Jochen Raedle

Mutations in the MutSα interaction interface of MLH1 can abolish DNA mismatch repair

MutLα, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSα and assembles and controls further repair enzymes. We tested if the interaction of MutLα with DNA-bound MutSα is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLα for MutSα is located on the edge of an extensive β-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLα–MutSα interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.

Hypothesis : Possible role of retinoic acid therapy in patients with biallelic mismatch repair gene defects

A boy showing symptoms of a Turcot-like childhood cancer syndrome together with stigmata of neurofibromatosis type I is reported. His brother suffers from an infantile myofibromatosis, and a sister died of glioblastoma at age 7. Another 7-year-old brother is so far clinically unaffected. The parents are consanguineous. Molecular diagnosis in the index patient revealed a constitutional homozygous mutation of the mismatch repair gene PMS2. The patient was in remission of his glioblastoma (WHO grade IV) after multimodal treatment followed by retinoic acid chemoprevention for 7xa0years. After discontinuation of retinoic acid medication, he developed a relapse of his brain tumour together with the simultaneous occurrence of three other different HNPCC-related carcinomas. We think that retinoic acid might have provided an effective chemoprevention in this patient with homozygous mismatch repair gene defect. We propose to take a retinoic acid chemoprevention into account in children with proven biallelic PMS2 mismatch repair mutations being at highest risk concerning the development of a malignancy.

Growth differentiation factor 15—an early marker of abnormal function of the Fontan circuit in patients with univentricular hearts

BACKGROUNDnIn patients after the Fontan procedure, assessment of ventricular function is difficult and amino-terminal pro-B-type natriuretic peptide levels failed to be directly related to echocardiographic measures of systolic ventricular function. The aim of the study was to evaluate growth differentiation factor 15 (GDF-15), a marker of various stress pathways in the heart and extracardiac tissues.nnnMETHODSnPlasma GDF-15 levels were measured in 38 consecutive patients after the Fontan procedure and compared to clinical, echocardiographic, and laboratory data; liver tissue stiffness; and venous hepatic flow velocities.nnnRESULTSnMean GDF-15 levels were 987.2±440.5 pg/mL in patients with an ejection fraction (EF)<50% as compared to 520.2±143.1 pg/mL in those with an EF≥50% (P<.001). Growth differentiation factor 15 levels were significantly related to the EF of the single ventricle (r=-0.66, P<.001), New York Heart Association functional class (r=0.43, P=.008), and γGT levels (r=0.50, P=.002) but weakly to liver tissue stiffness. According to receiver operating characteristic curve analysis, an EF<50% was best predicted by GDF-15 levels (area under the curve [AUC] 0.90, P<.001), peak venous hepatic flow at deep inspiration (AUC 0.89, P=.002), and age at Fontan operation (AUC 0.86, P=.001). Growth differentiation factor 15 and age at Fontan operation proved to be independent predictors in the multivariate analysis. The optimal cutoff of GDF-15 for the prediction of an EF<50% was calculated to be 613 pg/mL with a sensitivity of 90.0% and specificity of 85.7%.nnnCONCLUSIONSnGrowth differentiation factor 15 might be helpful in detecting early abnormal function of the Fontan circuit in patients with univentricular hearts. In patients with GDF-15 levels exceeding 613 pg/mL, further cardiac evaluation should be considered because impaired systolic function of the single ventricle may be present.

Absence of the interstitial cell of Cajal network in mitochondrial neurogastrointestinal encephalomyopathy

Abstractu2002 Mitochondrial neurogastrointestinal enceph‐alomyopathy (MNGIE) is a rare autosomal‐recessive multisystemic disorder with predominant gastrointestinal involvement, presenting with variable degrees of gut dysmotility up to frank chronic intestinal pseudoobstruction. Despite major advances in understanding its basic molecular pathogenesis in recent years, the distinct mechanisms and pathoanatomical substrate underlying MNGIE‐associated gastrointestinal dysmotility are still widely unknown. As yet, though their critical role in proper gastrointestinal transit in terms of spontaneous pacemaker activity and enteric neurotransmission is well established, the population of the interstitial cells of Cajal (ICC) has not been investigated in MNGIE. Therefore, we examined small bowel samples of a well‐characterized MNGIE patient by using conventional histology and immunohistochemistry techniques. The ICC network was studied by immunohistochemistry for the tyrosine kinase Kit (CD117), known to reliably detect ICCs, while mucosal mast cells served as an internal and normal small bowel specimen as external controls. At a light microscopic level, no gross structural alteration of the bowel wall composition and its neuromuscular elements was noted. However, a complete absence of Kit immunoreactive cells could be demonstrated in regions where ICCs are normally abundant, while internal and external controls retained strong Kit positivity. In conclusion, our preliminary results provide a first evidence for an alteration of the ICC network in MNGIE, and support the notion that ICC loss might be an early pathogenetic event in MNGIE‐associated gut motor dysfunction before significant myopathic and/or neuropathic structural changes occur.

Evaluation of the MLH1 I219V alteration in DNA mismatch repair activity and ulcerative colitis.

Background: Inflammatory bowel diseases (IBDs; ulcerative colitis, UC, and Crohns disease, CD) show familial clustering suggestive of a genetic background. A linkage susceptibility region for these diseases (IBD9) lies on chromosome 3p and includes the DNA mismatch repair gene MLH1. Loss of MLH1 confers the characteristic microsatellite instability (MSI) phenotype which is also frequently found in the mucosa of IBD patients. A common germline alteration of MLH1 (655A>G) results in the amino acid exchange MLH1 I219V. Conflicting data exist on its effect on the function of the protein and it has recently been reported to cosegregate with refractory UC, suggesting that this alteration may impair mismatch repair activity and thereby contribute to certain forms of UC. Methods: We analyzed the MLH1 I219V alteration using in silico and biochemical analyses and assessed its appearance in 67 well‐classified UC patients in comparison to 40 healthy individuals. Results: The analyses showed that I219 is a conserved, buried hydrophobic residue, and that I219V is unlikely to abolish MLH1 function but may modulate it. Quantitative biochemical evaluation showed identical stability and activity of the protein. Furthermore, the alteration occurred equally frequently in analyzed patients and healthy volunteers. Conclusions: The MLH1 I219V alteration does not directly contribute to the etiology of UC through an impairment of mismatch repair. A putative linkage disequilibrium of MLH1 I219V with the causative gene(s) of the IBD9 locus is rather distant.

MUTYH hotspot mutations in unselected colonoscopy patients: MUTYH hotspot mutations in unselected colonoscopy patients

Aimu2002 Biallelic MutY human homologue (MUTYH) germline mutations predispose to recessively inherited adenomatous polyposis, designated MUTYH‐associated polyposis (MAP), and colorectal cancer (CRC). The hotspot mutations p.Y179C and p.G396D account for the majority of pathogenic variants of MUTYH in Caucasians. Our aim was to evaluate the prevalence of MUTYH mutations in a prospective cohort of unselected patients with different colorectal diseases.

Adenoma development in a patient with MUTYH-associated polyposis (MAP): new insights into the natural course of polyp development.

PurposeBiallelic germ-line mutations in MUTYH have recently been found to predispose for MUTYH-associated polyposis (MAP). Affected patients present with a wide range of clinical phenotypes at the time of diagnosis, but there is little precise information about the natural course of this disease.ResultsFourteen years of colonoscopic surveillance of an MAP patient (compound heterozygous p.Y165C/p.G382D) showed that adenoma development was slow after initial diagnosis of a single colorectal carcinoma at the age of 44, but then the annual number of new adenomas increased substantially in the patient’s early fifties.ConclusionThis course of the disease, with a strong subsequent acceleration of polyp development, may explain the wide range of polyp numbers counted in newly diagnosed MAP patients as a result of the time of observation. Therefore, MAP should also be considered in younger patients (35–55xa0years) with only few adenomas or colorectal cancer. The high frequency of medium and severe dysplasia in the patient’s preferential small adenomas suggests accelerated progression from adenoma to carcinoma in MAP, but this observation must be confirmed by further studies.

Multidisciplinary treatment of desmoid tumours in Gardner’s syndrome due to a large interstitial deletion of chromosome 5q

BACKGROUND AND AIMSnClassic autosomal-dominant familial adenomatous polyposis (FAP) is clinically defined by the development of hundreds to thousands of colorectal adenomas beginning in childhood and adolescence. A variant of FAP characterized by polyposis in combination with osteomas or soft tissue tumours is called Gardners syndrome. FAP is caused by germline inactivation of the APC (adenomatous polyposis coli) tumour-suppressor gene located on the long arm of chromosome 5 (5q21-5q22). Cytogenetically visible deletions of chromosome 5q encompassing APC have very rarely been reported. Here, we aimed to phenotypically and genetically characterize a patient with a heterozygous 5q deletion resulting in Gardners syndrome.nnnMETHODS AND RESULTSnA 26-year-old female patient with mild mental handicap and dysmorphic features due to a cytogenetically visible deletion on chromosome 5q (microscopically estimated region 5q14-5q23) presented at our tertiary referral centre because of mild adenomatous polyposis (<500 polyps). Twenty months after prophylactic proctocolectomy with definitive ileostomy, three rapidly growing desmoids were observed. Tumour-associated complications necessitated a multidisciplinary approach including medical treatment, surgery and radiation therapy. The characterization of the deletion by comparative genomic hybridization identified a large 5q deletion expanding over a 20-Mb region (5q21.3-5q23.3) including the APC gene.nnnCONCLUSIONnChromosome deletions must be suspected in patients presenting with FAP together with mental handicap and dysmorphic features. This case also impressively shows that FAP-associated desmoids need multimodal treatment taking into account the patients individual symptoms, disease progression and tumour location.

Chronic Diarrhea Responding to Proton Pump Inhibitors: A Clinical Sign of Zollinger-Ellison Syndrome

In July 2003, a 43-year-old man presented for workup of hronic painless diarrhea of 3 years’ duration. Passing 3 to watery stools per day without pathologic admixtures, he ad lost 3 kg of weight. At the onset of symptoms, colonosopy had been performed without pathologic findings, and rritable bowel syndrome was assumed. He did not take any egular medication, and family history was negative for ndocrinopathies or cancer. On admission, physical examination was unremarkable, s was lower gastrointestinal endoscopy including serial iopsies. Upper endoscopy revealed low-grade esophagitis, iffuse gastritis with prominent mucosal folds, and multiple uperficial postbulbar ulcers (Figure). A gastric fluid specmen demonstrated a pH less than 2. Gastric histology ndicated Helicobacter pylori negative, hyperplastic gastriis, whereas duodenal biopsies revealed villous atrophy and rypt hyperplasia, however, with granulocytic infiltration. eliac serology, including anti-transglutaminase and antiliadin immunoglobulin-A, was negative. Fasting serum astrin before administration of antisecretory medication as discretely elevated to 217 pg/mL (assay-specific normal alue 150), as was chromogranin A (2290 g/L, noral 120). Of note, for the first time in 3 years, the paient’s diarrhea completely ceased after institution of proton ump inhibitor (PPI) therapy at double standard dose. In onjunction with the highly suggestive endoscopic findings, his was in support of our clinical diagnosis of Zollingerllison syndrome (ZES) despite ambiguous gastrin levels. owever, after PPI withdrawal, another 2 serum gastrin easurements were normal (124 and 147 pg/mL), and seretin stimulation test failed to unmask excessive gastrin roduction (peak gastrin increase by 91 pg/mL after 2 IU/kg ntravenous secretin. However, endoscopic ultrasound was uspicious for a duodenal wall gastrinoma measuring 10 mm in