Bi-allelic IARS mutations in a child with intra-uterine growth retardation, neonatal cholestasis, and mild developmental delay.

Recently, bi-allelic mutations in cytosolic isoleucyl-tRNA synthetase (IARS) have been described in three individuals with growth delay, hepatic dysfunction, and neurodevelopmental disabilities. Here we report an additional subject with this condition identified by whole-exome sequencing. Our findings support the association between this disorder and neonatal cholestasis with distinct liver pathology. Furthermore, we provide functional data on two novel missense substitutions and expand the phenotype to include mild developmental delay, skin hyper-elasticity, and hypervitaminosis D.

Aldose reductase gene polymorphisms and susceptibility to diabetic nephropathy in Type 1 diabetes mellitus.

AIMS: To investigate association and linkage between DNA sequence variants in the aldose reductase (AR) gene on chromosome 7q35 and diabetic nephropathy (DN) in Type 1 diabetes mellitus. METHODS: By sequencing the promoter region and 10 exons in eight DN cases and eight controls, a frequent bi-allelic polymorphism (C-106T) was discovered. This polymorphism and the known 5'ALR2 dinucleotide repeat polymorphism were genotyped in unrelated cases with advanced nephropathy (n = 221) and unrelated controls with normoalbuminuria (n = 193). For a family based study, 166 case-trios (case and both parents) and 83 control-trios (control and both parents) were also genotyped. RESULTS: In the case-control study, carriers of the Z-2 allele of the 5'ALR2 polymorphism had a significantly higher risk of DN than non-carriers (odds ratios: 1.6 for heterozygotes and 2.1 for homozygotes, P<0.05 for each). The same was true for carriers of the T allele of the C-106T polymorphism (odds ratios: 1.6 for heterozygotes and 1.9 for homozygotes, P<0.05 for each). Moreover, the haplotype carrying both risk alleles was in excess in DN cases. In the family study, transmission of risk alleles from heterozygous parents was consistent with the case-control study, excess transmission in case-trios and deficient in control-trios. CONCLUSIONS: Association between DN and two DNA sequence variants in the promoter region of the AR gene implicates the polyol pathway in the development of kidney complications in Type 1 diabetes mellitus. Further examination of the molecular mechanisms underlying these findings may provide insight into the pathogenesis of DN.

Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus.

The helix-loop-helix (HLH) protein NEUROD1 (also known as BETA2) functions as a regulatory switch for endocrine pancreatic development. In mice homozygous for a targeted disruption of Neurod, pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene (Ins2). NEUROD1, following its heterodimerization with the ubiquitous HLH protein E47, regulates insulin gene (INS) expression by binding to a critical E-box motif on the INS promoter. Here we describe two mutations in NEUROD1, which are associated with the development of type 2 diabetes in the heterozygous state. The first, a missense mutation at Arg 111 in the DNA-binding domain, abolishes E-box binding activity of NEUROD1. The second mutation gives rise to a truncated polypeptide lacking the carboxy-terminal trans-activation domain, a region that associates with the co-activators CBP and p300 (refs 3,4). The clinical profile of patients with the truncated NEUROD1 polypeptide is more severe than that of patients with the Arg 111 mutation. Our findings suggest that deficient binding of NEUROD1 or binding of a transcriptionally inactive NEUROD1 polypeptide to target promoters in pancreatic islets leads to the development of type 2 diabetes in humans.

Hepatocyte nuclear factor-4gamma: cDNA sequence, gene organization, and mutation screening in early-onset autosomal-dominant type 2 diabetes.

The aim of this study was to investigate whether mutations in hepatocyte nuclear factor (HNF)-4gamma, a transcription factor homologous to HNF-4alpha, contribute to the etiology of early-onset type 2 diabetes. Linkage between diabetes and two polymorphic markers at the HNF-4gamma locus (D8S286 and D8S548) was evaluated in 32 multigenerational families with early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young genes. Total logarithm of odds (LOD) scores were strongly negative (-50.3 at D8S286 and -46.2 at D8S548), but linkage could not be excluded in 15 families having LOD scores >-2.0. To screen these pedigrees for HNF-4gamma mutations, the gene structure was defined. Because reverse transcriptase-polymerase chain reaction experiments indicated that the first 1,674 bp of the published cDNA sequence (3,248 bp) were a cloning artifact, the correct cDNA sequence was determined by 5' rapid amplification of cDNA ends (RACE) and primer extension assay. Based on the new cDNA sequence (1,731 bp), 11 exons were found. After screening the 5' flanking region and all coding exons for mutations, we identified several polymorphisms, one of which affected the amino acid sequence (M190I). However, no mutations segregating with diabetes could be found in these families. We conclude that genetic variability in the HNF-4gamma gene is unlikely to play a major role in the etiology of early-onset autosomal-dominant type 2 diabetes.

Identification of new mutations in the hepatocyte nuclear factor 4alpha gene among families with early onset Type 2 diabetes mellitus.

AIMS: Mutations in hepatocyte nuclear factor (HNF)-4alpha gene located on chromosome 20q have been found to be responsible for the development of early onset Type 2 diabetes mellitus (DM). Through a national campaign, 53 families with autosomal dominant, early onset Type 2 DM (n=654) were assembled to determine the frequency of mutations in the HNF-4alpha gene and their contribution to the development of diabetes. METHODS: Twelve exons and the promoter region of the HNF-4alpha gene were screened in probands of the families by a double gradient, denaturing gradient gel electrophoresis (DG-DGGE) protocol combined with automated bi-directional sequencing of the PCR products of all heterozygous individuals. RESULTS: We detected two new mutations in the HNF-4alpha gene that changed the amino-acid sequence. The first mutation was a Gly-->Ser substitution in codon 115 within a highly conserved DNA binding domain, and all six carriers of this mutation had diabetes and low insulin secretion. The second mutation was an Ile-->Val substitution in codon 454 within the transactivation domain. It was carried by four family members, two of whom also carried a mutation in the HNF-1alpha gene. Of those having only the mutation in HNF-4alpha one had diabetes and the other had normal glucose tolerance and both were obese and hyperinsulinaemic. Thus, it is uncertain that this mutation is responsible for any of the diabetes in this family. CONCLUSION: We have found that mutations in the HNF-4alpha gene account for a small proportion, about 2-4%, of families with early onset, autosomal dominant, Type 2 DM in US Caucasians.

Major susceptibility locus for nephropathy in type 1 diabetes on chromosome 3q: results of novel discordant sib-pair analysis.

Diabetic nephropathy (DN) clusters in families with type 1 diabetes and the degree of clustering suggests that a major gene having a common disease allele may be responsible. To investigate the chromosomal regions containing genes for the renin-angiotensin system, we performed a linkage study using pairs of siblings with type 1 diabetes who were discordant for DN. Theoretical considerations supported by simulation studies indicated that such discordant pairs, rather than the usual concordant pairs, would be more effective in detecting a major susceptibility gene for DN. We applied this novel strategy to test for linkage between DN and chromosomal regions containing genes for the ACE, angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1). Two polymorphic markers were genotyped in the vicinity of each of the three loci in 66 discordant sib pairs and were analyzed with multipoint methods. The regions containing ACE and AGT loci were not linked with DN, while the region containing the AT1 locus showed linkage with DN. As a result of these positive findings, eight additional polymorphic markers spanning a 63-cM region around AT1 locus were genotyped. Linkage was demonstrated between DN and a 20-cM region that includes AT1 (P = 7.7 x 10(-5)), an obvious candidate gene for DN. To investigate whether AT1 could account for the observed linkage, we sequenced all exons, splicing junctions, and the promoter region and examined the identified polymorphisms/mutations for association with DN using the transmission disequilibrium test. Four new polymorphisms in the gene were found, but neither these nor previously described polymorphisms were associated with DN. Thus, while our study does not implicate AT1 itself in the etiology of DN, it provides very strong evidence that a 20-cM region around AT1 contains a major locus for susceptibility to DN.

Effects of the antihypertensive dilevalol on aortic compliance in anesthetized dogs.

The present study examined the actions of dilevalol, an antihypertensive beta-adrenoceptor blocker with arterial vasodilator actions, on aortic compliance (AC) in anesthetized dogs. AC was measured by sonomicrometric determination of the ratio of aortic systolic-diastolic diameters (mm) and arterial pulse pressure (mm Hg). One AC unit (ACU) equals 10(-3) mm/mm Hg. Dilevalol (0.032, 0.1, and 3.2 mg/kg intravenously, i.v.) significantly (p less than .05) increased AC by 1.4 +/- 0.3, 3.7 +/- 1.4, and 4.5 +/- 1.2 ACU from basal values of 4.7 +/- 0.4-5.6 +/- 0.4 ACU while reducing blood pressure by 20 +/- 2, 31 +/- 9, and 41 +/- 10 mm Hg, respectively (p less than 0.05). Increases in AC were not the passive result of altered blood pressure. Proximal mechanical aortic occlusion dropped systolic blood pressure as much as 70 mm Hg without altering AC. Hydralazine also (0.3 and 1.0 mg/kg i.v.) lowered blood pressure significantly (p less than 0.05) by 14 +/- 3 and 40 +/- 4 mm Hg but increased AC only at 1.0 mg/kg (+ 1.6 +/- 0.4 ACU, p less than 0.05). Phenylephrine (1-30 micrograms/kg i.v.) significantly raised blood pressure 25 +/- 3-95 +/- 8 mm Hg but decreased AC significantly by 1.2 +/- 0.3-2.4 +/- 0.3 ACU. Isoproterenol (ISO) (0.01-1.0 micrograms/kg) produced effects on AC similar to those of dilevalol. Propranolol pretreatment attenuated dilevalol and ISO-induced increases in AC. Propranolol (0.32 and 1.0 mg/kg) did not significantly change AC.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of verapamil, diltiazem and nifedipine on aortic compliance in anesthetized dogs.

We tested the ability of verapamil (V), diltiazem (DTZ) and nifedipine (NIF) to alter aortic compliance upon i.v. administration in anesthetized dogs. Sonomicrometry was used to measure aortic systolic (SD) and diastolic (DD) diameters. Aortic distention (AD) was calculated from SD-DD. Aortic compliance was computed from the ratio of AD to arterial pulse pressure. One aortic compliance unit (ACU) is defined as 10(-3) mm/mmHg. V (0.1-3.0 mg/kg) reduced blood pressure, SD and DD dose-dependently. DD was more affected than SD and AD rose significantly. Since pulse pressure was not significantly altered, V induced a dose-related increase in aortic compliance. The 3.0 mg/kg dose increased aortic compliance by 10.3 +/- 1.5 ACU from a basal value of 4.7 +/- 0.5 ACU. DTZ (0.1-3.0 mg/kg) produced effects on blood pressure, aortic dimensions and aortic compliance similar to those seen with V. Infusion of NIF (3.2-32 micrograms/kg/min) exhibited a similar profile, i.e., reduced blood pressure, SD, DD and increased AD and aortic compliance. NIF, however was limited in its ability to increase aortic compliance, i.e., increases of 3.2 +/- 1.0 or 3.8 +/- 0.6 from basal values of 4.9 +/- 1.0 ACU were obtained with 10 and 32 micrograms/kg/min NIF, respectively. Although these 3 prototype calcium entry blockers share a common profile of action, V and DTZ appear to have a greater efficacy on aortic compliance than NIF.

Effects of spiraprilic acid, an angiotensin converting enzyme inhibitor, on large artery compliance in anesthetized dogs.

The present study examined the actions of spiraprilic acid, a new non-sulfhydryl angiotensin converting enzyme (ACE) inhibitor upon aortic compliance (AC) in anesthetized dogs. Enalaprilic acid was examined for comparative purposes. AC was determined via sonomicrometric determination of the ratio of aortic systolic-diastolic diameter (mm) and arterial pulse pressure (mmHg). One AC unit (ACU) equals 10(-3) mm/mmHg. In non-thiazide pretreated animals, spiraprilic acid (1 mg/kg i.v.), caused a sustained hypotensive response of 21 +/- 3 mmHg (P less than .05). At the same time, aortic systolic and diastolic dimensions were reduced from basal values of 8.17 +/- 0.45 and 7.95 +/- 0.40 mm by 0.19 +/- 0.07 (P less than .05) and 0.29 +/- 0.10 mm (P less than .05), respectively. The aortic systolic-diastolic dimension rose significantly by 0.10 +/- 0.01 mm. Since pulse pressure was unchanged, AC rose from a basal value of 4.6 +/- 0.8 ACU by 1.7 +/- .3 ACU (P less than .05). Similar effects were observed with enalaprilic acid. In dogs pretreated orally with 5 mg/kg hydrochlorothiazide (HCTZ) twice daily for 3 days, spiraprilic and enalaprilic acids caused slightly greater falls in blood pressure (34 +/- 7 and 30 +/- 4 mmHg, respectively) than in nonpretreated dogs. However, the onset of the hypotension effect was more rapid for both ACE inhibitors in HCTZ-pretreated dogs. Effects of spiraprilic and enalaprilic acid upon aortic dimensions were similar to those observed in non-HCTZ pretreated animals. The data illustrate that the ACE inhibitors spiraprilic and enalaprilic acids not only lower blood pressure but also enhance large artery compliance.

Effects of the antihypertensive dilevalol on large artery compliance in anesthetized dogs.

The present study examined the actions of dilevalol, an antihypertensive beta-adrenoceptor blocker with arterial vasodilator actions, upon aortic compliance (AC) in anesthetized dogs. AC was determined via sonomicrometric determination of the ratio of aortic systolic-diastolic diameters (mm) and arterial pulse pressure (mm Hg). One AC unit (ACU) equals 10(-3) mm/mm Hg. Dilevalol (0.032, 0.1, and 3.2 mg/kg i.v.) significantly (p less than 0.05) increased AC by 1.4 +/- 0.3, 3.7 +/- 1.4, and 4.5 +/- 1.2 ACU, respectively, from basal values of 4.7 +/- 0.4-5.6 +/- 0.4 ACU, while reducing blood pressure by 20 +/- 2, 31 +/- 9, and 41 +/- 10 mm Hg, respectively (p less than 0.05). Increases in AC were not the passive result of altered blood pressure. Proximal mechanical aortic occlusion dropped systolic blood pressure as much as 70 mm Hg without altering AC. Propranolol (0.32 and 1.0 mg/kg) did not significantly change AC, but propranolol pretreatment attentuated dilevalol-induced increases in AC. Pindolol (0.1-1.0 mg/kg i.v.) lowered blood pressure 17 +/- 3 (p less than 0.05) to 34 +/- 7 mm Hg (p less than 0.05), while significantly increasing AC by 1.2 +/- .2 to 2.7 +/- .7 ACU. The data show that dilevalol, unlike propranolol, increases AC substantially at antihypertensive doses. The inhibition of dilevalol-induced AC increases by propranolol illustrates a beta-adrenoceptor agonist activity in large arteries. Since large artery compliance is impaired in hypertension, dilevalol may afford a vasular protective action for known risk factors for evolving systemic arterial disease.