Monique G. Zaahl

Analysis of genes implicated in iron regulation in individuals presenting with primary iron overload.

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C→G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.

Fat Mass and Obesity-Associated (FTO) Gene Polymorphisms Are Associated with Physical Activity, Food Intake, Eating Behaviors, Psychological Health, and Modeled Change in Body Mass Index in Overweight/Obese Caucasian Adults

The fat mass and obesity-associated (FTO) gene is currently recognized as the most robust predictor of polygenic obesity. We investigated associations between the FTO rs1421085 and rs17817449 polymorphisms and the FTO rs1421085–rs17817449 haplotype and dietary intake, eating behavior, physical activity, and psychological health, as well as the effect of these associations on BMI. N = 133 treatment seeking overweight/obese Caucasian adults participated in this study. Genotyping was performed from whole blood samples. Weight and height was measured and a non-quantified food frequency questionnaire was completed to assess food group intake. Validated questionnaires were completed to assess physical activity (Baecke questionnaire), psychological health (General Health questionnaire, Rosenburg self-esteem scale and Beck Depression Inventory), and eating behavior (Three Factor Eating questionnaire). The risk alleles of the FTO polymorphisms were associated with poorer eating behaviors (higher hunger, internal locus for hunger, and emotional disinhibition scores), a higher intake of high fat foods and refined starches and more depressive symptoms. The modeled results indicate that interactions between the FTO polymorphisms or haplotypes and eating behavior, psychological health, and physical activity levels may be associated with BMI. The clinical significance of these results for implementation as part of weight management interventions needs further investigation.

Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.

A recently developed strip‐assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel‐based mutation‐screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip‐assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse‐hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African‐specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip‐based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

The −237C→T promoter polymorphism of the SLC11A1 gene is associated with a protective effect in relation to inflammatory bowel disease in the South African population

The purpose of this study was to assess the likelihood that variation in the promoter region of the solute carrier family 11 member 1 gene (SLC11A1) contributes to inflammatory bowel disease (IBD) susceptibility in the South African population. The study cohort included 102 IBD patients, 47 with Crohns disease (CD) and 55 with ulcerative colitis, and 192 population-matched controls. Mutation analysis revealed two novel alleles for the 5′-(GT)n repeat polymorphism, t(gt)5ac(gt)5ac(gt)6ggcaga(g)6 (allele 8) and t(gt)5ac(gt)5ac(gt)8ggcaga(g)6 (allele 9), and one previously documented point mutation −237C→T. A significantly decreased frequency of the −237C→T promoter polymorphism was observed in the patient group with IBD (p<0.001) and CD (p<0.0006) compared with the population-matched control group. These findings may be related to previous in vitro studies, which demonstrated that the point mutation at nucleotide position −237 represents a functional polymorphism that affects regulation of the upstream 5′-(GT)n repeat polymorphism differentially upon iron loading. Our findings raise the possibility that iron dysregulation mediated by allelic effects of SLC11A1 may contribute to IBD susceptibility.

Significance of novel endothelin-B receptor gene polymorphisms in Hirschsprung's disease: predominance of a novel variant (561C/T) in patients with co-existing Down's syndrome.

Several genes have been implicated in the pathogenesis of Hirschsprungs disease (HSCR). In a previous study performed, five novel (V202M, E480K, IVS10-2A/G, D771N, IVS19-9C/T) mutations and one previously described mutation (P937L) have been identified in the RET proto-oncogene in 20% of the study population. To further investigate the involvement of other genes, mutation analysis of the endothelin-B receptor (EDNRB) gene was performed in 52 unrelated sporadic HSCR patients, including 38 non-syndromic and 14 patients with HSCR and Downs syndrome. Six novel (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and IVS4 + 3A/G) sequence variants and one previously described (831G/A) polymorphism were identified. Statistically significant differences were achieved for six (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and 831G/A) of these variants. The T-allele of the 561C/T polymorphism was over represented in the HSCR/Downs syndrome patient group (36% representing 5 of 14) compared to normal controls (6% representing 5 of 84) (p < 0.002, chi(2) with Yates correction = 12.14), suggesting that the 561C/T variant is associated with a low penetrance effect in patients with this complex phenotype. Detection of the 178G/A polymorphism in only non-syndromic HSCR patients, provide further support for an important role of specific sequence variants in the EDNRB gene in the HSCR/Downs syndrome phenotype.

Clinical and genetic differences in total colonic aganglionosis in Hirschsprung's disease

UNLABELLED Although apparently the same condition as Hirschsprungs disease (HSCR), total colonic aganglionosis (TCA) patients (2%-14% congenital aganglionosis) display clinical, histopathologic, and genetic differences that may account for altered clinical presentations. PATIENTS AND METHODS Clinical, radiologic, and histologic features of 22 TCA patients of 114 HSCR cases (including 16 kindreds) were retrospectively evaluated by chart review. With ethical permission, DNA mutation analysis of the RET and EDNRB genes was carried out. Polymerase Chain Reaction (PCR) products were screened for genetic variation of by Hetroduplex Single Strand conformation polymorphism (HEX/SSCP) analysis and compared with 60 normal population control samples (20/ethnic groups). The SSCP variants were validated with automated sequencing techniques showing conformational variants in acrylamide gel. RESULTS Of the 22 patients, 12 (55%) presented within the first 28 days of extrauterine life, but 10 presented later with 3 (14%) presenting more than 6 months of age. The TCA patients evaluated differed clinically, radiologically, and histologically, and misdiagnosis occurred in 23% (5/22). Seven patients (32%) were familial-the remainder being nonrelated. Histologic features varied, and difficulties in diagnosis occurred in 5 (24%), with unclear histologic condition delaying diagnosis in one and a mistaken aganglionic level, requiring repeat surgery in two. RET variations were detected in 82% (18/22)of TCA as opposed to 33% short segment (S-HSCR) with multiple genetic RET variations in 5 (28%). Genetic variations included exon 2 SNPs but less than in S-HSCR. One had an isolated RET A4 variation with no other abnormalities. Intronic RET variations occurred in intron 6 (2 patients) (IVS6+56delG) and intron 16 (2 patients) (IVS16-38delG). A cysteine radical mutation (C620R) (2 patients) was related to Multiple Endocrine Neoplasia Type 2 (MEN2) in the family. In contrast to S-HSCR, genetic variations in TCA aggregated to the important tyrosine kinase (intracellular) region in 5 patients suggesting a possible pathogenetic link. EDNRB variations occurred in 7 patients (32%) all within exon 4 of the gene. CONCLUSIONS Total colonic aganglionosis differs clinically from other HSCR phenotypes and may lead to misdiagnosis. Potential disease-related RET gene mutations include exon 17-21 genetic variations that suggest the possibility of disrupted downstream signaling pathways from vital gene recruitment sites as possible TCA contributing factors.

Intronic RET gene variants in Down syndrome-associated Hirschsprung disease in an African population

BACKGROUND Clinical association between Hirschsprung disease (HD) and Down syndrome (DS) is well established. RET promoter and intron 1 variations have been shown to interfere with RET function, increasing the risk of HD pathogenesis. The intronic single-nucleotide polymorphism 2 (SNP2 [rs2435357]) has been associated with DS-associated HD (DS-HD). This study focuses on variations of specific RET intron, 1 SNPs (viz, SNP1 [rs2506004] and SNP2 [rs2435357]) in DS-HD. PATIENTS AND METHODS DNA was extracted from paraffin-embedded tissue samples and whole blood in 14 patients with DS with histologically proven HD. Polymerase chain reaction products of RET intron 1 were screened for genetic variation and matched to DS and controls from the general population. RESULTS Thirty-seven blood and/or tissue from 14 patients with DS-HD were investigated. RET intronic variations (SNP1 [rs2506004] or SNP2 [rs2435357]) were detected in all patients. SNP1 was detected in all patients, was heterozygous in 9, and homozygous in 5 samples (all aganglionic and 1 total colonic aganglionosis). SNP2 was absent in 6 patients, heterozygous in 6, and homozygous in 3. Three DS controls had a heterozygous SNP1. Homozygous intronic SNP RET variations were related to aganglionic tissue but not normally ganglionated or transitional zone from the same individual. CONCLUSIONS Potential disease-related RET mutations were identified in the intron region in 80% of patients with DS-HD investigated, suggesting a causal relationship. The presence of a homozygous form in the aganglionic tissue probably represents a somatic mutation, which suggests local microenvironmental factors in HD pathogenesis.

A review of genetic mutation in familial Hirschsprung's disease in South Africa: towards genetic counseling

BACKGROUND Hirschsprungs disease (HSCR) represents a complex disorder of signaling molecules, resulting from the effects of at least 9 known susceptibility genes. Affected families carry 200 times higher risk, but genetic counseling via pedigree analysis is difficult and the significance of genetic variations is unclear. This study evaluated a set of patients affected by HSCR with familial recurrence to evaluate factors of greatest value in genetic counseling. PATIENTS AND METHODS One hundred twenty patients with HSCR (including 18 kindreds) were screened for genetic variations of the 2 major susceptibility genes (RET and endothelin B receptor [EDNRB]) and compared with 60 control samples (20 per ethnic group). Familial recurrence patterns were studied for patient sex, pattern of recurrence, presence of associated syndromic features, and genetic features of major susceptibility genes. Polymerase chain reaction and HEX-SSCP analysis were performed on DBA extracted from blood/microdissected tissue samples. SSCP variants were validated and automated sequencing techniques performed on polymerase chain reaction products showing conformational variants in acrylamide gel. RESULTS Familial cases had a male-female ratio of 1.5:1, male-to-male transmission (n = 10; 2 father to son), female-to-male transmissions (n = 4; 3 female carriers, female-to-female (n = 4; 2 mother to daughter), and 1 paternal RET deletion-female with very long segment aganglionosis. Increasing gene penetrance occurred in 3 pedigrees. An increased incidence of long segment HSCR was noted in families with recurrence and appeared important. No consistent mendelian trends or specific genetic sites were observed, but 3 suggested autosomal dominant and recessive in a further 3. Identified genetic variations included deletions, frame shifts, and missense mutations, as well as a number of significant single nucleotide polymorphism variations. Transmitted RET mutations occurred in 5 (30%) of 16 kindreds. Splice RET mutation plus variants of exon 17 (973L) affected 2 children with identical total colonic aganglionosis. In a 3-generation family, variations in RET exons 6, 13, and 18 (928) affected 3 male children with increasing penetration to recur as total intestinal aganglionosis in a grandchild. CONCLUSIONS Mendelian transmission appears mediated by the RET proto-oncogene. EDNRB mutations suggest haplotypic gene-gene interaction. Genetic counseling remains a challenge in HSCR because of its multfactorial etiology.

The Hirschsprung's-multiple endocrine neoplasia connection

The risk of patients with Hirschsprungs disease later developing multiple endocrine neoplasia remains a matter of concern. The multiple endocrine neoplasia 2–Hirschsprungs disease association has been shown to cosegregate in Hirschsprungs disease patients with both short- and long-segment aganglionosis, although patients with long-segment aganglionosis a to carry the greatest risk. The Hirschsprungs disease–medullary thyroid carcinoma relationship also appears to be bi-directional, and activation or suppression of the rearranged during transfection gene appeared to vary over succeeding generations within the same family. Rearranged during transfection gene variations are associated with both conditions. The cosegregation of Hirschsprungs disease and multiple endocrine neoplasia 2 is particularly interesting as it involves both “switch off” and “switch on” of the rearranged during transfection proto-oncogene in the same patient. This cosegregation mostly relates to the cysteine-rich area on RET-620 (the “Janus gene”). The mechanism whereby rearranged during transfection influences gene activation in multiple endocrine neoplasia 2 is complex, but genetic variations impair the rearranged during transfection tyrosine kinase response to tyrosine kinase activation, thus appearing to dictate downstream signaling cascade responses. Better understanding of the RET-620 relationship allows for a more cost-effective method of identifying those at risk by focusing rearranged during transfection gene testing to this specific area as a “hot spot”. The clinical awareness of possible medullary thyroid carcinoma has led to timely intervention and early treatment of this chemo- and radioresistant tumor with poor prognosis. Establishment of “risk” by genetic testing has become a classic model of molecular medicine being integrated into patient care and offering rearranged during transfection-directed prophylactic surgical management. In addition, novel approaches to treatment based on this genetic knowledge have already shown early promise in randomized clinical trials.

Tissue specific somatic mutations and aganglionosis in Hirschsprung's disease

BACKGROUND RET proto-oncogene intron 1 variations [e.g. SNP1 (rs2506004) and SNP2 (rs 2435357)] have been shown to be etiologically important in the pathogenesis of Hirschsprungs disease (HSCR). Although activating somatic RET rearrangements have been identified in certain tumours, this is the first study to confirm somatic gene variation in HSCR. METHODS DNA was extracted from 53 paraffin embedded tissue samples (HSCR patients n=33, multiple levels n=17), and controls (n=3). Patients were grouped into aganglionic (Group 1), ganglionated (group 2), and transitional (group 3). PCR products of RET intron 1 were screened for genetic variation by semi-automated bi-directional sequencing analysis and matched to unaffected controls from the general population. Comparison was by Fishers exact test. P <0.05 was regarded as significant. RESULTS HSCR patients included short segment (n=26), long segment colonic [(n=4 (24%)], and total colonic aganglionosis (n=3). RET intronic variations [SNP1 (rs2506004) or SNP2 (rs 2435357)] showed somatic homozygous in affected tissue in 9/12 (75%) Group 1 (aganglionic tissue) compared with 2/5 (40%) and 1/10 (10%) of groups 2 and 3 (P<0.001). Homozygous SNP2 variation was observed in all long segment versus 4/10 short segment. 50% of the short segment cases showing homozygous SNP 1 variation. CONCLUSION We report somatic mutations in the RET intron 1 region of affected HSCR tissue, confirming for the first time that somatic mutations are present in aganglionic tissue and may promote local aganglionosis through deregulated receptor activity. Detailed understanding of the somatic genetic events that drive congenital aganglionosis may have bearing on diagnosis and therapy.