledge has not been described before, apart from a larger chromosomal deletion detected by fluorescent in situ hybridization encompassing the COL1A1 gene in a patient with mild osteogenesis imperfecta and other phenotypic abnormalities. Microarray analysis in three of the four families showed that it did not concern a founder mutation., Conclusion: The clinical picture of complete COL1A1 allele deletions is a comparatively mild type of osteogenesis imperfecta. As such, multiplex ligation-dependent probe amplification analysis of the COL1A1 gene is a useful additional approach to defining the mutation in cases of suspected osteogenesis imperfecta type I with no detectable mutation.

In 1979 Sillence proposed a classification of Osteogenesis Imperfecta (OI) in OI types I, II, III and IV. In 2004 and 2007 this classification was expanded with OI types V-VIII because of distinct clinical features and/or different causative gene mutations. We propose a revised classification of OI with exclusion of OI type VII and VIII since these types have been added because of genetic criteria (autosomal recessive inheritance) while the clinical and radiological features are indistinguishable from OI types II-IV. Instead, we propose continued use of the Sillence criteria I, II-A, II-B, II-C, III and IV for clinical and radiological classification of OI with additional mentioning of the causative mutated gene to this classification. OI type V and VI are still part of this revised classification, because of the distinguishing clinical/radiological and/or histological features observed in these types., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

We report two families in which the probands have compound-heterozygous Marfan syndrome (MFS). The proband of family 1 has the R2726W FBN1 mutation associated with isolated skeletal features on one allele and a pathogenic FBN1 mutation on the other allele. The phenotype of the compound-heterozygous probands appears to be more severe than that of their heterozygous family members which underlines the possibility that certain trans-located FBN1 mutations might act as modifiers of phenotype explaining some of the intrafamilial variability in Marfan syndrome.

In 1994, the molecular basis of pachyonychia congenita (PC) was elucidated. Four keratin genes are associated with the major subtypes of PC: K6a or K16 defects cause PC-1; and mutations in K6b or K17 cause PC-2. Mutations in keratins, the epithelial-specific intermediate filament proteins, result in aberrant cytoskeletal networks which present clinically as a variety of epithelial fragility phenotypes. To date, mutations in 20 keratin genes are associated with human disorders. Here, we review the genetic basis of PC and report 30 new PC mutations. Of these, 25 mutations were found in PC-1 families and five mutations were identified in PC-2 kindreds. All mutations identified were heterozygous amino acid substitutions or small in-frame deletion mutations with the exception of an unusual mutation in a sporadic case of PC-1. The latter carried a 117 bp duplication resulting in a 39 amino acid insertion in the 2B domain of K6a. Also of note was mutation L388P in K17, which is the first genetic defect identified in the helix termination motif of this protein. Understanding the genetic basis of these disorders allows better counseling for patients and paves the way for therapy development.

Objectives: Elucidation of the genetic background of familial abdominal aortic aneurysm (AAA) suggests a genetic etiology., Methods and Results: We carried out a genome-wide scan in three Dutch families with four or five affected siblings. Suggestive loci were further studied by subsequent fine mapping of the locus performed in 101 affected sib-pairs. The genome-wide scan was performed with 400 DNA markers and results were given as non-parametric, multipoint linkage scores (NPL). We observed a suggestive linkage for AAA (NPL score 3.25 at D19S902, 72.72 cM) on chromosome 19q in the three families. After fine mapping on chromosome 19, the NPL score became nominal in the 101 affected sib-pairs. A separate analysis of the three families with fine mapping revealed a peak with significant evidence for linkage (NPL score 3.95 at D19S904, 78.08 cM) on chromosome 19q. This peak was situated to the right compared to the region found in a previously published article for familial AAA on chromosome 19q., Conclusions: Our results identified a candidate locus in three Dutch families with AAA at chromosome 19q13.3. Separate analysis of these three families provides evidence for genetic heterogeneity.

Objectives: to examine possible involvement of several candidate genes in the aetiology of familial abdominal aortic aneurysm (AAA)., Design: after reviewing the literature on the genetics of familial AAA, betaine homocysteine methyltransferase (BHMT), collagen type Ialpha2 (COL1A2) and cathepsin H (CTSH), were selected as potential candidate genes, which influence structure, strength, elasticity and mechanical resistance of the aortic wall., Materials: forty-eight families with 110 family members and AAA were included in the affected sib-pair analysis. One large family of three generations was analysed separately because in this family also other clinical symptoms were involved., Methods: genetic linkage analysis was performed with DNA markers in the region of BHMT, COL1A2 and CTSH., Results: In the overall sib-pair analysis, the LOD scores for BHMT, COL1A2 and CTSH were 0.7, 0.2 and -0.7, whereas in the large family these numbers were -0.6, -2.2 and -2.7, respectively., Conclusions: none of the candidate genes selected showed a suggestive linkage with AAA. Exclusion of the COL1A2 and CTSH genes was possible in the large family that was analysed separately.

Objective: This study investigated a large number of families in which at least two individuals were diagnosed with abdominal aortic aneurysms to identify the relationship of the affected relatives to the proband., Subjects and Methods: Families for the study were recruited through various vascular surgery centers in the United States, Finland, Belgium, Canada, the Netherlands, Sweden, and the United Kingdom and through our patient recruitment website (www.genetics.wayne.edu/ags)., Results: We identified 233 families with at least two individuals diagnosed with abdominal aortic aneurysms. The families originated from nine different nationalities, but all were white. There were 653 aneurysm patients in these families, with an average of 2.8 cases per family. Most of the families were small, with only two affected individuals. There were, however, six families with six, three with seven, and one with eight affected individuals. Most of the probands (82%) and the affected relatives (77%) were male, and the most common relationship to the proband was brother. Most of the families (72%) appeared to show autosomal recessive inheritance pattern, whereas in 58 families (25%), abdominal aortic aneurysms were inherited in autosomal dominant manner, and in eight families, the familial aggregation could be explained by autosomal dominant inheritance with incomplete penetrance. In the 66 families where abdominal aortic aneurysms were inherited in a dominant manner, 141 transmissions of the disease from one generation to another were identified, and the male-to-male, male-to-female, female-to-male, and female-to-female transmissions occurred in 46%, 11%, 32%, and 11%, respectively., Conclusion: Our study supports previous studies about familial aggregation of abdominal aortic aneurysms and suggests that first-degree family members, male relatives, in particular, are at increased risk. No single inheritance mode could explain the occurrence of abdominal aortic aneurysms in the 233 families studied here, suggesting that abdominal aortic aneursyms are a multifactorial disorder with multiple genetic and environmental risk factors.

Background: Familial clustering of the abdominal aortic aneurysm (AAA) is clear, 12-19% of AAA patients have one or more first-degree relatives with an aneurysm and 4-19% is detected with ultrasound screening., Objectives: To review the genetic background of AAA. DESIGN, METHODS AND MATERIALS: Computer searches of the MEDLINE, EMBASE, SUMsearch database and the Cochrane Library and searched reference lists of English language articles concerning the genetics of AAA, candidate gene approach and linkage analysis., Results: Brothers of AAA patients are at high risk to develop an AAA. The candidate gene approach was performed to detect defects in one of the components of the connective tissue, i.e. type I and III collagen, elastin and fibrillin, the inflammatory cell-derived matrix metalloproteinase, there inhibitors, auto-immune components and components related to atherosclerosis., Conclusion: These studies give us insight in the pathology but do not lead to the specific genetic factor(s) responsible for (familial) AAA. Considering the supposed autosomal dominant inheritance, a gene mutation in one of the structural proteins of the connective tissue is expected. In the future, linkage analysis may resolve the genetic background of AAA.

Objective: This study was undertaken to assess frequencies of the methylenetetrahydrofolate reductase gene mutations cytosine-to-thymine substitution at base 677 (C677T) and adenine-to-cytosine substitution at base 1298 (A1298C) and their interactions with homocysteine and vitamin levels among Dutch women with preeclampsia., Study Design: Mutations were studied in the following 5 groups: 47 consecutive women with preeclampsia, 49 women with preeclampsia and with hyperhomocysteinemia, 36 women with preeclampsia but without hyperhomocysteinemia, 127 women with familial preeclampsia (typed for C677T mutations only), and 120 control subjects. Plasma levels of homocysteine, folate, and vitamin B12 were measured., Results: Although 10.6% of the consecutive women with preeclampsia had strictly defined hyperhomocysteinemia (values >97.5th percentile), neither mutation was found in excess relative to the control group. Women with preeclampsia who had mild hyperhomocysteinemia (values >75th percentile) had a significant excess of the TT genotype (homozygosity for C677T mutation) relative to the women with preeclampsia who did not have hyperhomocysteinemia (odds ratio, 8.2; 95% confidence interval, 1.8-39). They also had significantly lower vitamin levels., Conclusion: Hyperhomocysteinemia in women with preeclampsia was associated with mutations in the gene for methylenetetrahydrofolate reductase, but the high frequency of hyperhomocysteinemia itself cannot be explained by these mutations alone.

Objectives: type III collagen is responsible for the tensile strength of the aorta-wall. To determine if genetic defect in the type III collagen production is associated with familial clustering of AAA., Methods: fifty-six patients with AAA and 82 first-degree family members participated. The medical and family histories were obtained. All these relatives were screened by ultrasound for AAA. In 58 relatives of 20 families, skin biopsies were taken for protein analysis to measure type III collagen production in cultured fibroblasts., Results: only one new AAA was detected in a brother of a patient. Four other relatives were already known with AAA. Three AAA patients had a type III collagen deficiency, but type III collagen was normal in all family members., Conclusion: type III collagen deficiency does not appear to be an aetiological factor in the development of AAA., (Copyright 2000 Harcourt Publishers Ltd.)

Objectives: to evaluate the prevalence of familiar abdominal aortic aneurysm (AAA) and the role of type III collagen deficiency., Methods: fifty-six consecutive patients coming for aneurysm repair were asked if one or more first-degree relatives had an AAA. During operation, a skin biopsy was taken from the patients for protein analysis to measure the type III collagen production in cultured fibroblasts., Results: a positive family history was found in 28.6% of the AAA patients. Six (10.7%) of the AAA patients had a type III collagen deficiency (mean 4.3% (S.D.+/-0.5)). In this group three men, mean age 65.3 years (S.D.+/-5.0), had a positive family history and a type III collagen deficiency. Segregation analysis with an intragenic marker in the type III collagen gene in a single family was in favour of linkage with the gene for type III procollagen (COL3A1) locus., Conclusions: the high prevalence of familial AAA suggests a genetic aetiology. A small group of patients have a type III collagen deficiency. Linkage with the COL3A1 gene could not be proven or excluded in the families studied., (Copyright 1999 W.B. Saunders Company Ltd.)

Objectives: Our purpose was to determine the prevalence of BRCA1 and BRCA2 germline mutations among patients from ovarian cancer families and to evaluate age at diagnosis, histologic diagnosis, and International Federation of Gynecology and Obstetrics stage in this group., Study Design: We reviewed 50 ovarian cancer patients from 30 ovarian cancer families and compared relevant clinical characteristics with those of a cancer registry reference group. BRCA1 (exons 2 to 24) and BRCA2 (exon 11) germline mutations were detected by a protein truncation test and sequencing of BRCA1 exon 2 (185delAG mutation) in 25 of the 30 families., Results: Ten (40%) of 25 families tested revealed a germline BRCA1 or BRCA2 mutation. Patients with ovarian cancer from the study group were young with an advanced International Federation of Gynecology and Obstetrics stage at diagnosis and had a relatively high frequency of serous adenocarcinoma., Conclusion: Direct mutation analysis of BRCA1 and BRCA2 revealed a high frequency of germline mutations in ovarian cancer families. Some clinical characteristics of hereditary ovarian cancer may differ from those of sporadic disease.

Chronic Helicobacter pylori gastritis has been put forward as a risk factor for development of gastric mucosal atrophy and gastric cancer. The purpose of our study was to investigate the long-term effects of H pylori gastritis on the gastric mucosa. We prospectively studied 49 subjects negative for H pylori and 58 positive subjects for a mean follow-up of 11.5 years (range 10-13 years). Serum samples were obtained at the initial and follow-up visits for determination of H pylori IgG antibodies. Gastroscopies with biopsy sampling were done in all patients at both visits. Biopsy specimens were used for assessment of H pylori infection and histology. Development of atrophic gastritis and intestinal metaplasia occurred in 2 (4%) uninfected and 16 (28%) infected subjects. Regression of atrophy was noted in 4 (7%) infected subjects. Development of atrophic gastritis and intestinal metaplasia was significantly associated with H pylori infection (p = 0.0014; odds ratio 9.0, 95% CI 1.9-41.3). The proportion of atrophic gastritis in the study population showed an annual increase of 1.15% (0.5-1.8%). We conclude that H pylori infection is a significant risk factor for development of atrophic gastritis and intestinal metaplasia. Our findings support strongly the causative role of this infection in gastric carcinogenesis.

The prevalence of Helicobacter pylori antibodies increases with age, but it is unknown whether this is due to a constant rate of infection in different age groups, or whether most infection occurs in childhood. Follow-up data on infection rates and the course of infection in an untreated population are scarce. We measured H pylori IgG antibody concentrations in patients who were seen at our endoscopy unit between 1979 and 1983. 115 of 164 eligible patients (70%) participated in the study. H pylori IgG antibody concentrations were measured in two serum samples taken with a mean interval of 11.5 years. 56 patients tested positive at the first visit. During follow-up, 2 patients became infected (annual infection rate 0.30%, 95% Cl 0.04-1.08%). Evidence of infection disappeared in 6 patients: after gastric surgery in 3 and due to an unknown cause in the remaining 3 patients. A non-significant decrease of infection was shown in different age cohorts during follow-up. Antibody concentrations did not increase with age. These results strongly support the concept of dominant infection rates in childhood. Elimination of infection may occur in a few patients without eradication therapy.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.

The localization of pepsinogens (PG A and PG C) was studied intracellularly in human gastric biopsies embedded in Lowicryl K4M, using affinity-purified antibodies and protein A-gold. The homogeneous secretory granules of the chief cells contained both PG A and PG C, as was proved by serial sections. Identical reaction was also seen in the core of the biphasic mucous neck cell granules, whereas the mantle did not label. The rough endoplasmic reticulum (RER) and Golgi complex of the chief cells and mucous neck cells contained ample label. Transitional cells identified by the presence of granules of both chief cells and mucous neck cells were recognized. This type of mucous neck cell is thought to transform into a chief cell. However, an increase of RER that could explain an increase of the pepsinogen production was not observed. A mixture of these granules was also found in cells morphologically characterized as young parietal cells, suggesting a common precursor for these three cell types. These observations make the transformation from mucous neck to chief cells questionable. Antral gland cells contained only PG C, as was shown in serial section, too.