Janine Genschel

Genetic basis for increased intestinal permeability in families with Crohn’s disease: role of CARD15 3020insC mutation?

Background and aim: A genetically impaired intestinal barrier function has long been suspected to be a predisposing factor for Crohn’s disease (CD). Recently, mutations of the capsase recruitment domain family, member 15 (CARD15) gene have been identified and associated with CD. We hypothesise that a CARD15 mutation may be associated with an impaired intestinal barrier. Methods: We studied 128 patients with quiescent CD, 129 first degree relatives (CD-R), 66 non-related household members (CD-NR), and 96 healthy controls. The three most common CARD15 polymorphisms (R702W, G908R, and 3020insC) were analysed and intestinal permeability was determined by the lactulose/mannitol ratio. Results: Intestinal permeability was significantly increased in CD and CD-R groups compared with CD-NR and controls. Values above the normal range were seen in 44% of CD and 26% of CD-R but only in 6% of CD-NR, and in none of the controls. A household community with CD patients, representing a common environment, was not associated with increased intestinal permeability in family members. However, 40% of CD first degree relatives carrying a CARD15 3020insC mutation and 75% (3/4) of those CD-R with combined 3020insC and R702W mutations had increased intestinal permeability compared with only 15% of wild-types, indicating a genetic influence on barrier function. R702W and G908R mutations were not associated with high permeability. Conclusions: In healthy first degree relatives, high mucosal permeability is associated with the presence of a CARD15 3020insC mutation. This indicates that genetic factors may be involved in impairment of intestinal barrier function in families with IBD.

Frameshift and nonsense mutations in the gene for ATPase7B are associated with severe impairment of copper metabolism and with an early clinical manifestation of Wilson's disease.

Wilsons disease (WD) is an autosomal recessive disorder of copper metabolism. The clinical phenotype of the disease is varied. It is proposed that this variation may be a result of differential functional disruption of ATPase7B (ATP7B) resulting from mutations in the gene ATP7B. We aimed to assess the relationship between specific mutational defects in ATP7B and divergence in the phenotypic expression of WD. One hundred and forty‐two patients with clinically, biochemically and genetically diagnosed WD were included in the study. The phenotypic expression of WD was compared between patients with different types of mutations in ATP7B, detected by direct sequencing of exons 1–21 of the gene. Twenty‐six mutations were identified in ATP7B; eleven of them were mutations predicted to result in the absence of a full‐length normal protein [frameshift/nonsense mutations; classified as ‘severe’ mutations (SMs)], 14 were missense mutations (MMs) and one was a splice site mutation. Patients with one or two SMs on their alleles had lower serum copper and ceruloplasmin and were younger when the first symptoms of the disease appeared, compared with individuals with two MMs. The effect of SMs on the WD phenotype was dose‐dependent. It is concluded that mutations within ATP7B are very heterogeneous. Frameshift and nonsense mutations are associated with a severe phenotype of WD.

Mutations in the LMNA gene encoding lamin A/C

Very recently, mutations within the LMNA gene on chromosome 1q21.2 were shown to result in forms of muscular dystrophy, conduction‐system disease, cardiomyopathy, and partial lipodystrophy. The LMNA gene encodes for the nucleophilic A‐type lamins, lamin A and lamin C. These isoforms are generated by different splicing within exon 10 of LMNA. Thus lamin A/C is, besides emerin, the first known nucleophilic protein which plays a role in human disease. To date, 41 different mutations, predominantly missense, in the LMNA gene are known causing variable phenotypes. Twenty‐three different mutations of LMNA have so far been shown to cause autosomal‐dominant Emery‐Dreifuss muscular dystrophy (EDMD2), three mutations were reported to cause limb‐girdle muscular dystrophy (LGMD1B), eight mutations are known to result in dilated cardiomyopathy (CMD1A), and seven mutations were reported to cause familial partial lipodystrophy (FPL). The reports of lamin mutations including the corresponding phenotype are of great interest in order to gain insights into the function of lamin A/C. Here we summarize the mutations published to date in LMNA encoding lamin A/C. Hum Mutat 16:451–459, 2000.

p.H1069Q mutation in ATP7B and biochemical parameters of copper metabolism and clinical manifestation of Wilson's disease

We compared the effect of the p.H1069Q mutation and other non‐p.H1069Q mutations in ATP7B on the phenotypic expression of Wilsons disease (WD), and assessed whether the clinical phenotype of WD in compound heterozygotes depends on the type of mutation coexisting with the p.H1069Q. One hundred forty‐two patients with clinically, biochemically, and genetically diagnosed WD were studied. The mutational analysis of ATP7B was performed by direct sequencing. A total number of 26 mutations in ATP7B were identified. The p.His1069Gln was the most common mutation (allelic frequency: 72%). Seventy‐three patients were homozygous for this mutation. Of compound heterozygotes, 37 had frameshift/nonsense mutation, and 20 had other missense mutation on one of their ATP7B alleles. Twelve patients had two non‐p.H1069Q mutations. Patients homozygous for the p.H1069Q mutation had the less severe disturbances of copper metabolism and the latest presentation of first WD symptoms. The most severely disturbed copper metabolism and the earliest age at initial disease manifestation was noticed in non‐p.H1069Q patients. In compound heterozygotes, the type of mutation coexisting with the p.H1069Q to a small extent influenced WD phenotype. The phenotype of WD varied considerably among patients with the same genotype. The p.H1069Q mutation is associated with late WD manifestation and with a mild disruption of copper metabolism. In compound heterozygotes, the phenotype of WD to a small extent depends on the type of mutation coexisting with the p.H1069Q. Besides genotype, additional modifying factors seem to determine WD manifestations.

DLG5 Variants in Inflammatory Bowel Disease

OBJECTIVES:Genetic variants within DLG5 were recently reported to be associated with inflammatory bowel disease (IBD). The aim of our study was to test for allelic and haplotype associations of six DLG5 variants in 668 IBD patients from two European populations. Furthermore, we evaluated whether DLG5 variants alter gastrointestinal permeability in Crohns disease (CD).METHODS:Six DLG5 variants (p.R30Q, p.P1371Q, p.G1066G, rs2289308, DLG_e26, p.D1507D) were genotyped in two study populations: (1) German IBD patients (CD n = 250; ulcerative colitis (UC) n = 150) and German healthy controls (n = 422); (2) Hungarian IBD patients (CD n = 144; UC n = 124) and Hungarian healthy controls (n = 205). Subtyping analysis was performed in respect of CARD15 mutations and clinical characteristics. We also tested for differences within DLG5 genotypes in German CD patients with respect to gastroduodenal and intestinal permeability measured by triple-sugar-test.RESULTS:Allele as well as genotype frequencies of DLG5 variants did not differ between IBD patients and controls in either study population. Indeed, the p.R30Q polymorphism was found more frequently in controls than in patients. The distribution of DLG5 genotypes in German and Hungarian CD patients with CARD15 mutations was not different from patients without mutated CARD15. We did also not observe any association between DLG5 variants and clinical parameters. Importantly, DLG5 variants were not associated with gastroduodenal or intestinal permeability.CONCLUSIONS:We could not replicate that DLG5 is a relevant disease susceptibility gene for IBD in German or Hungarian subjects. In addition, we have no evidence that DLG5 variants are involved in altered gastrointestinal permeability in CD.

Introducing Genetic Testing for Adult-Type Hypolactasia

Background and Aims: To evaluate genotyping for two DNA variants (c.1993+327C>T and c.1438+117G>A), recently found to be associated with adult-type hypolactasia, in the diagnosis of lactose intolerance. Methods: In total, 166 consecutive patients with gastrointestinal symptoms mimicking hypolactasia admitted to the clinic between March 2002 and December 2002 were included. Genotyping for the two DNA variants (c.1993+327C>T and c.1438+117G>A) and standard H2 breath test was performed. Results: Among 116 patients with positive H2 breath test, the c.1993+327C variantwas detectablein 106 (91.4%) patients. Among 50 patients with negative H2 breath test, the c.1993+327Cvariantwas seen in 2 patients. Sensitivity, specificity, positive and negative predictive values for the c.1993+327C variant were 91.4, 96.0, 98.1 and 82.8%, respectively. Genotyping for the c.1438+117G variant did not bring any additional information. Among 4 of the 10 patients with positive H2 breath test but negative for the c.1993+327C and the c.1438+117G variant,further evaluation revealed other diseases known to cause secondary hypolactasia such as celiac disease and short bowel syndrome. Conclusion: In symptomatic patients, genotyping for the DNA variant c.1993+327C is a reliable test for adult-type hypolactasia with high sensitivity and specificity and thus provides a new tool in the diagnostic workup of hypolactasia.

Genetic and phenotypic analysis of dilated cardiomyopathy with conduction system disease: Demand for strategies in the management of presymptomatic lamin A/C mutant carriers

One‐third of cases of dilated cardiomyopathy (DCM) is of familial aetiology. Several genes have been reported to cause the autosomal dominant form of DCM.

FH clinical phenotype in Greek patients with LDL-R defective vs. negative mutations.

Background  Familial hypercholesterolaemia (FH) is caused by mutations in the low‐density lipoprotein receptor gene and the gene encoding apolipoprotein B‐100, affecting one in 500 individuals.

Quantification of Lipoprotein(a): Comparison of an Automated Latex-Enhanced Nephelometric Assay with an Immunoenzymometric Method

Several studies indicate the relevance of lipoprotein(a) (Lp(a)) in the genesis of premature coronary artery disease. A simple method for determining the concentration of Lp(a) is therefore of great interest for assessing the risk of coronary artery disease in patients. We compared a new latex-enhanced immunonephelometric assay (Behringwerke AG, Marburg, Germany), using the Behring Nephelometer System 100, with an established immunoenzymometric assay (Immuno, Heidelberg, Germany). A total of 163 patients was studied. Intra- and inter-assay coefficients of variation were between 2.2% and 7.1%, and between 3.4% and 8.6%, depending on the concentration of Lp(a). The correlation between the studied assays was excellent (r = 0.93, y = 0.98x -1.57, Spearman rank, Passing & Bablok). When values above 1000 mg/l for Lp(a) were excluded, the correlation was even higher. Increased light scattering with particle size, which hitherto has been a disadvantage of the nephelometric technique, seems to be negligible using the improved latex-enhanced approach. In patients with triacylglycerol values above 4.5 mmol/l (n = 19) there was no interference with the Behring system, i.e. the results of the nephelometric method were not increasing, and they agreed with those of the immunoenzymometric assay. In conclusion, this new latex-enhanced nephelometric immunoassay represents a rapid and precise method for the quantification of Lp(a).

A new LMNA mutation causing limb girdle muscular dystrophy 1B.

Sirs: We describe a family with adult-onset limb girdle muscular dystrophy 1B (LGMD1B) due to a new mutation in LMNA encoding for lamin A/C. Lamins A/C maintain nuclear shape and provide a structural support for chromosomes [7]. Mutations in LMNA cause a variety of diseases, now called laminopathies: autosomaldominant and autosomal-recessive Emery-Dreifuss muscular dystrophy (AD-EDMD and AR-EDMD) [1], limb girdle muscular dystrophy 1B (LGMD1B) [8], dilated cardiomyopathy (DCM) with conduction defect [3], familial partial lipodystrophy type Dunnigan (FPLP) [11], CharcotMarie-Tooth neuropathy 2B1 (CMT2B1) [10], and several progeria syndromes [2, 9].The molecular mechanisms leading to these different clinical phenotypes are not understood. It appears that different parts of the molecule play different roles in their interaction with other molecules and stability of the protein [4, 7]. The 48 year old female index patient was well until she was 35 years of age when she noticed difficulty in climbing stairs. The muscular weakness was slowly progressive and was accompanied by mild dyspnea. She denied any stiffness or rigidity of her back, elbows or ankles. The family history revealed that the father had died at a young age because of lung cancer but muscle weakness could not be recalled. Her mother was in good health. On physical examination manual muscle testing revealed a moderate limb-girdle weakness with the pelvic girdle being predominantly affected (hip-flexor paresis 3–4/5). There were no contractures (Fig. A and B). The 19 year old daughter had minimal limb girdle weakness without contractures. Cardiac examination was normal. The creatinkinase was 10-times above normal. Metabolically, there were no signs of insulin-resistance, hyperlipidemia or other abnormalities typical of lipodystrophy The muscle specimen showed signs of a mild dystrophy with a normal immunohistochemical analysis. Cardiac examination revealed permanent atrial fibrillation and AV-block III° after cardioversion. A pacemaker was implanted at age 49. Cardiac MRI was performed using a phased array cardiac surface coil. On CINE-MRI images wall motion abnormalities could be detected at the apex and inferoseptal wall of the left ventricle, as well as a delayed contrast enhancement in the same regions (Fig. C). The phenomenon of delayed myocardial enhancement on MRI was first used to detect areas of non-viable myocardium in chronic myocardial infarction [5]. To our knowledge, we are the first to describe this phenomenon in a patient with muscular dystrophy. For mutation analysis of LMNA all exons and the promoter region were amplified [3]. The analysis was confirmed by sequencing in both directions (Fig. D) and subsequently by restriction enzyme analysis (Fig. E). The identified missense mutation exchanges tryptophan at position 498 for cysteine (W498C). The mutation was found in the index patient and in both of her children. Her mother did not carry the mutation. LGMD1B is rare within the LMNA-associated neuromuscular disorders. Whereas only eight mutations have been found leading to LGMD1B the EDMD phenotype is caused by more than 40 mutations spread out over the entire gene [6, 12–14]. On the protein level, tryptophan 498 is located at the C-terminus of betastrand 6 within the globular domain of lamin A/C. At this position there is a conserved aromatic residue in lamins. The side chain of tryptophan 498 is located at the center of a cavity, surrounded by mainly hydrophobic side chains of different beta-strands that are not close in the lamin sequence but in tertiary structure. Interestingly, another mutation associated with the rare LGMD1B phenotype is also found in the globular domain of lamin A/C, Y481H [6]. Y481 is located on beta 5 in a cavity close to W498 (Fig. E). This might be coincidental but could also represent a mutational hotspot in LMNA for LGMD1B.