Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications.

The spondylo-meta-epiphyseal dysplasia [SMED] short limb-hand type [SMED-SL] is a rare autosomal-recessive disease, first reported by Borochowitz et al. in 1993.(1) Since then, 14 affected patients have been reported.(2-5) We diagnosed 6 patients from 5 different consanguineous Arab Muslim families from the Jerusalem area with SMED-SL. Additionally, we studied two patients from Algerian and Pakistani ancestry and the parents of the first Jewish patients reported.(1) Using a homozygosity mapping strategy, we located a candidate region on chromosome 1q23 spanning 2.4 Mb. The position of the Discoidin Domain Receptor 2 (DDR2) gene within the candidate region and the similarity of the ddr2 knockout mouse to the SMED patients' phenotype prompted us to study this gene(6). We identified three missense mutations c.2254 C > T [R752C], c. 2177 T > G [I726R], c.2138C > T [T713I] and one splice site mutation [IVS17+1g > a] in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene. The results of this study will permit an accurate early prenatal diagnosis and carrier screening for families at risk.

Arginine and clonidine stimulation tests for growth hormone deficiency revisited--do we really need so many samples?

Growth hormone (GH) reserve is defined biochemically by the peak serum concentration after stimulation with a known secretagogue. Arginine and clonidine stimulation tests are currently performed with 5 timed blood samples. We evaluated the diagnostic utility of taking fewer samples by retrospectively analyzing 289 tests (202 arginine and 87 clonidine) performed in a single hospital. 123/202 (60.9%) arginine tests and 46/87 (52.9%) clonidine tests had at least one sample above 10 ng/ml. These were defined as negative for GH deficiency and studied further. For arginine tests, three samples taken at 0', 45' and 90' would have provided an acceptable false positive rate of 4.5%. For clonidine tests, two samples taken at 60' and 90' provided a false positive rate of 4.3%. Addition of either a 0' or 120' sample further reduced the false positive rate to 2.2%. Both the arginine and clonidine stimulation tests can be reliably performed with fewer samples.

Role of a founder c.201_202delCT mutation and new phenotypic features of congenital lipoid adrenal hyperplasia in Palestinians.

CONTEXT: Congenital lipoid adrenal hyperplasia (CLAH), caused by mutations in steroidogenic acute regulatory protein (StAR), is most frequent in Japanese and Palestinians. We report eight Palestinians from four unrelated families with CLAH. OBJECTIVE: The objective of the study was to identify the mutation(s) in StAR, correlate genotype with phenotype, and determine whether the common mutation represents a founder mutation. PATIENTS AND SETTING: Clinical, histopathological, and molecular genetic characterization was performed in these eight patients. RESULTS: All affected individuals (three XY, five XX) presented neonatally with undetectable adrenocortical hormones and are responding to replacement therapy. Only two sisters had neurodevelopmental deficits. Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells. Significantly, already at 1 yr of age, positive placental alkaline phosphatase and octamer binding transcription factor staining indicated neoplastic potential. Sequence analysis of StAR revealed homozygosity for c.201_202delCT mutation in all eight cases, causing premature termination of the StAR protein. This mutation was confirmed to be a founder mutation using both an intragenic microsatellite and several single nucleotide polymorphism markers. Screening of 100 normal Jerusalem Palestinians detected no carriers of this mutation. CONCLUSION: CLAH is rare in the general Palestinian population. In most Palestinian cases, a founder c.201_202delCT mutation in StAR is the cause. The observed early neonatal presentation may reflect the major StAR protein truncation caused by this mutation. A crucial role for StAR in the central nervous system was not supported with normal neurological examinations in six of eight cases. Finally, we advocate early gonadectomy in XY CLAH cases, given the early onset of neoplastic changes observed histologically.

GLP-1-RA Corrects Mitochondrial Labile Iron Accumulation and Improves ß-Cell Function in Type 2 Wolfram Syndrome.

CONTEXT: Type 2 Wolfram syndrome (T2-WFS) is a neuronal and ß-cell degenerative disorder caused by mutations in the CISD2 gene. The mechanisms underlying ß-cell dysfunction in T2-WFS are not known, and treatments that effectively improve diabetes in this context are lacking. OBJECTIVE: Unraveling the mechanisms of ß-cell dysfunction in T2-WFS and the effects of treatment with GLP-1 receptor agonist (GLP-1-RA). DESIGN AND SETTING: A case report and in vitro mechanistic studies. PATIENT AND METHODS: We treated an insulin-dependent T2-WFS patient with the GLP-1-RA exenatide for 9 weeks. An iv glucose/glucagon/arginine stimulation test was performed off-drug before and after intervention. We generated a cellular model of T2-WFS by shRNA knockdown of CISD2 (nutrient-deprivation autophagy factor-1 [NAF-1]) in rat insulinoma cells and studied the mechanisms of ß-cell dysfunction and the effects of GLP-1-RA. RESULTS: Treatment with exenatide resulted in a 70% reduction in daily insulin dose with improved glycemic control, as well as an off-drug 7-fold increase in maximal insulin secretion. NAF-1 repression in INS-1 cells decreased insulin content and glucose-stimulated insulin secretion, while maintaining the response to cAMP, and enhanced the accumulation of labile iron and reactive oxygen species in mitochondria. Remarkably, treatment with GLP-1-RA and/or the iron chelator deferiprone reversed these defects. CONCLUSION: NAF-1 deficiency leads to mitochondrial labile iron accumulation and oxidative stress, which may contribute to ß-cell dysfunction in T2-WFS. Treatment with GLP-1-RA and/or iron chelation improves mitochondrial function and restores ß-cell function. Treatment with GLP-1-RA, probably aided by iron chelation, should be considered in WFS and other forms of diabetes associated with iron dysregulation.

Relative expression of a dominant mutated ABCC8 allele determines the clinical manifestation of congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is most commonly caused by mutations in the ß-cell ATP-sensitive K(+) (K(ATP)) channel genes. Severe CHI was diagnosed in a 1-day-old girl; the mother's cousin and sister had a similar phenotype. ABCC8 gene sequencing (leukocyte DNA) revealed a heterozygous, exon 37, six-base pair in-frame insertion mutation in the affected patient and aunt but also in her unaffected mother and grandfather. In expression studies using transfected COSm6 cells, mutant sulfonylurea receptor 1 (SUR1) protein was expressed on the cell surface but failed to respond to MgADP even in the heterozygous state. mRNA expression in lymphocytes determined by sequencing cDNA clones and quantifying 6FAM-labeled PCR products found that although the healthy mother predominantly expressed the normal transcript, her affected daughter, carrying the same mutant allele, primarily transcribed the mutant. The methylation pattern of the imprinting control region of chromosome 11p15.5 and ABCC8 promoter was similar for all family members. In conclusion, differences in transcript expression may determine the clinical phenotype of CHI in this maternally inherited dominant mutation. The use of peripheral lymphocytes as a peripheral window to the ß-cell transcription profile can serve in resolving ß-cell phenotypes. The severe, dominant-negative nature of the 1508insAS mutation suggests that it affects the functional stoichiometry of SUR1-regulated gating of K(ATP) channels.

A novel severe N-terminal splice site KISS1R gene mutation causes hypogonadotropic hypogonadism but enables a normal development of neonatal external genitalia.

BACKGROUND: Kisspeptin 1 receptor (KISS1R) gene mutations are rare but have recently become an important etiology of normosmic isolated hypogonadotropic hypogonadism (IHH). OBJECTIVES: To characterize the genetic defect, the phenotype, and response to therapy of three IHH siblings with a novel severe KISS1R mutation. PATIENTS AND METHODS: Three siblings (16- and 22-year-old sisters and their 20-year-old brother) born to consanguineous parents with normal neonatal external genitalia presented with no pubertal development, normosmia, and a low response to GNRH stimulation. Homozygosity mapping, KISS1R gene sequencing, and RNA expression were performed. RESULTS: The females' basal low estradiol level (50 pmol/l) failed to rise in response to human chorionic gonadotropin (hCG). The brother's low testosterone (1.87 nmol/l) responded to combined hCG and human menopausal gonadotropin (hCG) and HMG therapies, but the testes remained small (1-2 ml). Secondary sexual characteristics were attained by exogenous sex steroid replacement. SNP array studies revealed shared homozygosity for a chromosome 19 region encompassing KISS1R. Sequencing revealed a novel homozygous KISS1R mutation at the nt-1 canonical acceptor splice site of intron 1 in affected siblings. The mother (menarche at 14 years) was heterozygous. cDNA sequencing showed that the G>A mutation results in skipping of exon 2 and a premature stop codon at residue 151. CONCLUSIONS: The novel severe N-terminal KISS1R splice site (c.245-1G>A) mutation results in IHH. Heterozygous female carriers may manifest a subtle fertile phenotype. The subnormal gonadal response to hCG in patients may implicate a direct role of KISS1R in gonadal function. The normal neonatal virilization in a male homozygous to this severe mutation challenges the hypothesis that KISS1R is required for fetal development of male external genitalia.

Autosomal recessive familial neurohypophyseal diabetes insipidus: onset in early infancy.

BACKGROUND: Familial neurohypophyseal diabetes insipidus (FNDI), usually an autosomal dominant disorder, is caused by mutations in the arginine vasopressin (AVP)-neurophysin II preprohormone leading to aberrant preprohormone processing and gradual destruction of AVP-secreting cells. Patients typically present between 1 and 6 years of age with polyuria and polydipsia. OBJECTIVE: Clinical, biochemical, and genetic studies of three new cases of autosomal recessive FNDI presenting in early infancy. PATIENTS: Three Palestinian cousins presented with failure to thrive, vomiting, irritability, and fever. The parents were asymptomatic. Patients developed hypernatremia (154-163 mmol/l) and serum hyperosmolality (>320 mOsm/kg), while urine osmolality remained between 73 and 229 mOsm/kg. Plasma AVP levels were low, and the posterior pituitary bright spot was absent on magnetic resonance imaging (MRI). All patients responded to desmopressin. RESULTS: Patients were homozygous and parents were heterozygous for microsatellite markers flanking the AVP gene. All patients were homozygous for the P26L (proline to leucine) substitution affecting mature AVP. A founder effect with the single original kindred carrying the P26L mutation was confirmed by microsatellite analysis, but patients in that family presented only at 2 years of age. In microsatellite analysis, the new kindred patients were not homozygous and did not share a single allele at the aquaporin 2 and vasopressin receptor-2 genes locuses. CONCLUSION: This is the first description of autosomal recessive FNDI presenting in the neonatal period. The unusual early clinical and radiological (MRI) presentation argues against gradual destruction of AVP-secreting neurons as the pathophysiological mechanism. Factors beside allelism of AVP-related genes must influence the age of FNDI presentation given the founder effect demonstrated for the P26L mutation.

Novel de novo mutation in sulfonylurea receptor 1 presenting as hyperinsulinism in infancy followed by overt diabetes in early adolescence.

OBJECTIVE: Congenital hyperinsulinism, usually associated with severe neonatal hypoglycemia, may progress to diabetes, typically during the 4th decade of life in nonpancreatectomized patients. We aimed to genotype the ATP-sensitive K(+) channel in a 10.5-year-old girl presenting with overt diabetes following hyperinsulinism in infancy. RESEARCH DESIGN AND METHODS: A female aged 10.5 years presented with new-onset, antibody-negative diabetes (A1C 10.6%). She was born large for gestational age (5 kg) to a nondiabetic mother and developed frequent hypoglycemic episodes, which persisted until age 3 years and responded initially to intravenous glucose and later to oral sweets. Currently, she is fully pubertal and obese (BMI 30.2 kg/m(2)), with a partially controlled convulsive disorder (since age 1 year) and poor school performance. Glucose levels were >11.1 mmol/l throughout 72 h of continuous glucose monitoring, with low insulin secretion during intravenous glucose tolerance testing. KCNJ11 and ABCC8 mutation analysis was performed, and the mutation identified was characterized in COSm6 cells. RESULTS: A novel, de novo heterozygous ABCC8 sulfonylurea receptor (SUR)1 mutation (R370S) was identified in the patient's DNA but not in that of either parent. Cotransfection of Kir6.2 and mutant SUR1 demonstrate that the mutated protein is expressed efficiently at the cell surface but fails to respond to MgADP, resulting in minimal channel activity. Interestingly, the heterozygous channel (WT:R370S) responded well to glibenclamide, a finding that lead to the successful initiation of sulfonylurea therapy. CONCLUSIONS: This new ABCC8 mutation is associated with neonatal hyperinsulinism progressing within 10 years to insulinopenic diabetes. Consistent with in vitro findings, the patient responded to sulfonylurea treatment. The mechanism causing the relatively rapid loss in beta-cell function is not clear, but it may involve mutation-induced increased beta-cell apoptosis related to increased metabolic demand.

Prenatal diagnosis of congenital lipoid adrenal hyperplasia (CLAH) by estriol amniotic fluid analysis and molecular genetic testing.

OBJECTIVE: Congenital lipoid adrenal hyperplasia is an autosomal recessive condition due to mutations in Steroidogenic Acute Regulatory Protein (StAR) associated with the inability to synthesize all adrenal and gonadal steroids, resulting in adrenal insufficiency and failure to develop male genitalia in affected 46,XY individuals. We used two independent methods of prenatal diagnosis for CLAH. METHOD: CLAH was diagnosed prenatally by measuring amniotic fluid estriol, adrenal, and gonadal hormone levels, and by mutation analysis for the c.201-202 delCT mutation in StAR. RESULTS: Prenatal testing diagnosed one affected and three unaffected fetuses in two families at risk for CLAH. CONCLUSION: Prenatal testing for CLAH is effective, and targeted molecular genetic analysis should be considered in Palestinian families with a fetus with discordant 46,XY karyotype and female genitalia on ultrasound, and low maternal serum estriol.

Impaired glucose-stimulated insulin secretion is coupled with exocrine pancreatic lesions in the Cohen diabetic rat.

OBJECTIVE: The Cohen diabetes-sensitive rat develops postprandial hyperglycemia when fed a high-sucrose, copper-poor diet, whereas the Cohen diabetes-resistant rat maintains normoglycemia. The pathophysiological basis of diabetes was studied in the Cohen diabetic rat centering on the interplay between the exocrine and endocrine compartments of the pancreas. RESEARCH DESIGN AND METHODS: Studies used male Cohen diabetes-sensitive and Cohen diabetes-resistant rats fed 1-month high-sucrose, copper-poor diet. Serum insulin and glucose levels were measured during glucose and insulin tolerance tests. The pancreas was evaluated for weight, insulin content, macrophage, and fat infiltration. Glucose-stimulated insulin secretion (GSIS) was determined in isolated perfused pancreas and in islets. RESULTS: Hyperglycemic Cohen diabetes-sensitive rats exhibited reduced pancreatic weight with lipid deposits and interleukin-1beta-positive macrophage infiltration in the exocrine pancreas. Islet morphology was preserved, and total pancreatic insulin content did not differ from that of Cohen diabetes-resistant rats. Lipids did not accumulate in skeletal muscle, nor was insulin resistance observed in hyperglycemic Cohen diabetes-sensitive rats. Intravenous glucose-tolerance test revealed markedly elevated glucose levels associated with diminished insulin output. Insulin release was induced in vivo by the non-nutrient secretagogues arginine and tolbutamide, suggesting a selective unresponsiveness to glucose. Decreased GSIS was observed in the isolated perfused pancreas of the hyperglycemic Cohen diabetes-sensitive rat, whereas islets isolated from these rats exhibited glucose-dependent insulin secretion and proinsulin biosynthesis. CONCLUSIONS: The association of the in vivo insulin secretory defect with lipid accumulation and activated macrophage infiltration in the exocrine pancreas suggests that changes in the islet microenvironment are the culprit in the insulin secretory malfunction observed in vivo.

The role of reactive oxygen species in diabetes-induced anomalies in embryos of Cohen diabetic rats.

The role of the antioxidant defense mechanism in diabetes-induced anomalies was studied in the Cohen diabetes-sensitive (CDs) and -resistant (CDr) rats, a genetic model of nutritionally induced type 2 diabetes mellitus. Embryos, 12.5-day-old, of CDs and CDr rats fed regular diet (RD) or a diabetogenic high-sucrose diet (HSD) were monitored for growth retardation and congenital anomalies. Activity of superoxide dismutase (SOD) and catalase-like enzymes and levels of ascorbic acid (AA), uric acid (UA), and dehydroascorbic acid (DHAA) were measured in embryonic homogenates. When fed RD, CDs rats had a decreased rate of pregnancy, and an increased embryonic resorption. CDs embryos were smaller than CDr embryos; 46% were maldeveloped and 7% exhibited neural tube defects (NTDs). When fed HSD, rate of pregnancy was reduced, resorption rate was greatly increased (56%; P < .001), 47.6% of the embryos were retrieved without heart beats, and 27% exhibited NTD. In contrast, all the CDr embryos were normal when fed RD or HSD. Activity of SOD and catalase was not different in embryos of CDs and CDr rats fed RD. When fed HSD, levels of AA were significantly reduced, the ratio DHAA/AA was significantly increased, and SOD activity was not sufficiently increased when compared to embryos of CDr. The reduced fertility of the CDs rats, the growth retardation, and NTD seem to be genetically determined. Maternal hyperglycemia seems to result in environmentally induced embryonic oxidative stress, resulting in further embryonic damage.