Mutation surveyor: software for DNA sequence analysis.

Advances in high-throughput sequencing techniques had presented a significant challenge to the processing capabilities of genetic laboratories. However, recent developments in the field of semi-automated mutation detection have revolutionised the task of mutation detection.This chapter provides user information for one commercially available program, Mutation Surveyor. The software is manufactured by SoftGenetics (Pennsylvania, USA) and provides an accurate and efficient program for detecting sequence variants. The chapter focuses on the methodology of setting up GenBank files as reference files and provides information on analysis parameters and data processing.

Homozygous mutations in NEUROD1 are responsible for a novel syndrome of permanent neonatal diabetes and neurological abnormalities.

OBJECTIVE: NEUROD1 is expressed in both developing and mature beta-cells. Studies in mice suggest that this basic helix-loop-helix transcription factor is critical in the development of endocrine cell lineage. Heterozygous mutations have previously been identified as a rare cause of maturity-onset diabetes of the young (MODY). We aimed to explore the potential contribution of NEUROD1 mutations in patients with permanent neonatal diabetes. RESEARCH DESIGN AND METHODS: We sequenced the NEUROD1 gene in 44 unrelated patients with permanent neonatal diabetes of unknown genetic etiology. RESULTS: Two homozygous mutations in NEUROD1 (c.427_ 428del and c.364dupG) were identified in two patients. Both mutations introduced a frameshift that would be predicted to generate a truncated protein completely lacking the activating domain. Both patients had permanent diabetes diagnosed in the first 2 months of life with no evidence of exocrine pancreatic dysfunction and a morphologically normal pancreas on abdominal imaging. In addition to diabetes, they had learning difficulties, severe cerebellar hypoplasia, profound sensorineural deafness, and visual impairment due to severe myopia and retinal dystrophy. CONCLUSIONS: We describe a novel clinical syndrome that results from homozygous loss of function mutations in NEUROD1. It is characterized by permanent neonatal diabetes and a consistent pattern of neurological abnormalities including cerebellar hypoplasia, learning difficulties, sensorineural deafness, and visual impairment. This syndrome highlights the critical role of NEUROD1 in both the development of the endocrine pancreas and the central nervous system in humans.

Sequencing of candidate genes selected by beta cell experts in monogenic diabetes of unknown aetiology.

CONTEXT: Approximately 39% of cases with permanent neonatal diabetes (PNDM) and about 11% with maturity onset diabetes of the young (MODY) have an unknown genetic aetiology. Many of the known genes causing MODY and PNDM were identified as being critical for beta cell function before their identification as a cause of monogenic diabetes. OBJECTIVE: We used nominations from the EU beta cell consortium EURODIA project partners to guide gene candidacy. SUBJECTS: Seventeen cases with permanent neonatal diabetes and 8 cases with maturity onset diabetes of the young. MAIN OUTCOME MEASURES: The beta cell experts within the EURODIA consortium were asked to nominate 3 "gold", 3 "silver" and 4 "bronze" genes based on biological or genetic grounds. We sequenced twelve candidate genes from the list based on evidence for candidacy. RESULTS: Sequencing ISL1, LMX1A, MAFA, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, SOX2, SREBF1, SYT9 and UCP2 did not identify any pathogenic mutations. CONCLUSION: Further work is needed to identify novel causes of permanent neonatal diabetes and maturity onset diabetes of the young utilising genetic approaches as well as further candidate genes.

Wolcott-Rallison syndrome is the most common genetic cause of permanent neonatal diabetes in consanguineous families.

CONTEXT AND OBJECTIVE: Mutations in EIF2AK3 cause Wolcott-Rallison syndrome (WRS), a rare recessive disorder characterized by early-onset diabetes, skeletal abnormalities, and liver dysfunction. Although early diagnosis is important for clinical management, genetic testing is generally performed after the full clinical picture develops. We aimed to identify patients with WRS before any other abnormalities apart from diabetes are present and study the overall frequency of WRS among patients with permanent neonatal diabetes. RESEARCH DESIGN AND METHODS: The coding regions of EIF2AK3 were sequenced in 34 probands with infancy-onset diabetes with a clinical phenotype suggestive of WRS (n = 28) or homozygosity at the WRS locus (n = 6). RESULTS: Twenty-five probands (73.5%) were homozygous or compound heterozygous for mutations in EIF2AK3. Twenty of the 26 mutations identified were novel. Whereas a diagnosis of WRS was suspected before genetic testing in 22 probands, three patients with apparently isolated diabetes were diagnosed after identifying a large homozygous region encompassing EIF2AK3. In contrast to nonconsanguineous pedigrees, mutations in EIF2AK3 are the most common known genetic cause of diabetes among patients born to consanguineous parents (24 vs. < 2%). Age at diabetes onset and birth weight might be used to prioritize genetic testing in the latter group. CONCLUSIONS: WRS is the most common cause of permanent neonatal diabetes mellitus in consanguineous pedigrees. In addition to testing patients with a definite clinical diagnosis, EIF2AK3 should be tested in patients with isolated neonatal diabetes diagnosed after 3 wk of age from known consanguineous families, isolated populations, or countries in which inbreeding is frequent.

Clinical heterogeneity in patients with FOXP3 mutations presenting with permanent neonatal diabetes.

OBJECTIVE: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by FOXP3 mutations. We aimed to determine the prevalence, genetics, and clinical phenotype of FOXP3 mutations in a large cohort with permanent neonatal diabetes (PNDM). RESEARCH DESIGN AND METHODS: The 11 coding exons and the polyadenylation region of FOXP3 were sequenced in 26 male subjects with diabetes diagnosed before 6 months of age in whom common genetic causes of PNDM had been excluded. Ten subjects had at least one additional immune-related disorder, and the remaining 16 had isolated diabetes. RESULTS: We identified four hemizygous FOXP3 mutations in 6 of 10 patients with associated immune-related disorders and in 0 of 16 patients with isolated diabetes (P = 0.002). Three patients with two novel mutations (R337Q and P339A) and the previously reported L76QfsX53 developed classic IPEX syndrome and died within the first 13 months. The novel mutation V408M was found in three patients from two unrelated families and had a mild phenotype with hypothyroidism and autoimmune enteropathy (n = 2) or nephrotic syndrome (n = 1) and survival to 12-15 years. CONCLUSIONS: FOXP3 mutations result in approximately 4% of cases of male patients with permanent diabetes diagnosed before 6 months. Patients not only have classic IPEX syndrome but, unexpectedly, may have a more benign phenotype. FOXP3 sequencing should be performed in any male patient with the diagnosis of diabetes in the first 6 months who develops other possible autoimmune-associated conditions, even in the absence of full IPEX syndrome.

The impact of the angiotensin-converting enzyme insertion/deletion polymorphism on severe hypoglycemia in Type 2 diabetes.

The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene (ACE) is associated with altered serum ACE activity. Raised ACE levels and the ACE DD genotype are associated with a 3.2 to 6.8-fold increased risk of severe hypoglycemia in type 1 diabetes. This relationship has not been assessed in type 2 diabetes. We aimed to test for association of the ACE I/D polymorphism with severe hypoglycemia in type 2 diabetes. Patients with type 2 diabetes (n = 308), treated with insulin (n = 124) or sulphonylureas (n = 184), were classified according to whether or not they had previously experienced severe hypoglycemia. Samples of DNA were genotyped for the ACE I/D polymorphism using two alternative polymerase chain reactions to prevent mistyping due to preferential amplification of the D allele. Overall, 12% of patients had previously experienced one or more episodes of severe hypoglycemia. This proportion did not differ between genotype groups (odds ratio (95% confidence limits) for carriers of D allele relative to II homozygotes: 0.79 (0.35-1.78)). This study found no evidence for association of the ACE I/D polymorphism with severe hypoglycemia frequency in patients with type 2 diabetes. However, we cannot rule out a smaller effect (odds ratio

Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits.

The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.

Association studies of genetic variation in the WFS1 gene and type 2 diabetes in U.K. populations.

Mutations in the WFS1 gene cause beta-cell death, resulting in a monogenic form of diabetes known as Wolfram syndrome. The role of variation in WFS1 in type 2 diabetes susceptibility is not known. We sequenced the WFS1 gene in 29 type 2 diabetic probands and identified 12 coding variants. We used 152 parent-offspring trios to look for familial association; the R allele at residue 456 (P = 0.04) and the H allele at residue 611 (P = 0.05) as well as the R456-H611 haplotype (P = 0.032) were overtransmitted to affected offspring from heterozygous parents. In a further cohort of 327 type 2 diabetic subjects and 357 normoglycemic control subjects, the H611 allele and the R456-H611 haplotype were present in more type 2 diabetic subjects than control subjects (one-tailed P = 0.06 and P = 0.023, respectively). In a combined analysis, the H611 allele was present in 60% of all diabetes chromosomes and 55% of all control chromosomes (odds ratio [OR] 1.24 [95% CI 1.03-1.48], P = 0.02), and the R456-H611 haplotype was significantly more frequent in type 2 diabetic subjects than in control subjects (60 vs. 54%, OR 1.29 [95% CI 1.08-1.54], P = 0.0053). Our results provide the first evidence that variation in the WFS1 gene may influence susceptibility to type 2 diabetes.