Loss of thymidine phosphorylase activity disrupts adipocyte differentiation and induces insulin-resistant lipoatrophic diabetes.

BACKGROUND: Thymidine phosphorylase (TP), encoded by the TYMP gene, is a cytosolic enzyme essential for the nucleotide salvage pathway. TP catalyzes the phosphorylation of the deoxyribonucleosides, thymidine and 2'-deoxyuridine, to thymine and uracil. Biallelic TYMP variants are responsible for Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), an autosomal recessive disorder characterized in most patients by gastrointestinal and neurological symptoms, ultimately leading to death. Studies on the impact of TYMP variants in cellular systems with relevance to the organs affected in MNGIE are still scarce and the role of TP in adipose tissue remains unexplored. METHODS: Deep phenotyping was performed in three patients from two families carrying homozygous TYMP variants and presenting with lipoatrophic diabetes. The impact of the loss of TP expression was evaluated using a CRISPR-Cas9-mediated TP knockout (KO) strategy in human adipose stem cells (ASC), which can be differentiated into adipocytes in vitro. Protein expression profiles and cellular characteristics were investigated in this KO model. RESULTS: All patients had TYMP loss-of-function variants and first presented with generalized loss of adipose tissue and insulin-resistant diabetes. CRISPR-Cas9-mediated TP KO in ASC abolished adipocyte differentiation and decreased insulin response, consistent with the patients' phenotype. This KO also induced major oxidative stress, altered mitochondrial functions, and promoted cellular senescence. This translational study identifies a new role of TP by demonstrating its key regulatory functions in adipose tissue. CONCLUSIONS: The implication of TP variants in atypical forms of monogenic diabetes shows that genetic diagnosis of lipodystrophic syndromes should include TYMP analysis. The fact that TP is crucial for adipocyte differentiation and function through the control of mitochondrial homeostasis highlights the importance of mitochondria in adipose tissue biology.

Genetic contribution of ABCC2 to Dubin-Johnson syndrome and inherited cholestatic disorders.

BACKGROUND AND AIMS: The ABCC2 gene is implicated in Dubin-Johnson syndrome (DJS), a rare autosomal recessive liver disorder. The primary aim of this study was to determine the diagnostic value of ABCC2 genetic testing in the largest cohort of DJS reported to date. The high number of patients with cholestatic manifestations in this series prompted us to evaluate the genetic contribution of rare, potentially pathogenic ABCC2 variants to other inherited cholestatic disorders. METHODS: The cohort study included 32 patients with clinical DJS diagnosis, and 372 patients referred for the following disorders: low phospholipid-associated cholelithiasis (LPAC) syndrome, intrahepatic cholestasis of pregnancy (ICP) and benign recurrent intrahepatic cholestasis (BRIC). ABCC2 was screened by next-generation sequencing. RESULTS: Most patients with clinical DJS had positive genetic diagnosis (n = 30; 94%), with a great diversity of point mutations and copy number variations in ABCC2. Strikingly, eight (27%) of these patients showed transient cholestatic features at presentation: four neonatal cholestasis, two ICP, one contraceptive-induced cholestasis and one sporadic cholestasis. Conversely, the frequency of rare, heterozygous, potentially pathogenic ABCC2 variants in patients with LPAC, ICP or BRIC did not differ significantly from that of the general population. CONCLUSIONS: This large series reveals that DJS is a highly homogeneous Mendelian disorder involving a large spectrum of ABCC2 variants. Genetic testing is crucial to establish early DJS diagnosis in patients with atypical presentations, such as neonatal cholestasis. This study also provides no evidence for the contribution of rare, potentially pathogenic ABCC2 variants to other inherited cholestatic disorders.

Clinical characteristics and genetic profiles of young and adult patients with cholestatic liver disease.

BACKGROUND AND AIMS: heterozygous ABCB4, ABCB11 and ATP8B1 sequence variants were previously reported to be associated with low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy, benign recurrent intrahepatic cholestasis and biliary lithiasis. The present study aimed to identify the presence of sequence variations in genes responsible for Mendelian liver disorders in patients with cholestatic liver disease. METHODS: targeted massive parallel sequencing of a panel of genes involved in bile acid homeostasis was performed in 105 young and adult patients with cholestatic liver disease in our laboratory for molecular diagnosis. The effects of novel variants were evaluated using bioinformatics prediction tools and the Protter and Phyre2 software programs were used to create 2D, 3D topology protein modeling. Genotype-phenotype correlation was established according to molecular analysis and clinical records. RESULTS: twenty novel heterozygous ABCB4 sequence variations, one heterozygous ABCB4 large intragenic deletion and only one novel missense variant in ABCB11 and ATP8B1 were identified. Interestingly, heterozygous and homozygous SLC4A2 missense variants were detected in patients with low phospholipid-associated cholelithiasis. Two patients harbored heterozygous GPBAR1 variants. Common variants such as homozygous ABCB11 p.Val444Ala and heterozygous ABCG8 p.Asp19His were also identified in 12 cases. CONCLUSIONS: forty-eight variants were identified in five genes including ABCB4, ABCB11, ATP8B1, SLC4A2 and GPBAR1, twenty-five of which were novel. This study expands the phenotypic and mutational spectrum in genes involved in bile acid homeostasis and highlights the genetic and phenotypic heterogeneity in patients with inherited liver disorders.

Metachronous colorectal cancer risk in Lynch syndrome patients-should the endoscopic surveillance be more intensive?

PURPOSE: Regular follow-up for patients with Lynch syndrome (LS) is vital due to the increased risk of colorectal (50-80%), endometrial (40-60%), and other cancers. However, there is an ongoing debate concerning the best interval between colonoscopies. Currently, no specific endoscopic follow-up has been decided for LS patients who already have an index colorectal cancer (CRC). The aim of this study was to evaluate the risk of metachronous cancers (MC) after primary CRC in a LS population and to determinate if endoscopic surveillance should be more intensive. METHODS: A prospective cohort of patients with a confirmed diagnosis of hereditary CRC since 2009 was included. Patients with LS and a primary CRC were the cohort of choice. RESULTS: One hundred twenty-one patients were included with a median age of 44 years(16-70). At least one MC occurred in 39 patients (32.2%), with a median interval of 67 months (6-300) from index cancer. Fifteen (38.5%) developed two or more MCs during follow-up, with a median number of two (2-6) tumors occurring. Metachronous CRC were diagnosed after a median interval of 24 (6-57) months since last colonoscopy and were more commonly seen in MSH2 mutation carriers (58 vs. 35%, p = 0.001). After a median follow-up of 52.9 (3-72) months, no cancer-related deaths were recorded. CONCLUSION: Patients with LS have an increased risk of MC, especially CRCs. With a median time period of 24 months between colonoscopy and metachronous CRC, the interval between surveillance colonoscopies following primary CRC should not exceed 18 months, especially in patients with MSH2 mutation.

LMNA-associated partial lipodystrophy: anticipation of metabolic complications.

BACKGROUND: Type-2 familial partial lipodystrophy (FPLD2) is a rare autosomal dominant lipodystrophic disorder due to mutations in LMNA encoding lamin A/C, a key epigenetic regulator. FPLD2 severity is determined by the occurrence of metabolic complications, especially diabetes and hypertriglyceridaemia. We evaluated the disease history and severity over generations. METHODS: This retrospective study of the largest cohort of patients with FPLD2 reported to date investigates 85 patients from 24 families comprising three generations (G1: n=39; G2: n=41; G3: n=5). RESULTS: Lipodystrophy appears with the same characteristics and at the same age in first generation (G1;18.6±1.5 years) and second generation (G2;15.9±0.8 years). Despite similar body mass index (23.7±0.6 vs 23.8±0.6 kg/m2), the mean delay between the onset of lipodystrophy and diabetes was far shorter in G2 (10.5±2.4 years) than in G1 (29.0±3.5 years) (p=0.0002). The same is true for the delay preceding hypertriglyceridaemia (G2: 4.5±1.4; G1: 19.3±3.2 years) (p=0.002), revealing an anticipation phenomenon. Observations in G3, and analysis within each family of disease history and diagnostic procedures, confirmed this result. CONCLUSIONS: This study is a rare example of anticipation unrelated to a trinucleotide expansion. Discovery of this early occurrence of metabolic complications in young generations underlines the utility of presymptomatic genetic diagnosis, with careful metabolic screening and preventive lifestyle in all at-risk individuals.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

BACKGROUND: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex). METHODS: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins. RESULTS: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS. CONCLUSIONS: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.

Global molecular analysis and APOE mutations in a cohort of autosomal dominant hypercholesterolemia patients in France.

Autosomal dominant hypercholesterolemia (ADH) is a human disorder characterized phenotypically by isolated high-cholesterol levels. Mutations in the low density lipoprotein receptor (LDLR), APOB, and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes are well known to be associated with the disease. To characterize the genetic background associated with ADH in France, the three ADH-associated genes were sequenced in a cohort of 120 children and 109 adult patients. Fifty-one percent of the cohort had a possible deleterious variant in LDLR, 3.1% in APOB, and 1.7% in PCSK9. We identified 18 new variants in LDLR and 2 in PCSK9. Three LDLR variants, including two newly identified, were studied by minigene reporter assay confirming the predicted effects on splicing. Additionally, as recently an in-frame deletion in the APOE gene was found to be linked to ADH, the sequencing of this latter gene was performed in patients without a deleterious variant in the three former genes. An APOE variant was identified in three patients with isolated severe hypercholesterolemia giving a frequency of 1.3% in the cohort. Therefore, even though LDLR mutations are the major cause of ADH with a large mutation spectrum, APOE variants were found to be significantly associated with the disease. Furthermore, using structural analysis and modeling, the identified APOE sequence changes were predicted to impact protein function.

PIK3R1 mutations cause syndromic insulin resistance with lipoatrophy.

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.

Bone imaging findings in genetic and acquired lipodystrophic syndromes: an imaging study of 24 cases.

OBJECTIVE: To describe the bone imaging features of lipodystrophies in the largest cohort ever published. MATERIALS AND METHODS: We retrospectively examined bone imaging data in 24 patients with lipodystrophic syndromes. Twenty-two had genetic lipodystrophy: 12/22 familial partial lipodystrophy (FPLD) and 10/22 congenital generalized lipodystrophy (CGL), 8 with AGPAT2-linked CGL1 and 2 with seipin-linked CGL2. Two patients had acquired generalized lipodystrophy (AGL) in a context of non-specific autoimmune disorders. Skeletal radiographs were available for all patients, with radiographic follow-up for two. Four patients with CGL1 underwent MRI, and two of them also underwent CT. RESULTS: Patients with FPLD showed non-specific degenerative radiographic abnormalities. Conversely, CGL patients showed three types of specific radiographic alterations: diffuse osteosclerosis (in 7 patients, 6 with CGL1 and 1 with CGL2), well-defined osteolytic lesions sparing the axial skeleton (7 CGL1 and 1 CGL2), and pseudo-osteopoikilosis (4 CGL1). Pseudo-osteopoikilosis was the sole bone abnormality observed in one of the two patients with AGL. Osteolytic lesions showed homogeneous low signal intensity (SI) on T1-weighted and high SI on T2-weighted MR images. Most of them were asymptomatic, although one osteolytic lesion resulted in a spontaneous knee fracture and secondary osteoarthritis in a patient with CGL1. MRI also showed diffuse fatty bone marrow alterations in patients with CGL1, with intermediate T1 and high T2 SI, notably in radiographically normal areas. CONCLUSIONS: The three types of peculiar imaging bone abnormalities observed in generalized lipodystrophic syndromes (diffuse osteosclerosis, lytic lesions and/or pseudo-osteopoikilosis) may help clinicians with an early diagnosis in pauci-symptomatic patients.

Metabolic and cardiac phenotype characterization in 37 atypical Dunnigan patients with nonfarnesylated mutated prelamin A.

BACKGROUND: Laminopathies are associated with a broad spectrum of clinical manifestations, from lipodystrophy to cardiac diseases. The purpose of this study was to assess genotype-phenotype correlations in a lipodystrophic laminopathy caused by the Lamin A (LMNA) mutation T655fsX49. This mutation leads to synthesis of nonfarnesylated-mutated prelamin A that does not undergo the physiologic lamin A maturation process. METHODS AND RESULTS: We studied 35 patients originating from Reunion Island who carried the LMNA T655fsX49 mutation. Comparisons of cardiac and endocrinologic features were made between homozygous and heterozygous patients. Homozygous patients presented more overlapping syndromes with severe cardiac phenotypes, defined by cardiolaminopathy, early atheroma with coronary heart disease (CHD) and high-degree conduction disorder compared with heterozygous (40% vs 4%; P = .016). Moreover, homozygous patients had earlier onset (49.6 vs 66 years old; P = .0002). Left ventricle lowered ejection fraction associated with heart failure was more frequent in homozygous than in heterozygous patients (40% vs 0%, respectively). Lipodystrophic traits were more marked in the homozygous group but only reached statistical significance for L4 subcutaneous fat measurement (2.8 ± 2.16 vs 18.7 ± 8.9 mm; P = .008) and leptin levels (2.45 ± 1.6 vs 11.26 ± 7.2 ng/mL; P = .0001). CONCLUSIONS: Our results suggest that there is a relationship between mutated prelamin-A accumulation and the severity of the phenotypes in homozygous familial partial lipodystrophy type 2 patients who harbor the LMNA T655fsX49 mutation. A dose-dependent effect seems likely.

Perilipin deficiency and autosomal dominant partial lipodystrophy.

Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.

Peroxisome proliferator-activated receptor-γ mutations responsible for lipodystrophy with severe hypertension activate the cellular renin-angiotensin system.

OBJECTIVE: Inactivating peroxisome proliferator-activated receptor-γ (PPARγ) mutations lead to a syndrome of familial partial lipodystrophy (FPLD3) associated with early-onset severe hypertension. PPARγ can repress the vascular renin-angiotensin system (RAS) and angiotensin II receptor 1 expression. We evaluated the relationships between PPARγ inactivation and cellular RAS using FPLD3 patients' cells and human vascular smooth muscle cells expressing mutant or wild-type PPARγ. Approach and Results- We identified 2 novel PPARG mutations, R165T and L339X, located in the DNA and ligand-binding domains of PPARγ, respectively in 4 patients from 2 FPLD3 families. In cultured skin fibroblasts and peripheral blood mononuclear cells from the 4 patients and healthy controls, we compared markers of RAS activation, oxidative stress, and inflammation, and tested the effect of modulators of PPARγ and angiotensin II receptor 1. We studied the impact of the 2 mutations on the transcriptional activity of PPARγ and on the vascular RAS in transfected human vascular smooth muscle cells. Systemic RAS was not altered in patients. However, RAS markers were overexpressed in patients' fibroblasts and peripheral blood mononuclear cells, as in vascular cells expressing mutant PPARγ. Angiotensin II-mediated mitogen-activated protein kinase activity increased in patients' fibroblasts, consistent with RAS constitutive activation. Patients' cells also displayed oxidative stress and inflammation. PPARγ activation and angiotensin II receptor 1 mRNA silencing reversed RAS overactivation, oxidative stress, and inflammation, arguing for a role of angiotensin II receptor 1 in these processes. CONCLUSIONS: Two novel FPLD3-linked PPARG mutations are associated with a defective transrepression of cellular RAS leading to cellular dysfunction, which might contribute to the specific FPLD3-linked severe hypertension.

Will students pass a competitive exam that they failed in their dreams?

We tested whether dreams can anticipate a stressful exam and how failure/success in dreams affect next-day performance. We collected information on students' dreams during the night preceding the medical school entrance exam. Demographic, academic, sleep and dream characteristics were compared to the students' grades on the exam. Of the 719 respondents to the questionnaire (of 2324 total students), 60.4% dreamt of the exam during the night preceding it. Problems with the exam appeared in 78% of dreams and primarily involved being late and forgetting answers. Reporting a dream about the exam on the pre-exam night was associated with better performance on the exam (p=.01). The frequency of dreams concerning the exam during the first term predicted proportionally higher performance on the exam (R=0.1, p=.01). These results suggest that the negative anticipation of a stressful event in dreams is common and that this episodic simulation provides a cognitive gain.

What the genetics of lipodystrophy can teach us about insulin resistance and diabetes.

Genetic lipodystrophic syndromes are rare diseases characterized by generalized or partial fat atrophy (lipoatrophy) associated with severe metabolic complications such as insulin resistance (IR), diabetes, dyslipidemia, nonalcoholic fatty liver disease, and ovarian hyperandrogenism. During the last 15 years, mutations in several genes have been shown to be responsible for monogenic forms of lipodystrophic syndromes, of autosomal dominant or recessive transmission. Although the molecular basis of lipodystrophies is heterogeneous, most mutated genes lead to impaired adipogenesis, adipocyte lipid storage, and/or formation or maintenance of the adipocyte lipid droplet (LD), showing that primary alterations of adipose tissue (AT) can result in severe systemic metabolic and endocrine consequences. The reduced expandability of AT alters its ability to buffer excess caloric intake, leading to ectopic lipid storage that impairs insulin signaling and other cellular functions ("lipotoxicity"). Genetic studies have also pointed out the close relationships between ageing, inflammatory processes, lipodystrophy, and IR.

Perinatal, metabolic, and reproductive features in PPARG-related lipodystrophy.

OBJECTIVE: The adipogenic PPARG-encoded PPARγ nuclear receptor also displays essential placental functions. We evaluated the metabolic, reproductive, and perinatal features of patients with PPARG-related lipodystrophy. METHODS: Current and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre. RESULTS: 26 patients from 15 unrelated families were studied (18 women, median age 43 years). They carried monoallelic PPARG variants except a homozygous patient with congenital generalized lipodystrophy. Among heterozygous patients aged 16 or more (n = 24), 92% had diabetes, 96% partial lipodystrophy (median age at diagnosis 24 and 37 years), 78% hypertriglyceridaemia, 71% liver steatosis, and 58% hypertension. The mean BMI was 26 ± 5.0 kg/m2. Women (n = 16) were frequently affected by acute pancreatitis (n = 6) and/or polycystic ovary syndrome (n = 12). Eleven women obtained one or several pregnancies, all complicated by diabetes (n = 8), hypertension (n = 4), and/or hypertriglyceridaemia (n = 10). We analysed perinatal data of patients according to the presence (n = 8) or absence (n = 9) of a maternal dysmetabolic environment. The median gestational age at birth was low in both groups (37 and 36 weeks of amenorrhea, respectively). As expected, the birth weight was higher in patients exposed to a foetal dysmetabolic environment of maternal origin. In contrast, 85.7% of non-exposed patients, in whom the variant is, or is very likely to be, paternally-inherited, were small for gestational age. CONCLUSIONS: Lipodystrophy-related PPARG variants induce early metabolic complications. Our results suggest that placental expression of PPARG pathogenic variants carried by affected foetuses could impair prenatal growth and parturition. This justifies careful pregnancy monitoring in affected families.

Screening for Lynch syndrome in colorectal cancer: are we doing enough?

PURPOSE: The purpose of this study was to assess the efficacy of screening for the detection of Lynch syndrome (LS) in an unselected population undergoing surgery for a colorectal cancer. METHODS: A total of 1,040 patients were prospectively included between 2005 and 2009. LS screening modalities included the Bethesda criteria, immunochemistry (IHC) for MLH1, MSH2, and MSH6, and microsatellite instability (MSI) by using pentaplex markers. Promoter methylation was assessed in tumors with a loss of MLH1 expression. Gene sequencing was offered to patients with abnormal IHC or MSI status without promoter methylation. RESULTS: A total of 105 patients had an abnormal result: 102 (9.8%) exhibited a loss of protein on IHC and 98 (9.4%) had MSI. A discordant result was observed in 10 patients with eventual proven LS in 6 patients. Loss of MLH1 (n = 64) was due to promoter methylation in 43 patients (67.2%). Overall, of 62 patients with an abnormal result, 38 had genetic sequencing leading to 25 (65.8%) identified with a germ-line mutation. Loss of MSH2 on IHC was associated with a mutation in 78.3% (18 of 23) of cases. Among the 62 patients with abnormal results, 23 (37.1%) did not meet the Bethesda criteria. CONCLUSIONS: Strict application of the Bethesda criteria does not lead to identification of all patients with LS. IHC and MSI testing are complementary methods and should be used in association to identify potential LS patients.

Methylation tolerance due to an O6-methylguanine DNA methyltransferase (MGMT) field defect in the colonic mucosa: an initiating step in the development of mismatch repair-deficient colorectal cancers.

BACKGROUND AND AIMS: O(6)-Methylguanine-DNA methyltransferase (MGMT) removes methyl adducts from O(6)-guanine. Known as methylation tolerance, selection for mismatch repair (MMR)-deficient cells that are unable to initiate lethal processing of O(6)-methylguanine-induced mismatches in DNA is observed in vitro as a consequence of MGMT deficiency. It was therefore hypothesised that an MGMT field defect may constitute a preneoplastic event for the development of MMR-deficient tumours displaying microsatellite instability (MSI). METHODS: MGMT expression was investigated by immunohistochemistry and the methylation status of the gene promoter by PCR in neoplastic, adjacent and distant mucosal tissues of patients with MSI or non-MSI (MSS) colorectal cancer (CRC). The cancers were familial (42 MSI, 13 MSS) or sporadic (40 MSI, 49 MSS) in origin, or arose in the context of inflammatory bowel disease (IBD; 13 MSI, 36 MSS). Colonic mucosa from patients with diverticulitis (n=20) or IBD (n=39 in 27 patients) without cancer served as controls. RESULTS: Loss of MGMT expression was more frequent in MSI than MSS CRC (p=0.047). In comparison with MSS tumours, MSI CRC occurred more frequently adjacent to patches of mucosa that lacked MGMT expression (p=0.002). Overall, loss of MGMT expression was associated with MGMT gene promoter methylation (p=0.03). CONCLUSION: MGMT field defects are more frequently associated with MSI than MSS CRC. These findings indicate that methylation tolerance may be a crucial initiating step prior to MMR deficiency in the development of MSI CRC in familial, sporadic and IBD settings.

Two Decades after Mandibuloacral Dysplasia Discovery: Additional Cases and Comprehensive View of Disease Characteristics.

Pathogenic variants in the LMNA gene cause a group of heterogeneous genetic disorders, called laminopathies. In particular, homozygous or compound heterozygous variants in LMNA have been associated with "mandibuloacral dysplasia type A" (MADA), an autosomal recessive disorder, characterized by mandibular hypoplasia, growth retardation mainly postnatal, pigmentary skin changes, progressive osteolysis of the distal phalanges and/or clavicles, and partial lipodystrophy. The detailed characteristics of this multisystemic disease have yet to be specified due to its rarity and the limited number of cases described. Here, we report three unrelated Egyptian patients with variable severity of MAD features. Next-generation sequencing using a gene panel revealed a homozygous c.1580G>A-p.Arg527His missense variant in LMNA exon 9 in an affected individual with a typical MADA phenotype. Another homozygous c.1580G>T-p.Arg527Leu variant affecting the same amino acid was identified in two additional patients, who both presented with severe manifestations very early in life. We combined our observations together with data from all MADA cases reported in the literature to get a clearer picture of the phenotypic variability in this disease. This work raises the number of reported MADA families, argues for the presence of the founder effect in Egypt, and strengthens genotype-phenotype correlations.

Biallelic CAV1 null variants induce congenital generalized lipodystrophy with achalasia.

OBJECTIVE: CAV1 encodes caveolin-1, a major protein of plasma membrane microdomains called caveolae, involved in several signaling pathways. Caveolin-1 is also located at the adipocyte lipid droplet. Heterozygous pathogenic variants of CAV1 induce rare heterogeneous disorders including pulmonary arterial hypertension and neonatal progeroid syndrome. Only one patient was previously reported with a CAV1 homozygous pathogenic variant, associated with congenital generalized lipodystrophy (CGL3). We aimed to further delineate genetic transmission, clinical, metabolic, and cellular characteristics of CGL3. DESIGN/METHODS: In a large consanguineous kindred referred for CGL, we performed next-generation sequencing, as well as clinical, imagery, and metabolic investigations. We studied skin fibroblasts from the index case and the previously reported patient with CGL3. RESULTS: Four patients, aged 8 months to 18 years, carried a new homozygous p.(His79Glnfs*3) CAV1 variant. They all displayed generalized lipodystrophy since infancy, insulin resistance, low HDL-cholesterol, and/or high triglycerides, but no pulmonary hypertension. Two patients also presented at the age of 15 and 18 years with dysphagia due to achalasia, and one patient had retinitis pigmentosa. Heterozygous parents and relatives (n = 9) were asymptomatic, without any metabolic abnormality. Patients' fibroblasts showed a complete loss of caveolae and no protein expression of caveolin-1 and its caveolin-2 and cavin-1 partners. Patients' fibroblasts also displayed insulin resistance, increased oxidative stress, and premature senescence. CONCLUSIONS: The CAV1 null variant investigated herein leads to an autosomal recessive congenital lipodystrophy syndrome. Loss of caveolin-1 and/or caveolae induces specific manifestations including achalasia which requires specific management. Overlapping phenotypic traits between the different CAV1-related diseases require further studies.